Wenfei Zhu

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

BACKGROUND Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus. METHODS We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses. RESULTS A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died. CONCLUSIONS Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Biological features of novel avian influenza A (H7N9) virus

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1β, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

Genetic tuning of the novel avian influenza A(H7N9) virus during interspecies transmission, China, 2013

A novel avian influenza A(H7N9) virus causing human infection emerged in February 2013 in China. To elucidate the mechanism of interspecies transmission, we compared the signature amino acids of avian influenza A(H7N9) viruses from human and non-human hosts and analysed the reassortants of 146 influenza A(H7N9) viruses with full genome sequences. We propose a genetic tuning procedure with continuous amino acid substitutions and reassorting that mediates host adaptation and interspecies transmission. When the early influenza A(H7N9) virus, containing ancestor haemagglutinin (HA) and neuraminidase (NA) genes similar to A/Shanghai/05 virus, circulated in waterfowl and transmitted to terrestrial poultry, it acquired an NA stalk deletion at amino acid positions 69 to 73. Then, receptor binding preference was tuned to increase the affinity to human-like receptors through HA G186V and Q226L mutations in terrestrial poultry. Additional mammalian adaptations such as PB2 E627K were selected in humans. The continual reassortation between H7N9 and H9N2 viruses resulted in multiple genotypes for further host adaptation. When we analysed a potential association of mutations and reassortants with clinical outcome, only the PB2 E627K mutation slightly increased the case fatality rate. Genetic tuning may create opportunities for further adaptation of influenza A(H7N9) and its potential to cause a pandemic.

Human infection with an avian influenza A (H9N2) virus in the middle region of China.

During the epidemic period of the novel H7N9 viruses, an influenza A (H9N2) virus was isolated from a 7‐year‐old boy with influenza‐like illness in Yongzhou city of Hunan province in November 2013. To identify the possible source of infection, environmental specimens collected from local live poultry markets epidemiologically linked to the human case in Yongzhou city were tested for influenza type A and its subtypes H5, H7, and H9 using real‐time RT‐PCR methods as well as virus isolation, and four other H9N2 viruses were isolated. The real‐time RT‐PCR results showed that the environment was highly contaminated with avian influenza H9 subtype viruses (18.0%). Sequencing analyses revealed that the virus isolated from the patient, which was highly similar (98.5–99.8%) to one of isolates from environment in complete genome sequences, was of avian origin. Based on phylogenetic and antigenic analyses, it belonged to genotype S and Y280 lineage. In addition, the virus exhibited high homology (95.7–99.5%) of all six internal gene lineages with the novel H7N9 and H10N8 viruses which caused epidemic and endemic in China. Meanwhile, it carried several mammalian adapted molecular residues including Q226L in HA protein, L13P in PB1 protein, K356R, S409N in PA protein, V15I in M1 protein, I28V, L55F in M2 protein, and E227K in NS protein. These findings reinforce the significance of continuous surveillance of H9N2 influenza viruses. J. Med. Virol. 87:1641–1648, 2015.

Two Outbreak Sources of Influenza A (H7N9) Viruses Have Been Established in China

ABSTRACT Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened. IMPORTANCE Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.

Human Infection with Highly Pathogenic Avian Influenza A(H7N9) Virus, China

The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient’s adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.

Mutations in Polymerase Genes Enhanced the Virulence of 2009 Pandemic H1N1 Influenza Virus in Mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.

Novel Avian Influenza A(H7N9) Virus in Tree Sparrow, Shanghai, China, 2013

In spring 2013, influenza A(H7N9) virus was isolated from an apparently healthy tree sparrow in Chongming Dongping National Forest Park, Shanghai City, China. The entire gene constellation of the virus is similar to that of isolates from humans, highlighting the need to monitor influenza A(H7N9) viruses in different species.

Genesis and Spread of Newly Emerged Highly Pathogenic H7N9 Avian Viruses in Mainland China

ABSTRACT The novel low-pathogenic avian influenza A H7N9 viruses (LPAI H7N9 viruses) have been a threat to public health since their emergence in 2013 because of the high rates of mortality and morbidity that they cause. Recently, highly pathogenic variants of these avian influenza A H7N9 viruses (HPAI H7N9 viruses) have emerged and caused human infections and outbreaks among poultry in mainland China. However, it is still unclear how the HPAI H7N9 virus was generated and how it evolved and spread in China. Here, we show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region and spread southward to the Pearl River Delta region, possibly through live poultry trade. After introduction into the Pearl River Delta region, the origin LPAI H7N9 virus acquired four amino acid insertions in the hemagglutinin (HA) protein cleavage site and mutated into the HPAI H7N9 virus in late May 2016. Afterward, the HPAI H7N9 viruses further reassorted with LPAI H7N9 or H9N2 viruses locally and generated multiple different genotypes. As of 14 July 2017, the HPAI H7N9 viruses had spread from Guangdong Province to at least 12 other provinces. The rapid geographical expansion and genetic evolution of the HPAI H7N9 viruses pose a great challenge not only to public health but also to poultry production. Effective control measures, including enhanced surveillance, are therefore urgently needed. IMPORTANCE The LPAI H7N9 virus has caused five outbreak waves in humans and was recently reported to have mutated into highly pathogenic variants. It is unknown how the HPAI H7N9 virus originated, evolved, and disseminated in China. In this study, we comprehensively analyzed the sequences of HPAI H7N9 viruses from 28 human and 21 environmental samples covering eight provinces in China that were taken from November 2016 to June 2017. The results show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region. However, the insertion of four amino acids into the HA protein cleavage site of an LPAI H7N9 virus occurred in late May 2016 in the Pearl River Delta region. The mutated HPAI H7N9 virus further reassorted with LPAI H7N9 or H9N2 viruses that were cocirculating in poultry. Considering the rapid geographical expansion of the HPAI H7N9 viruses, effective control measures are urgently needed.

A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity

BackgroundInfluenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated.ResultsBy measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system.ConclusionsThis approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.