Metagenomic Analysis of Environmental Samples from Wildlife Rescue Station at Poyang Lake, China / 生物医学与环境科学（英文）

OBJECTIVE@#To improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China.@*METHODS@#Ten smear samples were collected in March 2019. Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus.@*RESULTS@#We identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01-S09 and S10. In S01-S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01-S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B.@*CONCLUSION@#This study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds.

Spatial and temporal distribution characteristics of seasonal A(H3N2) influenza in China, 2014-2019 / 中华流行病学杂志

Objective: To analyze the spatial and temporal distribution characteristics of seasonal A(H3N2) influenza [influenza A(H3N2)] in China and to provide a reference for scientific prevention and control. Methods: The influenza A(H3N2) surveillance data in 2014-2019 was derived from China Influenza Surveillance Information System. A line chart described the epidemic trend analyzed and plotted. Spatial autocorrelation analysis was conducted using ArcGIS 10.7, and spatiotemporal scanning analysis was conducted using SaTScan 10.1. Results: A total of 2 603 209 influenza-like case sample specimens were detected from March 31, 2014, to March 31, 2019, and the influenza A(H3N2) positive rate was 5.96%(155 259/2 603 209). The positive rate of influenza A(H3N2) was statistically significant in the north and southern provinces in each surveillance year (all P<0.05). The high incidence seasons of influenza A (H3N2) were in winter in northern provinces and summer or winter in southern provinces. Influenza A (H3N2) clustered in 31 provinces in 2014-2015 and 2016-2017. High-high clusters were distributed in eight provinces, including Beijing, Tianjin, Hebei, Shandong, Shanxi, Henan, Shaanxi, and Ningxia Hui Autonomous Region in 2014-2015, and high-high clusters were distributed in five provinces including Shanxi, Shandong, Henan, Anhui, and Shanghai in 2016-2017. Spatiotemporal scanning analysis from 2014 to 2019 showed that Shandong and its surrounding twelve provinces clustered from November 2016 to February 2017 (RR=3.59, LLR=9 875.74, P<0.001). Conclusion: Influenza A (H3N2) has high incidence seasons with northern provinces in winter and southern provinces in summer or winter and obvious spatial and temporal clustering characteristics in China from 2014-2019.

Consideration on implementation of co-administration of Seasonal Influenza and COVID-19 vaccines during pandemic in China / 中华预防医学杂志

Influenza is an infectious respiratory disease caused by the influenza viruses. Older people, infants and people with underlying medical conditions could have a higher risk of severe influenza symptoms and complications. The co-infection of Coronavirus Diseases 2019 (COVID-19) with influenza viruses could lead to the complication of prevention, diagnosis, control, treatment, and recovery of COVID-19. Influenza vaccine and COVID-19 vaccine overlapped in target populations, vaccination time, and inoculation units. Although there was insufficient evidence on the immunogenicity and safety of co-administration of influenza vaccine and COVID-19 vaccine, World Health Organization and some countries recommended co-administration of inactivated influenza vaccine and COVID-19 vaccine. This review summarized domestic and international vaccination policies and research progress, and put forward corresponding suggestions in order to provide scientific support for the formulation of vaccination strategy on seasonal influenza vaccine and COVID-19 vaccine.

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.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine.@*METHODS@#Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured.@*RESULTS@#H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus.@*CONCLUSION@#CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Surveillance analysis of influenza in Mainland China during 2016-2017 season / 中国人兽共患病学报

To analyze and summarize the surveillance results and epidemic situation of influenza in mainland China during 2016-2017 surveillance season,we collected ILI monitoring data and influenza outbreak data download from"China Influenza Surveillance Information System"and"Public Health Emergency Information Management System"in mainland China during 2016-2017 surveillance season.The results showed that influenza activity did not appear obvious summer peak in southern provinces,and the autumn and winter flu seasons were earlier than that during last season in both southern and northern prov-inces,while the epidemic peak level was lower than 2015-2016 surveillance season.Throughout the year,influenza predomi-nant viruses throughout the country underwent the alternation of type B,A (H3N2)and H1N1,respectively.The total num-ber of outbreaks of ILI reported in the country was less than that in 2015-2016.According the surveillance,we suggested that all regions continue to attach great importance to recent influenza virus activity,pay attention to the global epidemic situation and that of our country,strengthen the analysis and use of surveillance data,trace the activity levels,mutation and resistance change of influenza virus,carry out the flu outbreak investigation and disposal work timely.

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>

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>

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.

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.

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>

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>

Virological characterization of influenza A(H3N2) virus in Mainland China during 2011-2012 / 病毒学报

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.

Research advances on thogoto virus of the family orthomyxoviridae / 病毒学报

Thogoto virus belongs to the family Orthomyxoviridae. It is a tick-borne arbovirus that can infect both human and animals. Thogoto virus's genetic constitution, replication and transcription, and the function of the translated proteins are similar to influenza virus. The studies on Thogoto virus are important for us to better understand the conservative sites of influenza virus. Moreover, the animal model of Thogo-to virus is expected to be an alternative model for highly pathogenic influenza viruses. In the past years, Thogoto virus attracted limited public attention and few studies were engaged in this area. The classification of Thogoto virus, the genetic constitution and evolution, and viral proteins were included in this review. The functions of M protein and ML protein were emphasized, which were translated from the sixth segment and played an important role in viral replication, the interaction between Thogoto virus and host were also highlighted.

An uncontrolled open-label, multicenter study to monitor the antiviral activity and safety of inhaled zanamivir (as Rotadisk via Diskhaler device) among Chinese adolescents and adults with influenza-like illness / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is the first multicenter clinical study in China to investigate zanamivir use among Chinese adolescents and adults with influenza-like illness (ILI) since 2009, when inhaled zanamivir (RELENZA(®)) was marketed in China.</p><p><b>METHODS</b>An uncontrolled open-label, multicentre study to evaluate the antiviral activity, and safety of inhaled zanamivir (as Rotadisk via Diskhaler device); 10 mg administered twice daily for 5 days in subjects ≥ 12 years old with ILI. Patients were enrolled within 48 hours of onset and followed for eight days. Patients were defined as being influenza-positive if the real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test had positive results.</p><p><b>RESULTS</b>A total of 400 patients ≥ 12 years old were screened from 11 centers in seven provinces from March 2010 to January 2011. Three hundred and ninety-two patients who took at least one dose of zanamivir were entered into the safety analysis. The mean age was 33.8 years and 50% were male. Cardiovascular diseases and diabetes were the most common comorbidities. All the reported adverse events, such as rash, nasal ache, muscle ache, nausea, diarrhea, headache, occurred in less than 1% of subjects. Mild sinus bradycadia or arrhythmia occurred in four subjects (1%). Most of the adverse events were mild and did not require any change of treatment. No severe adverse events (SAE) or fatal cases were reported. Bronchospasm was found in a 38 years old woman whose symptoms disappeared after stopping zanamivir and without additional treatment. All the 61 influenza virus isolates (43 before enrollment, 18 during treatment) proved to be sensitive to zanamivir.</p><p><b>CONCLUSIONS</b>Zanamivir is well tolerated by Chinese adolescents and adults with ILIs. There is no evidence for the emergence of drug-resistant isolates during treatment with zanamivir.</p>

Establishment of a mouse-lethal model for pandemic H1N1 influenza virus / 病毒学报

To establish the mouse-lethal model for pandemic H1N1 influenza virus, provide an animal model for studying the pathogenicity and host adaptation of 2009 pandemic H1N1 influenza virus, and find out the key amino acid mutations which may affect viral virulence and replication. A pandemic H1N1 influenza virus strain, A/Sichuan/SWL1/2009 (H1N1, SC/1) was passaged in mouse lung by 15 cycles with intranasal infection. The passaged viruses were all propagated in MDCK cells and sequenced. Based on the sequencing results, four mice in each group were inoculated with 6 selected viruses and their weight and survival rate were monitored during the following 14 days after infection. Additionally, SC/1-MA P14 and P15 viruses were sequenced after purification by Plague Assay. Viral virulence was increased after serial passages and the mortality of 100% was detected after 7 passages. Several amino acid residue mutations of passaged viruses which may contribute to the enhanced virulence were observed. The increased virulence of passaged viruses and mammalian host adaptation maybe associated with amino acid mutations in viral functional proteins. Finally, we established a mouse-lethal model.