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    Guidance: The Impact of Influenza Viruses on Human and Animal Husbandry
    WANG Xiu-rong, CHEN Hua-lan
    Scientia Agricultura Sinica    2015, 48 (15): 3038-3039.   DOI: 10.3864/j.issn.0578-1752.2015.15.013
    Abstract503)   HTML8)    PDF (210KB)(565)       Save
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    Genome Sequencing and Genetic Analysis of H4N8 Subtype Avian Influenza Virus Isolated from Duck
    ZHAO Qing-qing, LI Qun-hui, ZHU Jie, ZHONG Lei, LIU Jing-jing, GU Min, WANG Xiao-quan, LIU Wen-bo, LIU Xiu-fan
    Scientia Agricultura Sinica    2015, 48 (15): 3040-3049.   DOI: 10.3864/j.issn.0578-1752.2015.15.014
    Abstract513)   HTML6)    PDF (442KB)(585)       Save
    【Objective】 Based on the difference of hemagglutinin (HA) and neuraminidase (NA), avian influenza viruses (AIVs) are classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. According to the differences in pathogenicity, AIVs can be divided into highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV). H4 AIVs are low pathogenic influenza viruses which are generally produced asymptomatic infections in poultry. But H4 AIVs also has potential threats to both poultry and mammals. Strengthening investigation on H4 subtype avian influenza viruses is important for the study of evolution of AIVs. The objective of this experiment is to investigate the molecule characteristics and genetic evolution of H4 subtype avian influenza virus. 【Method】One H4N8 subtype avian influenza virus, designated as A/duck/Nanjing/1102/2010 (H4N8) (DK/NJ/1102), was isolated from a live poultry market in eastern China during epidemiological surveillance in 2010. The complete genome sequences of the strain was sequenced and analyzed. The virus was identified by HA/HI test and RT-PCR test. The gene was cloned into pGEM-Teasy vector for sequencing, respectively. BLAST the nucleotide identity in GeneBank. The genome sequences of H4 subtype influenza viruses available in GeneBank and some other reference sequences were downloaded for genetic analysis .【Result】The results showed that the HA gene of DK/NJ/1102 had the highest nucleotide sequence identity of 98.9% with A/duck/Mongolia/274/2007(H4N3), and the amino acid sequence at the cleavage region of the HA gene was “P-E-K-A-S-R-G”, which is typical for low pathogenicity AIVs. NA gene had the highest nucleotide sequence identity of 98.8% with a duck-origin virus A/Duck/Eastern China/n91/2009 (H3N8) isolated from eastern China, whereas PB1, PA and NP genes were all mostly related with H1 subtype avian influenza viruses. The M gene shared the greatest nucleotide sequence identities (over 99.9%) with A/wild duck/Korea/CSM4-12/2009(H5N1) while the NS gene was most closely related to H7N7 subtype AIV isolated from Korea in 2009 and there was no amino acids missing in NS1 protein at 80-84 sites.【Conclusion】These data indicated that the genome composition of the strain A/duck/ Nanjing/1102/2010(H4N8) was complicated, it may be a reassortant whose genes originated from different subtypes of AIV.
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    Cited: Baidu(2)
    Development of RT-PCR Technique for Detection of H7N9 Subtype Avian Influenza Virus
    WANG Yun-he, BAO Hong-mei, SUN Jia-shan, LI Yan-bing, XU Xiao-long, WANG Zi-long, SHI Jian-zhong, ZENG Xian-ying, WANG Xiu-rong, CHEN Hua-lan
    Scientia Agricultura Sinica    2015, 48 (15): 3050-3055.   DOI: 10.3864/j.issn.0578-1752.2015.15.015
    Abstract411)   HTML3)    PDF (884KB)(514)       Save
    【Objective】On March 2013, the National Health and Family Planning Commission of China announced that human infections with H7N9 subtype AIV had occurred in Shanghai and Anhui province, China. Because this novel reassortant (H7N9) virus had not previously been seen in either animals or people, the situation raises many urgent questions and global public health concerns. In this study, two pairs of RT-PCR primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, and a reverse-transcription PCR assay to rapidly detect the novel H7N9 subtype avian influenza virus was developed and evaluated. 【Method】 H7 and N9 primers were chosen based on the conserved regions of sequences that were designed and analyzed using DNASTAR and oligo 6.0. one-step RT-PCR assay for the detection of H7N9 virus was established with RNA extracted by using one-step access RT-PCR kit. The specificity of the RT-PCR assay was evaluated with H7N9 influenza virus as positive control and Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus and other avian respiratory viral pathogens as negative control. After specificity test by using clear background influenza virus and other pathogens, the detection limits of the assay were assessed with serial 10-fold dilutions of H7N9 influenza virus (106.5 EID50·mL-1). The specificity assays were evaluated using influenza A viruses of various genetic backgrounds and other avian pathogen. The sensitivity assays were determined using viral RNA extracted from serially diluted AIV-infected allantoic fluid. In addition, a blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. 【Result】The H7 HA can be detected by this assay, while other H1-H6 and H8-H15 subtype HA, as well as other avian pathogens were detected negative in specificity assay. Similarly, only the N9 NA related to the novel H7N9 virus was detected, the other N1-N9 NA were detected negative. Results of 10-fold dilution series of allantoic fluid by one step RT-PCR assays showed that detection limit of the assay was approximately 1.4×102.5 EID50 per reaction. Furthermore, the assays showed clinical specificity for identification cloacal swabs of H7N9 virus.【Conclusion】The RT-PCR assay established in this study can be used as a referee method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.
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    Cited: Baidu(5)
    The Adaptation of H9N2 Subtype AIV in Mouse and Analysis of Amino Acid Mutation
    DING Jie, GAO Yu-wei, SANG Xiao-yu, CHENG Kai-hui, YU Zhi-jun, ZHANG Kun, CHAI Hong-liang, WANG Tie-cheng, XIA Xian-zhu, HUA Yu-ping
    Scientia Agricultura Sinica    2015, 48 (15): 3056-3063.   DOI: 10.3864/j.issn.0578-1752.2015.15.016
    Abstract393)   HTML2)    PDF (3603KB)(309)       Save
    【Objective】H9N2 subtype avian influenza virus can spread between the guinea pig after passages in guinea pigs continuously for 9 generations, showing that the capacity of H9N2 subtype avian influenza virus infection in mammals as well as the spreading capacity between mammals are still very strong. Thus, this experiment using this strain virus A/Chicken/Jinan/Li-2/2010 (H9N2), JN, to study the molecular basis of H9N2 subtype influenza virus variation, and screen the pathogenicity related amino acid sites.【Method】An H9N2 subtype AIV was serial passaged in the lung of mice to acquire the variant strains JN-P9-2-M1, the mice were dissected, the lungs were removed, after grinding and centrifugation, the mice of next generation were inoculated intranasally, after nine generations of spreading, MDCK cells were used for virus multiplication. Then the full-length sequences of JN-P5-2-M1 and JN-P9-2-M1eight segments were amplified, cloned and analyzed. Amino acids encoded by each gene were deduced, compared to JN primary virus (P0) , and the nucleotide and amino acid changes in virus passages were obtained. The mice were dissected, the lung, liver, spleen, kidneys, brain and intestines were obtained, and the virus titer of tissues was titrated. Mice were anesthetized, and each virus dilution was used to inoculate intranasally three mice for detecting the survival rate and morbidity of mice. Lungs of mice were collected, then the pathological and immunohistochemical staining was made to compare the JN and JN-P9-2-M1 virus. 【Result】 JN-P9-2-M1 had high pathogenicity to mice, its MLD50 was 103.5EID50, when had a 106EID50, 105EID50, 104EID50 dose in mice, its survival rate was 0, at least 1000 times higher than JN, the JN in mice was not lethal. When the dose of JN-P9-2-M1 was 106-103EID50 for mice, the body weight was significantly reduced, and the clinical symptoms were obvious. After inoculation for 3-8 d the mice showed listless, hair messy, shortness of breath, arched, etc., but when the mice were inoculated with 106EID50 JN virus, the mice body weight change rates were similar to the negative group. JN-P5-2-M1, JN-P9-2-M1, and JN could binding to SAa-2,6Gal receptor, like B influenza virus. JN virus was detected only in the lungs of mice, but JN-P9-2-M1 not only in the lungs of mice but also could be detected in liver, spleen, kidney and brain. 【Conclusion】JN-P9-2-M1’s pathogenicity in mice significantly improved, compared with the original drug increased by at least 1 000 times. The three amino acid sites PB2 E627K, HA N313D, and HA N496S might be the reasons of preliminary improvement of the virus virulence in mice, and the two acid sites PA L342I and NA N218T might be possible to further improve the virus virulence in mice. 
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    Electrochemical Luminescence Immunoassay for the Detection of H9 Subtype Avian Influenza Virus
    QI Wen-bao, LI Fang, LI Hua-nan, HUANG Li-hong, HE Jun, MU Guang-hui, LUO Kai-jian, LIAO Ming
    Scientia Agricultura Sinica    2015, 48 (15): 3064-3070.   DOI: 10.3864/j.issn.0578-1752.2015.15.017
    Abstract446)   HTML1)    PDF (614KB)(887)       Save
    【Objective】 H9 subtype avian influenza is an important zoonosis. Their six internal genes (PB2, PB1, PA, NP, M, NS) of H7N9 and H10N8 subtypes influenza viruses were derived from endemic H9N2 influenza viruses circulating in poultry. The objective of the study is to establish a special detecting method for H9 subtype avian influenza virus by Electrochemical Luminescence Immunoassay (ECLIA). This ECLIA is significant for influenza surveillance.【Method】The monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were firstly labeled with [Ru (bpy)3]2+ and biotin, respectively. And the labeled efficient was evaluated by MPI-E system and HABA. The second step is the reaction between samples and monoclonal antibody symbolized with [Ru(bpy)3]2+. Then combine the antigen-antibody complex with rabbit polyantibodies labled by biotin-streptavidin linkage system. The chemiluminescence detection can be conducted within the electrochemical analysis system after addition of tripropylamine as substrate. The best working concentrations of the labeled monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were optimized. The sensitivity, specificity and repeatability were tested. Three days and 5 days after challenging, 88 clinical samples were detected by ECLIA and chicken embryo isolation method, and the result was compared and analyzed.【Result】The efficiency of the monoclonal antibody symbolized with [Ru(bpy)3]2+ was 1﹕21, and the efficiency of the rabbit polyantibodies labled by biotin-streptavidin linkage system was 1﹕6. Both labeled antibodies were active in IFA and Western blotting. The detection cutoff value was 28.3, with a suspicious interval of 23.4-33.2. Negative and positive coefficients of variant were both less than 10%. The LOD (limit of detection) was 5×104 EID50. ECLIA can specially detect H9 subtype AIV, no reaction with other influenza viruses (H1, H3, H4, H5 and H6) and avian pathogens (NDV, IBV and IBDV). The accordance rate of ECLIA with chicken embryo isolation method was 86.4%.【Conclusion】The ECLIA could be an available tool for diagnosis and control of H9 subtype avian influenza.
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    Cited: Baidu(2)
    Phylogenetic Analysis and Molecular Characteristics of an H1N1 Subtype Swine Influenza Virus
    XU Hui-yang, XU Bang-feng, CHEN Yan, SUI Jin-yu, YANG Huan-liang, YIN Hang, YANG Da-wei, QIAO Chuan-ling, CHEN Hua-lan
    Scientia Agricultura Sinica    2015, 48 (15): 3071-3078.   DOI: 10.3864/j.issn.0578-1752.2015.15.018
    Abstract416)   HTML5)    PDF (695KB)(476)       Save
    【Objective】 The objective of this study is to understand the molecular epidemiology of swine influenza virus, to provide scientific references for prevention of animal influenza in China. 【Method】Nasal swab samples collected from pigs were inoculated into SPF chicken embryos for virus isolation. The HA positive samples were further purified and proliferated in SPF chicken embryos. Eight gene fragments of the isolated virus were amplified by RT-PCR and then sequenced by Applied Biosystems 3500xL Genetic Analyzer. Nucleotide homology was analyzed by using DNASTAR. The phylogenetic trees of the genes were constructed using MEGA 6.0. 【Result】 The isolated virus was identified as an H1N1 subtype influenza virus, designated as A/swine/Zhejiang/245/2013(H1N1). Nucleotide homology analysis showed that eight gene fragments of this strain were highly homologous with the respective gene of the viruses recently isolated from China. Phylogenetic analysis revealed that the strain belonged to the avian-like swine H1N1 lineage, no gene reassortment occurred. Amino acid sequence at the HA cleavage site was IPSIQSR↓G, which had characteristics of low-pathogenic influenza viruses. And its receptor-binding sites contained 190D and 225E, which prefer sialic acid (SA)-2,6-Gal-terminated saccharides that are abundant in mammal upper respiratory epithelium. Among the six glycosylation sites, four sites were located in HA1 section and the rest in HA2. The isolates contained 271T, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker which may confer resistance to amantadine and rimantadine antivirals.【Conclusion】The sustained existence and continual mutation of avian-like swine H1N1 influenza virus suggested that intensive swine influenza surveillance should be carried out in the future.
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