Since February 2013, China experienced an outbreak of the novel H7N9 avian flu, causing 131 cases of infection, and a death toll of 39. This particular H7N9 strain is considered to be one of the most worrisome pathogens since the H5N1 pandemic in 1997; a reputation based on the virus’ ability to spread easily across species and to infect humans. According to the May 23, 2013 Science paper published by the Joint Influenza Research Centre (State Key Laboratory of Emerging Infectious Diseases, Shantou PR, China), Drs. Y. Guan and Y. Shu reported that H7N9 infects the upper respiratory tract of ferrets and pigs, and spreads via direct contact, suggesting that the rapid surge of H7N9 infections are likely caused by human’s direct contact with infected birds.

The source of the H7N9 virus is quite elusive, mainly because birds carrying the H7N9 virus appear to be unharmed by the infection. This is quite unlike the H5N1 outbreak in 1997, where H5N1-infected birds can succumb to the infection, and that the presence of dead ducks and poultry often indicates the presence of the H5N1 virus. 

The Elusive Origin of H7N9
To solve the puzzle of H7N9’s origin, a group of scientists lead by Dr. Kwok Yung Yuen (University of Hong Kong) discovered that H7N9 virus was derived from two origins of ducks, a study the was published in the June 1, 2013 issue of the Lancet. The conclusion was based on the sequencing the hemagglutinin (H7) and neuraminidase (N9) viral genes in human patients that were infected with H7N9 (throat swabs, Zhejiang, China), and extensive analysis to determine whether similar H7 and N9 sequences can be found in duck and poultry samples from the Asian wet markets. Using this approach, Yuen reported that the H7 sequence found in domestic ducks in Zhejiang, and the N9 sequence found in wild ducks in Korea, were the closest match to the respective H7 and N9 protein sequences in H7N9-infected patients in Zhejiang. Yuen further highlighted that in contrast to the H7 proteins from domestic ducks, the H7 proteins in H7N9-infected patients carry several amino-acid mutations including the Gln226Leu, Gly186Val substitutions (associated with their ability to infect humans) and PB2 Asp701Asn mutation (associated with mammalian adaptation). 

Further extrapolating the origin of H7N9 from an influenza sequence database (Global Initiative on Sharing Avian Influenza Data (GISAID) database), Dr. George Gao at the Chinese Center for Disease Control and Prevention (Beijing) demonstrated that the H7 gene is likely to have evolved from wild migratory ducks in Asia. Gao further found the H7N9 virus also shares internal genes that are similar to what is found in two lineages of H9N2 poultry influenza virus. In his report published in the June 1, 2013 issue of the Lancet, Gao further concluded that the H7N9 in humans is an evolutionary product that is likely created by multiple gene reassortment events between influenza viruses from at least four different migratory ducks and poultry lineages. According to Gao, H7N9 virus transmission and mutations in birds, and potentially in intermediate animals, should be watched closely to minimize the chances of a future pandemic.

The Ease of H7N9 Mutations and Human-Adaptation Points to the Possibility of a Future Pandemic
As quickly as the H7N9 outbreak emerged in China, the virus seemed to have rapidly disappeared, with no further cases of H7N9 infections since the end of May 2013. But whether the virus would reappear, and whether there might be a future pandemic, are crucial questions to which scientists can only surmise. According to Dr. Michael Gardam (Director of infection prevention and Control at University Health Network, Toronto General Hospital), a global pandemic is really triggered by human-to-human transmission.  “As long as it’s just in birds and hopping to humans on occasion, you’re going to get the same epidemiology that you have now. If [H7N9] can efficiently transfer from human to human, you’ll have a pandemic”, says Gardam. “Influenza is ‘very sloppy’ when it replicates itself – it’s always making mistakes. It could make a ‘good mistake’ for the virus and alter itself to make it easier to spread from person to person. It could also recombine with other viruses, picking up components, of say H3N1 or H1N1, making it easier for humans to contract,” Gardam further explains.

Despite the temporary absence of H7N9 infection in China, scientists are still concerned that the H7N9 viruses in asymptomatic birds could continue to evolve to give rise to viruses capable of human-to-human transmission. Indeed, this is exactly happened to the deadly H5N1 since its outbreak in 1997. According to the 2005 study published in the Journal of Virology, the World Health Organization reported that while the certain H5N1 strains can cause fatality in ducks, there are strains that remain asymptomatic in ducks, and contribute to the continual spread and evolution of the H5N1 strains in wild ducks and waterfowl in 10 Asian countries throughout late 2003 and early 2005 H5N1 in 10 Asian countries. The result was 53 human fatalities in Thailand, Vietnam, and Cambodia, and the death and slaughter of more than 150 million birds. A similar study published in 2005 in the New England Journal of Medicine suggests the possibility of human-to-human transmission of H5N1 in 2004-2005, specifically in house-hold clusters in Vietnam. Whether history would repeat itself in the evolution of H7N9 virus is a possibility that can have serious and immediate impact on public health. 

The melting pot of influenza viruses carried in ducks and poultry is very likely the place where H7N9 might mutate by “picking up” viral components from other avian influenza strains. However, scientists are aware that H7N9 can just as likely to mutate in mammals and humans that are coinfected with H7N9 and other influenza strains. Indeed, a recent paper published in the June 15th issue of The Lancet reported a case of human coinfection, where a boy from Jiangsu, China was found to be infected by both the H7N9 and H3N2. According to Dr. Yefei Zhu and colleagues (who authored the paper), “the dual infections are a potential source of reassortment between human and avian viral strains, which could raise the risk of human-to-human transmission”.

Further concerning scientists is the relative ease of H7N9 in breaking the barrier to achieving human-to-human transmission, and acquiring resistance to antiviral drugs. In the two recent Cell papers (June 20, 2013) published by Dr. Ram Sasisekharan at the Massachusetts Institute of Technology, Sasisekharan reported that H7N9 and H5N1 require merely a single amino acid mutation in the hemagglutinin to dramatically enhance their ability to infect humans, and possibly enhance human-to-human transmission. In another study by Dr. Zhenghong Yuan at the Key Lab of Medical Molecular Virology (Shanghai Medical College of Fudan University), a single amino acid mutation (Arg292Lys mutation) in the neuramindase gene of H7N9 contributed to its ability to resist with oseltamivir (Tamiflu, Roche). The study, published in the May 28th online issue of The Lancet, was based on 14 human cases of H7N9 infection in Shanghai, China. 

Indeed, the evidence so far indicates that the risk of a future H7N9 pandemic is very real. Yet despite scientist’s efforts in keeping track of the virus, there is really nothing scientists can do to stop a pandemic. According to Gardam, “If H7N9 is able to transmit from person to person, there really isn’t anything realistically that you can do, it’s just going to spread. I’ve likened it to a tidal wave – it’s about surfing the tidal wave than trying to stop the tidal wave.” Gardam further suggests that the most realistic strategy to “ride the tidal wave” is to take hygienic precautions to limit human-bird interactions, and to develop drugs and vaccines against the H7N9 virus.  

Riding the Avian Flu Tidal Wave: Avian Flu Vaccines in the Horizon
 On June 14th, 2013, the first DNA vaccine against H7N9 virus was created by Inovio, a pharmaceutical company based in Blue Bell, Pa. Preclinical testing of this vaccine can trigger immune response levels exceeding what are considered protective levels in other common influenza subtypes, protecting 100% of the mice from H7N9 infection. According to the company’s press release, 10 mice received two intramuscular doses of vaccine 3 weeks apart, and tested for antibodies against the H7N9 virus. The results showed that all 10 animals produced high antibody titers against the hemagglutination (H7). The company further affirms the effectiveness of this vaccine for humans, stating that the DNA vaccine is designed to target the HA influenza antigen from sequences collected from several infected H7N9 patients as a way to provide broad protection against all H7N9 viruses. The company expects to optimize and manufactured within 2 weeks, and to develop more broad acting vaccines in the future to afford protection against other forms of avian influenza including H1N1, H2N2, H3N2, and H5N1, as well as type B influenza.  

Lessons from Ducks 
According to Dr. Li Ning, a member of the Chinese Academy of Engineering and a professor at China Agricultural University, ducks (Anas platyrhynchos) are the natural reservoir for influenza A viruses. Unlike other birds that can succumb to influenza A infections, ducks can harbour infections from a wide range of influenza A viral strains, including 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes, all without any disease manifestation. The duck’s ability to remain unscathed during a flu infection makes them the ideal place for virus to thrive and evolve, earning the duck’s reputation as the silent carrier of the influenza A virus.  

How ducks actually survive the deadly avian flu infection (with the exception of certain H5N1 strains) remains a question that scientists have no clear answer, although there is a consensus that the ducks have a much stronger immune system. In a recent study published in the June 9th, 2013 issue of Nature Genetics, Ning attempted to use sequencing technology to solve the immunological riddle behind the duck’s uncanny ability to survive avian flu infections.  In this study, Ning and colleagues analysed the duck’s entire genome sequence, and then collected the immunological transcriptome of ducks that are infected with two different H5N1 viruses. The sequencing data revealed that ducks do not necessarily have a stronger immune system per se. Indeed, the duck genome does not appear to encode more copies of immune genes, nor does the immune transcriptome indicate that ducks express more immune genes during the course of H5N1 infection, compared to other animals. Rather, Ning found that ducks appear to express unique families of immune genes (beta-defensin and butyrophilin genes) that are thought to be involved in the host antiviral defense against influenza, genes that are not seen in other animals. Ning further concluded that the discovery of the duck’s unique defense mechanism against avian influenza might point the way to new antiviral drugs against H5N1, H7N9, and other avian flu outbreaks in humans. 

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