Dayan Wang

Human infection with a novel, highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings.

BACKGROUND AND OBJECTIVESnSevere 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnA 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.

Biological characterisation of the emerged highly pathogenic avian influenza (HPAI) A(H7N9) viruses in humans, in mainland China, 2016 to 2017

With no or low virulence in poultry, avian influenza A(H7N9) virus has caused severe infections in humans. In the current fifth epidemic wave, a highly pathogenic avian influenza (HPAI) H7N9 virus emerged. The insertion of four amino acids (KRTA) at the haemagglutinin (HA) cleavage site enabled trypsin-independent infectivity of this virus. Although maintaining dual receptor-binding preference, its HA antigenicity was distinct from low-pathogenic avian influenza A(H7N9). The neuraminidase substitution R292K conferred a multidrug resistance phenotype.

Visual detection of human infection with influenza A (H7N9) virus by subtype-specific reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

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.

A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.

Characteristics of oseltamivir-resistant influenza A (H1N1) pdm09 virus during the 2013–2014 influenza season in Mainland China

BackgroundIn this study, we analyzed the characteristics of oseltamivir-resistant influenza A (H1N1) pdm09 virus isolated from patients in mainland China during the influenza season from September 2013 through March 2014, and provide guidance on which antiviral to be used for clinical treatment.MethodsThe all viruses collected from September 1, 2013 through March 31, 2014 were obtained from the Chinese National Influenza Surveillance Network. A fluorescence-based assay was used to detect virus sensitivity to neuraminidase inhibitors (NAIs). The hemagglutinin (HA) and neuraminidase (NA) gene of the oseltamivir-resistant viruses were sequenced.ResultsA total of 24 (2.14xa0%) influenza A (H1N1) pdm09 viruses that were resistant to oseltamivir were identified. These 24 viruses were isolated from 23 patients and no epidemiological link among them could be identified. Except for one virus with the H275H/Y mixture substitution, all the other 23 viruses had H275Y substitution in the NA protein. Sequence analysis revealed that the amino acid substitutions in the HA protein of influenza A (H1N1) pdm09 viruses with H275Y substitution isolated from mainland China were similar to the viruses from clustered cases reported in the United States, and the amino acid substitutions in the NA protein were similar to the viruses reported in Sapporo, Japan in 2013–2014. All of the oseltamivir-resistant viruses in mainland China and Japan possessed additional substitutions N386K, V241I and N369K in the NA protein, while most (>89xa0%) resistant-viruses from the United States during the same period possess V241I and N369K and did not have the N386K substitution. The N386K substitution was also exist in most sensitive viruses during the same period in mainland China. The amino acid substitutions in both HA and NA protein differed from the clustered cases from Australia reported in 2011 with additional substitutions. The drug-resistant influenza A(H1N1) pdm09 viruses were from patients without any known NAIs medication history prior to sampling.ConclusionsDuring the influenza season from September 2013 through March 2014 in Mainland China, oseltamivir-resistant influenza A(H1N1)pdm09 viruses were much more frequently detected than ever since the appearance of the virus in 2009.

Post-mortem findings in a patient with avian influenza A (H5N6) virus infection

Avian influenza A (H5N6) has been found to infect humans, and has resulted in ten cases with six deaths in China since 2014. Here, we describe the systematic post-mortem pathology of a patient fatally infected with H5N6 virus and evaluate the associated pathogenesis compared with H1N1 pdm09 fatal cases. The most prominent histopathological features were diffuse alveolar damage and pulmonary vasculitis in the lungs of the patient. The virus disseminated to extrapulmonary organs, including the brain. Compared with H1N1 pdm09 fatal infection, H5N6 infection induced a more exacerbated immune response involving overt pulmonary inflammation, which led to alveolar damage and respiratory failure.

Characteristics of influenza H13N8 subtype virus firstly isolated from Qinghai Lake Region, China

BackgroundSince the highly pathogenic H5N1 influenza caused thousands of deaths of wild bird in this area in 2005, Qinghai Lake in China has become a hot spot for study of the influence of avian influenza to migratory wild birds. However, the ecology and evolution of low pathogenic avian influenza virus in this region are limited. This project-based avian influenza surveillance in Qinghai lake region was initiated in year 2012.MethodSamples of wild bird feces and lake surface water were collected in Qinghai Lake in year 2012.Virus isolation was conducted on embryonated chicken eggs. The influenza A virus was determined by rRT-PCR. Virus sequences were acquired by deep sequencing. The phylogenetic correlation and molecular characteristics of the viruses were analyzed. The virus growth and infection features, receptor binding preference were studied, and pathogenicity in vitro as well as.ResultsTwo H13N8 subtype influenza viruses were isolated. The viruses are phylogenetically belong to Eurasian lineage. Most of the genes are associated with gull origin influenza virus except PB1 gene, which is most probably derived from Anseriformes virus. The evidence of interspecies reassortment was presented. The two viruses have limited growth capacity on MDCK and A549 cells while grow well in embryonated eggs. The dual receptor binding features of the two viruses was shown up. The low pathogenic features were determined by trypsin dependence plaque formation assay.ConclusionsThe two H13N8 subtype influenza viruses are highly associated with gull origin. The interspecies reassortment of H13 subtype virus among Anseriforme sand Charadriiformes wild birds emphasizes the importance of strengthening avian influenza surveillance in this region. This study is helpful to understand the ecology, evolution and transmission pattern of H13 subtype influenza virus globally.

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses’ role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.

A sandwich ELISA for the detection of neuraminidase of avian influenza A(H7N9) virus

Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1400 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed. The immunoassay employed mouse monoclonal antibody (mAb) 3C1 to specifically capture the N9 and 3E9 for the detection. Its limit of detection is 6.25ng/ml for N9 protein of A/Anhui/1/2013(H7N9, AH1/2013) and 0.125HAU/50μL for live virus, AH1/2013 and A/Environment/Jiangxi/28/2009 (H11N9), respectively. When applied to test the five clinic throat swabs from H7N9 patients confirmed by nuclear acid testing (NAT) using quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), two samples showed positive result in sandwich ELISA while all were negative using commercial Flu A and H7 subtype rapid antigen tests (RAT). The ELISA using anti-N9 mAbs provided a valuable approach to detect H7N9 virus and quantify the N9 protein.

Identification of a novel reassortant A (H9N6) virus in live poultry markets in Poyang Lake region, China

Live poultry markets (LPM) are one of the most important sources of human infection with avian influenza virus (AIV). During our routine surveillance of AIV, we identified an H9N6 virus (JX-H9N6) in a LPM in Nanchang city, Jiangxi Province, China. Using Bayesian coalescent analysis, it was predicted that JX-H9N6 had originated from a reassortment event between H9N2 and H6N6 AIVs in early 2014, instead of being derived from an H9N6 virus reported previously. Mutations in HA, PB1, PA, M, and NS protein, which could increase mammalian transmission and virulence, were also detected. Currently, both H9N2 and H6N6 AIVs are widely distributed in poultry and contribute to the generation of novel reassortant viruses causing human infection. Our findings highlight the importance of enhanced surveillance in birds for early prediction of human infections.