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Emerging in December 2019, coronavirus disease 2019 (COVID-19) eventually became a pandemic and has posed a tremendous threat to global public health. However, the origins of SARS-CoV-2, the causative agent of COVID-19, remain to be determined. It has reported that a certain number of the early case clusters had a contact history with Huanan Seafood Market. Therefore, surveillance of SARS-CoV-2 within the market is of vital importance. Herein, we presented the SARS-CoV-2 detection results of 1380 samples collected from the environment and the animals within the market in early 2020. By SARS-CoV-2-specific RT-qPCR, 73 environmental samples tested positive for SARS-CoV-2 and three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.980% to 99.993% with the human isolate HCoV/Wuhan/IVDC-HB-01. In contrast, no virus was detected in the animal swabs covering 18 species of animals in the market. The SARS-COV-2 nucleic acids in the positive environmental samples showed significant correlation of abundance of Homo sapiens with SARS-CoV-2. In summary, this study provided convincing evidence of the prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stage of COVID-19 outbreak.

Influenza B virus (IBV) is one of the most important human respiratory viruses: it causes approximately one-third of the global influenza-related disease burden each year. However, compared with the several pathogenicity-related molecular markers that have been identified for influenza A virus (IAV), little is known about potential IBV pathogenicity-related markers. Here, although the IBV strain B/Anhui-Tunxi/1528/2014 (AH1528/14) exhibited a more efficient replication ability in vitro and higher pathogenicity in vivo compared with IBV strain B/Anhui-Baohe/127/2015 (AH127/15), only three amino acids differences (HAA390E, NAN342D and PB1V212I) were observed among their full genomes. The contributions of each amino acid difference to the virus pathogenicity were further investigated. Compared with the wild type IBV virus rAH127, the recombinant virus harbouring a single substitution of HAA390E had a similar phenotype, whereas the recombinant virus harbouring PB1V212I replicated to a moderately higher titre in both MDCK cells and in mice. Notably, the virus harbouring NAN342D showed significantly better growth properties in MDCK cells and higher fatality rates in mice. In addition, the presence of NAN342D dramatically enhanced the viral neuraminidase activity. In conclusion, our study identified a novel IBV molecular marker, NAN342D, that could significantly increase the virulence of IBV in mice.

Background A novel coronavirus (COVID-19) has raised world concern since it emerged in Wuhan, China in December 2019. The infection may result in severe pneumonia with clusters of illness onsets. Its impacts on public health make it paramount to clarify the clinical features with other pneumonias. Methods Nineteen COVID-19 and 15 other patients with pneumonia (non-COVID-19) in areas outside of Hubei were involved in this study. Both COVID-19 and non-COVID-19 patients were confirmed to be infected using throat swabs and/or sputa with/without COVID-2019 by real-time RT-PCR. We analyzed the demographic, epidemiological, clinical, and radiological features from those patients, and compared the differences between COVID-19 and non-COVID-19. Results All patients had a history of exposure to confirmed cases of COVID-19 or travel to Hubei before illness. The median (IQR) duration was 8 (6–11) and 5 (4–11) days from exposure to onset in COVID-19 and non-COVID-19 cases, respectively. The clinical symptoms were similar between COVID-19 and non-COVID-19. The most common symptoms were fever and cough. Fifteen (78.95%) COVID-19 but 4 (26.67%) non-COVID-19 patients had bilateral involvement while 17 COVID-19 patients (89.47%) but 1 non-COVID-19 patient (6.67%) had multiple mottling and ground-glass opacity on chest CT images. Compared with non-COVID-19, COVID-19 presents remarkably more abnormal laboratory tests, including AST, ALT, γ-GT, LDH, and α-HBDH. Conclusions The COVID-19 infection has onsets similar to other pneumonias. CT scan may be a reliable test for screening COVID-19 cases. Liver function damage is more frequent in COVID-19 than non-COVID-19 patients. LDH and α-HBDH may be considerable markers for evaluation of COVID-19.

Here, we report the identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge, Guyana. In March 2019, 14 employees of Chongqing Bosai Mining Company, China, working in a manganese mining of Guyana, had unexplained fever, and two of them died. We obtained lung and brain tissues as well as the blood samples from the two deceased cases (patient No. 1 and 2), and bronchoscopy lavages and cerebrospinal fluid samples from one severe case (patient No. 3), respectively. All samples were tested by pathological examination, high-throughput sequencing, and real-time PCR. Pathological detection showed the presence of spore-like structures in the lung tissue of patient No. 1, indicating a fungal infection in this patient. Nanopore sequencing identified the existing of H. capsulatum in the lung tissue sample within 13 h. Next-generation sequencing identified specific fragments of H. capsulatum in all of the samples tested (lung, brain and blood serum from the deceased cases, and plasma from the severe case). Real-time PCR assays did not reveal any viral infection related to transmission from bat feces. We conclude that H. capsulatum was the causative pathogen of this disease cluster based on epidemiologic, clinical, pathological and nucleic acid evidence., Highlights • Scientific question This study reported the identification of Histoplasma as the cause of an unexplained disease cluster in Matthews Ridge, Guyana. • Evidence before this study In March 2019, 14 Chinese employees from Chongqing Bosai Mining Company, China, were engaged in manganese mining in Guyana and presented with unexplained fever. Two of them died. After preliminary examination by the local hospital, some potential infectious pathogens were excluded, including Leptospira, HIV, influenza H1N1, Zika virus, Chikungunya virus, Dengue virus, and Influenza A and B viruses. Histoplasmosis is a fungal disease caused by members of the genus Histoplasma and is mainly prevalent in the American continents. Histoplasma is capable of survival in moist soils and can often be isolated from soils containing decaying feces of bats and birds. Human activities in the surface soil produce aerosols, which in turn are inhaled to cause infection. • New findings In response to the unexplained disease cluster, pathological examination, high through-put sequencing and real-time PCR were implemented. A TGS platform found Histoplasma within 13 hours. NGS was also successfully applied in response to this event. Compared with NGS, the main features of nanopore sequencing are long sequencing ability, simplicity of use, the fastest turn-around time, high portability and real-time analysis of sequencing data. Though NGS had a longer turnaround time (24 hours), it worked well with different sample types (lung tissue, brain tissue and serum from deceased cases and plasma from a severe case) and was more sensitive than nanopore sequencing. Real-time PCR assays did not reveal any infection by viruses related to bat feces transmission. Pathological detection results showed the presence of spore-like structures, indicating fungus infection in this patient. All the results were consistent with the NGS analysis, supporting the fungus infection. • Significance of the study We concluded that H. capsulatum is the causative pathogen for this disease cluster based on epidemiologic, clinical, pathological and nucleic acid supportive evidence.

Highlights • We reported a hospital cluster combined with family cluster of H7N9 infection. A poultry farm was the initially infectious source of the H7N9 virus infection. Airborne transmission may result in the hospital cluster., Summary Objectives To identify human-to-human transmission of H7N9 avian influenza virus, we investigated a hospital cluster combined with family cluster in this study. Methods We obtained and analyzed clinical, epidemiological and virological data from the three patients. RT-PCR, viral culture and sequencing were conducted for determination of causative pathogen. Results The index case presented developed pneumonia with fever after exposure to chicken in a poultry farm. Case A presented pneumonia with high fever on day 3 after she shared a hospital room with the index case. Case B, the father of the index case, presented pneumonia with high fever on day 15 after he took care of the index case. H7N9 virus circulated in the local farm to which the index case was exposed. Full genomic sequence of virus showed 99.8–100% identity shared between the index case and case A or case B. Compared to the earliest virus of Anhui, a total of 29 amino acid variation sites were observed in the 8 segments. Conclusions A hospital cluster combined with family cluster of H7N9 avian influenza infection was identified. Air transmission resulted in the hospital cluster possibly. A poultry farm was the initially infectious source of the cluster.

It has recently been proposed that the Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) is one of the most likely zoonotic viruses to cause the next influenza pandemic. Two main genotypes EA H1N1 viruses have been recognized to be infected humans in China. Our study finds that one of the genotypes JS1-like viruses are avirulent in mice. However, the other are HuN-like viruses and are virulent in mice. The molecular mechanism underlying this difference shows that the NP gene determines the virulence of the EA H1N1 viruses in mice. In addition, a single substitution, Q357K, in the NP protein of the EA H1N1 viruses alters the virulence phenotype. This substitution is a typical human signature marker, which is prevalent in human viruses but rarely detected in avian influenza viruses. The NP-Q357K substitution is readily to be occurred when avian influenza viruses circulate in pigs, and may facilitate their infection of humans and allow viruses also carrying NP-357K to circulate in humans. Our study demonstrates that the substitution Q357K in the NP protein plays a key role in the virulence phenotype of EA H1N1 SIVs, and provides important information for evaluating the pandemic risk of field influenza strains.

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

BackgroundCurrently, Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) are widely prevalent in pigs in China, with sporadic human cases reported as well. As one of the key molecular makers detected in avian H5N1 and H 7N9 viruses and pandemic H1N1 2009 virus, contributions of T271A in PB2 protein to the EA H1N1 viruses are still unknown. In this study, we investigated the effects of residue 271 in PB2 protein on the viral properties of EA H1N1 viruses.MethodsInfectivity, replication, virulence and pathogenicity of the recombinant viruses containing A or T in position 271 in PB2 protein were studied in cells and mice.ResultsThe results showed that the substitution PB2-T271A increased the viral replication in mammalian and avian cell lines. In addition, the mutation enhanced the viral infectivity, virulence and pathogenicity in mice. The viral titers of lung tissue in the rgHuN271A virus were higher than that of the rgHuN271T at 1, 4, and 7 dpi. The MID50 of the rgHuN271A and rgHuN271T virus were 101.1 TCID50 and 101.9 TCID50, respectively. Besides, the substitution of PB2-T271A enhanced the viral polymerase activity in mammalian cells.ConclusionsThe pathogenicity and replication of EA H1N1 virus containing 271A in PB2 protein were higher than the EA H1N1 virus containing 271T in PB2 protein in vivo and in vitro. Therefore, the PB2-T271A mutation should be continually monitored in influenza viruses circulating in pigs and humans.

Background: SARS-CoV-2 has caused a huge outbreak in China and multiple countries since it emerged in Wuhan, China in December, 2019 The virus caused diseases

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.

Severe influenza diseases with high mortality have been frequently reported, especially in those patients infected with avian influenza A (H5N1, H7N9 or H10N8) or during a pandemic. Respiratory distress, which is attributed to alveolar damage associated with immunopathological lesions, is the most common cause of death. There is a wealth of information on pathogenesis or treatment options. In this study, we showed that high levels of C-reactive protein (CRP) were induced and correlated with complement activation in patients infected with severe influenza A (H5N1, H7N9 or H10N8), and higher levels were induced in fatal patients than in survivors. CRP treatment enhanced the phagocytosis of monocytes THP-1 to H5N1 virus as well as the expression of proinflammatory cytokines or apoptosis-associated genes in THP-1 cells or pneumocytes A-549 respectively. CRP may link to proinflammatory mediators contributing to activation of complement and boosting inflammatory response in severe influenza infections. Compound 1,6-bis(phosphocholine)-hexane improved the severity and mortality of mice infected with lethal influenza virus significantly. These observations showed that CRP is involved in deterioration of severe influenza diseases, and indicated a substantial candidate molecule for immunotherapy of severe influenza diseases., Graphical Abstract Image 1, Highlights • CRP induces exacerbated immunoresponse toward overt pulmonary inflammation in severe influenza infections. • CRP may link to proinflammatory mediators contributing to activation of complement and boosting inflammatory response. • CRP stabilizer can alleviate the immunopathological lesions and mortality in mice infected with lethal influenza virus. Severe influenza diseases with high mortality have been frequently reported, especially in those patients infected with avian influenza A (H5N1, H7N9, or H10N8) or during a pandemic. Respiratory distress associated with immunopathological lesions is the most common cause of death in patients infected by these viruses. In this study, we found that CRP may be linked to exacerbated immunoresponse toward overt pulmonary inflammation, which led to alveolar damage and respiratory failure in severe influenza infection. Our data identified that CRP stabilizer can be used to alleviate the immunopathological lesions and mortality in mice infected with lethal influenza virus.

Background The first human infections of novel avian influenza A(H7N9) virus were identified in China in March 2013. Sentinel surveillance systems and contact tracing may not identify mild and asymptomatic human infections of influenza A(H7N9) virus. Objectives We assessed the seroprevalence of antibodies to influenza A(H7N9) virus in three populations during the early stages of the epidemic. Patients/Methods From March 2013—May 2014, we collected sera from the general population, poultry workers and contacts of confirmed infections in nine Chinese provinces reporting human A(H7N9) infections and, for contacts, second sera 2-3 weeks later. We screened for A(H7N9) antibodies by advanced hemagglutination inhibition (HI) assay and tested sera with HI titers ≥20 by modified microneutralization (MN) assay. MN titers ≥20 or four-fold increases in paired sera were considered seropositive. Results Among general population sera (n=1480), none were seropositive. Among poultry worker sera (n=1866), 28 had HI titers ≥20; two (0.11%, 95% CI: 0.02%-0.44%) were positive by MN. Among 61 healthcare and 117 non-healthcare contacts’ sera, five had HI titers ≥20, and all were negative by MN. There was no seroconversion among 131 paired sera. Conclusions There was no evidence of widespread transmission of influenza A(H7N9) virus during March 2013 to May 2014, although A(H7N9) may have caused rare, previously unrecognized infections among poultry workers. Although findings suggest there were few undetected cases of influenza A(H7N9) early in the epidemic, it is important to continue monitoring transmission as virus and epidemic evolve. This article is protected by copyright. All rights reserved.

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) have become predominant in pig populations in China and have recently been reported to have the most potential to raise the next pandemic in humans. The mutation D701N in the PB2 protein, which accounts for 31% of H1N1 SIVs, has previously been shown to contribute to the adaptation of the highly pathogenic H5N1 or H7N7 avian influenza viruses in mammals. However, little is known of the effects of this substitution on the EA H1N1 viruses. Herein, we investigated the contributions of 701N in the PB2 protein to an EA H1N1 SIV (A/Hunan/42443/2015(H1N1), HuN EA-H1N1), which had 701D in the PB2 protein. Our results found that viral polymerase activity, viral replication, and pathogenicity in mice were indeed enhanced due to the introduction of 701N into the PB2 protein, and the increased viral growth was partly mediated by the host factor importin-α7. Thus, substantial attention should be paid to the D701N mutation in pig populations.

H6 avian influenza viruses (AIVs), which are prevalent in domestic and wild birds in Eurasian countries, have been isolated from pigs, a dog and a human. Routine virological surveillance at live poultry markets or poultry farms was conducted in southern China from 2009 to 2011. This study investigated the genetic and antigenic characteristics, analyzed the receptor-binding properties and evaluated the kinetics of infectivity of the AIVs in A549, MDCK and PK15 cells. A total of 14 H6N6 and 2 H6N2 isolates were obtained from four provinces in southern China. Genetic analysis indicated two distinct hemagglutinin lineages of the H6 strains cocirculating in southern China, and these strains facilitated active evolution and reassortment among multiple influenza virus subtypes from different avian species in nature. None of these isolates grouped with the novel Taiwan H6N1 virus responsible for human infection. Receptor-binding specificity assays showed that five H6 AIVs may have acquired the ability to recognize human receptors. Growth kinetics experiments showed that EV/HB-JZ/02/10(H6N2) and EV/JX/15/10(H6N6) initially reproduced faster and achieved higher titers than other viruses, suggesting that enhanced binding to α-2,6-linked sialic acids correlated with increased viral replication in mammalian cells. Overall, the results emphasize the need for continued surveillance of H6 outbreaks and extensive characterization of H6 isolates to better understand genetic changes and their implications.

Summary Background and objectives Severe infection with avian influenza A (H5N6) virus in humans was identified first in 2014 in China. Before that, it was unknown or unclear if the disease or the pathogen affected people. This study illustrates the virological and clinical findings of a fatal H5N6 virus infection in a human patient. Methods We obtained and analyzed the clinical, epidemiological, and virological data from the patient. Reverse transcription polymerase chain reaction (RT-PCR), viral culture, and sequencing were conducted for determination of the causative pathogen. Results The patient, who presented with fever, severe pneumonia, leucopenia, and lymphopenia, developed septic shock and acute respiratory distress syndrome (ARDS), and died on day 10 after illness onset. A novel reassortant avian-origin influenza A (H5N6) virus was isolated from the throat swab or trachea aspirate of the patient. The virus was reassorted with the HA gene of clade 2.3.4.4 H5, the internal genes of clade 2.3.2.1 H5, and the NA gene of the H6N6 avian virus. The cleavage site of the HA gene contained multiple basic amino acids, indicating that the novel H5N6 virus was highly pathogenic in chicken. Conclusions A novel, highly pathogenic avian influenza H5N6 virus with a backbone of H5N1 virus acquired from the NA gene from the H6N6 virus has been identified. It caused human infection resulting in severe respiratory disease.

The highly pathogenic avian influenza (HPAI) H5N1 virus poses a significant potential threat to human society due to its wide spread and rapid evolution. In this study, we present a comprehensive antigenic map for HPAI H5N1 viruses including 218 newly sequenced isolates from diverse regions of mainland China, by computationally separating almost all HPAI H5N1 viruses into 15 major antigenic clusters (ACs) based on their hemagglutinin sequences. Phylogenetic analysis showed that 12 of these 15 ACs originated in China in a divergent pattern. Further analysis of the dissemination of HPAI H5N1 virus in China identified that the virus's geographic expansion was co-incident with a significant divergence in antigenicity. Moreover, this antigenic diversification leads to global antigenic complexity, as typified by the recent HPAI H5N1 spread, showing extensive co-circulation and local persistence. This analysis has highlighted the challenge in H5N1 prevention and control that requires different planning strategies even inside China.

We compared complete genome sequences of two strains of an avian influenza A (H5N6) virus isolated from a patient in Anhui Province with those of other strains from GenBank and Global initiative on sharing all influenza data (GISAID). The HA gene of the isolated virus shared homology with that of A/chicken/Zhejiang/727155/2014 (H5N6) at the level of similarity of 98%. The six internal genes of the Anhui strains were close to those of H9N2 viruses from Zhejiang, Shandong, and Guangdong provinces, with a similarity of 99%. In addition, the similarity between the internal antigens (NP and MP) of the isolated H5N6 virus and H7N9 and H10N8 viruses was 99%. Based on the data of phylogenetic analysis, the H5N6 influenza virus isolated in Anhui Province belonged to clade 2.3.4.4. The virus was shown to have molecular characteristics of highly pathogenic avian influenza viruses, including eight glycosylation sites and an amino acid sequence of the HA protein cleavage site, PLRERRRKKR/GLF, containing multiple basic amino acids. Additionally, the stalk domain of the NA protein was found to have a deletion in NA stalk region (11 amino acids in N6, positions 58–68). Our study demonstrated that the H5N6 virus from Anhui Province represented a triple-reassortant virus and could be highly pathogenic to humans. The prevalence of this virus should be closely monitored.

Summary: The 2016–2017 epidemic of influenza A (H7N9) virus in China prompted concern that a genetic change may underlie increased virulence. Based on an evolutionary analysis of H7N9 viruses from all five outbreak waves, we find that additional subclades of the H7 and N9 genes have emerged. Our analysis indicates that H7N9 viruses inherited NP genes from co-circulating H7N9 instead of H9N2 viruses. Genotypic diversity among H7N9 viruses increased following wave I, peaked during wave III, and rapidly deceased thereafter with minimal diversity in wave V, suggesting that the viruses entered a relatively stable evolutionary stage. The ZJ11 genotype caused the majority of human infections in wave V. We suggest that the largest outbreak of wave V may be due to a constellation of genes rather than a single mutation. Therefore, continuous surveillance is necessary to minimize the threat of H7N9 viruses. : The largest epidemic of influenza A (H7N9), wave V, prompted concerns of a pandemic. Zhu et al. analyze H7N9 viruses from all five waves and argue that the viruses entered a relatively stable stage. One gene constellation that emerged in H7N9 viruses is identified. Keywords: influenza A (H7N9) viruses, NP gene, gene constellation, PB2 A588V, the fifth wave

Infection with the novel H10N8 virus in humans has raised concerns about its pandemic potential worldwide. We report the results of a cross-sectional study of avian influenza viruses (AIVs) in live poultry markets (LPMs) in Nanchang, China, after the first human case of H10N8 virus infection was reported in the city. A total of 201 specimens tested positive for AIVs among 618 samples collected from 24 LPMs in Nanchang from December 2013 to January 2014. We found that the LPMs were heavily contaminated by AIVs, with H9, H10, and H5 being the predominant subtypes and more than half of the LPMs providing samples that were positive for the H10 subtype. Moreover, the coexistence of different subtypes was common in LPMs. Of the 201 positive samples, 20.9% (42/201) had mixed infections with AIVs of different HA subtypes. Of the 42 mixed infections, 50% (21/42) showed the coexistence of the H9 and H10 subtypes, with or without H5, and were from chicken samples. This indicated that the H10N8 virus probably originated from segment reassortment of the H9 and H10 subtypes.

Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans. IMPORTANCE Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.

The highly pathogenic avian influenza (HPAI) H5N1 virus poses a significant potential threat to human society due to its wide spread and rapid evolution. In this study, we present a comprehensive antigenic map for HPAI H5N1 viruses including 218 newly sequenced isolates from diverse regions of mainland China, by computationally separating almost all HPAI H5N1 viruses into 15 major antigenic clusters (ACs) based on their hemagglutinin sequences. Phylogenetic analysis showed that 12 of these 15 ACs originated in China in a divergent pattern. Further analysis of the dissemination of HPAI H5N1 virus in China identified that the virus’s geographic expansion was co-incident with a significant divergence in antigenicity. Moreover, this antigenic diversification leads to global antigenic complexity, as typified by the recent HPAI H5N1 spread, showing extensive co-circulation and local persistence. This analysis has highlighted the challenge in H5N1 prevention and control that requires different planning strategies even inside China.

Summary Background Human infections with different avian influenza viruses—eg, H5N1, H9N2, and H7N9—have raised concerns about pandemic potential worldwide. We report the first human infection with a novel reassortant avian influenza A H10N8 virus. Methods We obtained and analysed clinical, epidemiological, and virological data from a patient from Nanchang City, China. Tracheal aspirate specimens were tested for influenza virus and other possible pathogens by RT-PCR, viral culture, and sequence analyses. A maximum likelihood phylogenetic tree was constructed. Findings A woman aged 73 years presented with fever and was admitted to hospital on Nov 30, 2013. She developed multiple organ failure and died 9 days after illness onset. A novel reassortant avian influenza A H10N8 virus was isolated from the tracheal aspirate specimen obtained from the patient 7 days after onset of illness. Sequence analyses revealed that all the genes of the virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses. The aminoacid motif GlnSerGly at residues 226–228 of the haemagglutinin protein indicated avian-like receptor binding preference. A mixture of glutamic acid and lysine at residue 627 in PB2 protein—which is associated with mammalian adaptation—was detected in the original tracheal aspirate samples. The virus was sensitive to neuraminidase inhibitors. Sputum and blood cultures and deep sequencing analysis indicated no co-infection with bacteria or fungi. Epidemiological investigation established that the patient had visited a live poultry market 4 days before illness onset. Interpretation The novel reassortant H10N8 virus obtained is distinct from previously reported H10N8 viruses. The virus caused human infection and could have been associated with the death of a patient. Funding Emergency Research Project on human infection with avian influenza H7N9 virus, the National Basic Research Program of China, and the National Mega-projects for Infectious Diseases.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

A rapid and sensitive H7 and N9 subtype-specific reverse transcription loop-mediated isothermal amplification assay was developed respectively for visual detection of human-infected influenza A (H7N9) virus. The reaction was performed in one step in a single tube at 63°C for 60 min with the addition of hydroxynaphthol blue dye before amplification. The detection limits of both subtype-specific assays were comparable to those of validated H7 and N9 real-time PCR assays respectively and no cross-detection was observed with influenza A pandemic H1N1, H3N2, H5N1, H9N2 or influenza B virus. The assays were evaluated further with H7N9 virus-infected clinical specimens.

Objectives The first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. Methods We sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. Results All 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. Conclusions Classical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong.

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.

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.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.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.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.).

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.

Background Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses. Methods Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted. Results Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests. Conclusion Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0149-2) contains supplementary material, which is available to authorized users.

H9N2 avian influenza virus circulates widely in poultry and has been responsible for sporadic human infections in several regions. Few studies have been conducted on the pathogenicity of H9N2 AIV isolates that have different genomic features. We compared the pathology induced by a novel reassortant H9N2 virus and two currently circulating H9N2 viruses that have different genomic features in ferrets. The results showed that the three viruses can induce infections with various amounts of viral shedding in ferrets. The novel H9N2 induced respiratory infection, but no pathological lesions were observed in lung tissues. The other two viruses induced mild to intermediate pathological lesions in lung tissues, although the clinical signs presented mildly in ferrets. The pathological lesions presented a diversity consistent with viral replication in ferrets.

A fatal case caused by an H5N1 virus infection was investigated. In addition to serials of clinical chemistry assays, we tested the patient’s peripheral blood mononuclear cells (PBMC) and stool by reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and/or immunofluorescent assay. Our results suggested that PBMC can carry the virus and help the H5N1 virus spread to the human gut, resulting in infection with inflammation and viral discharge. * PBMC : peripheral blood mononuclear cells RT-PCR : reverse transcription polymerase chain reaction ARDS : acute respiratory distress syndrome OB : occult blood IgM : immunoglobulin M H5 : hemagglutinin N1 : neuraminidase PCR : polymerase chain reaction WHO : World Health Organization PBS : phosphate buffered saline FITC : fluorescein isothiocyanate NP : nucleoprotein HPAI : highly pathogenic avian influenza

The recent influenza pandemic, caused by a novel H1N1 influenza A virus, as well as the seasonal influenza outbreaks caused by varieties of influenza A and B viruses, are responsible for hundreds of thousands of deaths worldwide. Few studies have evaluated the utility of real-time reverse transcription-PCR to detect influenza virus RNA from formalin-fixed, paraffin-embedded tissues obtained at autopsy. In this work, respiratory autopsy tissues from 442 suspect influenza cases were tested by real-time reverse transcription-PCR for seasonal influenza A and B and 2009 pandemic influenza A (H1N1) viruses and the results were compared to those obtained by immunohistochemistry. In total, 222 cases were positive by real-time reverse transcription-PCR, and of 218 real-time, reverse transcription-PCR-positive cases also tested by immunohistochemistry, only 107 were positive. Although formalin-fixed, paraffin-embedded tissues can be used for diagnosis, frozen tissues offer the best chance to make a postmortem diagnosis of influenza because these tissues possess nucleic acids that are less degraded and, as a consequence, provide longer sequence information than that obtained from fixed tissues. We also determined that testing of all available respiratory tissues is critical for optimal detection of influenza virus in postmortem tissues.