Comparison of epidemiological characteristics of human infection with avian influenza A (H5N1) virus in five countries of Asia and Africa / 中华预防医学杂志

Objective@#To understand characteristics of demographic, seasonal and spatial distribution of H5N1 cases in major countries of Asia (Indonesia, Cambodia, Vietnam, China) and Africa (Egypt).@*Methods@#Through searching public data resource and published papers, we collected cases information in five countries from May 1st, 1997 to November 6th, 2017, including general characteristics, diagnosis, onset and exposure history, etc. Different characteristics of survived and death cases in different countries were described and χ2 test was used to compare the differences among death cases and odds ratio (OR) and 95%CI value was used to compare death risk in different countries.@*Results@#A total of 856 cases were reported in five countries with Egypt had the most cases (44.3%). The highest number of cases were reported in 2015 (18.3%). 53% cases were reported from January to March, and 96.1% of cases had the history of poultry exposure. 64.2% (43 cases) cases in China had live poultry market exposure, but the sick/dead poultry exposure was the major exposure for cases in other four countries. 452 death cases were reported in five countries, and the fatality rate was 52.8%. With Egypt as the reference group, the highest death risk was seen in Indonesia (OR (95%CI): 11.52 (7.46-17.77)), followed by Cambodia (OR (95%CI): 4.27(2.37-7.69)) and China (OR (95%CI): 2.87 (1.73-4.74)). The age distribution of death cases among 5 countries was statistically significant, and the highest fatality rate was in 15-54 years group in Egypt (83.6%, 102 cases), while in Cambodia the highest fatality rate was in 0-14 years group (76.9%, 30 cases). The highest number of deaths were reported in 2006, and 48.3% were reported from January to March. There was difference in exposure routes among 5 countries (χ2=43.85, P=0.001), 63.2% (24 cases) of the death cases in China had live poultry market exposure. 92.9% (79 cases), 83.3% (40 cases) and 100.0% (38 cases) death cases in Indonesia, Vietnam and Camodia had sick/dead poultry exposure, respectively;and 81.6% (31 cases) of the death cases in Egypt had backyard poultry exposure.@*Conclusion@#The geographical distribution, seasonal age, gender, exposure matter and outcome of H5N1 cases in five countries were different.

Evolution of the HA, NA and NS genes of H5N1 avian influenza viruses from sewage in live bird markets in Changsha, 2014 / 中国人兽共患病学报

The aim of this study is to analyze the evolutional and molecular characteristics of Hemagglutinin (HA),Neuraminidase(NA)and non-structural (NS) genes of H5N1 avian influenza virus (AIV) from sewage in live bird markets (LBMs) in Changsha,2014.Five hundred and one specimens were collected from environment in LBMs in Changsha,2014,and real-time RT-PCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.Sequencing were used for the positive of single H5.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The phylogenetic trees for HA,NA and NS genes and the ClustalW Multiple alignments of amino acids were constructed using MEGA 5 and BioEdit software,respectively.Results showed that of 501 environmental samples,177 (35.33 ％) samples were positive for influenza A viruses and H5 subtype.Eight H5N1 subtype AIV were confirmed by sequencing from the samples of the positive of single H5.Phylogenetic analysis indicated that most of HA genes of the H5N1 subtype AIV strains isolated in Changsha city were located in 2.3.2 and clustered into new subclade,and the most of NA and NS genes in this study were clustered into subclade 2.3.2.1b.QSG of the HA protein of the receptor binding site were found in these H5N1 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of AIV.In strains from this study,we did not found amino acid substitutions of the NA protein at H275Y and N295S,and sensitive to neuraminidases,and the high pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.In conclusion,molecular characteristics of the HA,NA and NS of these H5N1 subtype viruses in this study showed high pathogenicity,but that may not facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.

Interactions of highly pathogenic avian influenza H5N1 nucleoprotein with NF-κB-inducing kinase(NIK) / 军事医学

Objective To study the interactions between the highly pathogenic avian influenza H5N1 nucleoprotein (H5N1 NP) and NF-κB-inducing kinase (NIK),and to reveal the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Methods The gene encoding NIK protein was amplified by RT-PCR from total RNA of HeLa cell line.Eukaryotic expression plasmid pCMV-Myc-NIK and prokaryotic expression plasmid pGEX-4T-1-NP (GST-NP) were constructed by cloning from HeLa cell cDNA and pcDNA3-Flag-NP vector,respectively.Co-immunoprecipitation (co-IP) and GST pull-down were used to test the interactions between H5N1 NP and NIK.Dual-luciferase reporter gene analysis system was used to test the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Results Co-IP and GST pull-down showed that pCMV-Myc-NIK and pGEX-4T-1-NP (GST-NP) could express Myc tagged NIK protein and GST tagged NP protein in HEK293T cells and E.coli,respectively,and that H5N1 NP was associated with NIK in vivo and in vitro.Dual-luciferase reporter gene analysis suggested that H5N1 could inhibit NIK-induced NF-κB transcriptional activity.Conclusion H5N1 NP interacts with NIK and inhibits NIK-induced NF-κB transcriptional activity.This finding can facilitate further study of H5N1.

Recent outbreaks of highly pathogenic avian influenza viruses in South Korea

Outbreaks of H5 highly pathogenic avian influenza viruses (HPAIVs) have caused economic loss for the poultry industry and posed a threat to public health. In South Korea, novel reassortants of HPAIVs such as H5N6 and H5N8 had been circulating in poultry. Here, we will discuss the identity of recent novel reassortants of Korean H5 HPAIVs and the recent advances in vaccine development, which will be useful for controlling HPAIV transmission in poultry and for effectively preventing future epidemics and pandemics.

Optimized clade 2.3.2.1c H5N1 recombinant-vaccine strains against highly pathogenic avian influenza

A/Puerto Rico/8/34 (PR8)-derived recombinant viruses have been used for seasonal flu vaccines; however, they are insufficient for vaccines against some human-fatal H5N1 highly pathogenic avian influenza (HPAI) viruses (HPAIV) due to low productivity. Additionally, the polymerase basic 2 (PB2) protein, an important mammalian-pathogenicity determinant, of PR8 possesses several mammalian-pathogenic mutations. We previously reported two avian PB2 genes (01310 and 0028) related to efficient replication in embryonated chicken eggs (ECEs) and nonpathogenicity in BALB/c mice. In this study, we generated PR8-derived H5N1 recombinant viruses harboring hemagglutinin (attenuated) and neuraminidase genes of a clade 2.3.2.1c H5N1 HPAIV (K10-483), as well as the 01310 or 0028 PB2 genes, and investigated their replication and immunogenicity. Compared with a control virus harboring six internal PR8 genes (rK10-483), the recombinant viruses possessing the 01310 and 0028 PB2 genes showed significantly higher replication efficiency in ECEs and higher antibody titers in chickens. In contrast to rK10-483, none of the viruses replicated in BALB/c mice, and all showed low titers in Madin-Darby canine kidney cells. Additionally, the recombinant viruses did not induce a neutralization antibody but elicited decreased protective immune responses against K10-483 in mice. Thus, the highly replicative and mammalian nonpathogenic recombinant H5N1 strains might be promising vaccine candidates against HPAI in poultry.

Metrology and visualized analysis of H5N1 virus:a research of literature / 军事医学

Objective To analyze domestic and foreign papers on H5N1 virus research using literature metrology and visualized knowledge mapping.Methods Pajek, Bibexcel,and VoSviewer software were used based on Web of Knowledge platform.Results Study on H5N1 virus has been a research focus wide since 2005 and a series of institutions and investi-gators have appeared, with US being the leader in this area and followed by China.The most popular topics of studies on H5N1 included its virology, epidemiology, basic research, anti-virus drugs and vaccines.Conclusion Studies on H5N1 have received more attention, which suggests that more efforts have to be made in China in terms of communication and co-operation with key institutions and famous researchers so as to achieve more success in key areas.

Influenza h5n1 nnext pandemic? / Influenza h5n1: próxima pandemia? / Influenza h5n1: pandemia próximo?

Since 1990 there have been cases of patients infected by influenza caused by Low Pathogenic Avian Influenza. However, since 1997, highly pathogenic viruses, such as A/H5N1, have caused an increase in the number of people infected via zoonotic transmission. Infected patients have constitutional and mainly respiratory symptoms, hematological disorders and liver dysfunction. About half of patients are under 20 years and about 90% under 40 years. In recent studies in mammals it seems that only few mutations in viral genome are necessary to confer the virus the ability to infect mammalian cells. In response to these studies, the Centers for Disease Control (CDC) propose continuous researches without alerting the health care system, because, at present, there is not a global threat. Moreover, in the future, this issue would take place as a major concern from the point of view of public health; it could be the next pandemic. Due to the high human mortality rate occurring in cases of patients infected with this virus, an outbreak or pandemic would require proper knowledge of the biology of the avian influenza virus, the determinants of pathogenicity, prevention through vaccination and possible new treatment regimens.

Desde 1990 se han reportado casos de pacientes infectados por influenza causadas por virus de baja patogenicidad, sin embargo, desde el año 1997, virus altamente patógenos como el A/H5N1 han provocado un aumento en el número de personas infectadas por transmisión zoonotica. Los pacientes infectados presentan síntomas constitucionales y respiratorios, trastornos hematológicos y disfunción hepática. Cerca de la mitad de casos son pacientes menores de 20 años, y aproximadamente el 90% son menores de 40 años. Recientes estudios en mamíferos indican que son necesarias algunas mutaciones en el genoma viral para que el virus tenga la capacidad de infectar células mamíferas. En respuesta a estos estudios el centro de control de enfermedades (por sus siglas en ingles Centers for Disease Control), ente regulador en la dinámica epidemiológica mundial propone continuar con las investigaciones sin alertar a los sistemas de salud, pues en este momento no constituye una amenaza mundial. Sin embargo, este aspecto toma lugar como una preocupación importante desde el punto de vista de la salud pública, por cuanto podría constituir la próxima pandemia. Debido a la alta mortalidad en humanos que se presenta en los casos de pacientes infectados con este virus, un brote o una pandemia exigirán por tanto un conocimiento apropiado de la biología del virus de la influenza aviar, los determinantes de su patogenicidad, la prevención mediante vacuna y los posibles esquemas de tratamiento.

Desde 1990 se têm reportado casos de pacientes infectados por influenza causadas por vírus de baixa patogenicidade, porém, desde o ano 1997, vírus altamente patógenos como o A/H5N1 têm provocado um aumento no número de pessoas infectadas por transmissão zoonótica. Os pacientes infectados apresentam sintomas constitucionais e respiratórios, transtornos hematológicos e disfunção hepática. Cerca dametade de casos são pacientes menores de 20 anos, e aproximadamente o 90% são menores de 40 anos. Recentes estudos em mamíferos indicam que são necessárias algumas mutações no genoma viral para que o vírustenha a capacidade de infectar células mamíferas. Em resposta a estes estudos o centro de controle de doenças (por suas siglas em inglês: Centers for Disease Controle), ente regulador na dinâmica epidemiológica mundial propõe continuar com as pesquisassem alertar aos sistemas de saúde, pois em este momento não constituiu maameaça mundial. Porém, este aspecto toma lugar como uma preocupação importante desde o ponto de vista dasaúde pública, já que poderia constituir a próxima pandemia. Devido à alta mortalidade em humanos que se apresenta nos casos de pacientes infectados com este vírus, um surto ou uma pandemia, exigirão por tanto um conhecimento apropriado da biologia do vírus da influenza aviar, os determinantes dasua patogenicidade, a prevenção mediante vacinae os possíveis esquemas de tratamento.

Abibliometric analysis of the international literatures on avian influenza / 第二军医大学学报

Objective To conduct a bibliometric analysis of international scientific literatures on research of avian influenza. Methods We performed a search in the Web of Science (WoS) database and analyzed the relevant records with Thomson Data Analyzer (TDA) and citation trees. Results Due to the rapid growth in publications by the United States and China, the number of publications on research of avian influenza has increased remarkably since 2005. The publication of Japan and Netherlands were of high quality due to support of international research programs. The researches about H5N1 virus mutation by the United States, Japan and Netherlands were the typical achievement of the area but also triggered arguments. Conclusion More international efforts arewarranted on research of H7N9 and other influenza viruses.

Construction and identification of a eukaryotic expression vector of fusion protein containing avian H5N1 influenza virus hemagglutinin antigen and the cholera toxin B subunit / 中华传染病杂志

Objective To construct a eukaryotic vector which contains avian H5N1 influenza virus hemagglutinin (HA) antigen and the cholera toxin B subunit (CTB) and to investigate its expression in COS7 cells,and the ability to induce specific immune responses in vivo in different periods.Methods After cloned by polymerase chain reaction (PCR),CTB and HA genes were digested with BamH Ⅰ and connected into CTB-HA gene with T4 ligase.The connected gene was referred to as CH.After double digestion,CH gene was inserted into a eukaryotic recombinant plasmid pCI-neo.The pCI-CH plasmid was then transfected into COS7 cells.Western blot was used to detect the expression of HA antigen.After New Zealand white rabbits were immunized,the titer of HA antigen-specific antibody in serum and its specificity with other strains such as H1N1,H9N1,H3N2 and influenza B virus were determined by indirect enzyme-linked immunosorbent assay.Results The pCI-CH vector (DNA vaccine) was successfully constructed,which could be efficiently expressed in COS7 cells and induce specific antibodies against pCI-CH in rabbits.Cross reactions indicated that DNA vaccine pCI-CH specific antisera could not only react with H5N1 strain (P/N＞2.1),but also H1N1,H9N1 and H3N2 strains,but did not cross react with influenza B virus.Conclusion The newly constructed avian H5N1 influenza virus nucleic acid vaccine has good immunogenicity.

Genetic diversification of avian influenza H5N1 virus in boundary areas of Yunnan province / 中华流行病学杂志

Objective To elucidate the genetic diversifications of avian influenza subtype H5N1 viruses in the boundary regions of Yunnan province during 2009 to July,2011.Methods Swab samples were collected from foreign poultry and wild birds in boundary regions of Yunnan province during 2009 to July,2011 and tested by H5/N1 subtype-specific multiplex RT-PCR.The HA genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD 18-T vectors for sequencing.Both alignment and phylogenetic analysis were performed with sequences of the known reference strains.Results Fifteen different HA sequences were obtained from 36 representative positive samples and could be divided into 2 distinct Clades (2.3.2 and 2.3.4).Through phylogenetic analysis,Clade 2.3.2 and 2.3.4 could then be further divided into 3 ( Ⅱ -1 to Ⅱ -3 ) and 2 smaller clades ( Ⅰ -1 and Ⅰ -2),respectively.The viruses of Clade 2.3.2 Ⅱ -1 and Ⅱ-2 were new variant strains of H5N 1 virus.The cleavage sites of HA from positive samples all possessed molecular characterization of highly pathogenic avian influenza virus.Mutation of key amino acids had been found among receptor binding sites,potential glycosylation sites,neutralizing epitopes and others.Conclusion It seemed evident that the H5N1 subtype viruses showed genetic diversifications and had undergone the evolution progress of multi-clade (2.3.2,2.3.4) to single caide (2.3.2) in the boundary regions of Yunnan province,during 2009 to July,2011.

Molecular characteristics of the hemagglutinin genes of avian influenza virus subtype H5N1 in the boundary region of Yunnan province / 中国人兽共患病学报

To elucidate the molecular characteristics of the hemagglutinin (HA) genes of H5N1 subtype of avian influenza viruses in the boundary region of Yunnan province. Of 420 samples were collected from the foreign poultry in boundary region of Yunnan province during 2003 to 2008 and these samples were subjected to screening by H5/N1 subtype-specific and multiplex RT-PCR. testing. The HA genes of H5N1 viruses from positive samples were amplified by RT-PCR and cloned into vector pMD18 T for subsequent sequencing. The alignment with sequences of the known reference strains and phylogenetic analysis were then performed. The genes from 21 representative positive samples with 4 different sequences at the cleavage site were obtained and all of them possessed the molecular characteristic of highly pathogenic avian influenza virus. The mutation of key amino acids had been found among receptor-binding sites, potential glycosylation sites and neutralizing epitopes.-Phylogenetic analysis showed those positive samples could be divided into 5 distinct clades, including clade 1, 2.4. 2.3.2, 2.3.4 and 7. It is evident that H5N1 viruses from the foreign boundary region of Yunnan province in 2003 to 2008 show genetic divergence and clade 2,3,4 is the dominant clade in this region.

Study of immunoadjuvant effect induced by three types of bacterial genomes and CpG ODN to avian influenza virus H5N1 subtype inactivated oil emulsion vaccine / 中国免疫学杂志

0.05) or significantly enhanced(P0.05).In this case,the immunoadjuvant capability of CpG ODN was better than FQ68 genomes.Conclusion:CpG ODN or FQ68 genome enhances humoral and Th1 type of immune responses when packed into AIV oil emulsion vaccine,while the adjuvants do not show better adjuvanticity when separated from the oil emulsion vaccine.

Two cases of Avian Influenza A(HSNI) in Anhui province of China / 中华传染病杂志

Objective To analyze the clinical features,epidemiologic information and outcome in confirmed cases of human infection with a highly pathogenic influenza A(H5N1).Methods The clinical features and epidemiologic findings in 2 confirmed cases of avian influenza A(H5N1)in Anhui province,in November 2005.Clinical data on vital signs,physical findings,laboratory tests and roentgenology were obtained by means of retrospective review of the hospital records.Epidemiologic data were collected through interviews of the patients and their relatives.Results In both cases,the diagnosis of influenza A(H5N1)was confirmed by means of viral culture and real-time polymerase chain reaction with specific primers for H5 and N1 in samples obtained from tracheal aspiration.All patients were previously healthy young women and resided in village.They had a clear history of di- rect contact with sick/dead poultry and prepared dead chickens at home for eating(removed feathers, washed,cut)hut no report of confirmed HSN1 animals in the village.The time between exposure and onset of illness were 5 days,no one else in family sick.The time between the onset of illness and hos- pitalization were 5 days and 6 days,respectively.Two patients have initial symptoms of high fever (typically a temperature more than 38℃).The prominent clinical features were those of influenza syndrome,including fever,cough,and shortness of breath.Upper respiratory tract symptoms were absent.The platelet counts were decreased.In both patients,there were marked abnormalities on chest radiography,radiographic changes include bilateral and unilateral lobular consolidations with air bronchograms,and had dramatic worsening of findings.Two patients developed acute respiratory dis- tress syndrome(ARDS)complicated multiple organ dysfunction syndrome(MODS)and died of pro- gressive respiratory failure.Conclusions Exposure to dead poultry within a week before the onset of illness was associated with Influenza A(H5N1)infection in humans,but no definitive evidence of hu man-to-human transmission has been found yet.Influenza A(HSNI)infection,characterized by fe- ver,an influenza-like illness with lower respiratory tract symptoms,carries a high risk of death.

Research progress on the mechanism of transmission,pathopoiesis,treatment and prevention of avian influenza virus(H5N1) / 解放军医学杂志

The outbreak of high-pathogenicity avian influenza viruses causing severe illness and death has severely disrupted poultry production and trade and posed a significant threat to public health.In recent thirty years,the virus has been endemic in Southeast Asia,China,Europe and Africa.Although the virus shows little ability of transmission between humans,a process of genetic reassortment could occur in a human co-infected with avian influenza A virus and a human strain of influenza A virus.So H5N1 represents a potential source of the next pandemic influenza.Direct avian-human H5N1 virus transmission is the predominant means of human infection,through direct contact with infected birds and virus-contaminated fomites or with fertilizer containing poultry feces.After several rounds of infection the viruses acquire the mutations required for crossing species barrier and infect a range of mammalian species including humans.Currently,vaccination is still the key strategy to prevent severe illness and death from pandemic influenza.Antiviral drugs such as NA inhibitors play an important role in antiviral treatment.The features of avian influenza virus and its mechanism of transmission in animals and humans are herewith reviewed,and the pathopoiesis of the virus,the current status of vaccine development and antiviral treatment are also discussed.