Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice / 生物医学与环境科学（英文）

Objective@#Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection .@*Methods@#We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of -/- and wild-type mice.@*Results@#-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of -/- mice during acute influenza infection.@*Conclusions@#Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in -/- mice.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A (H5N1) Virus / 生物医学与环境科学（英文）

Objective@#To recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features.@*Methods@#We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled.@*Results@#Two BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages, , V 1-69-D2-15-J 4 (2-12D5) and V 1-2-D3-9-J 5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in V 1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively.@*Conclusions@#Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform.</p><p><b>METHODS</b>Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance.</p><p><b>RESULTS</b>The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus.</p><p><b>CONCLUSION</b>The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.</p>

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012 / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization.</p><p><b>METHODS</b>RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed.</p><p><b>RESULTS</b>Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus.</p><p><b>CONCLUSION</b>Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.</p>

A novel reassortant H2N3 influenza virus isolated from China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.</p><p><b>METHODS</b>PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed.</p><p><b>RESULTS</b>The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.</p><p><b>CONCLUSION</b>A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.</p>

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.</p><p><b>METHODS</b>Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.</p><p><b>RESULTS</b>The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.</p><p><b>CONCLUSION</b>The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.</p>

Preliminary study of a universal vaccine based on the HA2 protein of the H5N1 influenza virus / 病毒学报

Fragments encoding amino acids 76-130 in the linear conserved region (LCR) of A/Hubei/1/2010 (H5N1) HA2 was fused to hepatitis B core antigen (HBc) to generate a LCR-HBe virus-like particle (VLP). Results showed that the fusion protein of LCR-HBc was highly expressed in this prokaryotic expression system. The purified LCR-HBc particle stimulated high levels of IgG production in mice with a titer of > 1:12 800, and provided 50% cross-protection against lethal challenge by H1N1 viruses.

Advances in research and development of universal influenza vaccines / 病毒学报

Vaccination is the primary strategy for the prevention and control of pandemic influenza. Because influenza virus is highly variable across strains, universal influenza vaccines need to be developed to address this problem. This review describes the research progress in conserved epitopes of influenza virus, the advances in the research and development of universal influenza vaccines based on the relatively conserved sequences of NP, M2e, HA2, and headless HA, the mechanisms of cross-protection, and the methods to improve cross-protection.

Complete genome phylogenetic analysis of five H9N2 avian influenza viruses isolated from poultry flocks in Qinghai lake region / 病毒学报

Five H9N2 avian influenza virus strains were isolated from the environmental samples in live poultry market in Qinghai Lake region from July to September, 2012. To evaluate the phylogenetic characteristics of these H9N2 isolates, the eight gene segments were amplified by RT-PCR and sequenced. The phylogenetic and molecular characteristics of the five strains were analyzed. The results showed that the HA genes of five strains shared 93. 2%-99. 1% nucleotide identities with each other, and the NA genes shared 94. 5%-99. 8% nucleotide identities. The HA cleavage site sequence of the A/environment/qinghai/ 017/2012 isolate was PSKSSRGLF, and the HA cleavage site sequences of the other four strains were all PSRSSRGLF. The HA receptor-binding site had the Q226L mutation. The M1 gene segment had the N30D and T215A mutations. The phylogenetic analysis showed that the five strains were similar to the virus A/chicken/Hunan/5260/2005 (H9N2) isolated in Hunan Province, China and were reassortant genotype viruses; the HA, NA, and NS genes belonged to the Y280-like lineage; the MP gene belonged to the G1-like lineage; the NP, PB1, PB2, and PA genes belonged to the F98-like lineage.

An overview of surveillance of avian influenza viruses in wild birds / 病毒学报

Wild birds (mainly Anseriformes and Charadriiformes) are recognized as the natural reservoir of avian influenza viruses (AIVs). The long-term surveillance of AIVs in wild birds has been conducted in North America and Europe since 1970s. More and more surveillance data revealed that all the HA and NA subtypes of AIVs were identified in the wild ducks, shorebirds, and gulls, and the AIVs circulating in wild birds were implicated in the outbreaks of AIVs in poultry and humans. Therefore, the AIVs in wild birds pose huge threat to poultry industry and human health. To gain a better understanding of the ecology and epidemiology of AIVs in wild birds, we summarize the transmission of AIVs between wild birds, poultry, and humans, the main results of surveillance of AIVs in wild birds worldwide and methods for surveillance, and the types of samples and detection methods for AIVs in wild birds, which would be vital for the effective control of avian influenza and response to possible influenza pandemic.

Development of multi-pathogen detection techniques for respiratory viruses / 病毒学报

Viral respiratory tract infection is among the leading causes of mortality and morbidity worldwide. Rapid screening methods for multiple detection of a wider range of pathogens become very important for diagnosis of respiratory infection. This article describes conventional detection technologies and several emerging multiplex assays that have potential applications in the diagnosis and monitoring of respiratory viral infections. These techniques include new rapid culture system, multiplex reverse transcription-PCR, real-time reverse transcription PCR, solid and suspension microarrays, mass spectrometry as well as metagenomics methods. The development and application of these techniques will not only improve the ability of rapid detection and control of viral respiratory infection, but play pivotal roles in the rapid characterization of new viral pathogens.

A review of H7 subtype avian influenza virus / 病毒学报

Since 2002, H7 subtype avian influenza viruses (AIVs) have caused more than 100 human infection cases in the Netherlands, Italy, Canada, the United States, and the United Kingdom, with clinical illness ranging from conjunctivitis to mild upper respiratory illness to pneumonia. On March 31st, three fatal cases caused by infection of a novel reassortant H7N9 subtype were reported in Shanghai City and Anhui Province in China. With the ability of H7 subtype to cause severe human disease and the increasing isolation of subtype H7 AIVs, we highlighted the need for continuous surveillance in both humans and animals and characterization of these viruses for the development of vaccines and anti-viral drugs.

Virological characterization of influenza B virus in mainland China during 2011-2012 / 病毒学报

In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.

Emerged Pdm09 influenza virus increased purifying selection of seasonal H1N1 influenza virus / 病毒学报

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P<0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.

A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic duck farm in Poyang Lake area / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011.</p><p><b>METHODS</b>The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted.</p><p><b>RESULTS</b>The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice.</p><p><b>CONCLUSION</b>The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.</p>

Reverse genetics platform construction of influenza pandemic virus strain / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Reverse genetics was used to construct the platform of flu pandemic strain A/California/07/2009 (H1N1).</p><p><b>METHODS</b>Eight genes fragements were amplified and ligated with bidirectional vector, recombinant plasmids were co transfected to the 293 T cells and rescued the virus. Gene sequencing, antigenic analysis and growth property were used to evaluate the rescued virus.</p><p><b>RESULTS</b>Rescued virus show the genes sequence correct, keep the same antigenicity and similar growth property compared with wild type virus.</p><p><b>CONCLUSION</b>The pandemic virus reverse genetics platform of A/California/07/2009 (H1N1) were built. Based on this platform, rescued virus hold the similarity of antigenicity and growth ability with wild type virus.</p>

Selection pressure analysis of H3N2 influenza virus from China between 1992 and 2012 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to investigate the relationship between selection pressure and the prevalence of antigenic clusters, we sequenced and analyzed the H3N2 influenza virus from China between 1992 and 2012.</p><p><b>METHODS</b>The H3N2 influenza virus (n = 1206) in China from 1992 to 2012 was analyzed, include global selection pressure and sites positive selection pressure analysis.</p><p><b>RESULTS</b>Considering all the H3N2 influenza viruses during these 21 years, a total of four amino acid sites subject to positive selection. The global selection pressure varies with the variation of different antigenic clusters and three years with peak bottom selection pressure were identified.</p><p><b>CONCLUSION</b>The global selection pressure rise from the peak bottom, a new antigenic clusters will appear andprevalent in the population, indicating the best time to replace the vaccine strain.</p>

Accessing the features of surface neuraminidase (N1) of influenza A virus presenting on the platforms for anti-NA Abs screening / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand if the Neuraminidase (N1) of Influenza A virus at the surface of yeast-displaying system, eukaryotic expression system and the infected cells could be used for anti-NA Abs screening, their activities and bindings to five candidate Abs were assayed.</p><p><b>METHODS</b>The surface NA expression was obtained by transfecting by recombinant NA constructors with specific tag-labels or live virus infection. The functional activity was measured by the fluorescent assay. Their bindings to the Abs were detected by flow cytometry.</p><p><b>RESULTS</b>The surface NAs presenting on the yeast-displaying system and eukaryotic expression system exhibited functional NA activities as the NA at the surface of virus-infected cells which showed affinities to Ab1, 4, and 5. The same bindings to Abl and 5 were found in the surface NA expressed by eukaryotic expression system while minor binding was observed in the yeast displayed-NA.</p><p><b>CONCLUSION</b>The epitopes of yeast-displayed NA may be different from the NAs present at eukaryotic expression system and the infected cells which more likely suitable for the screening of anti-NA Abs.</p>

Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.</p><p><b>METHODS</b>Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.</p><p><b>RESULTS</b>This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.</p><p><b>CONCLUSION</b>The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.</p>