H7n9 Genetic Analysis Raises Concern Over Pandemic Potential

H7N9 Bird Flu Epidemic In China Worst Since 2013 H7N9 can infect poultry without causing clinical symptoms, which makes its spread difficult to monitor. Ethical Issues in Genetic Testing. Testing should be discouraged when the health care provider determines that potential harms of genetic testing in children and adolescents outweigh the potential benefits. A health care provider has no obligation to provide a medical service for a child or adolescent that is not in the best interest of the.

• H7n9 Genetic Analysis Raises Concern Over Pandemic Potential Energy
• H7n9 Genetic Analysis Raises Concern Over Pandemic Potential 2017

Apr 12, 2013 (CIDRAP News) – A new analysis of H7N9 genetic sequences from the first Chinese patients infected with the virus and from poultry markets found more signals that the virus can attach and replicate efficiently in the airways of humans and other mammals, raising concerns about the virus's pandemic potential. The new findings, published late yesterday in Eurosurveillance, are the first detailed comparison of both the human and market sequences. Results are similar to the genetic details of samples from the first three cases reported by Chinese scientists yesterday in the New England Journal of Medicine. The new results also affirm early observations from some experts that the novel virus has adapted to infect mammals, yielding more information that health officials need to gauge the pandemic threat from the new virus.

The research team from Japan includes Yoshihiro Kawaoka, DVM, PhD, who heads a group at the University of Wisconsin that has done extensive genetic studies on the H5N1 virus, and Masato Tashiro, MD, PhD, director of the World Health Organization Collaborating Center for Reference and Research on Influenza at Japan's National Institute of Infectious Diseases in Tokyo. Their look at sequences from influenza databases included human samples from the first two patients from Shanghai, as well as from a woman from Anhui province and a man from Hangzhou province. All of the patients died. Samples from a market in Shanghai include isolates from a pigeon, a chicken, and an environmental sample. Phylogenetic analysis of the four human samples suggest they have a common ancestor, with the hemagglutinin (HA) gene part of the Eurasian avian influenza lineage and closely resembling HA genes of low-pathogenic H7N3 viruses detected in 2011 in Zhejiang province, south of Shanghai. The group reported that the neuraminidase (NA) gene closely resembles a low-pathogenic H11N9 virus found in the Czech Republic in 2010. Internal genes of the H7N9 virus were closely related to H9N2 avian flu viruses that recently circulated in poultry in Shanghai, as well as Zhejiang and Jiangsu provinces, according to the report.

Researchers said the findings strongly suggest that the new viruses are reassortants that got their HA and NA genes (the H7 and N9) from avian influenza viruses and the rest of their genes from recent H9N2 poultry viruses. When they compared the nucleotides from the four human specimens, they found that one of the Shanghai samples and the ones from Anhui and Hangzhou were 99% similar, despite the fact that they came from cities that were several hundred kilometers apart. They found differences between the two Shanghai samples and noted other patterns with the human and market samples that suggest five of the viruses came from a closely related infection source, while one of the Shanghai samples and the one from the pigeon came from different sources. The Japan group's findings appear to echo the report from Chinese researchers yesterday that there have been at least two introductions into humans. The Japanese researchers also detected mutations increase binding to human receptors, a key marker health officials use to gauge the infectivity of new flu viruses. They found that the two Shanghai strains and the Anhui strain had mutations that increase the binding of H5 and N7 viruses to human-type receptors.

One was the Q226L mutation, also flagged by Chinese researchers yesterday. It has been linked to the spread of respiratory droplets in ferrets and was a finding in two controversial studies—one by Kawaoka's group—in 2012 involving lab-modified H5N1 strains. 'The finding of mammalian-adapting mutations in the RBS receptor-binding site of these novel viruses is cause for concern,' the investigators wrote. The isolate from the Hangzhou patient had a genetic marker (isoleucine at position 226) found in seasonal H3N2 flu viruses. All seven of the viruses had an HA substitution seen in other recently circulating H7 viruses that has been linked to increased binding to human-type receptors, according to the report. In the polymerase PB2 protein, they found a marker in the human samples that is essential for efficient replication and has been seen in H5N1 viruses and in an H7N7 sample that was isolated from a fatal case in the Netherlands in 2003.

When they looked for mutations that influence sensitivity to antiviral medications, they projected that all of the human H7N9 samples should be sensitive to neuraminidase inhibitors, except for one of the Shanghai samples. The exception has a R294K mutation in the NA protein that has been linked to resistance in N2 and N9 flu subtypes, which is concerning, the team wrote. Neuraminidase inhibitors are the most common types of flu antiviral drugs prescribed and include oseltamivir (Tamflu) and zanamivir (Relenza). Researchers also found some virulence markers, including one at the NA stalk and the other in the PB1-F2 protein; however, they said the human sequences so far lack the N66S mutation that was associated with increased pathogenicity of the 1918 pandemic virus and the H5N1 virus. The mutation they saw at the NA stalk can occur when the virus adapts to terrestrial birds, which suggests the novel H7N9 virus or their ancestors may have circulated in terrestrial birds before infecting humans. So far, the host of the virus has not been identified, and health officials are considering a range of animals.

The H7N9 virus sequences also showed an NS1 protein sequence pattern that might attenuate the viruses in mammals. Taken together, the findings present a somewhat clearer picture of the threat the new virus could pose, the group concluded. 'These viruses possess several characteristic features of mammalian influenza viruses, which are likely to contribute to their ability to infect humans and raise concerns regarding their pandemic potential,' they wrote. Kageyama T, Fujisaki S, Takashita E, at al.

Genetic analysis of novel avian influenza A (H7N9) influenza viruses isolated from patients in China, February to April 2013. Eurosurveill 2013 Apr 11;18(15) See also: Apr 11 CIDRAP News story '.

Yoshihiro Kawaoka 1Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 575 Science Drive, Madison, WI 53711, USA 2ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama 332-0012, Japan 3Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan 5Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. Avian influenza viruses rarely infect humans, but the recently emerged avian H7N9 influenza viruses have caused sporadic infections in humans in China, resulting in 440 confirmed cases with 122 fatalities as of May 16, 2014. In addition, epidemiologic surveys suggest that there have been asymptomatic or mild human infections with H7N9 viruses. These viruses replicate efficiently in mammals, show limited transmissibility in ferrets and guinea pigs, and possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. Here, we summarize the characteristic features of the novel H7N9 viruses and assess their pandemic potential. Influenza A virus as a zoonotic pathogen Influenza A viruses are maintained in wild waterfowl, poultry, humans, pigs, and horses; in addition, infection of dogs, marine mammals, and several other mammalian species has been reported (reviewed in ). Influenza A viruses are divided into subtypes according to the antigenicity of their two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); to date, 18 HA and 11 NA subtypes have been identified –.

In humans, only viruses of three HA subtypes (H1, H2, and H3) and two NA subtypes (N1 and N2) have caused annual epidemics and sporadically occurring pandemics in the last and current centuries. Influenza A viruses of all subtypes (except for viruses of the H17N10 and H18N11 subtypes, whose genomic material has been identified in bats, ) have been detected in waterfowl, which is considered the natural host of influenza A viruses. In waterfowl, most influenza A viruses replicate in the intestinal tract and spread to other birds via the fecal-oral route, whereas the respiratory tract is the major site of influenza A virus replication in mammals. Despite the wide host range of influenza A viruses, their transmission from avian to mammalian species or vice versa is rare due to host range restrictions. Influenza A viruses circulating in avian species (so-called ‘avian influenza viruses’) rarely infects humans, and influenza A viruses circulating in humans (‘human influenza viruses’) rarely infect avian species –. Recently, however, avian influenza A viruses of the H5N1 and H7N9 subtypes have caused hundreds of cases of human infections.

So far, sustained human-to-human transmission of these viruses has not been reported. Nonetheless, additional adaptive mutations and/or reassortment with circulating human viruses may enable H5N1 or H7N9 viruses to efficiently infect humans and transmit among them.

Because humans lack protective antibodies against these viruses, human-transmitting H5N1 or H7N9 viruses could spread worldwide, resulting in an influenza pandemic. In this review, we focus on the avian H7N9 influenza viruses that recently infected humans in China , describing their biological features and pandemic potential. Human infections with avian H7N9 influenza viruses in China in 2013–2014 To date, two sizable waves of human infection with H7N9 viruses have been documented. The first wave started with a human case of H7N9 influenza virus infection in Shanghai on February 19, 2013 (this case was officially reported on March 31, 2013).

In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China by the end of April. Most cases were reported from the provinces of Jiangsu, Zhejiang, and Shanghai, which are all located in the eastern part of China ( and ). The detection of H7N9 viruses in live poultry markets –, and epidemiological data suggesting that contact with poultry or contaminated environments in live bird markets was the likely source of many (although not all) human cases (see ‘Epidemiology of human H7N9 virus infections’) prompted the Chinese government to close live poultry markets in several provinces in mid-April, 2013,. This measure most likely led to the rapid decline in new human H7N9 cases during the following two weeks,.

The second wave of human infections with H7N9 viruses started in the fall of 2013 , perhaps spurred by the lower fall temperatures and/or the reopening of poultry markets. The number of confirmed human H7N9 virus infections spiked in January and February of 2014, with more than 30 new cases over several consecutive weeks. Since February 2014, the number of new human cases has declined, although new infections continue to be reported. The second, ongoing wave is characterized by a larger number of human H7N9 virus infections, and by more extensive geographic spread. While most human cases during the first wave were reported in Eastern China, the majority of human H7N9 virus infections during the second wave occurred in the southern province of Guangdong.

As of May 16, 2014, a total of 440 human infections with H7N9 viruses have been confirmed with 122 associated deaths (unofficial statement; ); 425 of the cases occurred in China , whereas the remaining 15 were exported cases. Number of confirmed human cases of H7N9 influenza virus infection in 2013–2014. Number of laboratory-confirmed cases of human infection with H7N9 influenza virus by week of onset of illness (A) and by Chinese provinces by wave (B). Blue and red bars indicate the number of human cases of H7N9 virus infection detected in the first and second waves, respectively.

Provinces are categorized into two groups: northern and southern regions of China. (C) Human cases of H7N9 influenza virus infection by age- and gender-groups. Data for graphs (A) and (C) are based on FluTrackers 2013/14 Human Case List of Provincial/Ministry of Health/Government Confirmed Influenza A (H7N9) Cases with Links. Number of cases per province in (B) is based on the data shown at the CIDRAP website. Epidemiology of human H7N9 virus infections Epidemiological studies have shown that H7N9 virus infections have affected mainly middle-aged or older individuals (; i.e., the median age at infection is 63 years) –. Interestingly, two-thirds of the infected individuals have been male, –, –.

The high number of cases among elderly men may reflect socio-economical differences among age groups and genders since elderly men may have frequent work-related or non-job-related contact with poultry. Most H7N9 influenza patients exhibit general influenza-like symptoms, including fever and cough, and more than half of the infections typically progress to severe pneumonia, acute respiratory distress syndrome (ARDS), and multi-organ failure, –. Most H7N9 virus-infected patients possessed at least one underlying medical condition, such as chronic obstructive pulmonary disease (COPD), diabetes, hypertension, obesity, and/or chronic lung and heart disease, –, suggesting that these comorbidities may increase the risk of severe H7N9 virus infection. Genesis of H7N9 influenza viruses Phylogenetic analyses have revealed that the novel H7N9 viruses likely emerged via reassortment of at least four avian influenza A virus strains. The HA gene of the H7N9 viruses belongs to the Eurasian lineage of avian influenza viruses and is closely related to that of avian H7N3 viruses isolated from ducks in Eastern China in 2010–2011, –.

The NA gene of the novel H7N9 viruses is closely related to that of avian H2N9 and/or H11N9 influenza viruses isolated from wild migratory birds along the East Asian flyway, –. The remaining six viral genes likely originated from two distinct subgroups of an H9N2 sub-lineage (formed by the reassortment of a major H9N2 lineage in China with a Eurasian wild bird virus) circulating in Eastern China, –. This genetic heterogeneity suggests that several reassortment events occurred during the generation and ongoing evolution of H7N9 viruses, –. Genesis of H7N9 influenza virus. The novel H7N9 viruses likely resulted from the reassortment of at least four avian influenza A virus strains. The HA gene of the H7N9 viruses is closely related to the Eurasian lineage of avian influenza viruses and to that of the avian H7N3 viruses recently isolated from ducks in Eastern China. The NA gene of the novel H7N9 viruses is closely related to that of avian H2N9 and/or H11N9 influenza viruses isolated from wild migratory birds along the East Asian flyway.

The remaining six viral genes likely originated from two distinct subgroups of an H9N2 sub-lineage circulating in poultry in Eastern China. The virus encircled by the dashed line represents a possible precursor of H7N9 avian influenza viruses. Although the H7N9 viruses currently circulating in birds do not encode determinants of mammalian adaptation (i.e., PB2-627K, PB2-701N, or PB2-591K/R and HA-226L/I), such mutations can arise during H7N9 virus replication in humans. This figure was created by modification of a figure in. HA determines viral receptor-binding specificity Influenza virus infections are initiated by the binding of HA to receptors on host cells. Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal), which is the predominant sialyloligosaccharide species expressed on epithelial cells in the upper respiratory tract of humans (reviewed in ).

In contrast, avian influenza viruses preferentially recognize sialic acid-α2,3-galactose (SAα2,3Gal), which is the major sialyloligosaccharide species expressed in the intestinal tract of waterfowl, where avian viruses replicate efficiently. Typically, avian influenza viruses exhibit low affinity for SAα2,6Gal (i.e., ‘human-type’ receptors) and therefore, a shift of HA receptor-binding specificity from SAα2,3Gal to SAα2,6Gal is thought to be critical for avian influenza viruses to replicate and transmit efficiently in humans.

Previous studies revealed that the amino acid at position 226 (HA numbers in this article refer to the amino acid position in H3 HA after the removal of the signal peptide) of H3 HAs affects binding to human-type receptors. Although sequence analyses of most H7N9 viruses revealed leucine or isoleucine residues at HA position 226 (i.e., the human virus-type residue) , purified H7N9 HAs bearing these human virus-type residues at position 226 still preferentially bound to SAα2,3Gal in studies using soluble recombinant trimeric HAs expressed in mammalian cells. Therefore, the H7N9 viruses may require additional adaptive mutations in HA, such as a Gly-to-Ser mutation at position 228 of HA, to efficiently bind to the human-type receptors. Nonetheless, several groups have demonstrated that whole H7N9 viruses possessing leucine or isoleucine at position 226 of HA bind to both SAα2,3Gal and SAα2,6Gal –, whereas an H7N9 virus encoding glutamine (i.e., the avian virus-type residue) preferentially bound to SAα2,3Gal, suggesting that the binding of H7N9 virions to human-type receptors might be affected by other viral components (i.e., the neuraminidase).

DAmino acid positions of NA are based on NA numbering of human-infecting H7N9 viruses (avian N9 NA numbering is shown in parentheses). In addition, although the leucine residue at position 226 of HA is characteristic of human influenza viruses, it is encoded by most avian H7N9 virus isolates, indicating that it did not arise during H7N9 virus replication in humans. Therefore, the leucine or isoleucine residue at position 226 of HA in H7N9 viruses likely emerged during virus replication in poultry, and may now facilitate the infection of mammalian cells. PB2 determines viral replicative ability The PB2 protein is one of the three subunits of the viral polymerase complex, which catalyzes viral replication and transcription in the nucleus of infected cells. A lysine residue at position 627 of PB2 (as found in most human influenza viruses) confers efficient replication to avian influenza viruses in mammals. By contrast, glutamic acid at this position, as is found in most avian influenza viruses, significantly restricts avian influenza virus replication in mammals.

Currently, the mechanism through which the amino acid at position 627 of PB2 directs viral replicative ability is thought to involve interactions with other viral and/or host proteins –, most likely in a temperature-sensitive manner. Avian body temperature is 41°C, whereas the temperatures in the human lung and upper respiratory tract are generally considered to be 37°C and 33°C, respectively. Consequently, most avian influenza viruses replicate more efficiently at 41°C than at 33°C. Having a lysine at position 627 of PB2 confers efficient replication to avian influenza viruses at 33°C and 37°C, enabling them to establish robust infections in mammals. Additionally, the PB2-627K mutation has recently been shown to increase the transmissibility of avian influenza viruses –.

H7n9 Genetic Analysis Raises Concern Over Pandemic Potential Energy

Sequence analyses have shown that H7N9 viruses isolated from avian hosts possess glutamic acid at position 627 of PB2, whereas many human H7N9 viruses encode lysine at this position ; this finding suggests that the PB2-627K mutation likely emerges during virus replication in humans, as occurred in the human cases of infection with H7N7 avian influenza viruses in the Netherlands in 2003. Interestingly, some human H7N9 virus isolates that lack PB2-627K possess other potentially mammalian-adapting amino acid changes in PB2 that may compensate for the lack of the mammalian-adapting lysine residue at position 627. Some of the human H7N9 virus isolates that encode PB2-627E possess an aspartic acid-to-asparagine mutation at position 701 of PB2, a mutation known to improve avian virus replication in mammalian cells. Moreover, a PB2-D701N mutation has been shown to increase the replicative ability and virulence in mice of an H7N9 virus encoding PB2-627E. Another human H7N9 isolate lacking PB2-627K acquired a glutamine-to-lysine mutation at position 591 of PB2. A basic amino acid at this position was shown to compensate for the lack of PB2-627K in pandemic 2009 H1N1 viruses,. A comparison of viruses encoding PB2-627E or PB2-627E/591K showed higher virus titers and virulence in mice for the latter virus.

Together, these findings demonstrate that the H7N9 viruses currently circulating in birds do not encode strong determinants of mammalian adaptation in PB2 (namely, PB2-627K, PB2-701N, or PB2-591K/R), but that such mutations arise easily during H7N9 virus replication in humans. Contribution of amino acids in PA to H7N9 virulence Viral proteins other than HA and PB2 also contribute to H7N9 virulence, albeit to a lesser extent than these major virulence determinants. Previous studies have suggested a potential role for PA in the adaptation and pathogenicity of avian influenza viruses in a mammalian host –. Several computational and phylogenetic analyses have identified amino acid mutations in the H7N9 PA protein (another subunit of the viral polymerase complex) that are typically found in human-, but not in avian-influenza viruses, –. Indeed, experimental testing revealed that some of these amino acids (i.e., A100V, R356K, and N409S) affect H7N9 replicative ability and virulence.

Interestingly, however, replacement of human-type amino acids in PA with the amino acids commonly found in avian influenza viruses slightly increased their replicative ability in human cells and their virulence in mice, contrary to the expected attenuating effect. Although the exact reason for this unexpected finding is unknown, it may be that these mutations are introduced to optimize virus replication in different environments. Amino acid changes in the HA of viruses recovered from contact ferrets in the human-infecting H7N9 virus groups. Shown is the three-dimensional structure of A/Anhui/1/2003 (H7N9) HA (PDB ID: 4BSE) in complex with human receptor analogues and a close-up view of the globular head. Mutations shown in cyan (i.e., A138S, G186V, and Q226L/I) are known to increase the binding of avian H5 and H7 viruses to human-type receptors. Mutations that emerged in HA of human-infecting H7N9 viruses during replication and/or transmission in ferrets are shown in green (see also ).

The human receptor analogue is shown in orange. Images were created with MacPymol. DThe H7 HA possesses amino acid insertions corresponding to the position between residues 158 and 159 in the H3 HA. In patients treated with NA inhibitors, resistant H7N9 variants have been detected encoding the R292K mutation in NA that confers resistance to oseltamivir –. Oseltamivir-resistance mutations frequently reduce viral fitness (reviewed in ), although compensatory amino acid changes can restore it. The NA-R292K mutation was found to reduce H7N9 viral fitness in cultured cells in one study; however, another study did not detect an effect of the NA-R292K mutation in an H7N9 virus on virulence in mice or transmissibility in guinea pigs, suggesting that oseltamivir-resistant H7N9 viruses may be competitive in nature. Natural isolates of highly pathogenic avian H5N1 viruses do not transmit among ferrets (reviewed in ), whereas novel H7N9 have limited transmissibility in mammalian models.

Thus, the pandemic potential of novel H7N9 viruses appears to be greater than that of highly pathogenic H5N1 viruses. Combined with the emergence of already partially adapted phenotypes and the relatively high fitness of oseltamivir-resistant H7N9 viruses, these novel viruses pose a significant pandemic threat. Concluding remarks To date, the novel H7N9 influenza viruses have not caused a pandemic in humans due to their inability to support sustained human-to-human transmission.

H7n9 Genetic Analysis Raises Concern Over Pandemic Potential 2017

However, these viruses exhibit high replicative ability and limited transmissibility in mammals, have acquired mammalian-adapting amino acid changes, may reassort with circulating human viruses (based on the reported coinfection of a patient with H7N9 and human H3N2 viruses), and readily acquire resistance to the NA inhibitor oseltamivir,. Moreover, humans lack protective immunity to H7N9 infection and frequently show relatively weak antibody responses when infected with these viruses,. Therefore, a better understanding of the molecular mechanisms of pathogenicity, transmissibility, and immunogenicity of the novel H7N9 influenza viruses, combined with continued surveillance in avian and human populations, will be vital to develop countermeasures against H7N9 infections in humans.