Further defense of the Chinese H1N1 – H5N1 study

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.

Influenza H5N1 x H1N1 reassortants: ignore the headlines, it’s good science

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):

h1n1 h5n1 reassortants

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.

TWiV 229: Partly cloudy with a high of H7N9

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.microbe.tv/twiv.

First human infections with avian influenza H7N9 virus

comingled birdsFourteen 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.

Pandemic H1N1 influenza virus outcompetes seasonal strains in ferrets

ferret-h1n1-coinfectionWhen more than one influenza A virus subtype is circulating in humans, as has been the case since 1977, there are several possible outcomes. The viruses might co-circulate, one virus might out-compete another, or co-infection of cells with two viruses can lead to the production of genetically distinct viruses by the process of reassortment of viral RNAs. Experiments have been done in ferrets to determine how the 2009 pandemic H1N1 strain interacts with seasonal H3N2 and H1N1 viruses.

Ferrets were intranasally co-infected with an H1N1 pandemic strain [Ca/04] and either a seasonal H1N1 virus [BR/59] or a seasonal H3N2 virus [BR/10].  One uninfected ferret was placed in the same cage (to allow contact transmission) and a second in another cage separated by a wire mesh (to allow aerosol transmission). They determined whether the viruses replicated in the animals by detecting viral RNA in nasal washes taken 1 day after infection using polymerase chain reaction (PCR), or by hemagglutination-inhibition assays to measure serum antibodies.

The results are striking. Both viruses replicate well in co-infected ferrets – look at panel C of the image above. The figure is a photograph of the DNA products of the PCR, separated by gel electrophoresis. Each lane shows the DNA corresponding to the individual influenza viral RNA. Panels A and B show the amplified DNAs from the nasal washes of two animals who were infected by contact. In these animals, the pandemic CA/04 virus replicates well (right half) while the seasonal H1N1 strain [BR/59] does not (left half).

When the nasal washes from the respiratory droplet contact ferrets were used to infect a new set of ferrets, only the pandemic CA/04 virus was detected; there was no evidence of the seasonal BR/59 or BR/10 viruses.

The pattern of the DNAs are distinct for each virus. For example, the PB2 DNA of BR/59 migrates more slowly on the gel than the PB2 DNA of CA/04 (panel C). Given these differences in migration of the DNAs, it would be easy to determine if there were reassortants in the co-infected ferrets. None can be detected by this analysis.

If these results were directly applicable to humans, we would predict that the 2009 H1N1 pandemic strain is not likely to reassort with the seasonal strains; and that it will out-compete those strains, which will eventually disappear. But we are not ferrets, and we don’t know whether these findings apply to humans. Nevertheless, the authors are allowed to speculate:

Although we must be cautious interpreting studies in the ferret model, it is reasonable to speculate that this prototypical pandemic strain, Ca/04, has all the makings of a virus fully adapted to humans.

In this case ‘fully adapted’ means that the pandemic strain replicates better than the seasonal strains or any reassortants that might arise in co-infections.

What did the press learn from this work? Reuters concluded:

And while a new study in ferrets suggested the virus spreads more quickly and causes more severe disease than seasonal flu, the good news is that it does not appear likely to mutate into a “superbug” as some researchers had feared.

Virologists consider mutation and reassortment to be two distinct phenomena. The mutation rate of the virus is determined by error-prone RNA synthesis. The host applies the selection pressure that enriches for a particular phenotype. The results of these studies reveal nothing about the ability of the virus to mutate.

Perez, D., Sorrell, E., Angel, M., Ye, J., Hickman, D., Pena, L., Ramirez-Nieto, G,, Kimble, B., & Araya, Y. (2009). Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection PLoS Currents RRN1011.2.

Reassortment of the influenza virus genome

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genomes, such as influenza virus, have another mechanism for generating diversity: reassortment.

When an influenza virus infects a cell, the individual RNA segments enter the nucleus. There they are copied many times to form RNA genomes for new infectious virions. The new RNA segments are exported to the cytoplasm, and then are incorporated into new virus particles which bud from the cell.

If a cell is infected with two different influenza viruses, the RNAs of both viruses are copied in the nucleus. When new virus particles are assembled at the plasma membrane, each of the 8 RNA segments may originate from either infecting virus. The progeny that inherit RNAs from both parents are called reassortants. This process is illustrated in the diagram below, which shows a cell that is co-infected with two influenza viruses L and M. The infected cell produces both parental viruses as well as a reassortant R3 which inherits one RNA segment from strain L and the remainder from strain M.

influenza-reassortment

One example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. For example, the 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses, as illustrated.

influenza-h1n1-reassortment

Reassortment can only occur between influenza viruses of the same type. Why influenza A viruses never exchange RNA segments with influenza B or C viruses is not understood. However, the reason is probably linked to the packaging mechanism that ensures that each influenza virion contains at least one copy of each RNA segment.

Trifonov, V., Khiabanian, H., & Rabadan, R. (2009). Geographic Dependence, Surveillance, and Origins of the 2009 Influenza A (H1N1) Virus New England Journal of Medicine DOI: 10.1056/NEJMp0904572