On episode #233 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review aerosol transmission studies of influenza H1N1 x H5N1 reassortants, H7N9 infections in China, and the MERS coronavirus.

You can find TWiV #233 at www.twiv.tv.

Robert Herriman of The Global Dispatch interviewed me this week on the H1N1 – H5N1 reassortant study that has been in the headlines:

There was much written concerning the research published earlier this month in Science, where researchers from China’s Harbin Veterinary Research Institute reported creating an  avian H5N1 (highly pathogenic) and pandemic 2009 H1N1 (easily transmissible) hybrid, that according to them, achieved airborne spread between guinea pigs.

Read the rest of the article at The Global Dispatch.

This discussion of West Nile virus was recorded at the headquarters of the American Society for Microbiology during a “Microbes After Hours” event on May 6, 2013. The speakers are Dr. Lyle Petersen Lyle R. Petersen, M.D., M.P.H., director of the Division of Vector-Borne Diseases at CDC, and Dr. Roberta DeBiasi, MD, FIDSA, Associate Professor of Pediatrics at George Washington University School of Medicine, Acting Chief and Attending Physician in the Division of Pediatric Infectious Diseases at Children’s National Medical Center, and investigator at Children’s Research Institute in the Center for Translational Science in Washington, D.C.

MWV Episode 70 – Microbes After Hours – West Nile Virus from microbeworld on Vimeo.

I joined Buddhini Samarasinghe, Scott Lewis, Tommy Leung, and William McEwan for a discussion of the avian influenza H5N1 virus transmission experiments done in ferrets.

On episode #232 of the science show This Week in Virology, Vincent meets up with Roberto, Reuben, Lou, and Leslie at the University of Minnesota to talk about their work on HIV-1, APOBEC proteins, measles virus, and teaching virology to undergraduates.

You can find TWiV #232 at www.twiv.tv.

Those of you with an interest in virology, or perhaps simply sensationalism, have probably seen the recent headlines proclaiming another laboratory-made killer influenza virus. From The Independent: ‘Appalling irresponsibility: Senior scientists attack Chinese researchers for creating new strains of influenza virus’; and from InSing.com: ‘Made-in-China killer flu virus’. It’s unfortunate that the comments of several scientists have tainted what is a very well done set of experiments. Let’s deconstruct the situation with an analysis of the science that was done.

It is known that avian influenza H5N1 viruses can occasionally infect but not transmit among humans, while the 2009 pandemic H1N1 virus (which continues to circulate) readily transmits from person to person. The investigators asked whether reassortants of the two viruses – which could arise in nature – might confer transmissibility to H5N1 virus. To answer this question they produced 127 different reassortants of the two viruses, and tested their ability to transmit by aerosol among guinea pigs. The latter have been used for transmission studies on influenza, notably to understand the seasonality of infection. Ferrets have been more famously used for influenza virus transmission studies.

Rather than describe the results, I’ve made an illustration that shows what I believe to be the most important conclusions of the study (click for a larger version):

The H5N1 virus (red RNAs) is not transmissible among guinea pigs, while the H1N1 virus (green RNAs) has highly efficient transmission. Exchange of the H5N1 RNA coding for PA or NS from H1N1 produces a highly transmissible virus. Exchange of the H5N1 RNA coding for NA or M produces a less efficiently transmitted virus. These are interesting and novel findings. It will be of great interest to determine how the PA, NS, NA, or M genes mechanistically enhance aerosol transmission. This is important information because our understanding of the determinants of transmission is very poor.

All the reassortant viruses shown in the figure have the H5 HA; when only the H1 of the H1N1 virus was substituted with the H5 HA, the reassortant virus transmitted efficiently among guinea pigs. In ferrets the H5 HA is not compatible with aerosol transmission. Therefore guinea pigs are clearly different from ferrets with respect to the determinants of transmissibility.

I cannot understand why some scientists have called these experiments ‘appallingly irresponsible’ and of no scientific use. I can only assume that they are not familiar with the literature on viral transmission and do not appreciate how the results advance our understanding of the field. It also seems irresponsible to predict that these viruses, should they escape from the laboratory, could kill millions of people. If you accept guinea pigs as a predictor of human pathogenicity – which I do not – then there is no reason for fear because none of the reassortants were lethal. I do not believe that any animal model predicts what will occur in humans, and so I am even less concerned about the safety of these experiments. I firmly believe that laboratory-constructed viruses do not have what it takes to be a human pathogen: only viral evolution in nature can produce the right combination of RNA segments and mutations. I also believe that scientists are quite responsible when it comes to safe handling of pathogens. If we worry about every type of transmission experiment involving influenza H5N1 virus, we will never make progress in understanding why this virus does not transmit among humans. The moratorium on H5N1 transmission research is over; let’s move beyond the sensational headlines and get back to the science.

In summary, I believe that these are well designed experiments which show that single RNA exchanges with H1N1 virus can produce an H5N1 virus that transmits via aerosol among guinea pigs. The relevance of these findings to humans is not known; nevertheless understanding how the individual viral proteins identified in this study enhance transmission may be mechanistically informative. I believe that the news headlines depicting these experiments as irresponsible and dangerous are based on uninformed statements made by scientists who are not familiar with the literature on influenza virus transmission. I wonder if they even read the paper in its entirety before making their comments.

On episode #231 of the science show This Week in Virology, Vincent meets up with Amit, Lan, and Ian to discuss their discovery of hepaciviruses and pegiviruses in bats and rodents.

You can find TWiV #231 at www.twiv.tv.

I joined Buddhini Samarasinghe and Scott Lewis on a Science Sunday Hangout on Air to talk about my career in virology: how I came to be interested in viruses, and what goes on in my laboratory. You can find hangouts and more at the ScienceSunday community.

On episode #230 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review H7N9 infections in China, the debate over patenting genes, and receptor-binding by ferret-transmissible avian H5 influenza virus.

You can find TWiV #230 at www.twiv.tv.

Results of a study of four patients in Zhejiang, China, who developed influenza H7N9 virus infection suggests sporadic poultry-to-human transmission:

We diagnosed avian influenza A H7N9 in all four patients (who were epidemiologically unlinked), two of whom died and two of whom were recovering at the time of writing. All patients had histories of occupational or wet market exposure to poultry. The genes of the H7N9 virus in patient 3′s isolate were phylogenetically clustered with those of the epidemiologically linked wet market chicken H7N9 isolate. These findings suggest sporadic poultry-to-person transmission.

The four patients had occupational contact with poultry: one was a chef, one slaughtered and cooked live market poultry, and two bought live market poultry. Each had contact with poultry 3-8 days before onset of disease, and all were positive for influenza H7N9 virus by polymerase chain reaction of sputum or throat swab samples (virus was cultured from three of the four patients). Two of five pigeons and four of 20 chickens from two different wet markets were also positive for influenza H7N9 virus. Sequence analysis of virus recovered from patient 3 revealed that the HA and NA genes are nearly identical with those of two viruses isolated from epidemiologically linked chickens (1673 of 1683 bases for HA, 1394 of 1398 bases for NA).

While these H7N9 infections might have been acquired from poultry, the origin of other infections in different areas of China (>100) is unclear. According to the Ministry of Agriculture, as of 26 April 2013, only 46 of the 68,060 samples collected from poultry markets, habitats, farms and slaughterhouses across the country have tested positive for H7N9 virus, and none of these positive samples have been from poultry farms.

Virologist Hilary Koprowski died on 11 April 2013 at the age of 96. His main accomplishments are nicely summarized in the New York Times, but for a more comprehensive overview of his life, I highly recommend his biography Listen to the Music by Roger Vaughan. I did not have many opportunities to interact with Dr. Koprowski, but I did follow his work on poliovirus vaccines and I have a few reminiscences.

In the 1930s Max Theiler had found that propagating yellow fever virus in an unnatural host – the chick embryo – dramatically reduced its capacity to cause disease in humans. Theiler’s work (which garnered him a Nobel Prize) lead to the production of the infectious, attenuated yellow fever vaccine which helped to vastly reduce the global incidence of yellow fever. Koprowski was inspired by Theiler’s work and decided to take a similar approach to developing a poliovirus vaccine – his first efforts involved passage of a type 2 strain of poliovirus in mice and then in cotton rats. After passage in  rodents, the virus did not cause paralysis in monkeys. Koprowski tested the candidate vaccine strain in humans, and ultimately produced two other attenuated poliovirus strains. By the 1960s these attenuated poliovirus vaccine candidates had been tested in millions of humans. However, they were never licensed for use in the US. While Koprowski was carrying out his work, Albert Sabin was also developed attenuated vaccine strains of poliovirus. Both Sabin’s and Koprowski’s strains were tested side by side in a monkey neurovirulence test carried out by Joseph Melnick at Baylor University. Sabin’s virus strains were slightly more attenuated, and in 1961-62 those were selected for licensing in the US. Sabin’s oral poliovirus vaccines (OPV) have been the mainstay of the World Health Organization in its polio eradication campaign.

Koprowski’s polio vaccines were tested by human clinical trials, notably in the former Belgian Congo in 1957-58. It was subsequently suggested that this clinical trial initiated the AIDS pandemic. The idea, first proposed by Tom Curtis (19 March 1992 “The Origin of AIDS: A startling new theory attempts to answer the question, ‘Was it an act of God or an act of man?” Rolling Stone pp. 54–9, 61, 106, 108) and subsequently by Edward Hooper in ‘The River‘, was that Koprowski had propagated the vaccine strains in kidney cell cultures produced from locally captured chimpanzees. If these animals were infected with the precursor of HIV-1, simian immunodeficiency virus (SIV), then the virus might have entered the human population during the polio vaccine trials. This hypothesis was subsequently shown to be incorrect as phylogenetic analysis showed that the main group of HIV-1 viruses, the M group, clearly crossed from chimpanzees to humans in the early 1900s.

A committee was established to investigate the virological aspects of the HIV-polio vaccine controversy, and towards the end of its work I was asked to join. When it was discovered that samples of Koprowski’s polio vaccines were frozen at the Wistar Institute in Philadelphia, it was decided to determine whether these vaccines had been propagated in rhesus monkey or chimpanzee cells.

I was given the job of dividing and coding the samples. I met a representative of the Wistar Institute in the parking lot of a restaurant just off the New Jersey Turnpike, halfway between New York and Philadelphia. He handed me a white styrofoam box, packed with ice, that contained  vials of the Koprowski vaccine. To the uninformed observer, it might have looked like a drug exchange.

I took the vials back to the lab (see photo), thawed them, separated them into aliquots, and gave each a code. I then returned them to the Wistar in the same way, after a second trip on the New Jersey Turnpike. The samples were sent to three different laboratories where experiments were done to determine the mitochondrial DNA type of the cells in which the viruses had been propagated (although the samples were free of cells, some mitochondrial DNA would still be present from virus induced cell lysis). The results, which have been published (reference one, two), clearly showed that the vaccines had not been grown in chimpanzee cells. I was pleased to have played a small role in this story.

Although I had spoken with Dr. Koprowski several times on the telephone, I did not meet him until 2005 when he presented a talk on the history of rabies in the History of Science series at Columbia University Medical Center. I was his host for that visit, during which I was photographed with Dr. Koprowski and Dr. Rich Kessin, another Columbia professor. We invited Dr. Koprowski to dinner after the seminar but he declined, but he did want to have a drink together. After being warned by his driver not to keep him too late, we walked to a local bar and Dr. Koprowski ordered a Boodles gin martini. The bartender noted that he didn’t receive many calls for that brand. Dr. Koprowski said it was his favorite gin. We talked for a while and then returned Dr. Koprowski to his car for the trip back to Philadelphia. During his visit I had Dr. Koprowski autograph my copy of Listen to the Music (photo). He wrote: “To my friend Vince, with warmest regards, Hilary, 4/14/05.” It was the first and last time I saw him.

I would like to relate one last story which has nothing to do with me, but is irresistible. It takes place in the opening pages of Listen to the Music. Koprowski and his technician Thomas Norton are about to drink an early version of his attenuated polio vaccine. The virus has been passaged in rats and appears to be attenuated in monkeys. On a January day in 1948 Koprowski and Norton are in a laboratory at Lederle Laboratories in Pearl River, NY., where they are blending the brains and spinal cords of rats that had been infected with the vaccine strain virus. They both drink a milliliter of the cold, greasy, mix which flows thickly over their tongues and is difficult to swallow.

Here is the best part:

When he can speak, Norton asks, “Have another?”

“Better not,” Koprowski says. “I’m driving.”

The World Health Organization has been publishing weekly reports on the avian influenza A(H7N9) outbreak which include the geographical location of each case, the cumulative number of cases, and the epidemiological curve. Go to this page at the WHO website for an archive of the weekly reports (there you will also find other useful information on the H7N9 outbreak). Images for report #3 of 24 April 2013 are reproduced below. Click each image for a larger view.

From the Centers for Disease Control in Taiwan:

In the late afternoon of April 24, 2013, the Central Epidemic Command Center (CECC) confirmed the first imported case of H7N9 avian influenza in a 53-year-old male Taiwanese citizen who worked in Suzhou, Jiangsu Province, China prior to illness onset. He developed his illness three days after returning to Taiwan. Infection with avian influenza A (H7N9) was confirmed on April 24, 2013. The patient is currently in a severe condition and being treated in a negative-pressure isolation room.

It’s not clear how the patient acquired the infection in China; he had no contact with birds or poultry and did not eat undercooked poultry or eggs.

The patient has had contact with 139 others, and all but 3 have used the appropriate personal protective equipment to prevent infection.

There is still no evidence for human-to-human transmission of avian influenza H7N9 virus in China. If this trend continues in Taiwan there should be no spread of the virus to others.

As long as people are allowed to travel from China, this probably won’t be the only imported case.

During my visit to Berkeley, CA to record TWiV #228, I met Deb Sklut, an artist who is inspired by the power of science. I recorded a brief conversation with Deb which you can view below. Her work can be found at SqueakySqueegeeArt.etsy.com.

On episode #229 of the science show This Week in Virology, Vincent, Rich, Dickson, and Alan review the current status of human infections with avian influenza H7N9 virus.

You can find TWiV #229 at www.twiv.tv.

An outbreak of high-pathogenicity avian influenza H7N7 virus that took place on 255 poultry farms in the Netherlands during 2003 has been used to provide clues about the current avian influenza H7N9 viruses in China. During the Dutch outbreak 453 humans showed symptoms of illness and 89 were confirmed to have infection with the virus. Some interesting observations from that outbreak:

• Conjunctivitis (inflammation of the membranes surrounding the eyelids) was observed in many of the human cases, as well as in later human infections with H7 influenza viruses. Apparently these viruses replicate well in the eye, which bears alpha-2,3 sialic acid receptors. From there the viruses could reach the nasal cavity via the nasolacrimal duct. 
• There was one fatal infection during the Dutch outbreak, and virus isolated from this individual contained the amino acid change E627K in viral protein PB2, which is associated with higher replication of avian influenza viruses in mammals. This change likely arose during replication of virus in the patient as it was not observed in other isolates. The recent H7N9 isolates from China all have the PB2 E627K mutation.
• In the Dutch outbreak there was no evidence for human to human transmission of H7N7 viruses. This conclusion is in part supported by phylogenetic analysis of viral sequences, which showed that during the outbreak the viruses diversified into multiple lineages with human strains at the ends of the trees (Figure; click to enlarge. Credit: Eurosurveillance).

So far the H7N9 virus does not appear to be spreading from human to human. This observation suggests that the virus is widespread in poultry in China, and that there have been multiple introductions into humans. It seems likely that these novel viruses arose relatively recently in China and some time thereafter had to opportunity to infect humans.

The question on everyone’s mind is whether the avian influenza H7N9 viruses will acquire the ability to transmit among humans. On this subject the authors have the following comment:

Although human infections with H7 influenza viruses have occurred repeatedly over the last decades without evidence of sustained human-to-human transmission, the absence of sustained human-to-human transmission of A(H7N9) viruses does not come with any guarantee.

It is possible that during replication in birds or humans, the H7N9 viruses might randomly acquire a mutation that allows for transmission. In the right place at the right time, such a virus could spread through the human population. Alternatively, such a transmission-facilitating mutation might interfere with the overall fitness of the virus, thereby preventing it from spreading. I favor the latter hypothesis because the H7N9 viruses have been transmitting since at least February 2013; they have undergone many replication cycles without such a mutation arising. If the virus has not entered wild birds, culling poultry could eradicate it from China – assuming that it has not gone elsewhere.

from Brian Foley:

18th International BioInformatics Workshop on Virus Evolution and Molecular Epidemiology
University of Florida, Emerging Pathogens Institute
Gainesville, Florida, USA
August 25th – August 30th, 2013
Bioinformatics Methods Applied to Virology and Epidemiology

Announcing the organization of the international workshop on Virus Evolution and Molecular Epidemiology (VEME) in 2013, hosted by the Emerging Pathogens Institute in the warm city of Gainesville and sponsored by several local partners.

We plan to organize a ‘Phylogenetic Inference’ module that offers the theoretical background and hands-on experience in phylogenetic analysis for those who have little or no prior expertise in sequence analysis. An ‘Evolutionary Hypothesis Testing’ is targeted to participants who are well familiar with alignments and phylogenetic trees, and would like to extend their expertise to likelihood and Bayesian inference in phylogenetics, coalescent and phylogeographic analyses (‘phylodynamics’) and molecular adaptation. A ‘Large Dataset Analysis’ module will cover the more complex analysis of full genomes, huge datasets of pathogens including Next Generation Sequencing data, and combined analyses of pathogen and host. Practical sessions in these modules will involve software like, PHYLIP, PAUP*, PHYML, MEGA, PAML or HYPHY, TREE-PUZZLE, SplitsTree, BEAST, MrBayes Simplot and RDP3.

We recommend participants to buy The Phylogenetic Handbook as a guide during the workshop, and to bring their own data set.

For further information and applications check this website.

Abstract and application deadline is April 30th.

Selections will be made by end of May 2013.

The registration fee of 1000 USD covers attendance, lunches and coffee breaks.

Participation is limited to 25 scientists in each module and is dependent on a selection procedure based on the submitted abstract and statement of motivation. A limited number of grants are available for scientists who experience difficulties to attend because of financial reasons.

There have been over 60 human infections with avian influenza virus H7N9 in China, and cases have been detected outside of Shanghai, including Beijing, Zhejiang, Henan, and Anhui Provinces. Information on the first three cases has now been published, allowing a more detailed consideration of the properties of the viral isolates.

The first genome sequences reported were from the initial three H7N9 isolates: A/Shanghai/1/2013, A/Shanghai/2/2013, and A/Anhui/1/2013. These were followed by genome sequences from A/Hongzhou/1/2013 (from a male patient), A/pigeon/Shanghai/S1069/2013), A/chicken/Shanghai/S1053/2013), and A/environment/Shanghai/S1088/2013, the latter three from a Shanghai market.

Analysis of the viral genome sequences reveals that all 8 RNA segments of influenza A/Shanghai/1/2013 virus are phylogenetically distinct from A/Anhui/1/2013 and A/Shanghai/2/2013, suggesting that the virus passed from an animal into humans at least twice. Similar viruses have been isolated from pigeons and chickens, but the source of the human infections is not known. There is as yet no evidence for human to human transmission of the H7N9 viruses, and it seems likely that all of the human infections are zoonotic – transmission of animal viruses to humans. Since the H7N9 viruses are of low pathogenicity in poultry, infected animals may not display disease symptoms, further facilitating transmission to humans.

The RNA sequences reveal that the H7N9 viruses isolated from humans are all triple reassortants, which means that they contain RNA segments derived from three parental viruses. The gene encoding the hemagglutinin protein (HA) is most closely related to the HA from A/duck/Zhejiang/12/2011 (H7N3), while the NA gene is most similar to the NA gene from A/wild bird/Korea/A14/2011 (H7N9). The remaining 6 RNA segments are most related to genes from A/brambling/Beijing/16/2012-like viruses (H9N2). The type of animal(s) in which the mixed infections took place is unknown.

Some observations on the relatedness of these sequences:

• A/Shanghai/2/2013, A/Anhui/1/2013, and A/Hangzhou/1/2013 were isolated in distant cities yet have over 99% identity. The pigeon, chicken, and environmental isolates are also very similar except for one gene of A/pigeon/Shanghai/S1069/2013. Long-range shipping of infected poultry might explain these similarities.
• There are 53 nucleotide differences between A/Shanghai/1/2013 and A/Shanghai/2/2013. Perhaps A/Shanghai/1/2013 and the remaining viruses originated from different sources.

When the gene sequences of these human viral isolates are compared with closely related avian strains, numerous differences are revealed. The locations of the proteins in the influenza virion are shown on the diagram; click for a larger version (figure credit: ViralZone).

• All seven H7N9 viruses do not have multiple basic amino acids at the HA cleavage site. The presence of a basic peptide in this location allows the viral HA to be cleaved by proteases that are present in most cells, enabling the virus to replicate in many organs. Without this basic peptide, the HA is cleaved only by proteases present in the respiratory tract, limiting replication to that site. This is one reason why the H7N9 viruses  have low pathogenicity in poultry.
• All seven viruses have a change at HA amino acid 226 (Q226L) which could improve binding of the viruses to alpha-2,6 sialic receptors, which are found throughout the human respiratory tract. Avian influenza viruses prefer to bind to alpha-2,3 sialic acid receptors. This observation suggests that the H7N9 isolates should be able to infect the human upper respiratory tract (alpha-2,3 sialic acid receptors are mainly located in the lower tract of humans). However, viruses which bind better to alpha-2,3 sialic acids still bind to alpha-2,6 receptors and can infect humans.
• All seven viruses have a change at HA amino acid 160 from threonine to alanine (T160A). This change, which has been identified in other circulating H7N9 viruses, prevents attachment of a sugar to the HA protein and could lead to better recognition of human (alpha-2,6 sialic acid) receptors.
• Five amino acids are deleted from the neuraminidase (NA), the second viral glycoprotein, in all seven viruses. In avian H5N1 influenza virus this change may influence tropism for the respiratory tract and enhance viral replication, and might regulate transmission in domestic poultry. This change is believed to be selected upon viral replication in terrestrial birds.
• One of the viruses (A/Shanghai/1/2013) has an amino acid change in the NA glycoprotein associated with oseltamivir resistance (R294K).
• An amino acid change in the PB1 gene, I368V, is known to confer aerosol transmission to H5N1 virus in ferrets.
• An amino acid change in the PB2 gene, E627K, is associated with increased virulence in mice, higher replication of avian influenza viruses in mammals, and respiratory droplet transmission in ferrets.
• Changes of P42S in NS1 protein, and N30D and T215A in M1 are associated with increased virulence in mice, but these changes are also observed in circulating avian viruses.
• All seven viruses have an amino acid change in the M2 protein known to confer resistance to the antiviral drug amantadine.
• All seven viruses lack a C-terminal PDZ domain-binding motif which may reduce the virulence of these viruses in mammals.

For the most part we do not know the significance of any of the amino acid changes for viral replication and virulence in humans.

I believe that these H7N9 viruses might take one of two pathways. If they are widespread in birds, they could spread globally and cause sporadic zoonotic infections, as does avian influenza H5N1 virus. Alternatively, the H7N9 viruses could cause a pandemic. Influenza H7N9 virus infections have not occurred before in humans, so nearly everyone on the planet is likely susceptible to infection. Global spread of the virus would require human to human transmission, which has not been observed so far. Some human to human transmission of avian H7N7 influenza viruses was observed during an outbreak in 2003 in the Netherlands, but those viruses were different from the ones isolated recently in China. Whether or not these viruses will acquire the ability to transmit among humans by aerosol is unknown and cannot be predicted. If a variant of H7N9 virus that can spread among humans arises during replication in birds or humans, it might not have a chance encounter with a human, or if it did, it might not have the fitness to spread extensively.

What also tempers my concern about these H7N9 viruses is the fact that the last influenza pandemic (H1N1 virus) took place in 2009.  No influenza pandemics in modern history are known to have taken place 4 years apart, although only 11 years separated the 1957 (H2N2) and 1968 (H3N2) pandemics. I suppose that is not much consolation, as there are always exceptions, especially when it comes to viruses.

Meanwhile a vaccine against this H7N9 strain is being prepared (it will be months before it is ready), surveillance for the virus continues in China and elsewhere, and health agencies ready for a more extensive outbreak. These are not objectionable courses of action. But should this be our response to every zoonotic influenza virus infection of less than 100 cases?

Sources

Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus.

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.

On episode #228 of the science show This Week in Virology, Vincent visits the University of California at Berkeley and speaks with Britt Glaunsinger and Eva Harris about their work on Kaposi’s sarcoma associated herpesvirus and dengue virus.

You can find TWiV #228 at www.twiv.tv.

Readers of this blog likely know how I feel about the importance of curiosity. It is what powers my ability to do research and to educate others about what I have learned.

Roger Ebert agrees and takes it one step further:

What I believe is that all clear-minded people should remain two things throughout their lifetimes: curious and teachable

Brilliant.

On episode #227 of the science show This Week in Virology, the complete TWiV team reviews the controversial publication of the HeLa cell genome, a missing vial of Guanarito virus in a BSL-4 facility, and human infections with avian influenza H7N9 virus.

You can find TWiV #227 at www.twiv.tv.

Fourteen people in China have been infected with avian influenza H7N9 virus, leading to five deaths. This avian influenza virus has never been isolated from humans.

Influenza A viruses with the H7 hemagglutinin protein circulate among birds, and some, such as H7N2, H7N3, and H7N7, have been previously found to infect humans. It is not known how the individuals in China acquired the H7N9 virus. Some of the infections have occurred in Shanghai, where a similar virus was found in pigeon samples collected at a marketplace in that city. It is not clear what types of pigeon samples tested positive for the virus, nor is it known whether the virus spread from poultry to pigeons or vice versa. In response the city has begun mass slaughter of poultry to stem further spread of the virus.

Influenza H7N9 virus is typically a low-pathogenicity virus, which means that infection of chickens causes mild respiratory disease, depression, and decrease in egg production. The virus does not have a basic peptide between HA1 and HA2. The presence of a basic peptide in this location allows the viral hemagglutinin glycoprotein to be cleaved by proteases that are present in most cells, enabling the virus to replicate in many organs. Without this basic peptide, the HA is cleaved only by proteases present in the respiratory tract, limiting replication to that site.

According to Brian Kimble on Google+, the nucleotide sequence reveals that the H7N9 human isolate is a reassortant* with 6 RNA segments encoding the internal proteins PB1, PB2, PA, NP, M, and NS derived from H9N2 virus, and the HA and NA from H7N9 virus. The significance of this observation is not clear, because I do not know if H7N9 viruses isolated from birds are also reassortants. One possibility is that reassortment produced a virus that can infect humans. It is known that reassortants of H9N2 viruses with the 2009 pandemic H1N1 strain can transmit via aerosols in ferrets.

An important question is whether this H7N9 virus isolated from humans has pandemic potential. So far there is no evidence for human to human transmission of the virus. There is no vaccine for this subtype of influenza virus, but the virus is susceptible to neuraminidase inhibitors oseltamivir and zanamivir. WHO has released the following statement:

Any animal influenza virus that develops the ability to infect people is a theoretical risk to cause a pandemic. However, whether the influenza A(H7N9) virus could actually cause a pandemic is unknown. Other animal influenza viruses that have been found to occasionally infect people have not gone on to cause a pandemic.

*Because the influenza virus genome occurs as 8 segments of RNA, when multiple viruses infect a single cell, new viruses can be produced with combinations of the parental segments, a process known as reassortment.

Update: Peter Palese notes that the human H7N9 isolates do not have a serine in position 61 (as does the 1918 virus). This change is a human virulence marker for some animal influenza viruses. Brian Kimble notes that the H7N9 isolates possess a L226 equivalent in the HA, which confers human-like receptor binding in other viruses. Human influenza viruses prefer to bind to alpha-2,6 sialic acid receptors, while avian strains bind alpha-2,3 sialic acids. If the human H7N9 viruses can bind alpha-2,6 sialic acid receptors then they are adapted to infect the human upper respiratory tract.

Clase #3, Genética y Genomas, es una discusión de los siete diferentes tipos de genomas virales, y su paso hacia RNA mensajeros, seguido de una revisión del análisis moderno de genética viral.

David Bhella, Ph.D., a structural virologist at the MRC Centre for Virus Research in Glasgow, accepts the Peter Wildy Prize for Microbiology Education, awarded annually by the Society for General Microbiology for an outstanding contribution to microbiology education.

In his laboratory David produces beautiful three-dimensional structures of viruses. In this video you will see how he uses this visual material to educate the public about viruses. David has also developed engaging course material for activities at the Glasgow Science Centre.

I was honored to receive the Wildy Prize in 2012 for my use of social media to teach microbiology.

On episode #226 of the science show This Week in Virology, Vincent and Dickson speak with Terry Dermody about his career in medicine and virology.

You can find TWiV #226 at www.twiv.tv.

On episode #53 of the science show This Week in Microbiology, Vincent, Laura, David, Kalin and Paul get together at the Society for General Microbiology meeting in Manchester, England to talk about next-generation approaches to antimicrobial therapy.

You can find the audio for TWiM #53, along with show notes, at microbeworld.org/twim. Watch video of the episode below.

Behind the scenes in Manchester

Earlier this month the European Molecular Biology Laboratory (EMBL) published the DNA sequence of the genome of HeLa cells, the cell line that is widely used for research in virology, cell biology, and many other areas. This cell line was produced from a tumor taken from Henrietta Lacks in 1951. Unfortunately the EMBL did not receive permission from Ms. Lacks’ family to publish her genome sequence, and have withdrawn the information from public databases.

The history of HeLa cells has been well chronicled in Johns Hopkins Magazine and by Rebecca Skloot in The Immortal Life of Henrietta Lacks. In early 1951, Ms. Lacks was found to have a malignant tumor of the cervix. During her examination at Johns Hopkins Hospital in Baltimore, MD, a sample of the tumor was removed and used to produce the HeLa cell line. But Ms. Lacks’ family never learned about the important cells that were derived from her until 24 years after her death.

It is quite clear that permission to publish the HeLa cell genome sequence should have been obtained from the Lacks family. This issue are discussed in an opinion piece by Rebecca Skloot in the New York Times.

I was honored to work with Rebecca Skloot during the preparation of Immortal Life, and I am flattered that Ms. Skloot thanked me in the afterward of the book. I have also written about my work with HeLa cells (that’s me in the photo with a spinner of the cells). You might also be interested in my conversation with Philip Marcus, who was the first to produce single cell clones of HeLa cells.

One of five vials of Guanarito virus is missing from the Galveston National Laboratory, which houses a BSL-4 laboratory designed to safely study the most dangerous pathogens.

Guanarito virus is a member of the family Arenaviridae, which includes enveloped viruses with a segmented, negative-strand RNA genome. It is found in Venezuela where it is transmitted to humans from rats. The natural host is believed to be the short-tailed cane mouse. In humans the virus causes hemorrhagic fever with a case fatality rate of 23%. The virus does not transmit among humans and is not known to replicate in US rodents. Other viruses that cause hemorrhagic fevers include filoviruses, bunyaviruses, and flaviviruses.

It is believed that the missing vial of virus was inadvertently destroyed within the facility.

For an inside view of a BSL-4 laboratory, including a discussion of how virus stocks are secured, don’t miss ‘Threading the NEIDL‘, a documentary that explores the working of a similar facility in Boston, MA.

MicrobeWorld and the Society for General Microbiology (UK) to live stream two events from their Spring Conference 2013 in Manchester, England, March 25-28.

Peter Wildy Prize for Microbiology Education
Monday, March 25, 2013 17:20 GMT (1:20 PM EST | 10:20 AM PST)  

David Bhella, Ph.D., will be accepting the Peter Wildy Prize for Microbiology Education, awarded annually by the Society for General Microbiology for an outstanding contribution to microbiology education. Bhella’s acceptance speech will be live streamed at 17:20 GMT (1:20 PM EST | 10:20 AM PST). Vincent Racaniello was awarded the Wildy Prize in 2012.

This Week in Microbiology
Wednesday, March 27, 2013 15:30 GMT (11:30 AM EST | 8:30 AM PST) 

Join Vincent Racaniello and co-host Laura Piddock, Ph.D., with guests Paul Williams, Ph.D., Kalin Vetsigian, Ph.D., and David Harper, Ph.D., for a live-streaming episode of This Week in Microbiology. The live stream starts at 15:30 PM GMT (11:30 AM EST | 8:30 AM PST) and you can watch it below. If you have any questions for Vincent or his guests during the broadcast you can tweet your question using the #sgmman hash tag or type it into the chat function of the video player.

If you live elsewhere in the world, please use www.everytimezone.com, to calculate when the live streams will start in your area.

On episode #225 of the science show This Week in Virology, Vincent, Rich, and Kathy read listener comments and questions on viral oncotherapy, science communication, a functional HIV cure in an infant, and much more.

You can find TWiV #225 at www.twiv.tv.

The theme of a recent New Media in Education Conference held at Columbia University was how digital media has reshaped the traditional academic publishing paradigm. I participated in a session entitled ‘New media publishing: Whither the textbook?‘ in which four panelists spoke about their experiences in this area. I spoke about how I use podcasting, blogging, and online courses to teach the public about virology. Paulette Bernd discussed the iPad dissection manual she developed for use in the Gross Anatomy laboratory at the College of Physicians & Surgeons of Columbia University. Grant Ackerman detailed the Business School’s integration of iPads in the MBA program and their use of the iBook as an extended learning resource. Mark Newton recounted how the Center for Digital Research and Scholarship partners with Columbia faculty to implement innovative digital tools and publishing platforms for content delivery and preservation.

Recently thousands of dead and decaying pigs were pulled from rivers in Shanghai and Jiaxing, China. Apparently farmers dumped the animals into the water after the pigs became ill. Porcine circovirus has been detected in the in pig carcasses and in the water.

Porcine circoviruses are small, icosahedral viruses that were discovered in 1974 as contaminants of a porcine kidney cell line. They were later called circoviruses when their genome was found to be a circular, single-stranded DNA molecule. Upon entry into cells, the viral ssDNA genome enters the nucleus where it is made double-stranded by host enzymes. It is then transcribed by host RNA polymerase II to form mRNAs that are translated into viral proteins. There is some evidence that circoviruses might have evolved from a plant virus that switched hosts and then recombined with a picorna-like virus.

Porcine circoviruses are classified in the Circoviridae family, which contains two genera, Circovirus and Gyrovirus. There are two porcine circoviruses, PCV-1 and PCV-2; only the latter causes disease in pigs. Infection probably occurs via oral and respiratory routes, and leads to various diseases including postweaning multisystemic wasting syndrome, and porcine dermatitis and nephropathy syndrome. Virions are shed in respiratory and oral secretions, urine, and feces of infected pigs. Other circoviruses may cause diseases of birds, including psittacine beak and feather disease, and chicken infectious anemia, the latter caused by the sole member of the Gyrovirus genus. There are also circoviruses that infect canaries, ducks, finches, geese, gulls, pigeons, starlings, and swans.

We have no good evidence that porcine or avian circoviruses can infect humans. In the United States, porcine circovirus sequences can be detected in human feces. These most likely originate from consumption of pork products, most of which also contain porcine circoviruses. Circovirus sequences have also been found in commonly eaten animals such as cows, goats, sheep, camels, and chickens. Outside of the United States, the circoviruses found in human stools do not appear to be derived by meat consumption and might cause enteric infections.

Recently both PCV-1 and PCV-2 sequences were detected in Rotarix and RotaTeq, vaccines for the prevention of rotavirus disease in infants. The source of the contaminant was trypsin, an enzyme purified from porcine pancreas, which is used in the production of cell cultures used for vaccine production. Use of these vaccines was temporarily suspended, but resumed when the Food and Drug Administration concluded that there is no evidence that porcine circoviruses pose a safety risk to humans.

The good news is that porcine circoviruses in Shanghai’s waters are no danger to humans. But it is not a good idea to have rotting pig carcasses in a river that supplies some of Shanghai’s drinking water.

In the past five days we released three science shows on the TWi* network.

On This Week in Microbiology (TWiM) episode #52, Vincent and Michael meet up with Ellen Jo Baron to talk about working in a clinical microbiology laboratory.

On This Week in Parasitism (TWiP) episode #52, Vincent and Dickson review the life cycle and pathogenesis of the giant kidney worm, Dioctophyme renale.

On This Week in Virology (TWiV) episode #224, Vincent, Alan, Kathy, and Dickson discuss identification of a cell receptor for the coronavirus-EMC, and the role of interferon-epsilon in protecting the female reproductive tract.

Thanks for listening.

Viruses are obligate intracellular parasites, which means that they must enter a cell to reproduce. As virions are too large to diffuse passively across the plasma membrane, cellular pathways for uptake of extracellular materials provide entry routes. The first step in entry is adherence of virus particles to the membrane, an interaction mediated by binding to one or more receptor molecules on the cell surface. Identification of cell receptors for viruses is an important objective because their study may lead to information about how the virus enters the cell, how it is targeted to specific tissues, and how it causes disease. The cell receptor for the recently identified coronavirus-EMC, which has so far infected 15 humans with 9 deaths, has been identified as dipeptidyl peptidase 4 (DPP4).

Enveloped viruses such as CoVs typically attach to cell receptors via spike glycoproteins embedded in the viral envelope. To identify the CoV-EMC receptor, part of the viral spike (S) glycoprotein was expressed as a fusion protein with the Fc domain of IgG antibody. The protein was mixed with lysates of cells known to be infected with CoV-EMC, and bound proteins were isolated by using agarose beads bound to protein A (which binds the Fc domain). A single polypeptide bound to the CoV-EMC spike protein was identified as DPP4. Four different lines of evidence indicate that DPP4 is a bona fide receptor for CoV-EMC:

• Soluble DPP4 blocks infection of susceptible cells with CoV-EMC
• Expression of DPP4 in non-susceptible cells renders them susceptible to infection
• Antibody to DPP4 blocks infection of cells with CoV-EMC
• Purified DPP4 protein binds CoV-EMC and inhibits infection

Considering that CoV-EMC was isolated in November 2012 from a sick patient, the identification of the cell receptor is indeed rapid progress.

DPP4 protein is expressed in primary human bronchiolar lung tissue and on primary bronchiolar epithelial cell cultures, consistent with the ability of the virus to infect the respiratory tract. The protein is also present on the epithelium of kidney, small intestine, liver and prostate. CoV-EMC has been detected in the respiratory tract and in urine. Whether the virus replicates in the respiratory tract, and then disseminates and replicates elsewhere (e.g. kidney) remains to be determined.

CoV-EMC is believed to have originated in bats. Consistent with this hypothesis, expression of bat DPP4 confers susceptibility to the virus, although not to the same extent as human DPP4. Once the bat precursor of CoV-EMC is identified, it will be interesting to determine how the viral spike glycoprotein has evolved to enable more efficient usage of human DPP4.

DPP4 is a transmembrane protein that regulates the activity of hormones and chemokines through proteolytic cleavage. The cell receptors for two other CoVs are also membrane-bound peptidases, but proteolytic activity is not needed for infection. A soluble form of DPP4 is also present in blood. The authors speculate that reduction of DPP4 protein levels by CoV-EMC infection could result in higher virus-induced disease. If DPP4 is important in regulating the activity of cytokines – major components of immune responses – their removal from the circulation could result in greater virus replication and more tissue damage. It will be important to study the levels of DPP4 in humans infected with CoV-EMC, and to determine whether levels of the receptor affect viral disease.

Studio 360 with Kurt Andersen is a radio show co-produced by Public Radio International and WNYC. The show for the week of 8 March 2013 is called ‘Going Viral‘ and includes seven segments entitled ‘Viruses at the movies’, ‘Does your zombie have rabies’, and ‘Playing against the virus’. They did speak with one virologist for a segment called ‘Reconstructing viruses‘.

To record this segment of Studio 360 I traveled down to the WNYC studios on Varick Street in New York. I sat in a glass-walled, silent room with headphones and before a large microphone. I spoke with the show’s host, Kurt Andersen, who was in a studio somewhere in Los Angeles. The sound quality was excellent and our conversation was wide-ranging, including a discussion on synthetic viruses, avian influenza H5N1, dual-use research, bioterrorism, and zombies. We spoke for 30 minutes but only a bit of that ended up being released. I think that a five minute discussion of science is far less than optimal – I favor long-form science discussions which can truly inform the listener.

You can listen to my segment below or over at the Studio 360 website.

On episode #223 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss new influenza virus NA inhibitors, detection of EEEV antibody and RNA in snakes, and replication of the coronavirus EMC in human airway epithelial cells.

You can find TWiV #223 at www.twiv.tv.

Last fall the science show This Week in Virology teamed up with MicrobeWorld (the public outreach website by the American Society for Microbiology) and Boston University School of Medicine to produce a documentary offering a rarely seen behind-the-scenes view of a biosafety level 4 (BSL-4) laboratory. Today I am pleased to announce the release of Threading the NEDIL: TWiV goes inside a BSL-4.

Constructed in 2009 in the highly populated South End neighborhood of Boston, Massachusetts, the National Emerging Infectious Diseases Laboratories (NEIDL) facility contains labs that operate at biosafety levels 2, 3 and 4. Due to its location the NEIDL has faced a raft of legal and regulatory hurdles that have prevented BSL-3 and BSL-4 labs from becoming functional.

Threading the NEIDL is a 1-hour documentary in which Assistant Director Ron Corley along with Elke Mühlberger and Paul Duprex, virologists that will be working the facility, lead the TWiV team on a walkthrough of the BLS-4 laboratory. They explore how the NEDIL is secured from unauthorized entry, what’s like to wear a BLS-4 level safety suit, how the facility is constructed to make it safe, and how workers carry out experiments with highly dangerous viruses such as Ebola virus and Lassa virus without jeopardizing their health or that of the surrounding community.

The documentary is a never before seen look at how one of America’s state of the art biodefense research facilities operates and the security measures put in place to keep it safe, even in the heart of a major urban center.

Threading the NEIDL from microbeworld on Vimeo.

In the past five days we released three science shows on the TWi* network.

On This Week in Microbiology (TWiM) episode #51, Vincent, Michael, and Elio meet up with Hazel Barton to talk about cave microbiology.

On This Week in Parasitism (TWiP) episode #51, special guest Anthony A. James joins Vincent and Dickson to discuss how mosquitoes can be genetically modified to control infectious diseases.

On This Week in Virology (TWiV) episode #222, the complete TWiV team discusses the amazing finding that cyclic GMP-AMP synthase is a cytosolic innate immune DNA sensor.

Thanks for listening.

Each year as I teach my undergraduate virology course, I record each lecture and put them online where they are freely accessible. You can find the 2013 lectures here at virology blog and on iTunes U. The complete 2012 course lectures are also available (virology blog and iTunes). And don’t forget Virologia en Español, a translation of my 2012 lectures. Apple announced Thursday that over 1 billion lectures have been downloaded from iTunes U, and I’m pleased to have contributed – my 2012 virology course has over 75,000 subscribers!

A student in my virology course approached me recently to thank me for making the lectures available online, and wondered why other professors did not so the same. To help out my teaching colleagues, I have prepared a brief video tutorial on how I record my lectures. As always I am happy to respond to questions: vincent@virology.ws.

I believe that professors should share their courses online free of charge. Such distribution is not likely to impact enrollment – indeed if the courses are great, it will encourage enrollment – and will help educate everyone, which is always a good outcome. So check out my video and start recording!

The US Office of Science and Technology Policy recently released proposed guidelines for maximizing the benefits and minimizing misuse of life sciences research. The measures establish oversight responsibilities for universities and other institutions that receive Federal funding:

Specifically, such institutions would be required to review their current life sciences research involving those pathogens or toxins deemed to be the most dangerous or most amenable to misuse, and then work with the researchers and funding agencies to develop appropriate risk mitigation plans.

This adds to a previously announced internal policy to identify DURC research and institute risk-reducing mitigation plans.

OSTP has requested comments on the proposed policy from researchers, institutions, consumers, security experts, and other stakeholders. The proposed policy can be found at this location (pdf), and instructions on how to submit comments can be found in the Federal Register.

Here is the gist of the proposal. It pertains to you if you get Federal money to work on the following organisms or toxins:

1. Avian influenza virus (highly pathogenic)
2. Bacillus anthracis
3. Botulinum neurotoxin
4. Burkholderia mallei
5. Burkholderia pseudomallei
6. Ebola virus
7. Foot-and-mouth disease virus
8. Francisella tularensis
9. Marburg virus
10. Reconstructed 1918 Influenza virus
11. Rinderpest virus
12. Toxin-producing strains of Clostridium botulinum
13. Variola major virus
14. Variola minor virus
15. Yersinia pestis

And if your research might have the following consequences:

1. Enhances the harmful consequences of the agent or toxin
2. Disrupts immunity or the effectiveness of an immunization against the agent or toxin without clinical and/or agricultural justification
3. Confers to the agent or toxin resistance to clinically and/or agriculturally useful prophylactic or therapeutic interventions against that agent or toxin or facilitates their ability to evade detection methodologies
4. Increases the stability, transmissibility, or the ability to disseminate the agent or toxin
5. Alters the host range or tropism of the agent or toxin
6. Enhances the susceptibility of a host population to the agent or toxin
7. Generates or reconstitutes an eradicated or extinct agent or toxin listed above

If any of this applies to you, it is necessary for you and your institution to develop and implement a risk mitigation plan which must be approved by the funding agency.

If any of this applies to you, but you do not receive Federal funds for the research, you are strongly encouraged to carry out similar oversight procedures.

On episode #221 of the science show This Week in Virology, Vincent, Dickson, and Kathy review two emerging bunyaviruses, SFTSV and SBV.

You can find TWiV #221 at www.twiv.tv.

The risk of being infected with human immunodeficiency virus type 1 (HIV-1) is substantially enhanced in individuals with other sexually transmitted diseases. For example, infection with herpes simplex virus type 2 (HSV-2) increases the risk ratio of acquiring HIV from 2 to 4. Explanations for this increased risk include direct inoculation of HIV-1 into the blood through genital ulcers, and the induction of inflammatory cells by HSV-2 which act as sites of replication for HIV-1. The results of infections carried out in cell culture suggest a biological mechanism for the enhancement of HIV-1 infection by HSV-2.

Langerhans cells (LC) are believed to one of the first cells in which HIV-1 replicates after sexual exposure. LCs are dendritic cells which patrol the mucosal epithelium, taking up and processing antigens and presenting them to T cells in the lymph nodes. These cells express the HIV-1 receptors CD4 and CCR5, but not CXCR4, and can therefore be infected with CCR5-tropic* but not CXCR4-tropic HIV-1. Individuals who do not express CCR5 are resistant to HIV infection. For these and other reasons CCR5-tropic HIV-1 viruses are believed to be ones that transmit infection from one individual to another.

In human skin explant cultures, which contain LCs, co-infection with HSV-2 substantially increased the number of HIV-1 cells. This observation could not be explained by co-infection of individual cells because very few of these were observed in the cultures. When applied to fresh cells, the supernatant of cultures infected with HSV-2 also stimulated the number of HIV-1 infected LCs. These observations suggested that HSV-2 infection stimulates the production of one or more substances from infected cells which in turn improve HIV-1 infection.

Human epithelial and epidermal cells are known to produce antimicrobial peptides such as defensins and cathelicidin. These are short, evolutionarily conserved peptides that inhibit the growth of bacteria, viruses, and fungi. HSV-2 infected keratinocytes were found to produce a number of antimicrobial peptides, but the most important one is called LL-37. This peptide enhanced the expression of HIV-1 receptors CD4 and CCR5 on LCs, leading to increased susceptibility of the cells to HIV-1. Removing LL-37 from the supernatant of HSV-2 infected cells reduces the ability of the medium to stimulate susceptibility to HIV-1.

These findings provide a plausible mechanism by which HIV-1 infection is enhanced by HSV-2. When HSV-2 infects the genital mucosa, the epithelial cells produce LL-37. This antimicrobial peptide enhances the production of CD4 and CCR5 on LCs, allowing more efficient infection by HIV-1. This mechanism is supported by the observation that elevated levels of LL-37 correlate with HIV-1 infection in sex workers.

I wonder why antimicrobial peptides up-regulate CD4 and CCR5. In addition to their antimicrobial properties, the cathelicidins possess chemotactic, immunostimulatory, and immunomodulatory effects, and the upregulation of CD4 and CCR5 are likely part of these activities.

These are exciting findings, and if they are further correlated in humans, they might lead to novel ways of interfering with HIV-1 infection, such as by antagonizing LL-37.

*CCR5 and CXCR4-tropic refer to HIV-1 virions that bind to chemokine receptors CCR5 or CXCR4, respectively, in addition to CD4, to initiate infection.

Harvard University is home to some of the world’s finest virologists. But apparently they do not communicate with the writers at Harvard Magazine, where a botched story on the avian H5N1 influenza virus has just been published.

The problems begin with the first paragraph:

But when Dutch researchers recently created an even more deadly strain of the virus in a laboratory for research purposes, they stirred grave concerns about what would happen if it escaped into the outside world.

Readers of virology blog will know by now that the Dutch researchers did not make an ‘even more deadly strain of the virus’ – they made one that could be transmitted by aerosol, but which had lost its lethality.

The title of the article, ‘The Deadliest Virus’, presumably refers to the H5N1 virus that transmits by aerosol among ferrets. This title is simply wrong, because the virus is not deadly to ferrets.

The first paragraph also contains an equally egregious statement by epidemiologist Marc Lipsitch:

If you make a strain that’s highly transmissible between humans, as the Dutch team did, it could be disastrous if it ever escaped the lab.

Dr. Lipsitch seems to be saying that the Dutch group created an H5N1 virus that transmits among humans. As far as I know, ferrets are not humans.

The article is accompanied by a photograph of two scientists working in BSL4 suits. The legend reads:

The modified H5N1 virus could infect a billion people if it escaped a biocontainment lab like the Canadian facility shown above.

And later Lipsitch is quoted as saying:

It could infect millions of people in the United States, and very likely more than a billion people globally, like most successful flu strains do. This might be one of the worst viruses—perhaps the worst virus—in existence right now because it has both transmissibility and high virulence.

For Lipsitch to say that the virus is both transmissible and of high virulence in humans is a misrepresentation of the Dutch group’s findings. He seems to be making up numbers and scenarios.

Perhaps Dr. Lipsitch does not know that ferret studies are not predictive of how viruses will behave in humans. With so many virologists at Harvard, the writer could have checked Dr. Lipsitch’s statements. But he did not, and the result looks as foolish as the New York Times.

In a brief letter to Biosecurity and Bioterrorism, Alan Zelicoff notes a problem with serosurveys for influenza H5N1 infection:

…peak titers after H5N1 infection occur at about 4 to 6 weeks postinfection and may drop by as much as 32-fold over the course of a year, probably decreasing the sensitivity of serologic testing for past asymptomatic infections. Micro-neutralization testing may be more sensitive.

He cites a serological survey carried out on poultry workers in South Korea, in which 9 of 2,500 subjects were found to have antibodies to H5N1 virus, in the absence of illness. These seropositive individuals carried antibodies that neutralize H5N1 virus infectivity. Assays for antibodies that block infection may be more specific for infection than hemagglutination-based assays. His conclusion:

One can anticipate additional serological surveys that will better inform public health practitioners of the threat to humans from circulating H5N1 clades….morbidity from novel influenza strains does not equate with an impending pandemic, let alone one with high mortality. It would appear likely that a systematic, prospective cohort study is in order to adequately capture the frequency of asymptomatic infection.

On episode #220 of the science show This Week in Virology, Vincent, Rich, Alan, and Kathy discuss regulation of influenza virus replication by splicing, and the bacteriophage T7 random walk.

You can find TWiV #220 at www.twiv.tv.

Tengo el gusto de anunciarles que mi curso de virologia esta ahora disponible en Español.

Este trabajo se realizó bajo la dirección de la Dra. Susana López, virologa del Departamento de Genética del Desarrollo y Fisiología Molecular del Instituto de Biotecnología de la Universidad Nacional Autónoma de Mexico. Las traducciones fueron realizadas gracias a la visión y buena disposición de Daniela Silva, Rosa Rubio, Liliana Sánchez, Alfonso Oceguera y Eugenio de la Mora. Quisiera agradecerles a estas personas por su gran esfuerzo, no solo en traducir mis palabras, sino tambien el material que ilustra cada clase.

Estas son las clases que presenté en mi curso de virología para licenciatura en la Universidad de Columbia en la primavera del 2012. Ustedes pueden encontrar las clases de virología en español en la página: virology.ws/virologia.

Este es otro paso para convertirme en el profesor de virologia del mundo.

My virology colleagues in Mexico have kindly translated my 2012 undergraduate virology lecture videos into Spanish! This has been done by a wonderful group (see photo) lead by virologist Dr. Susana López, in the Departamento de Genética del Desarrollo y Fisiología Molecular del Instituto de Biotecnología de la Universidad Nacional Autónoma de Mexico. Daniela Silva, Rosa Rubio, Liliana Sánchez, Alfonso Oceguera and Eugenio de la Mora translated both the slides and my spoken words. I’m really grateful for this arduous task as it helps spread information about virology even further. Many thanks to all of you for doing this!

The lectures will be posted every two weeks at virology.ws/virologia. You can also find them at the YouTube playlist. Below is the first, ¿Qué es un virus?

About 2% of the world’s population is chronically infected with hepatitis C virus (HCV). This enveloped, positive-strand RNA virus was discovered in 1989, but serological and phylogenetic evidence indicates that it has been infecting humans for hundreds of years, perhaps as long ago as the 14th century. All human viral infections most likely originated in non-human species, but the progenitor of HCV is not known. Recent evidence suggests that horses might have been the source of HCV in humans.

For many years there were no known non-human relatives of HCV until canine hepacivirus was discovered in dogs (we discussed this virus on TWiV #137). However two subsequent studies failed to reveal additional evidence for CHV infection of dogs. In one study, no antibodies to CHV were found in sera from 80 dogs in New York State, and in a second study, PCR failed to detect CHV nucleic acid sequences in 190 samples from dogs in Scotland. Samples from rabbits, deer, cows, cats, mice, and pigs were also negative for CHV. However both groups found evidence for infection of horses. These viruses have been called non-primate hepaciviruses (NPHV).

In one study carried out on horses in New York State, 8 of 103 samples were found to contain antibodies to NPHV. Complete viral genomes were identified from all 8 horses. Most are genetically distinct from CHV, but one viral sequence, obtained from a pool of sera from New Zealand horses, is nearly identical to CHV. NPHV was also detected by PCR in sera from 3 of 175 Scottish horses. Separate serum samples obtained from one horse 5 months apart were positive for viral RNA, indicating persistent infection. None of the horses had any evidence of clinical hepatitis or any other illness.

These results from geographically distinct areas suggest that horses are a reservoir of NPHV. It seems likely that dogs might acquire NPHV infection from horses, as there are opportunities for contact between the two animals on farms or in kennels. Additional NPHV isolates from horses must be studied to confirm this hypothesis.

It will be important to determine if horse NPHV was the source of human HCV. This is theoretically possible because horse products, such as serum containing antibodies to pathogens or toxins, have been injected into humans. There are six genotypes of HCV, each of which is believed to have emerged at different times and geographic locations. Whether their emergence represent different cross-species transmissions, as is the case with the different groups of HIV-1, remains to be determined.

I also wonder how horses originally acquired NPHV. Perhaps it was transmitted to them from another species via a vector bite, such as a mosquito – but from what species?

On episode #219 of the science show This Week in Virology, Vincent and Rich meet up with Anthony S. Fauci, MD, Director of the National Institute of Allergy and Infectious Diseases.

You can find TWiV #219 at www.twiv.tv.

The lethality of avian influenza H5N1 infections in humans has been a matter of extensive debate. The >50% case fatality rate established by WHO is high, but the lethality of the virus might be lower if there are many infections accompanied by mild or no disease. One way to answer this question is to determine how many individuals carry antibodies to the virus in populations that are at risk for infection. A number of such studies have been done, and some have concluded that the results imply a low but substantial level of infection (even less than one percent of millions of people is a lot of infections). The conclusion of a new meta-analysis of H5N1 serosurveys is that most of the studies are flawed, and that the frequency of H5 infections appears to be low.

Twenty-nine different H5N1 serological studies were included in this meta-analysis. None of these are particularly satisfactory according to the authors:

None of the 29 serostudies included what we would consider to be optimal, blinded unexposed controls in their published methodologies, i.e., including in the serology runs blinded samples from individuals with essentially no chance of H5N1 infection. Serological assays can easily produce misleading results, especially when paired sera are not available.

Some of the problems identified in the serological surveys include the possibility that many H5N1 positive sera are the result of false positives, that is, cross reaction with antigens from other influenza virus strains. In addition, many studies utilized H5N1 strains that are no longer circulating.

It is clear that most of the H5N1 serosurveys have not been done as well as they should have been. The authors conclude that “it is essential that future serological studies adhere to WHO criteria and include unexposed control groups in their laboratory assays to limit the likelihood of misinterpreting false positive results.”

Let’s not forget that a completely different way of assessing H5N1 infection – by looking for virus-specific T cells – has been reported. The results provide further evidence for subclinical H5N1 infection and are not subject to the caveats noted here for antibody surveys.

I come away from this meta-analysis with an uneasy sense that the authors are not being sufficiently objective, and that they firmly believe that there are no mild or asymptomatic H5N1 infections. One reason is the authors’ use of ‘only’ to describe their findings. For example: “Of studies that used WHO criteria, only [italics mine] 4 found any seropositive results to clades/genotypes of H5N1 that are currently circulating”. The use of ‘only’ in this context implies a judgement, rather than an objective statement of fact. Furthermore, despite the authors stated problems with all H5N1 serosurveys, they nonetheless conclude that there is little evidence for asymptomatic H5N1 infection. If the studies are flawed, how can this conclusion be drawn?

My concern about the authors’ objectivity is further heightened by the fact that they are members of the Center for Biosecurity at the University of Pittsburgh. These are individuals whose job it is to find dangerous viruses that could be used as weapons. On the front page of the website for the Center for Biosecurity is a summary of the meta-analyis article which concludes that “In the article, Assessment of Serosurveys for H5N1, Eric Toner and colleagues discuss their extensive review of past studies and conclude that there is little evidence to suggest that the 60% rate is too high.”

I would argue that if the H5N1 serosurveys are flawed, then do them properly; it is incorrect to simply assume that the H5N1 virus is as lethal as WHO suggests. The World Health Organization should call for and coordinate a study that satisfies criteria established by virologists and epidemiologists for a robust analysis of human H5N1 exposure.

Any questions?