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.

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.

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>

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.

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>

Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification / 病毒学报

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

Characterization of pseudotyped viruses coated with hemagglutinin of H5N1 avian influenza / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct pseudovirus bearing H5N1 HA based on a lentivirus vector system. Then we study the biological feature of the pseudovirus. With the newly established viral particles, we performed the serological tests.</p><p><b>METHODS</b>H5N1 avian influenza virus that isolated from human case was cloned to construct pLP-HA, then pLP-HA co-transfected with lentivirus vector plasmids pLP1, pLP2 and pEmGFP into 293T cells. The supernatant was harvested 48h post-transfection. Concentrated by super centrifuge, the pseudotyped viruses were analyzed by infection test, HA test and micro-neutralization test. At the same time, optimized HA gene and a Vietnam H5N1 HA gene were used to construct pseudotyped virus for comparison.</p><p><b>RESULTS</b>Pseudotyped virus particles can be observed with electronic microscope. Western-blot revealed that HA glycoprotein can be expressed in the virions. Our neutralization assay by using the pseudoviruses was comparable with the conventional microneutralization assay with wild-type viruses. A high degree of correlation was detected.</p><p><b>CONCLUSION</b>Pseudotyped Viruses coated with HA of H5N1 High Pathogenic Avian Influenza were successfully constructed; it can be used to for the microneutralization assay. The HA gene from different sources affect the efficiency of the packaging of the pseudovirus. But the optimized HA gene can not obviously improve packaging efficiency of the pseudovirus.</p>

The virus isolation analysis of the H5N1 subtype human avain influenza cases in mainland China from 2005 to 2009 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China.</p><p><b>METHODS</b>The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs.</p><p><b>RESULTS</b>Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens.</p><p><b>CONCLUSION</b>We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.</p>

Establishment of a method for rapid detection of the nucleic acid of the novel A (H1N1) influenza virus / 病毒学报

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China / 病毒学报

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

Laboratory diagnosis and molecular characterization analysis of the H5N1 influenza virus isolated from the first human case in Shenzhen, China / 病毒学报

The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza (firstly found in Shenzhen, China) were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient's tracheal aspirates on MDCK cell and was named A/Guangdong/2/06(H5N1). The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1 : 160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.

Detection of influenza viruses/avian influenza viruses and identification of virulence using a microarray / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence.</p><p><b>METHODS</b>Hemagglutinin (HA), neuramidinase (NA) and nucleoprotein(NP) genes were chosen simultaneously as targets for designing a microarray used for detection of viruses and identification virulence. The nucleic acid were amplified by single primer amplication (SPA). And then its specificity,sensitivity and reproducibility were evaluated.</p><p><b>RESULTS</b>The microarray was able to specially detect H1N1, H3N2, B influenza viruses and H5N1, H9N2 avian influenza viruses. Their limits were 8HAU, 16HAU, 32HAU, and 8HAU, 8HAU respectively. The limit for virulence was 32HAU. When samples were analyzed by both RT-PCR and microarray in parallel, the results agreed in 83.9% (47/56).</p><p><b>CONCLUSION</b>The microarray can detect and distinguish five tested viruses, and especially identify virulence. It not only supplies an assistant tool for clinical diagnosis and control of infectious disease, but also is valuable for controlling and preventing outbreak of avian influenza epidemic.</p>