Saturday, May 4, 2013

AVIAN INFLUENZA (51): H5N1/H1N1 HYBRIDS, TRANSMISSIBILITY IN GUINEA PIGS

A ProMED Mail Post:
Published Date: 2013-05-04 15:29:56
PRO/AH/EDR> Avian influenza (51): H5N1/H1N1 hybrids, transmissibility 
Date: Thu 2 May 2013
Source: CIDRAP News [abridged and edited]
http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/news/may0213h5n1.html


Chinese scientists report that lab-generated hybrid viruses combining genes from avian H5N1 and pandemic 2009 H1N1 (pH1N1) influenza viruses can achieve airborne spread between guinea pigs, a finding that seems likely to renew the debate about the risks of creating novel viruses that might be able to spark a human pandemic. Writing in Science, the researchers say that 5 of 127 hybrids they generated by shuffling genes [that is, genome subunits] from the 2 subtypes showed "highly efficient" transmission in guinea pigs. None of the guinea pigs died, but some mice that were infected with the reassortant strains did succumb.

Guinea pigs are not regarded as the best experimental model for human flu, a distinction that belongs to ferrets [that is, the most susceptible mammal]. The Chinese team did not test any of the hybrid viruses in ferrets, because a voluntary moratorium on "gain of function" research on H5N1 virus -- studies involving the creation of potentially dangerous new strains -- intervened in January 2012 and lasted a year. The moratorium was prompted by the controversy that erupted in late 2011 over 2 earlier studies in which researchers generated novel H5N1 strains that spread among ferrets via respiratory droplets. One of the studies involved an H5N1-H1N1 reassortant, while the other involved an H5N1 virus in which specific mutations were induced. The US National Science Advisory Board for Biosecurity (NSABB) sought to prevent publication of full details of the 2 studies but eventually reversed itself, and the studies were published in May and June of 2012. In a statement to reporters, the editors of Science said the new Chinese study "provides evidence that H5N1 viruses spread by air between mammals can be generated by reassortment. Reassortment, as it occurs in nature, however, is much slower."

In another study on H5N1 transmissibility, also published today [2 May 2013] in Science, another team of Chinese researchers examined how hemagglutinin (HA) from H5N1 attaches to human cell receptors. They identified a mutation, Q226L, that enables a mutant form of H5N1 HA to bind to both avian and human receptors.

In the reassortant study, the team focused on H5N1 and pH1N1 because both viruses can infect pigs, suggesting the possibility that reassortants could arise naturally. The researchers, led by Ying Zhang of the Harbin Veterinary Research Institute, conducted their experiments in enhanced animal biosafety level 3 (ABSL-3) conditions at that institute. They say all the experiments were done before the moratorium began. The scientists used an H5N1 virus isolated from a duck in China in 2001 and the 1st pH1N1 strain that was identified in China during the 2009 pandemic. Using reverse genetics, they generated 127 reassortants that combined the H5N1 virus's HA gene with all possible combinations of the other 7 genes from the pH1N1 isolate. The reassortants were easily generated, and all grew efficiently in chicken eggs, the report says. The authors used genetically modified mice to test the virulence of the hybrid viruses. They found that 54 of the reassortants had about the same virulence as the original H5N1 duck virus, killing some of the mice; 38 viruses were less pathogenic than the H5N1 strain; and 35 viruses were more virulent, killing all the mice.

The researchers say guinea pigs are comparable with ferrets as models of human flu transmission. Although guinea pigs have both avian and mammalian types of airway receptors, flu viruses that bind only to avian receptors (alpha2,3-linked sialic acids) don't spread by respiratory droplets in the animals, they report. The team used the original H5N1 and pH1N1 viruses and 19 of their reassortants to test for airborne transmission in guinea pigs. For each strain, 3 guinea pigs were dosed with the virus, and 3 other guinea pigs were placed in cages near them but not close enough for direct contact. The scientists found "highly efficient" airborne transmission of the pH1N1 virus in 5 of the 19 reassortants. The results indicated that the PA (acidic polymerase) and NS (nonstructural protein) genes of the pH1N1 virus can make H5N1 highly transmissible by respiratory droplets in guinea pigs and that the NA (neuraminidase) and M (matrix) genes from the pH1N1 also promote such transmission of H5N1. "These transmission studies indicate that many of the H5N1 hybrid viruses bearing one or more of the PA, NA, M, or NS genes of 2009/H1N1 were transmissible in guinea pigs," the report states.

The scientists say they previously showed that a mutant strain of a duck H5N1 isolate, with changes at HA positions 226 and 228, bound exclusively to human receptors (alpha2,6-linked sialic acids). They found that this mutant, which contains the same combination of mutations as reported in the ferret-transmissible H5N1 virus reported by Ron Fouchier, PhD and colleagues in 2012, did not transmit in guinea pigs. "Our studies provide evidence that H5N1 viruses that are capable of respiratory droplet transmission between mammals can be generated by reassortment between mammalian 2009/H1N1 and avian H5N1 viruses," the report concludes. "Since the internal genes of these reassortants can already replicate efficiently in mammalian hosts, we predict that similar reassortants could infect humans and subsequently acquire mutations that improve binding efficacy for alpha2,6-linked sialic acids," it states.

The Science statement says that studies in a more human-like animal model, such as ferrets, were halted by the research moratorium, "but these are the potential next steps if the influenza research community decides it is important to quantify the degree of threat from avian influenza viruses."

[These experiments are described in the following publications:

Zhang Y, Zhang Q, Kong H, et al. H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet. Science 2013 May 2 (Early online publication) [abstract http://www.sciencemag.org/content/early/2013/05/01/science.1229455]

Zhang W, Shi R, Lu X, et al. An airborne transmissible avian influenza H5 hemagglutinin seen at the atomic level. Science 2013 May 2 (Early online publication) [abstract http://www.sciencemag.org/content/early/2013/05/01/science.1236787]

The reactions
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The report drew varied reactions from other experts consulted by CIDRAP News, with one hailing it as a useful contribution and others questioning whether the findings were worth the risks involved in the experiments.

Andrew Pekosz, PhD, a virologist at Johns Hopkins Bloomberg School of Public Health in Baltimore, said the study provides new information about the kinds of genetic combinations that can lead to flu transmission, which is useful for flu surveillance. "The more we understand how wide-ranging those combinations of genetic changes are that can lead to this kind of transmission, the better off we'll be," he said. "That knowledge helps inform us what we should be looking for regarding viruses that are potential human pathogens." He commented that the study doesn't provide "any kind of lightning-strike or light-bulb observations, but it helps tell us what to be looking for in terms of viruses acquiring transmissibility."

Pekosz said he thinks the findings justify any risks involved in the experiments: "As far as I could tell from reading this and being familiar with the group and how they do their research, everything falls under the guidelines that apply to labs in the US regarding biocontainment, respiratory protection, and monitoring. ... I think the safety concerns are well taken care of." He also commented that it's difficult to make "direct leaps" from the guinea pig findings to what would happen in the human population, but the results suggest the possibility that the reassortants could spread in humans. He noted that guinea pigs are not as widely used as ferrets in flu transmission experiments because they've only been rediscovered in that regard in the past decade or so. "I think another important thing about this paper is this is a directed or systematic creation of reassortant viruses, which makes it hard to say if these would occur in natural infection," Pekosz said. "You're not allowing for the natural competition that would take place in an infected animal."

David Relman, MD, a microbiologist and infectious disease expert at Stanford University, expressed concern over the biosecurity implications of the study. "It clearly has biosecurity concerns," he said in an interview. "I would have liked to see these experiments discussed by a wider community of scientists and nonscientists before they were undertaken. ... I would have some grave questions about doing these without having a clearer idea of how exactly the results would lead to tangible real-time benefits. Having a BSL-3 lab and not working during the moratorium does not address the issue," he said.

Simon Wain-Hobson, PhD, a veteran HIV researcher and an opponent of gain-of-function experiments, was more sharply critical of the study. He is a professor at the Pasteur Institute in Paris and chairs the board of the Foundation for Vaccine Research, a privately funded group that recently asked President Obama's Commission for the Study of Bioethical Issues to review the ethics of experiments designed to increase the transmissibility of H5N1 viruses. In a written statement, Wain-Hobson said this study, along with the earlier ones by Fouchier and Yoshihiro Kawaoka, DVM, PhD, show that many different starting points and different experimental protocols can lead to a flu virus capable of airborne transmission. He said it's impossible to predict the pathway that might lead to a pandemic flu virus in nature. "Which evolutionary trajectory will nature take?" he said. "We don't know because there are simply too many. So what is the use [of the findings] for surveillance? What we learn [from the H5N1 transmissibility studies in general] is that, qualitatively, flu can exploit a huge fraction of sequence space and can adapt to almost anything given time," Wain-Hobson said. "But this we knew, not only from flu work, but also from RNA virology in general. ... The benefits are general knowledge which we basically knew, while the risks are increased by this work."

Wain-Hobson said the authors did "a super piece of work" from the science standpoint and undoubtedly meant well. But he labeled the study "very dangerous work disguised as big science. Given this, one wonders why it is published in Science."

[byline: Robert Roos]

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communicated by:
ProMED-mail
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[These experiments provide evidence that H5N1 viruses that are capable of respiratory droplet transmission between mammals can be generated by reassortment between mammalian 2009/H1N1 and avian H5N1 viruses. The authors conclude since the internal genes of these reassortants facilitate efficient replication in mammalian hosts, it is likely that similar reassortants could promote infection of humans and subsequently acquire mutations that improve binding efficacy to the alpha2,6-linked sialic acids receptors.

These are remarkable experiments in molecular genetics that provide better understanding of the determinants of host range of influenza viruses. Whether such experiments should have been carried out at all, even under BSL-3 conditions, is another matter. - Mod.CP]

http://www.promedmail.org/direct.php?id=20130504.1691399

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