A TIME'S MEMORY

Hong Kong, Additional overseas case of Severe Respiratory Disease associated with Novel Coronavirus closely monitored by DH (May 24 2013)

2013-05-24T08:36:00.001+02:00

[Source: Department of Health, Hong Kong PRC SAR, full text: (LINK).]

Additional overseas case of Severe Respiratory Disease associated with Novel Coronavirus closely monitored by DH

The Department of Health (DH) is today (May 24) closely monitoring an additional case of Severe Respiratory Disease associated with Novel Coronavirus reported to the World Health Organization (WHO) by the Kingdom of Saudi Arabia (KSA).

According to the WHO, the fatal case was reported from the central part of KSA and is not related to the cluster of cases reported from the eastern part of the country. The patient was a 63-year-old man with an underlying medical condition who was admitted to a hospital with acute respiratory distress on May 15 and died on May 20. Investigation into contacts of this case is ongoing.

Separately, the KSA authorities are also continuing the investigation into the outbreak that began in a health care facility in the beginning of April in the Eastern KSA. To date, a total of 22 patients including 10 deaths have been reported from this outbreak.

This brings the latest global number of confirmed cases of Severe Respiratory Disease associated with Novel Coronavirus to 44, including 22 deaths.

"The Centre for Health Protection (CHP) of the DH will seek more information on the case from the WHO and the relevant health authorities. The CHP will stay vigilant and continue to work closely with the WHO and overseas health authorities to monitor the latest developments of this novel infectious disease," a DH spokesman said.

Locally, the CHP will continue its surveillance mechanism with public and private hospitals, practising doctors and the airport for any suspected case of severe respiratory disease associated with novel coronavirus.

"No human infection with this virus has been identified so far in Hong Kong," the spokesman stressed.

"We would like to reassure the public that the Government will be as transparent as possible in the dissemination of information on cases of Severe Respiratory Disease associated with Novel Coronavirus. Whenever there is a suspected case, particularly involving patients with travel history to the Middle East and the affected areas, the CHP will release information to the public as soon as possible," the spokesman said.

In view of recent clusters reported in health-care facilities, health-care workers and hospitals are reminded to maintain vigilance against novel coronavirus and adhere to strict infection control measures while handling suspected cases in order to reduce the risk of transmission to other patients, health-care workers and visitors.

Health-care providers are advised to be vigilant among recent travellers returning from novel coronavirus-affected areas who develop severe acute respiratory infections. Patients' lower respiratory tract specimens should also be obtained for diagnosis when possible. Doctors are reminded that novel coronavirus infection should be considered even with atypical signs and symptoms, such as diarrhoea, particularly in patients who are immunocompromised.

Travellers returning from novel coronavirus-affected areas with respiratory symptoms are advised to wear face masks, seek medical attention and reveal their travel history to doctors.

The spokesman reminded members of the public to take heed of personal hygiene:

Wash hands before touching the eyes, nose and mouth;

Wash hands before eating or handling food;

Wash hands after using the toilet;

Wash hands after sneezing or coughing and cleaning the nose; and

Avoid direct contact with animals, birds or poultry.

Members of the public may visit the CHP's website for more information on Severe Respiratory Disease associated with Novel Coronavirus (www.chp.gov.hk/en/view_content/26511.html) or personal hygiene (www.chp.gov.hk/en/content/9/460/19899.html).

Ends/Friday, May 24, 2013
Issued at HKT 11:22
NNNN

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Earthquake M 8.2, Sea of Okhotsk (USGS, May 24 2013)

2013-05-24T08:31:00.001+02:00

[Source: USGS, full text: (LINK).]

Earthquake M 8.2, Sea of Okhotsk

‎24 May ‎2013

Friday, May 24, 2013 05:44:49 UTC
Friday, May 24, 2013 04:44:49 PM at epicenter
Depth: 601.80 km (373.94 mi)

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Infectivity, Transmission, and Pathology of Human H7N9 Influenza in Ferrets and Pigs (Science, abstract, edited)

2013-05-23T21:46:00.001+02:00

[Source: Science, full text: (LINK). Abstract, edited.]

Published Online May 23 2013

Science DOI: 10.1126/science.1239844

Report

Infectivity, Transmission, and Pathology of Human H7N9 Influenza in Ferrets and Pigs

H. Zhu¹,²,³,*, D. Wang⁸,*, D. J. Kelvin⁴,⁵,⁶, L. Li¹, Z. Zheng¹, S.-W. Yoon⁷, S.-S. Wong⁷, A. Farooqui⁴, J. Wang¹,³, D. Banner⁵, R. Chen¹, R. Zheng¹, J. Zhou¹,²,³, Y. Zhang¹, W. Hong¹, W. Dong⁴, Q. Cai¹, M. H. A. Roehrl⁵,⁶, S. S. H. Huang⁵,⁶, A. A. Kelvin⁴,⁵, T. Yao¹, B. Zhou², X. Chen², G. M. Leung³, L. L. M. Poon²,³, R. G. Webster⁷, R. J. Webby⁷, J. S. M. Peiris²,³, Y. Guan¹,²,³,†, Y. Shu⁸,†

Author Affiliations: ¹Joint Influenza Research Centre (SUMC/HKU), Shantou University Medical College, Shantou, PR China. ²State Key Laboratory of Emerging Infectious Diseases (HKU-Shenzhen Branch), Shenzhen Third People’s Hospital, Shenzhen, PR China. ³State Key Laboratory of Emerging Infectious Diseases/Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong SAR, PR China. ⁴Joint Vaccine Research Centre (SUMC/UHN). Shantou University Medical College, Shantou, PR China. ⁵University Health Network, Toronto, Canada. ⁶University of Toronto, Canada. ⁷Division of Virology, Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA. ⁸National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Key Laboratory for Medical Virology, National Health and Family Planning Commission, Beijing, PR China.

†Corresponding author. E-mail: yguan@hku.hk (Y.G.); yshu@cnic.org.cn (Y.S.)

* These authors contributed equally to this work.

Abstract

The emergence of the H7N9 influenza virus in humans in Eastern China has raised concerns that a new influenza pandemic could occur. Here, we used a ferret model to evaluate the infectivity and transmissibility of A/Shanghai/2/2013 (SH2), a human H7N9 virus isolate. This virus replicated in the upper and lower respiratory tracts of the ferrets and was shed at high titers for 6 to 7 days, with ferrets showing relatively mild clinical signs. SH2 was efficiently transmitted via direct contact, but less efficiently by airborne exposure. Pigs could be productively infected by SH2 and shed virus for 6 days but were unable to transmit the virus to other animals. Under appropriate conditions human-to-human transmission of the H7N9 virus may be possible.

Received for publication 30 April 2013. Accepted for publication 20 May 2013.

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Inefficient influenza H7N9 virus aerosol transmission among ferrets (Virology Blog, May 23 2013)

2013-05-23T21:38:00.001+02:00

[Source: Virology Blog, full text: (LINK).]

Inefficient influenza H7N9 virus aerosol transmission among ferrets

‎23 May ‎2013 | Vincent Racaniello

There have been 131 confirmed human infections with avian influenza H7N9 virus in China, but so far there is little evidence for human to human transmission. Three out of four patients report exposure to animals, ‘mostly chickens‘, suggesting that most of the infections are zoonoses. Whether or not the virus will evolve to transmit among humans is anyone’s guess. Meanwhile it has been found that one of the H7N9 virus isolates from Shanghai can transmit by aerosol among ferrets, albeit inefficiently.

(…)

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Cause of respiratory illness cluster in southeast Alabama determined (ADPH, May 23 2013, edited)

2013-05-23T20:59:00.001+02:00

[Source: Alabama Department of Health, full PDF document: (LINK). Edited.]

NEWS RELEASE

ALABAMA DEPARTMENT OF PUBLIC HEALTH, RSA Tower 201 Monroe Street, Suite 914 Montgomery, AL 36104, Phone 334-206-5300 Fax 334-206-5534, www.adph.org

Cause of respiratory illness cluster in southeast Alabama determined

FOR IMMEDIATE RELEASE

CONTACT: Mary McIntyre, M.D., M.P.H., (334) 206-5971, Corey Kirkland, (334) 792-9070

The Alabama Department of Public Health (ADPH) and the Houston County Health Department have determined that the cause of a respiratory illness cluster in southeast Alabama was a combination of influenza A, rhinovirus, the virus associated with the common cold, and bacterial pneumonia.

“This is good news. Testing has ruled out avian flu and novel coronavirus,” said State Health Officer Dr. Don Williamson.

Earlier this month, seven patients were admitted to the hospital with fever, cough and shortness of breath with no known cause for their illness. Public health officials began an epidemiological investigation to interview the families of the patients about travel and exposure. Specimens were requested and submitted to the ADPH Bureau of Clinical Laboratories in Montgomery. Of the seven patients whose specimens were submitted, six were found to be positive for either influenza A, rhinovirus or a combination of the two, and three patients were found to have bacterial pneumonia. Two of the seven patients eventually died.

Dr. Mary McIntyre, assistant state health officer for disease control and prevention, said, “While enhanced surveillance associated with this cluster is no longer necessary, health care providers are encouraged to continue routine year-round influenza surveillance activities and submit specimens to the state laboratory for testing.”

Health care providers should always use standard precautions when dealing with patients with respiratory illness.

If you or your family members have respiratory symptoms of fever, cough and shortness of breath, please contact your health care provider to be evaluated. In addition, everyone should take steps to prevent transmission of disease and are reminded to follow these precautions:

Cover your cough or sneeze with a sleeve or tissue.
Wash your hands often with soap and water, especially after you cough or sneeze. Alcoholbased hand sanitizers are also effective.
Avoid touching your mouth, eyes and nose with your hands. Try to avoid close contact with sick people.
If you get sick, stay home and limit contact with others to avoid infecting them.

-30- 5/23/13

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A comparison of rapid point-of-care tests for the detection of avian influenza A(H7N9) virus, 2013 (Euro Surveill., abstract, edited)

2013-05-23T19:05:00.001+02:00

[Source: Eurosurveillance, full text: (LINK). Edited.]

Eurosurveillance, Volume 18, Issue 21, 23 May 2013

Rapid communications

A comparison of rapid point-of-care tests for the detection of avian influenza A(H7N9) virus, 2013

C Baas¹^,2, I G Barr¹^,2, R A Fouchier³, A Kelso¹, A C Hurt ¹^,2

World Health Organization Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory (VIDRL), Melbourne, Victoria, Australia
Monash University, School of Applied Sciences, Churchill, Victoria, Australia
Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands

________

Citation style for this article: Baas C, Barr IG, Fouchier RA, Kelso A, Hurt AC. A comparison of rapid point-of-care tests for the detection of avian influenza A(H7N9) virus, 2013. Euro Surveill. 2013;18(21):pii=20487. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20487
Date of submission: 21 May 2013

________

Six antigen detection-based rapid influenza point-of-care tests were compared for their ability to detect avian influenza A(H7N9) virus. The sensitivity of at least four tests, standardised by viral infectivity (TCID₅₀) or RNA copy number, was lower for the influenza A(H7N9) virus than for seasonal A(H3N2), A(H1N1)pdm09 or other recent avian A(H7) viruses. Comparing detection limits of A(H7N9) virus with Ct values of A(H7N9) clinical specimens suggests the tests would not have detected most clinical specimens.

________

Human infections with influenza viruses derived directly from wild birds or poultry are relatively rare, although since 2003, over 600 human infections with influenza A(H5N1) viruses have been detected, many of which were fatal [1]. During the same period, a small number of influenza A(H7) virus infections worldwide have also occurred in humans upon contact with infected poultry, generally resulting in mild symptoms such as conjunctivitis with occasional respiratory involvement and one death [2-4]. In contrast, China announced in March 2013 human infections with a novel reassortant avian influenza A(H7N9) virus which caused severe pneumonia resulting in a number of deaths [5]. Cases have occurred predominantly in men over 60 years of age living in urban areas, and most cases had a history of recent contact with poultry or poultry products [5]. By 16 May 2013, 131 human cases of influenza A(H7N9) virus infection, in 10 provinces and municipalities in eastern China, had been reported to the World Health Organization (WHO), of which 32 had resulted in death [6]. To date there have not been any reports of sustained human-to-human transmission of the influenza A(H7N9) virus, but the rapid emergence of the virus has led to significant concerns that it could in the future acquire human transmissibility and spread globally, causing the next influenza pandemic.

Rapid testing and diagnosis of possible human influenza A(H7N9) virus infections is an important diagnostic and public health task. An accurate diagnosis will allow the timely administration of antiviral therapy [7,8] and may also enable the quarantining of infected cases to prevent further spread of the virus. Real-time PCR is now considered the gold standard laboratory-based assay for the detection of influenza virus infections due to its high sensitivity and specificity [6] and, although such assays have already been developed for the detection of influenza A(H7N9) virus [6], they require a high level of laboratory expertise and may not be available in all places where cases occur.

Point-of-care tests (POCTs) based on antigen detection, however, are simple to use and are designed for use in a medical clinic or outpatient setting, enabling the rapid testing of patient specimens within 15 minutes [9]. POCTs have mostly been licensed for detection of seasonal human influenza viruses, for which they generally have good specificity but low sensitivity [10]. Recently however, some POCTs have been specifically developed to utilise automated readers which have resulted in improved sensitivity. For public health purposes, it is important to determine whether the new or existing POCTs can detect the novel influenza A(H7N9) virus, particularly as previous studies have found that some POCTs had poorer sensitivity in detecting avian influenza strains compared to circulating human seasonal influenza strains [9]. If POCTs could reliably detect influenza A(H7N9) virus at clinically relevant levels, they would be a useful adjunct to real-time PCR in the detection of possible human cases, especially where technical resources are limited.

We evaluated six widely available POCTs that are based on detection of the nucleoprotein antigen (Table 1) for their ability to detect the avian influenza A(H7N9) virus A/Anhui/01/2013 [5], compared with three other low pathogenic avian influenza A(H7) viruses (A/Northern Shoveller/Egypt-EMC/1/2012, A/Mallard/Netherlands/4/2010 and A/Mallard/Lithuania-EMC/2/2010), two human seasonal influenza A(H3N2) (A/Sydney/506/2013 and A/Victoria/361/2011) and two influenza A(H1N1)pdm09 viruses (A/Auckland/1/2009 and A/Brisbane/292/2010).

________

Table 1. Details of influenza point-of-care tests evaluated in this study

________
Methods

All viruses were cultured in Madin-Darby Canine Kidney (MDCK) cells at a low multiplicity of infection for at least one passage before testing. All viruses were harvested at near full cytopathic effect (CPE), supernatant was centrifuged at low speed to remove cell debris, and viruses were frozen at -70°C prior to testing. A mean tissue culture infectious dose 50 (TCID₅₀) per mL was determined for each virus, based on at least three independent assays. Viruses were standardised to an infectivity titre of 1x10⁶ TCID₅₀/mL and then diluted in phosphate-buffered saline (PBS) in half-log₁₀ dilutions. Real-time RT-PCR analysis was conducted on each virus dilution to determine a cycle threshold (Ct) value and RNA copy number, using an Applied Biosystems 7500 Fast cycler and the real-time RT-PCR primer and probe set recommended by the United States Centers for Disease Prevention and Control (US CDC) for the detection of influenza A matrix genes (version 4 April 2006). RNA copy number was calculated using a standard curve of RNA standards (10-fold dilutions) of known copy number prepared from a pGEMT-A/California/7/2009 matrix plasmid using the Riboprobe In Vitro Transcription System (Promega, United States).

Each virus dilution was then tested in each POCT according to the manufacturer’s instructions and a limit of detection (LOD), based on either the TCID₅₀/mL or the RNA copy number/µL, was determined. Standardising viruses by viral infectivity (TCID₅₀/mL) is the most widely used method for the evaluation of POCTs, however it does not account for defective viral particles which may react in these antigen-detection assays. Therefore comparison of the LOD based on both TCID₅₀/mL and RNA copy number/µL (which accounts for both infective and defective viruses) can be informative. Half-log₁₀ dilutions of influenza A/Anhui/01/2013 virus were prepared in duplicate and both sets tested with the six POCTs. The number of available test kits was not sufficient to conduct duplicate testing of the other seven viruses. The duplicate sets of influenza A/Anhui/01/2013 virus concentrations gave highly comparable LOD data, therefore data for only the first set is presented. Four of the kits were read by eye, while two POCTs (Veritor and Sofia) utilised a mechanical reader (Table 1).

Results

Based on the TCID₅₀/mL, the LOD of five of the six POCTs for the A/Anhui/01/2013 influenza A(H7N9) virus ranged from 1x10⁵ to 1x10^5.5 TCID₅₀/mL, with the Sofia and Directigen EZ detecting virus at the lower limit. The Clearview POCT was unable to detect the influenza A(H7N9) virus at any of the concentrations tested (1x10⁶TCID₅₀/mL or lower) (Table 2). In comparison, the LOD of the POCTs for the other influenza A(H7) viruses tested was generally better than that seen with the A/Anhui/01/2013 virus, with some tests detecting virus levels as low as 1x10²TCID₅₀/mL. Seasonal influenza A viruses were also more easily detected by most POCTs than the influenza A(H7N9) virus, with the Sofia kit performing best: LOD ranging from 1x10² to 1x10³ TCID₅₀/mL for the human influenza A(H3N2) and A(H1N1)pdm09 viruses.

________

Table 2. TCID50 limit of detection of the influenza point-of-care tests evaluated in this study

________
Comparison of POCT LODs based on RNA copy number/µL showed similar results to those based on TCID₅₀/mL for four of the kits (Binax Now, Clearview, Veritor and Sofia). These POCTs were less sensitive for the detection of the influenza A(H7N9) virus compared to the seasonal or other influenza A(H7) viruses (Table 3). However, for the SD Bioline and the Directigen EZ tests, comparison of the LODs based on RNA copy number/µL showed that influenza A(H7N9) was detected at a similar sensitivity to the other viruses (Table 3).

________

Table 3. RNA copy number and Ct value limit of detection of the influenza point-of-care tests evaluated in this study

________
LODs based on RNA copy number/µL or Ct also allowed an estimate of the expected performance of the POCTs in detecting influenza A(H7N9) virus in clinical samples (Figure). Comparison of the published Ct values of clinical samples from patients with confirmed influenza A(H7N9) infection [11] suggested that five of the six POCTS would have detected only one of the four influenza A(H7N9)-positive clinical specimens, with the other three specimens being outside the LOD of these assays (Figure).

________

Figure. Mean Ct limit of detection for influenza A/Anhui/01/2013 in point-of-care tests compared with Ct values reported for four influenza A(H7N9) cases confirmed by RT-PCR

________
Discussion
For all viruses tested, the Sofia POCT, which uses an automated reader, had the highest sensitivity. The BD Veritor test, which also uses an automated reader, had comparable sensitivity to the BD Directigen EZ and the Binax Now tests, both of which are read by eye. The Clearview and SD Bioline POCTs demonstrated the poorest sensitivity.
It is important to note that both the Clearview and the BD Veritor tests are only approved for analysis of swab specimens, therefore the test method used here may not have been appropriate. Similarly, all POCT assays may perform better using a particular specimen type, which was not tested here. The collection of the virus sample used for the Clearview and the BD Veritor POCTs (dipping the swab into liquid and waiting at least 15 seconds for absorption) resulted in a sample volume of approximately 50 μL which, when combined with the recommended diluent volume, resulted in the lowest concentrations of virus used in this evaluation (Table 1).
Other limitations of this study include the use of only a single influenza A(H7N9) isolate A/Anhui/01/2013 (although this virus is genetically closely related to other human influenza A(H7N9) viruses for which sequences have been reported) and the fact that clinical specimens were not available for analysis. It is also important to note that these POCTs have not been primarily designed or licensed to detect influenza A(H7N9) viruses or other avian-derived viruses.
Nevertheless, this study does demonstrate that the sensitivity of at least four of the six evaluated POCTs is lower for the novel influenza A(H7N9) virus than for seasonal influenza viruses and the other avian influenza A(H7) viruses tested. Comparison with published Ct values for clinical specimens from influenza A(H7N9) patients suggested that these POCTs may not detect the majority of influenza A(H7N9) cases, particularly if samples are taken late in the course of disease. Therefore RT-PCR remains the diagnostic test of choice for the testing of suspected influenza A(H7N9) influenza cases.

________

Acknowledgements

The authors are grateful to Dr Yuelong Shu and Dr Dayan Wang, WHO Collaborating Centre for Reference and Research on Influenza, Chinese Center for Disease Control and Prevention, Beijing, China, for providing the A/Anhui/1/2013 A(H7N9) virus. We are grateful to Heidi Peck, WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, for preparing the plasmid used for RNA quantitation. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health and Ageing. RF was financed through NIAID-NIH contract HHSN266200700010C.

Conflict of interest

None declared

Authors’ contributions

Designed the study: CB, IB, AH. Analysed and interpreted the data: CB, RF, AK, IB and AH. Drafted the article: CB and AH. Revised the article: CB, RF, AK, IB and AH.

________

References

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Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA, Munster V, et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci U S A. 2004;101(5):1356-61.
http://dx.doi.org/10.1073/pnas.0308352100
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Centers for Disease Control and Prevention (CDC). Notes from the field: Highly pathogenic avian influenza A (H7N3) virus infection in two poultry workers--Jalisco, Mexico, July 2012. MMWR Morb Mortal Wkly Rep. 2012;61(36):726-7.
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Belser JA, Bridges CB, Katz JM, Tumpey TM. Past, present, and possible future human infection with influenza virus A subtype H7. Emerg Infect Dis. 2009;15(6):859-65.
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Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus. N Engl J Med. 2013;368(20):1888-97.
http://dx.doi.org/10.1056/NEJMoa1304459
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World Health Organization (WHO). Number of confirmed human cases of avian influenza A(H7N9) reported to WHO. Geneva: WHO. [Accessed: 16 May 2013]. Available from: http://www.who.int/influenza/human_animal_interface/influenza_h7n9/Data_Reports/en/index.html
Moscona A. Neuraminidase inhibitors for influenza. N Engl J Med. 2005;353(13):1363-73.
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Aoki FY, Macleod MD, Paggiaro P, Carewicz O, El Sawy A, Wat C, et al. Early administration of oral oseltamivir increases the benifits of influenza treatment. J Antimicrob Chemother. 2003;51(1):123-9.
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Sakai-Tagawa Y, Ozawa M, Tamura D, Le M, Nidom CA, Sugaya N, et al. Sensitivity of influenza rapid diagnostic tests to H5N1 and 2009 pandemic H1N1 viruses. J Clin Microbiol. 2010;48(8):2872-7.
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Hurt AC, Alexander R, Hibbert J, Deed N, Barr IG. Performance of six influenza rapid tests in detecting human influenza in clinical specimens. J Clin Virol. 2007;39(2):132-5.
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Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet. 2013; 25 April: pii: S0140-6736(13)60903-4.
http://dx.doi.org/10.1016/S0140-6736(13)60903-4

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Hong Kong, Update on number of suspected human cases of avian influenza A(H7) notified (May 23 2013)

2013-05-23T18:43:00.001+02:00

[Source: Centre for Health Protection, Hong Kong PRC SAR, full text: (LINK).]

Update on number of suspected human cases of avian influenza A(H7) notified

The Centre for Health Protection (CHP) of the Department of Health (DH) today (May 23) provided an update on the latest number of suspected human cases of avian influenza A(H7) notified to the CHP, including cases fulfilling reporting criteria and remaining ones not fulfilling reporting criteria.

From noon yesterday (May 22) to noon today, the CHP received notification of two cases which fulfilled reporting criteria, and two cases which did not fulfil reporting criteria. Cases are detailed in the Attachment.

This brings the total number of notifications received by the CHP since March 31 of cases fulfilling reporting criteria of suspected human cases of avian influenza A(H7) to 31, and the total number of notifications not fulfilling reporting criteria to 107.

A DH spokesman urged travellers not to visit wet markets with live poultry in the affected areas, and to avoid direct contact with poultry, birds or their droppings. If contact has been made, they should thoroughly wash hands with soap and water.

"Influenza A(H7) is a statutorily notifiable infectious disease in Hong Kong.’’

‘’Locally, no confirmed human cases of avian influenza A(H7N9) have been recorded so far," the spokesman stressed.

The spokesman reminded doctors to report to the CHP any suspected case of influenza A(H7). The Public Health Laboratory Services Branch of the CHP is ready to receive and test specimens whenever necessary.

The public may visit the CHP's avian influenza page (www.chp.gov.hk/en/view_content/24244.html) for further information.

Ends/Thursday, May 23, 2013
Issued at HKT 17:43
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Hong Kong, No new human case of avian influenza A(H7N9) on Mainland (May 23 2013)

2013-05-23T18:41:00.001+02:00

[Source: Centre for Health Protection, Hong Kong PRC SAR, full text: (LINK).]

No new human case of avian influenza A(H7N9) on Mainland

The Centre for Health Protection (CHP) of the Department of Health (DH) verified with the National Health and Family Planning Commission that there was no new human case of avian influenza A(H7N9) on the Mainland today (May 23).

As of 9pm today, there have been 130 laboratory confirmed cases of avian influenza A(H7N9) on the Mainland, including Zhejiang (46 cases), Shanghai (33 cases), Jiangsu (27 cases), Jiangxi (six cases), Fujian (five cases), Anhui (four cases), Henan (four cases), Shandong (two cases), Hunan (two cases) and Beijing (one case).

A DH spokesman stressed that the CHP is closely monitoring the situation and will continue to maintain close liaison with the Mainland health authorities for more information, as well as keeping a close eye on the latest advice from the World Health Organization.

The spokesman also reminded travellers, especially those returning with fever or respiratory symptoms from Shanghai, Jiangsu, Zhejiang, Anhui, Henan, Beijing, Shandong, Jiangxi, Fujian, Hunan or Guangdong, to wear facial masks immediately, seek medical attention, and reveal their travel history to doctors. Healthcare professionals should also pay special attention to those who might have had contact with birds, poultry or their droppings in affected areas.

"Locally, no confirmed human cases of avian influenza A(H7N9) have been recorded so far," the spokesman stressed.

"We would like to reassure the public that the Government will be as transparent as possible in the dissemination of information on human cases of avian influenza A(H7N9). Whenever there is a suspected case, the CHP will release information to the public as soon as possible," the spokesman added.

The spokesman urged travellers not to visit wet markets with live poultry in the affected areas and to avoid direct contact with poultry, birds or their droppings. If contact has been made, they should thoroughly wash their hands with soap and water.

Members of the public should remain vigilant and are reminded to take heed of the following preventive advice against avian influenza:

Poultry and eggs should be thoroughly cooked before eating;

Wash hands frequently with soap, especially before touching the mouth, nose or eyes, handling food or eating; after going to the toilet or touching public installations or equipment such as escalator handrails, elevator control panels or door knobs; or when hands are dirtied by respiratory secretions after coughing or sneezing;

Cover the nose and mouth while sneezing or coughing, and hold the spit with a tissue and put it into a covered dustbin;

Avoid crowded places and contact with fever patients; and

Wear a mask when respiratory symptoms develop or when taking care of fever patients.

The public may visit the CHP's avian influenza page (www.chp.gov.hk/en/view_content/24244.html) for further information.

Ends/Thursday, May 23, 2013
Issued at HKT 21:14
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Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus (Emerg Infect Dis., edited)

2013-05-23T18:31:00.001+02:00

[Source: Emerging Infectious Diseases Journal, full page: (LINK). Edited.]

Volume 19, Number 5—May 2013

Research

Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus

Matthew Cotten, Tommy T. Lam, Simon J. Watson, Anne L. Palser, Velislava Petrova, Paul Grant, Oliver G. Pybus, Andrew Rambaut, Yi Guan, Deenan Pillay, Paul Kellam , and Eleni Nastouli

Author affiliations: Wellcome Trust Sanger Institute, Hinxton, UK (M. Cotten, S.J. Watson, A.L. Palser, V. Petrova, P. Kellam); University of Oxford, Oxford, UK (T.T. Lam, O.G. Pybus); University College London, London, UK (D. Pillay, P. Kellam); University College London Hospitals,; London (P.Grant, E. Nastouli); University of Edinburgh, Edinburgh, Scotland, UK (A. Rambaut); Fogarty International Center–National Institutes for Health, Bethesda, Maryland, USA (A. Rambaut); The University of Hong Kong, Hong Kong (Y. Guan)

Abstract

A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient’s sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

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The ability of coronaviruses (CoVs) to infect multiple species and to rapidly change through recombination presents a continuing human health threat. The epidemic of severe acute respiratory syndrome (SARS) during 2003–2004 during which a CoV transmitted from bats to civet cats and then to humans demonstrated this potential (reviewed in [1,2]). Recently, a novel human betacoronavirus (betaCoV) was found to be associated with at least 13 human infections, 7 of which were fatal (3–7). One of the viruses, EMC/2012, has been sequenced, and its sequence similarity to several bat CoVs suggested an animal origin, but a definitive bat species of origin has not yet been identified (3). Additional genome sequences from this virus are needed to aid diagnostics, monitor population dynamics, identify the animal source, and characterize mechanisms of pathogenesis. The large size (30,000 nt) and high variability of CoV RNA genomes present a challenge for sequencing.

We describe a strategy for rapidly designing the primers necessary for reverse transcription and cDNA amplification of such diverse RNA viruses and report the full-genome determination of the novel CoV directly from patient sputum using next-generation short-read sequencing. Full genomes from 2 epidemiologically unlinked novel CoV infections separated in time by >2 months were analyzed to gain clues to 2 major questions: what are the precursors of the virus, and how long has the virus been circulating in its current form?

Materials and Methods

Primer Design

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Figure 1

Figure 1 - A) Primers designed for reverse transcription and overlapping PCR amplification of the novel coronavirus (CoV). Dots indicate the predicted binding site of each primer along the EMC/2012 genome...

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Fifteen betaCoV full genomes with the closest homology to EMC/2012 (GenBank accession no. JX869059) were analyzed to identify all possible primer-like sequences (melting temperature 58°C–60°C, guanine plus cytosine content 35%–60%) yielding 357,198 potential primers. The 30-kb JX869059 CoV genome was divided into fifteen 2.5-kb overlapping amplicons, and the 2 highest frequency sequences mapping in the 5′ and 3′ 250 bp of each amplicon were selected. Reverse complements of the 3′ mapping sequences were prepared, resulting in a set of 60 primers for the 15 amplicons (4 primers/amplicon). Three additional primers were added for the extreme termini of the genome. The algorithm also prepared a virtual PCR map of the predicted binding and amplicon size for the primer set on CoV genomes. A map of the primer mapping positions and the predicted PCR products using EMC/2012 as the target is shown in Figure 1, panel A. The primer sequences and details of their use are listed in the Technical Appendix [PDF - 71 KB - 2 pages] Table.

Sample History

The patient, originally from Qatar, visited Saudi Arabia, where he became unwell with severe respiratory disease and renal failure and was transferred to a hospital in London, UK (4). Viral RNA was extracted from a sputum sample collected on September 19, 2012, sixteen days after symptom onset. Real-time PCR was performed by using an assay targeting the upE gene (6) and confirmed by using primers and probe targeting the RNA-dependent RNA polymerase (RdRp) gene. RdRpTaq1: (5′-GAC TAA TCG CCA GTA CCA TCA G-3′), RdRpTaq2: (5′-GAA CTT TGT AGT ACC AAT GAC GCA-3′), RdRpProbe: (5′-FAM-ATG CTT AAG TCC ATG GCT GCA ACT CGT GGA G–BHQ-3′). The assays gave cycle threshold values of 17.83–18.01 for the upE and RdRp targets. A 100-μL sample of sputum was lysed with the addition of an equal volume of Qiagen Lysis Buffer AL (QIAGEN, Hilden. Germany). RNA was extracted from 200 µL of this material (final elution volume 60 µL) by using the EZ1 Virus Mini Kit v2.0 and EZ1 instrument (QIAGEN).

Reverse Transcription and PCR Amplification

Reverse transcription was performed at 50°C for 60 min by using the amplicon reverse primers. PCRs were then performed with forward and reverse primers for each amplicon (15 reactions) for 35 cycles (98°C, 10 sec; 54.5°C, 30 sec; 72°C, 2.5 min) with a final 10-min 72°C extension. A 3-µL aliquot of each reaction was analyzed by electrophoresis, and each reaction showed the expected 2–2.5-kb products (Figure 1, panel B). A detailed protocol is available on request from the authors.

Sequencing

The PCR products of amplicons were combined into 4 pools of approximately equal molarity and processed into standard Illumina multiplex libraries (Illumina, San Diego, CA, USA) with each pool bearing a unique bar code sequence. Libraries were sequenced by Illumina MiSeq generating 150-bp paired end reads. The resulting Illumina read sets were processed to remove adaptor and primer sequences and quality controlled to ensure a median read Phred quality score of 30 by using QUASR (8). The 5′- and 3′-most primers were derived directly from the first 27 nt or last 31 nt of the EMC/2012 genome. These primer sequences were not trimmed from the reads, which might mask sequence difference between EMC/2012 and England/Qatar/2012 at these positions.

Genome Assembly

Reference-based mapping and de novo assembly methods were applied to the data for assembly into viral genomes. Reference-based mapping was performed against the EMC/2012 genome by using the Burrows-Wheeler Aligner software package [9]). For de novo assembly, maximum contig lengths were obtained by using subsets of 30,000–60,000 reads. Therefore, random subsets of reads were extracted from the readset and assembled by using Velvet version 1.2.07 (10,11) and VelvetOptimiser (12). The de novo assembled sequences were used to confirm the validity of the reference-based sequence and showed that the de novo assembly and the reference-based mapping produced identical sequences, apart from several small gaps near the termini of the de novo assembled sequence (results not shown). The complete genome sequenced here is named England/Qatar/2012 and is available in GenBank (accession no. KC667074).

Sequence Alignment

The complete England/Qatar/2012 genome was combined with 46 previously published complete genomes of the α-(group 1), β-(group 2), γ-(group 3), and δ-(group 4) CoVs. The betaCoV complete genomes encompassed the a, b, c, and d lineages, as well as the 2 genomes of the novel human betaCoV, EMC/2012 (GenBank accession no. JX869059) and England1 (GenBank accession no. KC164505), the latter from the same patient as studied here. The sequences were aligned by using MUSCLE (13) within MEGA5 (14). Poorly aligned regions and the genes absent in some CoV genotypes were excluded from the alignment, resulting in a virtual concatenation of the open reading frame (ORF) 1ab, S, E, M, and N genes. A second alignment using a 396-bp region of the RdRp was generated similarly. This shorter alignment included strains that are genetically close to England/Qatar/2012 but lack complete genome sequences in GenBank.

Phylogenetic Methods

Maximum-likelihood (ML) phylogenetic trees were inferred from the sequence alignments by using PhyML version 3.0 (15). Phylogenies inferred from nucleotide and amino acid sequences used the general-time reversible and Whelan-and-Goldman substitution models, respectively. A discrete-Γ distribution of 4 rate categories (Γ₄) was used to model among-site heterogeneity. The robustness of the tree topology was assessed by bootstrap analysis of 1,000 pseudo-replicates of the sequences.

Molecular Clock Dating

The evolutionary time scale of 2 novel human betaCoVs, EMC/2012 and England/Qatar/2012, was estimated by using a strict clock model under the Bayesian Markov Chain Monte Carlo framework in BEAST version 1.7.4 (16). The Hasegawa-Kishino-Yano nucleotide substitution model was used with a discrete-Γ distribution of 4 rate categories (Γ₄) to enable among-site heterogeneity. A simple constant coalescent prior on the age of the divergence was used. With only 2 samples, estimating a rate of evolution was not possible, so we conditioned our date estimates on a range of fixed rates from 1.0 × 10⁻⁴ to 5.0 × 10⁻³ substitutions/site/year to enable comparison with plausible values. For each fixed rate value, a total of 10⁷ Bayesian Markov Chain Monte Carlo states were computed, with the first 10% discarded as burn-in.

Results

Comparison of England1, England/Qatar/2012, and EMC/2012

The index genome for this virus, EMC/2012, was originally obtained from a Saudi Arabian patient in July 2012 after 6 passages in cell culture (3). Our England/Qatar/2012 sequence was derived directly from the sputum of a Qatari patient receiving care in London in September 2012. A consensus genome from the same Qatari patient, but from a lower respiratory tract sample obtained later in the infection, is also available (England1, GenBank accession no. KC164505). The 3 genomes were aligned to determine sequence differences.

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Figure 2

Figure 2 - A) Sequence differences among EMC/2012, England/Qatar/2012 and England1. The sequences of the 3 genomes were aligned, and differences between the sequence of England/Qatar/2012 and England1 (upper row) or...
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The England/Qatar/2012 and England1 genomes show only nucleotide differences at the genome termini, with the England1 genome lacking 46 nt at the 5′ end and 42 nt at the 3′ end (Figure 2, panel A, Appendix). The England1 genome was generated by using 80 PCR products sequenced by Sanger dideoxy methods (17), and the high level of sequence identity between the England/Qatar/2012 and England1 sequences strongly validates the novel rapid sequencing methods described here.
The England/Qatar/2012 consensus genome has 30,021 (99.67%) of 30,119 nucleotides identical to the EMC/2012 genome. In addition to the single nucleotide differences, EMC/2012 shows an insertion of 6 nt starting at position 29639 and a single A insertion at 30661 relative to the England/Qatar/2012 genome. The sequence differences are evenly spread across the genome (Figure 2, panel A, Appendix; Table 1).
Deep sequencing data enable the generation of a consensus sequence from the majority nucleotide at each genome position and the identification of nonconsensus nucleotides at each position. The England/Qatar/2012 sample was sequenced to a high level of coverage (mean coverage = 4,444) compared with the previously reported England1 genome (2–11-fold), enabling insights into the variation consequent on within-host evolution. Setting a conservative estimate for total sequencing errors at 1% enables nucleotide variants present at >1% frequency to be considered true variants in the virus genome (Figure 2, panel B, Appendix). Variation is clearly detected across the virus genome with certain regions, such as nonstructural protein (NSP) NSP3, NSP5, NSP6, NSP12, and NSP14 showing increased levels of nonconsensus nucleotides (Table 2).
The predicted ORFs in England/Qatar/2012 are shown in Figure 2, panel C, Appendix. The ORF pattern is identical to EMC/2012, with 1 exception: England/Qatar/2012 has a G at position 27162 and a longer ORF 5, whereas EMC/2012 has an A (and the predicted ORF 5 is truncated). Van Boheemen et al. (3) had commented on mixed nucleotides (A–G) observed at this position. In addition, EMC/2012 had variation at position 11623 (U or G), whereas the England/Qatar/2012 genome has a U at this position.
An assessment of specific ORFs of the 2 viral genomes was performed because evolution of specific proteins is a key determinant of host range and defines cross-species transmission events that may be recent for this virus. As expected from the overall close homology of the 2 viruses, several ORFs show little or no change between the 2 genomes (Table 1). However, several ORFs show higher amino acid differences, including the nucleocapsid ORF N, and ORFs 3, 4a, and 8b (Table 1).
Of the 16 predicted nonstructural proteins encoded by ORF 1a and ORF 1b, 11 show no change at the amino acid level (Table 1), whereas 5 (NSP2, NSP3, NSP4, NSP13, and NSP15) show >0.3% difference (Table 1). As more sequences become available from this virus, such comparisons will yield clues about the adaptation to humans.

Close Phylogenetic Relationship with European Bat CoVs
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Figure 3

Figure 3 - Phylogenetic analyses of coronaviruses. A–F) Maximum-likelihood phylogenies of combined and each individual open reading frame (ORF), including ORF 1ab, S, E, M, and N. Previously defined viral lineages...
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A ML phylogenetic tree inferred from the whole genome alignment indicated that the 3 novel human betaCoVs sequences (England1, England/Qatar/2012, and EMC/2012) clustered closely, forming a monophyletic lineage that falls into group 2c (Figure 3, panel A, Appendix,). This novel human betaCoV lineage shares common ancestries with other group 2c bat CoV variants, including HKU4 and HKU5 strains isolated in southern People’s Republic of China (Figure 3, panel A, Appendix); however, the genetic distances between them remain substantial (≈70% similarity in nucleotide level). The novel human betaCoVs have even less similarity with group 2a, 2b, and 2d CoVs (<60% nt). Phylogenies constructed from individual ORFs including ORF 1ab, S, E, M, and N demonstrated a largely consistent phylogenetic position of the novel human betaCoVs, except for a small discordance in the order of branching between novel human betaCoV, HKU4, and HKU5 lineages (Figure 3, panels B–F, Appendix). Such phylogenetic incongruence could result from several possible evolutionary features of the virus, including evolutionary rate variation, homoplasy, and recombination, as well as from uncertainty in the alignment of distant sequences. Additional related sequences are needed to clarify this issue.
Because more CoV strains are available in GenBank for partial genomic sequences, we performed extensive BLAST searches (www.ncbi.nlm.nih.gov/blast) and identified several additional European bat CoV sequences sharing higher nucleotide sequence similarity (82.0%–87.7%) with the novel human betaCoVs. Because only partial RdRp sequences were available for these European bat CoVs, another ML phylogeny was constructed from this short region to examine their evolutionary relationships (Figure 3, panel G, Appendix). As noted, P.pipi/VM314/2008/NLD (GenBank accession no. GQ259977) identified in a Pipistrellus pipistrellus bat from the Netherlands (18) shows the closest sequence similarity (87.7%) and phylogenetic relationship to the novel human betaCoVs. In addition, 2 CoV sequences, H.sav/J/Spain/2007 (GenBank accession no. HQ184059; from Hypsugo savii, also known as P. savii) and E.isa/M/Spain/2007 (HQ184062; from Eptesicus isabellinus), obtained from bats from Spain (19) are also closely related to the novel human betaCoVs (Figure 3, panel G, Appendix).

Timing the Origin of Zoonosis
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Figure 4

Figure 4 - tMRCA analysis across a range of fixed evolutionary rates. The tMRCA of EMC/2012 and England/Qatar/2012 estimated from fixing a range of genomic evolutionary rates (1 × 10⁻⁴, 2...
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The time to the most recent common ancestor (tMRCA) of EMC/2012 (isolated June 13, 2012) and England/Qatar/2012 (isolated September 19, 2012) was estimated with a strict molecular clock model. A plot of estimated tMRCA of the 2 human strains as a function of the fixed rate of molecular evolution shows that with the fastest measured rate of human CoVs (2 × 10⁻³ substitutions/site/year for SARS-CoV [20]) the tMRCA (i.e., October 2011; 95% highest posterior density: August–December 2011) is older than the start of 2012, but with slower rates it is calculated to be much older (Figure 4 [21–23]). However, for evolutionary rates as high as those of human influenza A virus and HIV (e.g., 3–5 × 10⁻³ substitutions/site/year), the estimated tMRCA becomes compatible with the earliest disease report in Jordan in April 2012 (7).

Discussion
The reports of several human infections by similar strains of a novel betaCoV have raised global concern about a new SARS-like outbreak. Such worry is not misplaced, particularly when little is known about the origin and transmissibility of the virus and the natural history of the disease it causes. In this study, we presented a rapid deep sequencing method to obtain the complete genome sequence of the novel human betaCoV and its minor variants from a patient’s sputum sample, which provides a useful tool to study the origin, evolution, and detection of this novel CoV. Our detailed phylogenetic analyses of the viral genomes also provide additional clues to the emergence and evolution of this virus.
Mammalian zoonoses are often identified as the origin of new human CoV infections including OC43, NL63, and SARS-CoVs (24–27). In particular, bats, which maintain the largest diversity of CoVs (28), are thought to be a natural reservoir of the virus and thus a probable origin of the novel human betaCoV studied here. Indeed, the initial genetic study of the novel human betaCoV demonstrated its phylogenetic relatedness to the HKU5 and HKU4 bat CoVs in southern China (5). van Boheemen et al. found a bat CoV sequence from the Netherlands that clusters closely with EMC/2012 (3). In addition, most bat CoVs in the group 2c lineage, including the sequence from the Netherlands, were isolated from bat species of the Vespertilionidae family, whereas the SARS-like CoVs in group 2b lineage were mainly from bats from the Rhinolophidae family (29). In this study, we showed that 2 other CoVs from bats from Spain of Vespertilionidae again are also clustered near the novel human betaCoVs (Figure 3, panel G, Appendix), which further supports a European Vespertilionidae bat ancestry for this virus. However, such a genealogic link may be indirect and distant because a similar hypothesis for SARS-CoV has been under dispute: whether the CoV jumped from bats directly to humans or through civets or even through some other animals as intermediate hosts (30). Therefore, like SARS-CoV, the high dissimilarity between the bat CoVs and novel human betaCoVs makes it difficult to rule out the presence of other intermediate hosts transferring the virus from bats to humans and to confirm the geographic origin of the direct predecessor. With the current data, Europe and the Middle East would be plausible regions for more intensive pilot surveillance studies on bats and other animal reservoirs for this virus.
In the interest of public health, it is critical to determine whether these CoV infections in humans are the consequence of a single zoonotic event followed by ongoing human-to-human transmissions or whether the 3 geographic sites of infection (Jordan, Saudi Arabia, and Qatar) represent independent transmissions from a common nonhuman reservoir. The large genetic diversity of CoV maintained in animal reservoirs suggests that viruses that independently moved to humans from animals at different times and places are likely to be reasonably dissimilar in their genomes, possibly making the multiple transmission events model less likely. Further information is needed to confirm this point because the currently available data are limited. If we calibrate our molecular clock analysis using the evolutionary rate of Zhao et al. (20) estimated for SARS-CoV, we dated the tMRCA of EMC/2012 and England/Qatar/2012 viruses to early 2011. Therefore, if both sequenced viruses and the other cases descended from a single zoonotic event, then this tMRCA suggests that the novel virus has been circulating in human population for >1 year without detection and would suggest most infections were mild or asymptomatic. The rate would have to be considerably faster, of a magnitude observed for human influenza A virus, for the tMRCA to be compatible with the earliest known cases in April 2012. Perhaps more probable, therefore, is that the 13 known cases of this disease represent >1 independent zoonotic transmission from an unknown source. Viral sequence data from other patients infected with this novel human betaCoV will help to more accurately estimate the estimate a genomic evolutionary rate specific to this virus, which will then yield a tMRCA estimate closer to the actual time.
A major determinant of SARS pathology is the distribution of the host receptor that the virus has evolved to use for entry. A detailed analysis of EMC/2012 receptor use in cell culture (31) revealed that EMC/2012 was capable of infecting a range of mammalian cells, including human, pig, and bat cells, and the virus entry was independent of the ACE2 receptor. Comparing the S genes of England/Qatar/2012 predicts only 2 aa changes (in a 1,367-residue protein). Thus, this broad infection capability of EMC/2012 is likely to be valid for England/Qatar/2012 and would imply a higher possibility for the existence of other intermediate hosts transferring the virus from bats to humans.
Precise identification of the origin of this virus, defining its mode of evolution, and determining the mechanisms of viral pathogenesis will require full-genome sequences from all cases of human infection and substantially more sampling and sequencing from Vespertilionidae bats and other related animals. The sequencing method reported here markedly shortens the time required to process the clinical sample to genome assembly to 1 week and will provide a useful tool to study this novel virus.

The current number of confirmed novel CoV cases is 13, including 7 deaths (www.who.int/csr/don/2013_02_21/en/index.html). A third novel CoV genome sequence was posted on the Health Protection Agency website on February 18, 2013 (www.hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1317136246479). An analysis of the novel CoV genomes from the 3 dates suggests an evolution rate of 4 × 4e⁻⁴, which would give a tMRCA of early 2009. The analysis is available online at http://epidemic.bio.ed.ac.uk/coronavirus_analysis .

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Dr Cotten is a molecular virologist at the Wellcome Trust Sanger Institute in Hinxton, UK. His research interests are in virus evolution and development of novel deep sequencing technology and applying these interests to improving global health care.

Acknowledgments

We thank Bruce Macrae and Carmel Curtis for their assistance with the sample procurement and the Sanger Bespoke Illumina team for its support in rapidly sequencing England/Qatar/2012.

This work was supported by the Wellcome Trust and the European Community's Seventh Framework Programme (FP7/2007–2013) under the project EMPERIE, European Community grant agreement number 223498 and under the project PREDEMICS, grant agreement number 278433.

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Figures

Figure 1. . . . A) Primers designed for reverse transcription and overlapping PCR amplification of the novel coronavirus (CoV). Dots indicate the predicted binding site of each primer along the...
Figure 2. . . . A) Sequence differences among EMC/2012, England/Qatar/2012 and England1. The sequences of the 3 genomes were aligned, and differences between the sequence of England/Qatar/2012 and England1 (upper...
Figure 3. . . . Phylogenetic analyses of coronaviruses. A–F) Maximum-likelihood phylogenies of combined and each individual open reading frame (ORF), including ORF 1ab, S, E, M, and N. Previously defined...
Figure 4. . . . tMRCA analysis across a range of fixed evolutionary rates. The tMRCA of EMC/2012 and England/Qatar/2012 estimated from fixing a range of genomic evolutionary rates (1 ×...

Tables

Table 1. Nucleotide and amino acid differences between novel human betacoronaviruses EMC/2012 and England/Qatar/2012 major ORFs
Table 2. Nonconsensus variants detected in the sequence of a novel human betacoronavirus

Technical Appendix

Technical Appendix. [71 KB - 2 pages]

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Suggested citation for this article: Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, et al. Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus. Emerg Infect Dis [Internet]. 2013 May [date cited]. http://dx.doi.org/10.3201/eid1905.130057

DOI: 10.3201/eid1905.130057

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Laboratory Procedures - Serological detection of avian influenza A(H7N9) virus infections by turkey haemagglutination-inhibition assay, 23 May 2013 (WHO, edited)

2013-05-23T18:04:00.001+02:00

[Source: World Health Organization, full PDF document: (LINK). Edited.]

Laboratory Procedures - Serological detection of avian influenza A(H7N9) virus infections by turkey haemagglutination-inhibition assay, 23 May 2013

The WHO Collaborating Center for Reference and Research on Influenza at the Chinese National Influenza Center, Beijing, China, has made available attached laboratory procedures for serological detection of avian influenza A(H7N9) virus infections by turkey haemagglutination-inhibition assay.

For further information please contact us at: gisrs-whohq@who.int

SEROLOGICAL DETECTION OF AVIAN INFLUENZA A(H7N9) VIRUS INFECTIONS BY TURKEY HAEMAGGLUTINATION-INHIBITION ASSAY

INTRODUCTION

Serological diagnosis is an important approach when clinical specimens are unobtainable or when a laboratory does not have the resources required for virus isolation. The haemagglutination-inhibition (HAI) assay is a traditional method for assessing immune responses to influenza virus haemagglutinin (HA) and for identifying influenza virus field isolates. The HA protein on the surface of influenza virus agglutinates erythrocytes. Specific attachment of antibody to the antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on the erythrocytes. This effect inhibits haemagglutination and is the basis for the HAI. In general, a standardized quantity of HA antigen (4 HA units) is mixed with serially diluted anti-sera, and red blood cells are added to determine specific binding of antibody to the HA molecule. This assay is extremely reliable, relatively simple and inexpensive technique. Disadvantages of the HAI test include the need to remove nonspecific inhibitors which naturally occur in sera, to standardize antigen each time a test is performed, and the need for specialized expertise in reading the results of the test.

The receptor specificity of influenza virus HA correlates with the ability to agglutinate RBC from different animal species. Most avian influenza viruses preferentially bind to sialic acid receptors that contain N-acetylneuraminic acid α2,3-galactose (α2,3-Gal) linkages, while human influenza viruses preferentially bind to those containing N-acetylneuraminic acid α2,6-galactose (α2,6-Gal) linkages. We discovered that the novel H7N9 virus could bind to both avian-type (sialic acid α2, 3) and human-type (sialic acid α2, 6) receptors. The HAI assay, using turkey RBC that express a mixture of α2,3-Gal and α2,6-Gal linkages is a sensitive and relatively specific assay for detecting antibody response to current avian influenza A(H7N9) viruses in human sera following natural infection and vaccination and potentially, for detecting antibody to other avian subtypes. However, it is important to continuously monitor the specificity of this assay when new viruses are used, as there may be strain to strain variation in the detection of non-specific HAI antibody responses.

I. Materials Required

1. Virus strains

Live or beta-propriolactone (BPL)-inactivated viruses in allantoic fluid. Aliquot and store at -70oC.

2. Serum samples:

If serum is to be tested repeatedly, it is best to make several aliquots of the serum. Sera should not be repeatedly freeze-thawed and can be stored at -20 to -70oC. Both human and animal sera must be treated with receptor-destroying enzyme (RDE) to remove nonspecific inhibitors before use.

3. Buffers and Reagents

3.1. TRBC (Turkey red blood cell) in Acid Citrate Dextrose (ACD) solution

Preparation of ACD solution for collection of TRBC:

3.1.1. 22.0g/L sodium Citrate (Na3C6H5O7 . 2H2O)
3.1.2. 8.0g/L Citric acid (C6H8O7)
3.1.3. 24.5 g/L Dextrose
3.1.4. Sterile distilled water to 1L

3.2. Phosphate-buffered saline (PBS), 0.01M, pH 7.2.
3.3. Receptor destroying enzyme, RDE (II) “Seiken”, (Denka Seiken Co., Ltd, cat # 370013)

Note: Reconstitute each vial of RDE with 20 ml of PBS. Use immediately or freeze in single use aliquots at -20 oC or colder.

4. Equipment

4.1. 37oC water bath
4.2. 56oC water bath
4.3. Bench centrifuge
4.4. Class II Biological Safety Cabinet (BSC)
4.5. 4oC refrigerator
4.6. Freezer, - 70oC (for long term virus storage) or - 20oC (for serum storage)

5. Supplies

5.1. Centrifuge tubes (graduated conical 50ml and 2ml)
5.2. 96-well V bottom microtiter plates
5.3. Disposable reservoirs for multi-channel pipettes.
5.4. Assorted sterile pipettes and pipetting device; assorted microliter pipettes and disposable tips
5.5. 96-well PCR plate

Note that 96 sera can be treated in 1 plate.

II. Quality Control

A. Serum controls - make multiple aliquots of RDE-treated control sera and store at -20 to -70oC. Include both animal and if possible human negative and positive serum controls.

1. Positive serum control

1.1 For animal sera, use sera from infected ferrets or mice, or other (rabbit, sheep, goat) immunized animals.
1.2 For human sera, an optimal positive control would be acute and convalescent serum samples from a laboratory-confirmed H7N9 case that shows a > 4-fold rise in titer. Alternatively, a single high titered convalescent serum sample may also be used.

2. Negative serum control

2.1 For animal sera, use non-immune serum from same animal species used for negative control.
2.2 For human sera, use age-matched sera from individuals that have not been exposed to the particular virus strain in question to estimate the levels of non-specific antibody responses for a particular virus.
Screening of 50-100 sera from non-exposed anti-H7N9 antibody negative people is recommended to determine the specificity of the assay, each time a new virus is used.

B. Virus back titration

In each assay, include a virus back-titration of the working solution of the virus. Add 50μl of PBS to wells B-H of well 1 and 2 (duplicate). Add 100μl of the working solution of virus (= 8 HAU/50μl) to the first well (A1 and A2). Serially transfer 50μl down from well-A to H). Add 50μl of 1% TRBC and incubate 30min at RT.

C. RBC control

RBC controls allow adjustments in incubation times. There should be a RBC control on each plate, if possible.

III. PROCEDURE FOR HAI ASSAY

1. Generation of Stock Virus

Grow up virus to high titer in the allantoic cavity of 9 or 10-day old embryonated hens’ eggs.

Note: Any work with infectious highly pathogenic H7N9 virus must be performed in a BSL3 laboratory with enhancements according to CDC guidelines

1.1. Dilute virus sample (usually 1:100 to 1:50,000) in PBS + antibiotics (100 U/ml penicillin, 100 μg /ml streptomycin, and 100μg/ml gentamycin).
1.2. Pierce hole in the tops of eggs.
1.3. Inoculate eggs with diluted virus (100-200μl/egg) using a syringe fitted with a 22 gauge/1” needle. Seal hole in eggs with glue or paraffin wax.
1.4. Incubate eggs for 48 hr at 37°C.
1.5. Chill eggs (overnight at 4°C or 30-40 min at –20°C).
1.6. Remove tops of eggs and harvest allantoic fluid with pipette.
1.7. Clarify allantoic fluid by low-speed centrifugation (600 x g or 2000 rpm, 10 min).
1.8. Determine the HAU of the virus and aliquot virus in multiple ampoules (≈1.5ml/ampoules) and freeze at -70°C. H7N9 viruses should be inactivated by BPL. Complete inactivation should be confirmed before being brought into the BSL-2 laboratory for the HAI assay.

2. Treatment of reference anti-sera and human sera for inactivation of non-specific inhibitors

Non-antibody molecules present in serum are capable of binding to the influenza virus HA, resulting in nonspecific inhibition and leading to false interpretations. This effect is believed to occur because these serum components contain sialic acid residues that mimic the receptors of RBC, and compete with RBC receptors for the influenza HA. To perform a valid HAI test, one must ensure that the serum does not contain nonspecific inhibitors reactive with the virus antigen being tested. The inhibitors exhibit different levels of activity against the HA of different influenza strains. Several methods exist for inactivating nonspecific inhibitors in sera of different species. Treatment with RDE is a commonly used method. When nonspecific inhibitors of RDE-treated serum create a problem with interpretation in HAI test, alternative treatment methods need to be investigated.
2.1. Reconstitute the lyophilized RDE with 20 ml PBS, aliquot, and store at –20oC.
2.2. Add 3 volumes of RDE to 1 volume serum and incubate in 37oC water-bath for 16-18 hours.
2.3. Heat in a 56oC water-bath for 30 min to inactivate remaining RDE.
2.4. Add 6 volumes of PBS. The final dilution of sera is 1:10.

3. Detection of nonspecific agglutinins in treated sera and adsorption of Serum with RBCs to Remove Nonspecific Agglutinins

Nonspecific agglutinins must be removed from sera to prevent false negatives in the HAI test.

3.1. Add 25 μl PBS to each well except the first row.
3.2. Add 50 μl of each RDE-treated serum to the first wells.
3.3. Prepare serial 2-fold dilutions of the sera by transferring 25 μl from the first well to the successive wells in each column. Discard the final 25 μl after row H.
3.4. Add 25 μl PBS to all wells (instead of antigen) in all wells.
3.5. Add 50 μl 1% TRBC to all wells.
3.6. Mix using a laboratory shaker or by manually agitating the plates thoroughly.
3.7. Incubate the plates at room temperature for the appropriate time by checking the RBC control for complete settling of the cells. A total of 30 minutes is usually TRBCs.
3.8. Record and interpret the results. If the RBCs settle completely in the wells in a column containing diluted serum, that serum is acceptable for use in the HAI test. The presence of nonspecific agglutinins will be evident by any aemagglutination of the RBCs by the serum. In this case, the serum must be adsorbed with RBCs as follows:

3.8.1. To one volume of packed RBCs in a centrifuge tube add 20 volumes of RDE-treated serum.
3.8.2. Mix thoroughly and incubate at RT or 4°C for 1 hour, mixing at intervals to resuspend the cells.
3.8.3. Centrifuge in a microfuge at 600 x g (2000 rpm) for 5 minutes.
3.8.4. Carefully remove the adsorbed serum without disturbing the packed cells. It is expected that the total amount of diluted serum recovered will be similar to the volume of diluted serum added.
3.8.5. The final serum dilution after adsorption is still a 1:10. The removal of nonspecific agglutinins must be confirmed if the initial haemagglutination titer was ≥80 by combining the adsorbed serum with 1% TRBC as above and observing for the absence of agglutination. If the titer is still ≥20 then re-adsorb the serum with packed RBCs.

4. Collection of turkey blood and standardized RBC to 1%.

4.1. Collect turkey blood into a syringe with ACD solution (1 cc for 1 cc blood). Put the blood with ACD solution into a bottle and gently agitate it. Turkey blood should be store at 4oC and used as fresh as possible.
4.2. Add 5ml of blood into a 50ml conical centrifuge tube.
4.3. Fill tube with cold PBS to 50ml, mix gently.
4.4. Centrifuge at 2000 rpm for 5 min.
4.5. Remove supernatant and wash the partially packed cells with PBS two more times, discarding the final supernatant from the partially packed cells.
4.6. To determine the TRBC concentration, add 1.0 ml of partially packed previously washed TRBC into a 1.5 ml centrifuge tube, and centrifuge at 8000 rpm for 10 min. Estimate the volume of completely packed TRBC which should be appropriately at 60% to 75% of the original volume of partially packed TRBC. Discard the complete packed TRBC.
4.7. Dilute partially packed TRBC to final concentration of 1% in PBS. (1ml RBCs+99ml PBS)

5. HA titration of virus strains

5.1. Mark the V bottom plates with the names of tested viruses (duplicate)
5.2. Add 50μl of PBS to wells 2 through12.
5.3. Add 100 μl of each tested virus to the first well (duplicate, well-1A and well -1B).
5.4. Make serial 2-fold dilutions by transferring 50μl from the first well to successive well-11, discard the final 50μl from well-11. Well-12 contains only PBS as RBC control.
5.5. Add 50μl of 1% TRBC suspension to each well on the plate.
5.6. Gently mix the plates and incubate the plates at room temperature (RT) for 30min.
5.7. Record the titers of viruses after 30 min by tipping plates and reading RBC buttons that stream. The highest dilution of virus that causes complete haemagglutination is considered the HA titration end point. The HA titer is the reciprocal of the dilution of virus in the last well with complete haemagglutination.
5.8. Dilute virus in cold PBS to make a working solution containing 8HAU/50μl. Calculate the antigen dilution by dividing the HA titer (which is based on 50μl) by 8 because you wish to have 8 HAU/50μl. For example, an HA titer of 160 divided by 8 is 20. Mix 1 part of virus with 19 parts cold PBS to obtain the desired volume of standardized antigen. Keep a record of the dilution for the next HAI assay.
5.9. Perform a "back titration" to verify HA units (=8 HAU/50μl) by performing a second HA test. Store this working solution on ice and use within the same day (see Figure. 1).

Interpretation

Haemagglutination occurs when the RBC control has settled completely (well-12). This is recorded using a "+" symbol. When a portion of the RBC is partially agglutinated or partially settled, a "+/-" symbol is used. In the absence of haemagglutination, tear-shaped streaming of erythrocytes which flow at the same rate as RBC controls is observed.

Standardized working solution must have an HA titer of 8 HAU/50μl (= 4 HAU/25μl). This titer will haemagglutinate the first four wells of the back titration plate. If the working solution does not have an HA titer of 8 in 50μl, it must be adjusted accordingly by adding more antigen to increase units or by diluting to decrease units. For example, if complete haemagglutination is present in the fifth well, the virus now has a titer of 16 HAU/50μl and the test antigen should be diluted 2-fold. Conversely, if haemagglutination is only present to the third dilution, the antigen has a titer of 4 HAU/50μl and an equal volume of virus must be added to the test antigen as was used when the antigen was initially diluted. This will double the concentration of virus in the working solution to give a titer of 8 HAU/50μl. Continue adjusting the concentration of antigen until 4 HAU/25μl (= 8 HAU/50μl) is obtained.

6. HAI Assay

6.1. Label appropriate V bottom microtiter plates with serum numbers, antigens used, and plate numbers.
6.2. Add 25 μl cold PBS to wells B through H (B1-11 to H1-11) of each numbered column.
6.3. Add 50μl of each RDE-treated serum (1:10) to the first well (A1-11) of the appropriate numbered column.
6.4. Prepare serial 2-fold dilutions by transferring 25μl serum from the first well of numbered columns A1-11 to successive wells. Discard the final 25μl after row H.
6.5. Add 25μl of standardized virus containing 4 HAU to 2-fold diluted sera.
6.6. Gently tap the plates and incubate at RT (22o to 25oC) for 30 min.
6.7. Add 50μl of PBS to well-12 as a RBC control.
6.8. Add 50μl of standardized 1% TRBC to all wells. Mix as before.
6.9. Cover the plates and allow the RBC to settle at RT for the appropriate 30min.
6.10. Record HAI titers.

Interpretation

If an antigen/antibody reaction occurs, haemagglutination of the RBC will be inhibited. Symbols of "+" for complete haemagglutination, "+/-" for partial haemagglutination, and " - " for inhibition of haemagglutination are used. The HAI titer is the reciprocal of the last dilution of serum that completely inhibits haemagglutination.

To ensure optimal HAI results when diagnosing infections serologically, it is essential that test procedures be followed exactly. Occasionally, the HAI assay may be difficult to interpret, in such cases, consider the factors presented below.

1. Selecting virus isolated from same outbreak for optimal antigenic match or an antigenically equivalent strains used in HAI assay for maximum sensitivity.
2. Standardized virus working solution must contain 4 HAU/25μl. The antigen dilutions must be prepared and back titrated in each assay.
3. Incubation times must be strictly observed. Plates must be read promptly when the RBC control has completely settled. Elution of the RBC from the virus can occur with some virus strains. When this happens, the plates may be read earlier or placed at 4oC.
4. RBC suspension must be standardized in a consistent manner each time.
5. Test viruses must be handled and stored in the prescribed manner. To minimize freeze-thaws and to avoid bacterial contamination, dispense reagents in small volumes using sterile techniques.
6. All tested sera must be treated by RDE to remove nonspecific inhibitors.
7. The positive control antisera must be included in each diagnostic serologic assay, and should give consistent results when compared with previous test.
8. The turkey blood in ACD buffer should be used as fresh as possible.
9. Some human sera may contain nonspecific agglutinins and cause nonspecific haemagglutination of the TRBC, resulting in false diagnosis in sera containing low levels of HAI antibody. In this case, the sera must be adsorbed with RBC to remove the nonspecific agglutinins.

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Laboratory Procedures - Serological detection of avian influenza A(H7N9) infections by microneutralization assay, 23 May 2013 (WHO, edited)

2013-05-23T17:49:00.001+02:00

[Source: World Health Organization, full PDF document: (LINK). Edited.]

Laboratory Procedures - Serological detection of avian influenza A(H7N9) infections by microneutralization assay, 23 May 2013

For further information please contact us at: gisrs-whohq@who.int

Serological detection of avian influenza A(H7N9) infections by microneutralization assay

These procedures were adapted from the WHO Manual for the laboratory diagnosis and virological surveillance of influenza (Chapter 2G on page 63)(1).

INTRODUCTION

Serological methods rarely yield an early diagnosis of acute influenza virus infection. However, the demonstration of a significant increase in antibody titers (greater than or equal to 4-fold) between acute-phase and convalescent-phase sera may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative. Apart from their retrospective diagnostic value, serological methods such as virus neutralization and haemagglutination inhibition are the fundamental tools in epidemiological and immunological studies, as well as in the evaluation of vaccine immunogenicity.

The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses in human and animal sera, potentially including the detection of human antibodies to avian subtypes. Virus neutralization gives the most precise answer to the question of whether or not an individual has antibodies that can neutralize the infectivity of a given virus strain. The assay has several additional advantages in detecting antibodies to influenza virus. First, it primarily detects antibodies to the influenza viral HA protein and thus can identify functional strain-specific antibodies in human and animal sera. Second, since infectious virus is used, the assay can be carried out quickly once the emergence of a novel virus is recognized. Although conventional neutralization tests for influenza viruses (based on the inhibition of cytopathogenic effect formation in MDCK cell culture) are laborious and rather slow, a microneutralization assay using microtiter plates in combination with an ELISA to detect virus-infected cells can yield results within two days. The influenza virus microneutralization assay presented below is based on the assumption that serum-neutralizing antibodies to influenza viral HA will inhibit the infection of MDCK cells with virus. Serially diluted sera should be pre-incubated with a standardized amount of virus before the addition of MDCK cells. After overnight incubation, the cells are fixed and the presence of influenza A virus nucleoprotein (NP) protein in infected cells is detected by ELISA.

The absence of infectivity constitutes a positive neutralization reaction and indicates the presence of virus specific antibodies in the serum sample. In cases of influenza-like illness, paired acute and convalescent serum samples are preferred. An acute sample should be collected within seven days of symptom onset and the convalescent sample collected at least 14 days after the acute sample, and ideally within 1–2 months of the onset of illness. A 4-fold or great rise in antibody titer demonstrates a seroconversion and is considered to be diagnostic. With single-serum samples, care must be taken in interpreting low titers such as 20 and 40. Generally, knowledge of the antibody titers in an age-matched control population is needed to determine the minimum titer that is indicative of a specific antibody response to the virus used in the assay.

The microneutralization protocol is therefore divided into three parts:

Part I: Determination of the tissue culture infectious dose (TCID50).
Part II: Virus microneutralization assay.
Part III: ELISA.

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1. Materials required

1.1 Equipments

Class II biological safety cabinet.
Water baths, 37oC and 56oC.
Incubator, 37oC, 5% CO2.
Inverted microscope or standard microscope for the observation of cells.
Automatic ELISA reader with 492 nm filter.
Automatic plate washer (not essential but would be optimal).
Low speed, bench top centrifuge.
4oC refrigerator.
Freezer, - 70oC (for long term virus storage) or - 20oC (for serum storage).

1.2 Supplies

Cell culture flasks
96-well microtiter plates(flat-bottom)
Haemacytometer and haemacytometer coverslips
Cell counter
Multichannel pipette and tips
Pipettes
Tubes

1.3 Cell, media and buffers

MDCK cell culture monolayer – low passage (<25–30 passages) at low crowding (70–95% confluence)
D-MEM high glucose (1x) liquid, with L-glutamine and without sodium pyruvate (Invitrogen-cat.no 11965-092)
0.01 M PBS (pH 7.2)( Invitrogen-cat.no 20012-43)
HEPES buffer (1 M stock solution)( Invitrogen-cat.no 15630-080)
Citrate buffer capsules(Sigma cat.no P4922)
Water (distilled and deionized)
MDCK sterile cell culture maintenance medium(see below)
Virus diluent(see below)
Wash buffer(PBST)
Fixative solution(see below)
Stop solution(see below)

1.4 Reagents

Penicillin-streptomycin (stock solution contains 10000 U/ml penicillin; and 10000μg/ml streptomycin sulfate) Invitrogen-cat.no 15140-122
200 mM L-glutamine Invitrogen-cat.no 25030-081
Trypsin-EDTA (0.05% trypsin; 0.53 mM EDTA 4Na) Invitrogen-cat.no 25300-054
Trypsin – TPCK-treated (type XIII from bovinepancreas) Sigma-cat.no T1426
o-phenylenediamine dihydrochloride (OPD)Sigma-cat.no.P8287
Fetal bovine serum (FBS) Invitrogen cat. no. 10099-141
Bovine albumin fraction V (prepared as a 7.5% solution in water) Roche,70250224
Trypan blue stain (0.4%) Invitrogen-cat.no 15250-061
PBST
Acetone

1.5 Antibodies

1º-antibody: Anti-Influenza A NP mouse monoclonal antibody (millipore, mixed A1,A3), dilute 1:4000 in blocking buffer or at optimal concentration
2º-antibody: Goat anti-mouse IgG conjugated to HRP(KPL), dilute 1:2000 in blocking buffer or at optimal concentration

2. Preparation of media and solutions

2.1 MDCK sterile cell culture maintenance medium:

DMEM, 10% FBS, P/S
440 mL Dulbecco’s Modified Eagles Medium (DMEM)
5 mL 100 X P/S (100μ/mL penicillin, 100g/mL streptomycin)
5 mL 200 mM L-glutamine
50 mL FBS
FBS need to be heat-inactivated at 56°C for 30 min before use.

2.2 Virus diluent:

DMEM, 1% Bovine serum albumin (Roche,70250224), P/S – freshly prepared
415.4 mL DMEM
5 mL 100 X Antibiotics
67 mL 7.5% BSA; or 5 g BSA fraction V powder
12.5 mL HEPES buffer solution (1 M)
100ul Trypsin-TPCK-treated(2ug/ml) (type XIII from bovine pancreas)

2.3 Fixative: Cold 80% Acetone in PBS – freshly made

80 mL Acetone
20 mL PBS

2.4 Blocking Buffer: PBST, 1% BSA

1000 mL PBST
144 mL 7.5% BSA or 10 g BSA fraction V powder

2.5 Substrate: o-phenylenediamine dihydrochloride (OPD); For 20 mL of Citrate buffer, add 1 tablet of OPD (10 mg) just before use.

For citrate buffer, prepare as follows:

Prepare with Sigma Citrate Buffer capsules. Add1 capsule into 100 mL dH2O.

2.6 Stopping Solution: 1N Sulfuric acid. Add 28mL stock sulfuric acid [18M] into 1L dH2O

3.Quality Control

The procedure of Virus microneutralization assay is complicated. The changes of any factors involved in this assay such as virus, cell or sera may affect the final result. Thus quality control is necessary. Setting Positive and negative serum control and cell control in every test plates is requested, virus titer should be determined before virus microneutraliazation assay.
3.1 Serum controls:

Include serum samples, positive serum control and negative serum control. If samples are to be tested repeatedly, it is better to make aliquots. Sera should not be repeatedly freeze-thaw. Sera can be stored at -20 to -70℃. Human sera needs to be heat-inactivated at 56°C for 30 min and animal sera need be treated by RDE before use.

3.1.1 Positive (column1,2,infected or vaccinated) serum controls:

Include anti-sera to test viruses as positive control. For human sera, an optimal positive control would be acute and convalescent serum samples.

3.2.2 Negative (normal) serum control (column3,4): (FIGURE 2)

Include a normal serum to determine whether the virus is nonspecifically inactivated by serum components.
1) For human sera, use normal serum from a population not exposed to the particular virus subtype in question.
2) Use the normal serum at the same dilution as the matching viral antiserum.
Virus and cell controls include a virus back-titration and positive and negative cell controls with each assay.

3.2 Negative and positive cell controls:

Set up four wells as positive cell controls in column 12 (wells A-D) - VC (50μl medium + 50μl test dilution of virus + 100 μl of MDCK cells) and four wells as negative cell controls in column 12 (wells E-H) - CC (100μl medium + 100μl of MDCK cells) and assay in parallel with the microneutralization test. The cell controls should be included on each plate to control for plate to plate variation.

3.3 Virus titration check:

In each assay, include a back-titration of the test dilution of virus. Add 50μl of medium to wells A-H of column 11. Add 50μl of the test dilution of virus to the first well (A11). Serially transfer 50μl down the column 11 (7 wells, B to H). Add an additional 50μl of virus diluent to column 11. Add 100μl of MDCK cells (1.5x104/well) to column 11.

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Part I: Virus titration and determination of tissue culture infectious dose (TCID50) for microneutralization assay

For safety reasons, seasonal and low pathogenic avian viruses, and human serum samples, should be handled in a class-II biosafety cabinet in BSL-2 laboratories, while highly pathogenic avian influenza (including the novel H7N9 virus microneutralization assays should additionally be performed only in BSL-3+ laboratories

1. Virus dilution

Virus should be stored at –70℃; Determine TCID50 before use; never using freeze-thawed virus. Procedure of virus TCID50 assaywas described as below:

Virus can be diluted by log10 or ½ log10 dilution, ½ log10 dilution was introduced as below（FIGURE 3）:

1.1 Dilute virus 1/100 in dilution buffer (100 μl virus + 9.9 mL dilution buffer) as working stock.
1.2 Add 100 μl of virus diluent to all wells, except column A1-D1, of a 96-well tissue culture plate.
1.3 Add 146 μl virus of 1/100 working stock to column A1-D1. Transfer 46 μl serially from column 123…11 (½ log10 dilutions). Change tips in each dilution. Dilutions will be 10-2, 10-2.5, 10-3…10-7

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2. Preparation of MDCK cells

2.1 Check the MDCK cell monolayer (which should be 70–95% confluent).

Do not allow the cells to overgrow. Typically, a confluent T75 flask (approximately
2 x 107 cells/flask) should yield enough cells to seed 4–6 96-well microtiter plates.

2,2 When ready to perform the assay, wash 70–95% confluent cells with PBS to remove FBS.
2.3 Add 7 ml trypsin-EDTA to cover the cell monolayer.
2.4 Lie flask flat and incubate at 37°C until monolayer detaches (approximately 8–10 minutes).
2.5 Add 7 ml of virus diluent to each flask.
2.6 Wash cells twice with virus diluent to remove FBS.

— gently mix to resuspend and break up clumps of cells;
— fill tube to 50 ml with virus diluent;
— pellet cells by centrifugation at 1000rpm for 10 minutes;
— decant supernatant;
— perform one repeat of the previous 4 steps.

2.7 Resuspend cells in virus diluent (10 ml per trypsinized flask) and count cells with a haemacytometer as described below in FIGURE4, Determination of cell count and viability.
2.8 Adjust cell concentration to 1.5 x 105 cells/ml with virus diluents.
2.9 Add 100 μl diluted cells to each well of the microtiter plate.
2.10 Incubate cells for 18–20 hours at 37°C in 5% CO2.

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3.Fixation of cells

3.1 Remove medium from microtiter plate.
3.2 Wash each well with 200 μl PBS.
3.3 Remove PBS (do not allow wells to dry out) and add 100 μl/well of cold fixative.
3.4 Cover with lid and incubate at room temperature for 10–12 minutes.
3.5 Remove fixative and let the plate air dry.

4.Determination of TCID50 for microneutralization assay

4.1 Perform ELISA (see protocol below).
4.2 Calculate the mean absorbance (OD492) of the CCs.
4.3 Any test well with an OD492 greater than twice the average of OD492 of the CC wells is scored positive for virus growth.
4.4 Once all test wells have been scored positive or negative for virus growth, the TCID50 of the virus can be calculated as shown below by the Reed-Muench method (Reed & Muench, 1938).

Calculation (TABLE 2)

1. Record the number of positive values observed in column (1) and negative values in column (2) wells of the microtiter plates at each dilution.

2. Calculate the cumulative numbers of positive values in column (3) of TABLE 2. and negative values in column (4) of TABLE 2:

— column (3) – obtained by adding the numbers in column (1) starting at the bottom.
— column (4) – obtained by adding the numbers in column (2) starting at the top.

3. Calculate the ratios at each dilution in column (5) by dividing the number of positives in column (3) by the number of positives plus negatives in columns (3) + (4).

4. Calculate the percentage of positive wells in column (6) by converting each of the ratios in column (5) to percentages.

5. Calculate the proportional distance between the dilution showing >50% positives in column (6) and the dilution showing <50% positives in column (6) as follows:

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6. The virus working dilution is 200 times the log10 virus dilution at the cut-off point determined by the Reed-Muench method. 200x times the virus dilution at the cut-off point yields a virus working dilution that contains 100x TCID50 in 50 μl.

7. Calculate the microneutralization TCID50 by adding the proportional distance to the dilution showing >50% positive. In the above example, add 0.25 to 4.5 to obtain 10-4.75.The virus working dilution that is 200x the cut-off dilution is 10-4.75 x 200 = 10-4.75 +102.30 = 10-2.45 = 1/10 2.45 = 1:282. This dilution will give 100x TCID per 50 μl.

If other dilution series are used, other correction factors must be used. For example, in this case, the correction factor for a 2-fold dilution series would be 0.3; for a .log10 dilution series it would be 0.5; for a 5-fold dilution series it would be 0.7; and for a 10-fold dilution series it would be 1.0.

(...)

Part II: Virus microneutralization assay

1.Preparation of test sera

1.1 10 μl of sera are needed for each virus to be tested once. Sera should be tested in duplicate (requires 20 μl when possible).
1.2 Heat-inactivate sera for 30 min at 56°C.
1.3 Add 50 μl of diluent to each well of the plates.
1.4 Add an additional 40 μl of diluent to Row A (wells A1-A11).
1.5 Add 10μl of heat-inactivated sera including positive serum, negative serum and sera samples to Row A (1 serum/well except A12).
1.6 Perform 2-fold serial dilutions by transferring 50μl from row to row (ABC…H).

2. Addition of virus

2.1 Determine virus titer by TCID50.
2.2 Dilute virus to 100 TCID50 per 50 μl (200 TCID50 /100 μl) in virus diluent (5 mL/plate).
2.3 Add 50 μl diluted virus to all wells except CC (wells E12, F12, G12, and H12).
2.4 Add 50 μl diluent without virus to CC wells for negative cell control.
2.5 Set-up back-titration, start with the virus test dilution (100 TCID50), and prepare additional serial 2-fold dilutions with diluent.
2.6 Gently agitate the virus-serum mixtures, and incubate them and the virus back-titration for 1 hr at 37°C, 5% CO2.

3. Addition of MDCK cells:

3.1 Prepare MDCK cells as described above.
3.2 Add 100 μl cells to each well of plate (1.5x104 cells /well).
3.3 Incubate plates overnight at 37°C, 5% CO2 (18-20 hrs).

Note: To ensure even distribution of heat and CO2, stack plates only 4 to 5 high in incubator. Media color should always maintain an orange color (this indicates the desired pH best for sera, virus, and cells). To avoid pH change, work with fewer plates at one time.

4. Fixation of the plates:

4.1 Remove medium from plate.
4.2 Wash each well with 200 μl PBS.
4.3 Remove PBS (Do not let wells dry out) and add 100 μl/well of cold fixative.
4.4 Cover with lid and incubate at RT for 10 min.
4.5 Remove fixative and let plate air-dry.

Part III: ELISA

Based on the principle of antigen-antibody interaction, this test allows for easy visualization of results. In this assay, when the secondary antibody which is linked to an enzyme binding to the antigen-antibody complex formed in cell, a visible signal will be produced by adding an enzymatic substrate.

1. Wash plate(s) 3X with wash buffer. Fill wells completely with wash buffer for each wash.
2. Dilute 1°-antibody (anti-Influenza A pool; NP monoclonal) 1:4000 or at optimal concentration in blocking buffer.
3. Add diluted 1°-antibody to each well (100 μl / well).
4. Cover plate(s) and incubate for 1 h at RT.
5. Wash plate(s) 3X with wash buffer.
6. Dilute 2°-antibody (goat anti-mouse IgG; HRP conjugated) 1:2000 or at optimal concentration in blocking buffer.
7. Add diluted 2°-antibody to each well (100μl/well).
8. Cover plate(s) and incubate for 1 hr at RT.
9. Wash plate(s) 5X with wash buffer.
10. Add freshly prepared substrate (10 mg OPD to each 20 mL citrate buffer) to each well (100μl/well).
11. Incubate for 3 min (or until color change in VC is intense and before background CC begins to change color) at RT. Incubation time will vary between viruses.
12. Add stop solution (100 μl /well) to all wells.
13. Read absorbance (OD) of wells at 492 nm.
14. Data Analysis:

Calculations are determined for each plate individually.
14.1 Determine virus neutralizing antibody end point titer of each serum utilizing the equation below:

X = (Average OD of VC wells―Average OD of CC wells)/2 + (Average OD of CC wells)
where X = 50% of specific signal (i.e. 50% of the cells are infected). All values below this value are positive for neutralization activity.

14.2 The negative cell control (CC) should show OD < 0.2. The positive cell control (VC) should show OD > 0.8.
14.3 The virus test dose (100 TCID50) is confirmed by virus back-titration. In most cases, the test dose of virus is acceptable if the back-titration is positive in 5-7 wells containing the lowest dilution of test virus.
14.4 The serum positive controls should give titers within 2-fold of expected. The value of OD in the normal serum control should be similar to that observed in VC wells.

Cautions:

1. Human sera needs to be heat-inactivated at 56°C for 30 min and animal sera need be treated by RDE before use.
2. Sera should not be repeatedly freeze-thawed. If samples are to be tested repeatedly, it is better to make several aliquots of sera.
3. Virus should not be repeatedly use. In most cases, the test dose of virus is acceptable if the back-titration in positive in 5-7 wells, if not virus titer should be determined again by TCID50.
4. Check MDCK cell monolayer should be low passage (< 25-30 passages) at low crowding (70-95% confluent). Liquid nitrogen for cell storage should be performed within 10 passages.
5. Some factors in FBS can neutralize virus infectivity, do not use cell culture maintenance medium as diluent during the assay.
6. Plates should be strictly washed in each time to remove any proteins or antibodies that are not specifically bound in ELISA.
7. Properly control the incubation time after adding substrate in order to avoid a high background.
8. HEPES can stabilize cell culture maintenance medium when a large scale test is needed.

Occasionally, the microneutralization test may be difficult to interpret. In such cases, consider the factors presented in TABLE 3.

(...)

________

(1) http://www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_influenza/, accessed 23 May 2013

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Summary of status of development and availability of avian influenza A(H7N9) candidate vaccine viruses, 23 May 2013 (WHO, edited)

2013-05-23T17:33:00.001+02:00

[Source: World Health Organization, full PDF document: (LINK). Edited.]

Summary of status of development and availability of avian influenza A(H7N9) candidate vaccine viruses, 23 May 2013

[Parent virus - Candidate vaccine virus - Type of virus or reassortant - Developing institute - Available from]

A/Shanghai/2/2013 Synthetic HA&NA

IDCDC-RG32A* - Reverse genetics - CDC, USA - CDC, USA
NIBRG-267** - Reverse genetics - NIBSC, UK - NIBSC, UK
CBER-RG4A** - Reverse genetics - CBER, USA - CBER, USA

A/Anhui/1/2013

Wild type virus – … – … - WHO CCs
NIBRG-268** - Reverse genetics - NIBSC, UK - NIBSC, UK

(*) This virus is a candidate vaccine virus which has passed relevant safety testing and two-way haemagglutination inhibition (HI) test. It can be handled under BSL-2 enhanced containment(1).

(**) These are potential candidate vaccine viruses which have passed relevant safety testing and can be handled under BSL-2 enhanced containment1. Full virus characterization is yet to be finished.

________

Institutes contact details for candidate vaccine viruses orders/information:

CBER: Zhiping.Ye@fda.hhs.gov
CDC: rvd6@cdc.gov
NIBSC: standards@nibsc.hpa.org.uk or enquiries@nibsc.hpa.org.uk
WHO CCs: http://www.who.int/influenza/gisrs_laboratory/collaborating_centres/list/en/

For general enquiries, please contact gisrs-whohq@who.int

For other candidate vaccine viruses and potency testing reagents, please go to http://www.who.int/influenza/vaccines/virus/candidates_reagents/home/

_________

(1) http://www.who.int/biologicals/areas/vaccines/influenza/biosafety_risk_assessment_10may2013.pdf

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Update of the development and availability of candidate vaccine viruses for avian influenza A(H7N9), 20 May 2013 (WHO, edited)

2013-05-23T17:24:00.001+02:00

[Source: World Health Organization, full PDF document: (LINK). Edited.]

Update of the development and availability of candidate vaccine viruses for avian influenza A(H7N9), 20 May 2013

Safety testing of two potential candidate vaccine virus (CVV) for avian influenza A(H7N9) virus, IDCDC-RG32A(1), developed by the WHO Collaborating Centre for Reference and Research on Influenza in the Centers for Disease Control and Prevention, US, and NIBRG-2681, developed by the WHO Essential Regulatory Laboratory (ERL) in the National Institute for Biological Standards and Control (NIBSC), UK, using reverse genetics technology, have been completed following the recommended safety testing regimen as described in the ‘Update of WHO biosafety risk assessment and guidelines for the production and quality control of human influenza vaccines against avian influenza A(H7N9) virus’(2). The resulting data have been reviewed by an expert group convened by WHO and have been found to be satisfactory. Therefore, it was concluded that the virus IDCDC-RG32A and NIBRG-268 are attenuated compared to a closely related wild-type A(H7N9) virus, A/Anhui/1/2013.

In view of the above, the potential candidate vaccine viruses IDCDC-RG32A and NIBRG-268 can now be handled under BSL-2 enhanced(3) containment.

The expert group also concluded that CVVs that have HA and NA genes with sequences that are identical or nearly identical to those of a reassortant virus, in this case the IDCDC-RG32A, that has already been assessed, would be exempted from full safety testing(2). Therefore, two other potential CVVs of A(H7N9), NIBRG-267, developed by the WHO ERL in NIBSC, UK; and CBER-RG4A, developed by the WHO ERL at the Center for Biologics Evaluation and Research, US, can now also be handled in the same conditions as recommended for IDCDC-RG32A: BSL-2 enhanced containment.

It should be noted that these are potential CVVs that have not yet been fully characterized antigenically.

The summary table of candidate vaccine viruses of influenza A(H7N9) is updated on the WHO website(4).

_________

1. http://www.who.int/entity/influenza/vaccines/virus/candidates_reagents/summary_a_h7n9_cvv_20130510.pdf

2. http://www.who.int/biologicals/areas/vaccines/influenza/biosafety_risk_assessment_10may2013.pdf

3. WHO biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines, TRS No. 941, Annex 5 (2007), http://www.who.int/biologicals/publications/trs/areas/vaccines/influenza/Annex%205%20human%20pandemic%20influenza.pdf

4. http://www.who.int/influenza/vaccines/virus/candidates_reagents/summary_a_h7n9_cvv_20130520.pdf

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Pas de nouveaux cas signalés d’infection par le virus « Corona » (L'Economiste Maghrébin, May 23 2013)

2013-05-23T17:16:00.001+02:00

[Source: L’Economiste Maghrébin, full text: (LINK).]

Pas de nouveaux cas signalés d’infection par le virus « Corona »

‎23 May ‎2013 | L'Economiste Maghrébin

S’exprimant sur Express Fm, Noureddine Achour, directeur de l’Observatoire national des maladies nouvelles et émergentes, a démenti, aujourd’hui 23 mai, les informations selon lesquelles il y aurait deux nouveaux cas d’infection par le virus Corona. Noureddine Achour a précisé que suite au décès d’un Tunisien rentré des pays du Golfe, atteint par le virus Corona, [...]

Cet article Pas de nouveaux cas signalés d’infection par le virus « Corona » est apparu en premier sur L'Economiste Maghrébin.

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Epidemiological update: additional confirmed cases of Middle East respiratory syndrome coronavirus (novel coronavirus) in France, Saudi Arabia and Tunisia (ECDC, May 23 2013)

2013-05-23T17:12:00.001+02:00

[Source: European Centre for Disease Prevention and Control (ECDC), full page: (LINK). Edited.]

Epidemiological update: additional confirmed cases of Middle East respiratory syndrome coronavirus (novel coronavirus) in France, Saudi Arabia and Tunisia

23 May 2013

Since last ECDC update on 17 May 2013 [1], one probable and six new confirmed cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection have been reported.

One case was reported by the Ministry of Social Affairs and Health in France [2]. The patient shared the same hospital room in France with a previously reported MERS-CoV case with a recent history of travel to Dubai. Infection resulted most probably from human-to-human transmission.

Three cases were reported by the Ministry of Health of the Kingdom of Saudi Arabia [3-4]. Two of these cases, a 43-year-old woman with co-existing health conditions and a 45-year-old man, were healthcare workers associated with a cluster in Al-Hasa, Eastern Province of Saudi Arabia, and linked to the same healthcare facility [3]. The third case was a 63-year-old non-Saudi patient with an underlying medical condition who was admitted to hospital on 15 May and died on 20 May in the Al-Qaseem region, central Saudi Arabia [4-5].

Finally, three cases were reported by the Ministry of Health of Tunisia [6]. The index patient is a 66-year-old man with underlying health conditions and a recent travel history to Qatar and Saudi Arabia who died on 10 May 2013. Infection with MERS-CoV was not laboratory confirmed and the case is currently defined as probable. The two confirmed cases, a 34-year-old man and a 35-year-old woman, are the children of the index patient and travelled with the father during his journey. Both children had mild respiratory illness and did not require hospitalisation.

As of 23 May 2013, 44 confirmed MERS-CoV cases have been reported since the earliest case identified in April 2012 in Jordan, of which 22 are known to have died. To date, eight of these cases have been diagnosed in Europe: two in France [3], two in Germany [7] and four in the United Kingdom [8]. Overall, 31 cases have been reported in Saudi Arabia, 22 of these were associated with the cluster in Al-Hasa [6].

Due to the large number of guest workers in Saudi Arabia, WHO expressed concerns about possible importation of MERS-CoV into the Indian subcontinent and the Philippines, where investigating and controlling the virus transmission could be more challenging [9].

ECDC continues to monitor information on the situation of MERS-CoV worldwide. In the light of these new developments, the recommendations given in the rapid risk assessment of the 17 May 2013 are still valid [1].

References

ECDC updates Rapid Risk Assessment on Middle East respiratory syndrome coronavirus (novel coronavirus) http://ecdc.europa.eu/en/publications/Publications/Forms/ECDC_DispForm.aspx?ID=1121
Global Alert and Response. Novel coronavirus infection – update http://www.who.int/csr/don/2013_05_12/en/index.html
Global Alert and Response. Novel coronavirus infection – update
http://www.who.int/csr/don/2013_05_15_ncov/en/index.html
Saudi Arabia MoH: "A Novel Coronavirus Case Passed Away in Al-Qassim Region". http://www.moh.gov.sa/en/Ministry/MediaCenter/News/Pages/News-2013-05-22-002.aspx
Global Alert and Response. Novel coronavirus infection – update
http://www.who.int/csr/don/2013_05_23_ncov/en/index.html
Global Alert and Response. Novel coronavirus infection – update http://www.who.int/csr/don/2013_05_22_ncov/en/index.html
RKI. Aktualisierung der Risikoeinschätzung des RKI zu Erkrankungsfällen durch das neuartige Coronavirus (hCoV-EMC) 26 March 2013 http://www.rki.de/DE/Content/InfAZ/C/Corona/Risikoeinschaetzung.html
The Health Protection Agency (HPA) UK Novel Coronavirus Investigation team. Evidence of person-to-person transmission within a family cluster of novel coronavirus infections, United Kingdom, February 2013. Euro Surveill. 2013;18(11):pii=20427. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20427
http://www.cidrap.umn.edu/cidrap/content/other/sars/news/may2113corona.html

Related links
Confirmed cases of novel coronavirus infection reported in Europe as of 23 May 2013

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Novel coronavirus infection - update (Middle East respiratory syndrome- coronavirus) (WHO, May 23 2013, edited)

2013-05-23T16:27:00.001+02:00

[Source: World Health Organization, full page: (LINK). Edited.]

Novel coronavirus infection - update (Middle East respiratory syndrome- coronavirus)

23 May 2013

The Ministry of Health in Saudi Arabia has notified WHO of an additional laboratory-confirmed case of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV).

The fatal case was reported from Al-Qaseem region in the Central part of the country and is not related to the cluster of cases reported from Al-Ahsa region in the Eastern part of the country. The patient was a 63-year-old man with an underlying medical condition who was admitted to a hospital with acute respiratory distress on 15 May 2013 and died on 20 May 2013. Investigation into contacts of this case is ongoing.

The Saudi authorities are also continuing the investigation into the outbreak that began in a health care facility since the beginning of April 2013 in Al-Ahsa. To date, a total of 22 patients including 10 deaths have been reported from the outbreak.

Globally, from September 2012 to date, WHO has been informed of a total of 44 laboratory-confirmed cases of infection with MERS-CoV, including 22 deaths.

WHO has received reports of laboratory-confirmed cases from the following countries in the Middle East: Jordan, Qatar, Saudi Arabia, and the United Arab Emirates (UAE). France, Germany, Tunisia and the United Kingdom also reported laboratory-confirmed cases; they were either transferred for care of the disease or returned from Middle East and subsequently became ill. In France, Tunisia and the United Kingdom, there has been limited local transmission among close contacts who had not been to the Middle East but had been in close contact with the laboratory-confirmed or probable cases.

Based on the current situation and available information, WHO encourages all Member States to continue their surveillance for severe acute respiratory infections (SARI) and to carefully review any unusual patterns.

Health care providers are advised to maintain vigilance. Recent travellers returning from the Middle East who develop SARI should be tested for MERS-CoV as advised in the current surveillance recommendations. Specimens from patients’ lower respiratory tracts should be obtained for diagnosis where possible. Clinicians are reminded that MERS-CoV infection should be considered even with atypical signs and symptoms, such as diarrhoea, in patients who are immunocompromised.

Health care facilities are reminded of the importance of systematic implementation of infection prevention and control (IPC). Health care facilities that provide care for patients suspected or confirmed with MERS-CoV infection should take appropriate measures to decrease the risk of transmission of the virus to other patients, health care workers and visitors.

All Member States are reminded to promptly assess and notify WHO of any new case of infection with MERS-CoV, along with information about potential exposures that may have resulted in infection and a description of the clinical course. Investigation into the source of exposure should promptly be initiated to identify the mode of exposure, so that further transmission of the virus can be prevented.

WHO does not advise special screening at points of entry with regard to this event nor does it currently recommend the application of any travel or trade restrictions.

WHO continues to closely monitor the situation.

________

Note: To provide uniformity and facilitate communication about the disease, the Coronavirus Study Group of the International Committee on Taxonomy of Viruses has decided to call the new virus Middle East respiratory syndrome coronavirus (MERS-CoV). Reference: De Groot RJ, et al. Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group. J Virol. Published ahead of print 15 May 2013. doi:10.1128/JVI.01244-13.

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Hong Kong, Public urged to prevent hand, foot and mouth disease (May 23 2013)

2013-05-23T10:27:00.001+02:00

[Source: Centre for Health Protection, Hong Kong PRC SAR, full text: (LINK).]

Public urged to prevent hand, foot and mouth disease

The Centre for Health Protection (CHP) of the Department of Health today (May 23) called on people to maintain strict personal and environmental hygiene to prevent hand, foot and mouth disease (HFMD).

The appeal followed an outbreak of HFMD at a primary school in Kowloon City affecting 20 boys aged between 6 and 12.

The affected pupils have developed oral ulcers, fever, and rash on their hands or feet since April 12. All affected pupils sought medical consultation and no hospitalisation was required. Their current condition is stable.

The stool sample of one affected pupil tested positive for Coxsackie A virus.

Staff of the CHP have conducted a site visit and provided health advice to the staff. The school has been put under medical surveillance.

HFMD is a common disease in children and is usually caused by enteroviruses such as Coxsackie viruses and EV71.

To prevent HFMD, members of the public, and especially the management of institutions, should adopt the following measures:

Maintain good air circulation;

Wash hands before meals and after going to the toilet or handling diapers or other stool-soiled materials;

Keep hands clean and wash hands properly, especially when they are made dirty by respiratory secretions, such as after sneezing;

Cover the nose and mouth while sneezing or coughing and dispose of nasal and oral discharge properly;

Clean children's toys and other objects thoroughly and frequently with diluted household bleach (by adding one part of household bleach containing 5.25 per cent sodium hypochlorite to 49 parts of water), followed by rinsing/wiping with clean water;

Children who are ill should be kept out of school until their fever and rash have subsided and all the vesicles have dried and crusted; and

Avoid going to overcrowded places.

Ends/Thursday, May 23, 2013
Issued at HKT 15:28
NNNN

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Live-Animal Markets and Influenza A (H7N9) Virus Infection (N Engl J Med., extract, edited)

2013-05-22T23:23:00.001+02:00

[Source: The New England Journal of Medicine, full text: (LINK). Extract, edited.]

Correspondence

Live-Animal Markets and Influenza A (H7N9) Virus Infection

May 22, 2013 - DOI: 10.1056/NEJMc1306100

To the Editor:

A recent outbreak of a previously unrecognized novel reassortant avian influenza A (H7N9) virus in China had resulted in 131 documented cases and 36 deaths as of May 16. Although some patients had a history of contact with live poultry or visiting live-animal markets before the onset of illness, the source of infection remains unclear.

(…)

Chang-jun Bao, M.P.H., Lun-biao Cui, Ph.D., Ming-hao Zhou, Ph.D., Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China

Lei Hong, M.S., Nanjing Municipal Center for Disease Control and Prevention, Nanjing, China

George F. Gao, Ph.D., Chinese Center for Disease Control and Prevention, Beijing, China

Hua Wang, M.D., Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China hua@jscdc.cn

Supported in part by grants from the Innovation Platform for Public Health Emergency Preparedness and Response (ZX201109), the Jiangsu Province Key Medical Talent Foundation (RC2011084 and RC2011191), the “333” Projects of Jiangsu Province, and Jiangsu Province Science and Technology Support Program (BE2012769).

Drs. Bao and Cui contributed equally to this study.

The contents of this article are solely the responsibility of the authors and do not necessarily represent the views of the Jiangsu Provincial Center for Disease Control and Prevention or other organizations.

This letter was published on May 22, 2013, at NEJM.org.

Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.

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Clinical Findings in 111 Cases of Influenza A (H7N9) Virus Infection (N Engl J Med., abstract, edited)

2013-05-22T23:15:00.001+02:00

[Source: The New England Journal of Medicine, full text: (LINK). Abstract, edited.]

Original Article

Clinical Findings in 111 Cases of Influenza A (H7N9) Virus Infection

Hai-Nv Gao, M.D., Hong-Zhou Lu, M.D., Ph.D., Bin Cao, M.D., Bin Du, M.D., Hong Shang, M.D., Jian-He Gan, M.D., Shui-Hua Lu, M.D., Yi-Da Yang, M.D., Qiang Fang, M.D., Yin-Zhong Shen, M.D., Xiu-Ming Xi, M.D., Qin Gu, M.D., Xian-Mei Zhou, M.D., Hong-Ping Qu, M.D., Zheng Yan, M.D., Fang-Ming Li, M.D., Wei Zhao, M.D., Zhan-Cheng Gao, M.D., Guang-Fa Wang, M.D., Ling-Xiang Ruan, M.D., Wei-Hong Wang, M.D., Jun Ye, M.D., Hui-Fang Cao, M.D., Xing-Wang Li, M.D., Wen-Hong Zhang, M.D., Xu-Chen Fang, M.D., Jian He, M.D., Wei-Feng Liang, M.D., Juan Xie, M.D., Mei Zeng, M.D., Xian-Zheng Wu, M.D., Jun Li, M.D., Qi Xia, M.D., Zhao-Chen Jin, M.D., Qi Chen, M.D., Chao Tang, M.D., Zhi-Yong Zhang, M.D., Bao-Min Hou, M.D., Zhi-Xian Feng, M.D., Ji-Fang Sheng, M.D., Nan-Shan Zhong, M.D., and Lan-Juan Li, M.D.

May 22, 2013 - DOI: 10.1056/NEJMoa1305584

Abstract

Background

During the spring of 2013, a novel avian-origin influenza A (H7N9) virus emerged and spread among humans in China. Data were lacking on the clinical characteristics of the infections caused by this virus.

Methods

Using medical charts, we collected data on 111 patients with laboratory-confirmed avian-origin influenza A (H7N9) infection through May 10, 2013.

Results

Of the 111 patients we studied, 76.6% were admitted to an intensive care unit (ICU), and 27.0% died. The median age was 61 years, and 42.3% were 65 years of age or older; 31.5% were female. A total of 61.3% of the patients had at least one underlying medical condition. Fever and cough were the most common presenting symptoms. On admission, 108 patients (97.3%) had findings consistent with pneumonia. Bilateral ground-glass opacities and consolidation were the typical radiologic findings. Lymphocytopenia was observed in 88.3% of patients, and thrombocytopenia in 73.0%. Treatment with antiviral drugs was initiated in 108 patients (97.3%) at a median of 7 days after the onset of illness. The median times from the onset of illness and from the initiation of antiviral therapy to a negative viral test result on real-time reverse-transcriptase–polymerase-chain-reaction assay were 11 days (interquartile range, 9 to 16) and 6 days (interquartile range, 4 to 7), respectively. Multivariate analysis revealed that the presence of a coexisting medical condition was the only independent risk factor for the acute respiratory distress syndrome (ARDS) (odds ratio, 3.42; 95% confidence interval, 1.21 to 9.70; P=0.02).

Conclusions

During the evaluation period, the novel H7N9 virus caused severe illness, including pneumonia and ARDS, with high rates of ICU admission and death. (Funded by the National Natural Science Foundation of China and others.)

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Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies (Nature, abstract, edited)

2013-05-22T22:20:00.001+02:00

[Source: Nature, full text: (LINK). Abstract, edited.]

Nature | Letter

Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies

Masaru Kanekiyo,¹Chih-Jen Wei,¹Hadi M. Yassine,¹Patrick M. McTamney,^{1, 2}Jeffrey C. Boyington,¹James R. R. Whittle,¹Srinivas S. Rao,¹Wing-Pui Kong,¹Lingshu Wang¹& Gary J. Nabel^{1, 2}

Journal name: Nature - Year published: (2013) - DOI: doi:10.1038/nature12202

Received 28 August 2012 - Accepted 18 April 2013 - Published online 22 May 2013

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin–nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Affiliations

(1) Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA (Masaru Kanekiyo, Chih-Jen Wei, Hadi M. Yassine, Patrick M. McTamney, Jeffrey C. Boyington, James R. R. Whittle, Srinivas S. Rao, Wing-Pui Kong, Lingshu Wang & Gary J. Nabel)

Present addresses: MedImmune, 1 MedImmune Way, Gaithersburg, Maryland 20878, USA (P.M.M.); Sanofi, 640 Memorial Drive, Cambridge, Massachusetts 02139, USA (G.J.N.). Patrick M. McTamney & Gary J. Nabel

Contributions

M.K., J.C.B. and G.J.N. developed the concept of HA–ferritin nanoparticles; M.K., C.-J.W. and G.J.N. designed the research studies; M.K., C.-J.W., H.M.Y., P.M.M., J.C.B., J.R.R.W., W.-P.K., L.W. and G.J.N. performed the research and analysed data; M.K., C.-J.W., H.M.Y., P.M.M., J.C.B., J.R.R.W. and G.J.N. discussed the results and implications; S.S.R. assisted in animal studies and sample collection; M.K., C.-J.W., J.C.B. and G.J.N. wrote the paper and all authors participated in manuscript revisions.

Competing financial interests

G.J.N. is currently an employee of Sanofi, which produces commercial influenza vaccines. P.M.M. is currently an employee of MedImmune which also makes commercial influenza vaccines.

Corresponding author

Correspondence to: Gary J. Nabel

The authors declare that an intellectual property application has been filed by NIH based on data presented in this paper.

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Le coronavirus frappe la Tunisie (L'Economiste Maghrébin, May 22 2013)

2013-05-22T18:56:00.001+02:00

[Source: L’Economiste Maghrébin, full text: (LINK).]

Le coronavirus frappe la Tunisie

‎22 May ‎2013 | Meriem Ben Nsir

Au dernier bilan de l’OMS, celui du 22 mai, l’on apprend que le ministère tunisien de la Santé publique a rapporté deux cas d’infection par le nouveau coronavirus (NCoV) confirmés et un cas suspect. Les deux cas confirmés, frère et sœur et respectivement âgés de 34 et 35 ans , ont aux dernières nouvelles contracté [...]

Cet article Le coronavirus frappe la Tunisie est apparu en premier sur L'Economiste Maghrébin.

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Influenza A(H7N9) - China - Monitoring human cases (ECDC/CDTR, May 22 2013, edited)

2013-05-22T18:50:00.001+02:00

[Source: European Centre for Disease Prevention and Control (ECDC), full PDF document. (LINK). Extract, edited.]

COMMUNICABLE DISEASE THREATS REPORT

Week 20, 12-18 May 2013

(…)

Influenza A(H7N9) - China - Monitoring human cases

Opening date: 31 March 2013 Latest update: 16 May 2013

Epidemiological summary

On 31 March 2013, Chinese authorities announced the identification of a novel reassortant A(H7N9) influenza virus isolated from three unlinked fatal cases of severe respiratory disease in eastern China, two in Shanghai and one in Anhui province. The WHO Collaborating Centre for Reference and Research on Influenza at the Chinese Center for Disease Control and Prevention (CCDC) had subtyped and sequenced the viruses and found to be of almost identical low pathogenic avian origin.

Since 31 March 2013, 131 cases of human infection with influenza A(H7N9) have been reported from eastern China and Taiwan: Zhejiang (46), Shanghai (33), Jiangsu (27), Henan (4), Anhui (4), Beijing (1), Shandong (2), Fujian (5), Hunan (2), Jiangxi (6) and Taiwan (1). In addition, the virus has been detected in one asymptomatic case in Beijing. Onset of disease was between 19 February and 29 April 2013. The date of disease onset is currently unknown for fifteen patients. Most cases have developed severe respiratory disease. Thirty two patients have died (case-fatality ratio=24%). The median age is 61 years ranging between four and 91 years; 37 of 131 patients are female.

The Chinese health authorities are responding to this public health event with enhanced surveillance, epidemiological and laboratory investigation and contact tracing. The animal health sector has intensified investigations into the possible sources and reservoirs of the virus. The authorities reported to the World Organisation for Animal Health (OIE) that avian influenza A(H7N9) was detected in samples from pigeons, chickens and ducks, and in environmental samples from live bird markets ('wet markets') in Shanghai, Jiangsu, Anhui and Zhejiang provinces. Authorities have closed markets and culled poultry in affected areas.

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ECDC assessment

Influenza A(H7N9) is a zoonotic disease that has spread or is spreading in poultry in parts of eastern China causing a severe disease in humans. At this time there is no evidence of sustained person-to-person transmission. Close to 3 000 contacts have been followed-up and only four are reported to have developed symptoms, as part of three small family clusters.

At present, the most immediate threat to EU citizens is to those in China who are strongly advised to avoid live bird markets. The risk of the disease spreading to Europe via humans in the near future is considered low. However, it is likely that people presenting with severe respiratory infection in the EU and a history of potential exposure in the outbreak area will require investigation in Europe.

There is no specific guidance on blood or tissue donor deferral for exposure to avian influenza. The incubation period for A(H7N9) is assumed to be 10 days or less, and there is no reason to believe that infected people will be viraemic beyond the acute disease episode. Therefore, the risk of transmission through blood transfusion can be considered very low in the context of the current donor selection procedures.

The gradual geographical extension seems to have slowed down and there has been a decline in the number of cases during the last week possibly du to closing urban live bird markets in China. However, many unanswered questions remain regarding this outbreak e.g. the reservoir, the route of transmission, the spectrum of disease, the reason for the unusual age–gender imbalance.

Actions

ECDC is closely monitoring developments and is continuously re-assessing the situation in collaboration with WHO, the US CDC, the Chinese CDC and other partners.

ECDC published an updated Rapid Risk Assessment on 8 May 2013.

A case detection algorithm and an EU case definition has been developed and shared with EU Member states.

ECDC guidance for Supporting diagnostic preparedness for detection of avian influenza A(H7N9) viruses in Europe for laboratories was published on 24 April 2013.

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Novel Coronavirus (MERS-CoV) - Multistate - Severe respiratory syndrome (ECDC/CDTR, May 22 2013, edited)

2013-05-22T18:45:00.001+02:00

[Source: European Centre for Disease Prevention and Control (ECDC), full PDF document: (LINK). Extract, edited.]

COMMUNICABLE DISEASE THREATS REPORT

Week 20, 12-18 May 2013

(…)

Novel Coronavirus (MERS-CoV) - Multistate - Severe respiratory syndrome

Opening date: 24 September 2012 Latest update: 16 May 2013

Epidemiological summary

The first described case of MERS-CoV infection was a 60-year-old male resident of Saudi Arabia who died of severe pneumonia complicated by renal failure in June 2012. A previously unknown coronavirus isolated from this patient was identified.

As of 16 May, 40 laboratory confirmed cases have been reported by Saudi Arabia (30), Jordan (2), Germany (2), United Kingdom (4) and France (2). Twenty of these cases have died. All cases worldwide remain associated (including indirect association following secondary person-to-person transmission in the UK and France) with transmission in the Arabian Peninsula.The age of cases ranges from 24 to 94 years (age is unknown for 4 cases). Eight cases are female and 31 are male (gender is unknown for 1 case).

Since the beginning of May 2013, the Ministry of Health in Saudi Arabia reported 21 cases, seven of which were fatal. All cases were from the eastern Al HAsa governorate. The outbreak is primarily linked to a health care facility. Two patients are healthcare workers who were exposed to patients with confirmed MERS-CoV.

On 8 May 2013, there was a first case reported in France in a patient with recent travel history to the United Arab Emirates who presented with diarrhoea and fever. He was hospitalised on 23 April. On 12 May, France reported a second case. This patient shared a hospital room with the first patient from 27 to 29 April 2013. After the UK, France is the second country reporting local transmission in Europe.

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ECDC assessment

The additional recent coronavirus cases reported by the Saudi Arabian authorities indicate an ongoing source of infection present in the Arabian Peninsula.

The first French case who presented with diarrhoea is a reminder of the possibility that presentations may not include respiratory symptoms initially, especially in those with immunosuppression or underlying chronic conditions. This needs be taken into account when revising case-finding strategies. The imported case in France is the second nosocomial transmission in Europe following one in the UK in February 2013 highlighting the risk of onward transmissions in Europe, in particular in healthcare settings. Both French patients had underlying conditions, and a degree of immunosuppression. One of the transmissions in the UK was also to an immunosuppressed person. These underlying conditions may be increasing vulnerability and the risk of transmission. Information on many of the basic epidemiological indicators required for determining effective control measures are still missing for most cases that occurred in the Middle East, e.g. the reservoir of infection, risk groups, incubation period, period of infectivity, settings where infection has occurred.

The recent imported cases reported by Germany and France, following medical evacuation and travel, indicate that more cases may be expected to be identified in the EU in the immediate future.

Actions

ECDC has updated the rapid risk assessment, and published an epi-update on 7 May (Epidemiological update ECDC). The results of an ECDC coordinated survey on laboratory capacity for testing for the novel coronavirus in Europe were published in
EuroSurveillance. On 6 May, WHO posted technical guidance on infection prevention and control on their website. ECDC is closely monitoring the situation in collaboration with WHO and the European Union Member States.

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Novel Coronavirus, Saudi Arabia: An additional fatal confirmed case (Health Min., May 22 2013, edited)

2013-05-22T18:28:00.001+02:00

[Source: Saudi Arabia Ministry of Health, full page in Arabic: (LINK). Automatic translation.]

Novel Coronavirus, Saudi Arabia: An additional fatal confirmed case

May 22 2013

The Ministry of Health said in a statement that it was registered another case of the virus (Novel Coronavirus), a patient non-Saudi Qassim, who was admitted to hospital before several days; because of severe pneumonia, and died on Tuesday, may he rest in peace. It Should be noted that most of the injuries recorded so far have been in elderly patients who have multiple chronic diseases. In the province of Al-Ahsa did not record any new cases in the past five days, thankfully.

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Hong Kong, Update on number of suspected human cases of avian influenza A(H7) notified (May 22 2013)

2013-05-22T17:28:00.001+02:00

[Source: Centre for Health Protection, Hong Kong PRC SAR, full text: (LINK).]

Update on number of suspected human cases of avian influenza A(H7) notified

The Centre for Health Protection (CHP) of the Department of Health (DH) today (May 22) provided an update on the latest number of suspected human cases of avian influenza A(H7) notified to the CHP, including cases fulfilling reporting criteria and remaining ones not fulfilling reporting criteria.

From noon yesterday (May 21) to noon today, the CHP received no notification of cases which fulfilled reporting criteria, but two cases which did not fulfil reporting criteria. (...)

The total number of notifications received by the CHP since March 31 of cases fulfilling reporting criteria of suspected human cases of avian influenza A(H7) hence remains at 29, while the total number of notifications not fulfilling reporting criteria is now 105.

A DH spokesman urged travellers not to visit wet markets with live poultry in the affected areas, and to avoid direct contact with poultry, birds or their droppings. If contact has been made, they should thoroughly wash hands with soap and water.

"Influenza A(H7) is a statutorily notifiable infectious disease in Hong Kong.’’

‘’Locally, no confirmed human cases of avian influenza A(H7N9) have been recorded so far," the spokesman stressed.

The spokesman reminded doctors to report to the CHP any suspected case of influenza A(H7). The Public Health Laboratory Services Branch of the CHP is ready to receive and test specimens whenever necessary.

The public may visit the CHP's avian influenza page (www.chp.gov.hk/en/view_content/24244.html) for further information.

Ends/Wednesday, May 22, 2013
Issued at HKT 17:53
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