The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by se...The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.展开更多
[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 gen...[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.展开更多
[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu...[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.展开更多
[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the an...[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.展开更多
This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated...This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.展开更多
A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I...A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.展开更多
Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Metho...Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.展开更多
Background:Avian influenza viruses(AIVs)have been identified from more than 100 different species of wild birds around the globe.Wild migratory birds can act as potential spreaders for AIVs to domestic birds between d...Background:Avian influenza viruses(AIVs)have been identified from more than 100 different species of wild birds around the globe.Wild migratory birds can act as potential spreaders for AIVs to domestic birds between different countries.Egypt is situated on important migratory flyways for wild birds between different continents.While much is known about circulation of zoonotic potential H5N1 and H9N2 AIVs in domestic poultry in Egypt,little is known about the pivotal role of migratory birds in the maintenance and transmission of the viruses in Egypt.Methods:Targeted AIV surveillance has been conducted in 2017 in different wetlands areas in Northern and Eastern Egypt.Results:AIV of subtype H5 was detected in two bird species.In addition,a novel reassortant strain of the H6N2 subtype was identified which reveals the continuous risk of new influenza virus(es)introduction into Egypt.This novel virus possesses a reassortant pattern originating from different AIV gene pools.Conclusions:Intervention control strategies should be performed to minimize the possible contact of domestic birds with wild birds to lower the risk of virus transmission at this interface.In addition,constant monitoring of AIVs in migratory birds is essential in the early detection of influenza virus introduction into Egypt.展开更多
Low pathogenic Avian Influenza (AI) virus has the ability to evolve to high pathogenic viruses resulting in significant economic losses in the poultry sector. This study aims at assessing the impact of H9N2 viral pass...Low pathogenic Avian Influenza (AI) virus has the ability to evolve to high pathogenic viruses resulting in significant economic losses in the poultry sector. This study aims at assessing the impact of H9N2 viral passaging in broilers and its relatedness to pathogenicity and amino acid (a.a) sequences of the hemagglutinin (HA) cleavage site and neuraminidase (NA) stalk. The original H9N2 AI virus (P0) was used to challenge ten-21 days old broilers. Individual recovery of H9N2 virus from homogenates of trachea, lungs and airsacs was attempted in 9 days old chicken embryos, as a conclusion of the first passage (P1). Tracheal isolates of H9N2 were passaged for a second (P2) and a third (P3) time in broilers, followed by a similar embryonic recovery procedure. The a.a. sequence of a part of HA1 cleavage site and Neuraminidase stalk were compared among the differently passaged viruses;an assessement of the relatedness of the determined a.a. sequences to the pathogenicity in broilers, based on frequency of mortality, morbidity signs, gross and microscopic lesions at 3 days post challenge with the P1, P2, and P3-H9N2, is concluded. An increase in certain morbidity signs and specific lesions was observed in P2- and P3-H9N2 challenged broilers compared to birds challenged with P1-H9N2. A conserved R-S-S-R amino acid sequence at the HA1 cleavage site was observed in the differently passaged H9N2, associated with a variability in the NA stalk-a.a sequences. The passaging of the low pathogenic H9N2 virus in broilers leads to a trend of increase in pathogenicity, manifested in higher frequency of morbidity signs, and of specific gross and microscopic lesions of the examined organs. This passaging was associated with a conserved a.a. sequence of the hemaglutinin cleavage site and a variability in the sequence of the neuraminidase stalk. A detailed study of the potential of the detected variability in the neuraminidase stalk of H9N2 in induction of a higher pathogenicity in broilers will be the subject of future investigations.展开更多
Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylog...Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.展开更多
Background:Flying birds,especially those that hover,need to meet high energetic demands.Birds that meet this demand through nectarivory face the added challenges of maintaining homeostasis in the face of spikes in blo...Background:Flying birds,especially those that hover,need to meet high energetic demands.Birds that meet this demand through nectarivory face the added challenges of maintaining homeostasis in the face of spikes in blood sugar associated with nectar meals,as well as transporting that sugar to energetically demanding tissues.Nectarivory has evolved many times in birds and we hypothesized that the challenges of this dietary strategy would exert selective pressure on key aspects of metabolic physiology.Specifically,we hypothesized we would find convergent or parallel amino acid substitutions among different nectarivorous lineages in a protein important to sensing,regulating,and transporting glucose,glucose transporter 2(GLUT2).Methods:Genetic sequences for GLUT2 were obtained from ten pairs of nectarivorous and non-nectarivorous sister taxa.We performed PCR amplification of the intracellular C-terminal domain of GLUT2 and adjacent protein domains due to the role of this region in determination of transport rate,substrate specificity and glucosensing.Results:Our findings have ruled out the C-terminal regulatory region of GLUT2 as a target for selection by sugar-rich diet among avian nectarivores,though selection among hummingbirds,the oldest avian nectarivores,cannot be discounted.Conclusion:Our results indicate future studies should examine down-stream targets of GLUT2-mediated glucosensing and insulin secretion,such as insulin receptors and their targets,as potential sites of selection by nectarivory in birds.展开更多
Influenza type A, is an avian disease with a complicated ecology and transmission routes in verity of avian and mammalian species. The present study aimed to demonstrate the characteristic, clinical and experimental f...Influenza type A, is an avian disease with a complicated ecology and transmission routes in verity of avian and mammalian species. The present study aimed to demonstrate the characteristic, clinical and experimental features as well as pathogenecity of Avian Influenza Virus H5N2 through a laboratory-based experiment in western Iran. A post-mortem examination of experimentally chickens was undertaken in 2007. Overall 25 local native chickens including 15 layers and 10 roosters suspected with AI infection as well as 50 experimental chickens were studied. The virus was isolated from the embryonated specific pathogen-free （SPF） chicken eggs. There was an embryo mortality rate of 71% within 48 hours post inoculation （PI）. Hemagglutinin （HA） inhibition titres against AIV subtype H5N2 in the layers ranged from 4.20 to 4.75 （acute） and 6.21 to 7.82 （convalescent）. Accumulated mucous in trachea of the dissected birds, congested lungs, atrophied bursa, haemorrhagic cecal tonsils and inflamed thymus were the main clinical symptoms. Thickened and infected air sacs, pre hepatitis and enteritis signs were also observed, in experimental birds, the eyes＇ colour became red and the eyelashes were almost double in diameters after being infected. The AI virus found in the present study was classified as a highly pathogenic avian influenza.展开更多
Humpback whales are migratory, spending summers in cooler, high-latitude waters and mating and calving in tropical and subtropical waters in 14 identified district population segments. It may be possible that the coas...Humpback whales are migratory, spending summers in cooler, high-latitude waters and mating and calving in tropical and subtropical waters in 14 identified district population segments. It may be possible that the coastal areas are infected with low pathogenic avian influenza (LPAI) during the release of infected humpback whale feces. Therefore, humpback whales can be an effective reservoir of the avian influenza virus (AIV) from the Poles to the Continents to spread AIV to coastal animals. Strong ultraviolet (UV) exposure amidst CO2 emission increase and minimal sunspot number might cause mutations of aquatic virus and humpback whale in the Antarctic and the Arctic. LPAI or highly pathogenic avian influenza (HPAI) is expressed in the Continents under appropriate environmental factors. Since penguins are birds while humpback whales are marine mammals, the humpback whales infected by the mutant virus might cause interspecies transmission to a new host with evolutionary changes. The migration pattern is seasonally similar between migratory bird and humpback whale except: 1) different species of bird versus whale, 2) different landing area of land versus coast, 3) similar infection means of bird feces versus humpback whale feces. The contribution of AIV transmission by whales was several times larger than that by migratory birds. Therefore, the routes of humpback whales should be considered to prevent AIV outbreaks in addition to the flyways of migratory birds. Humpback whale stranding (y) along the Atlantic Coast of the USA was correlated with CO2 emissions (x) to have y = 0.3515x + 18.595 (R2 = 0.4069) during 1992-2016 while y = 0.0652x + 4.5847, (R2 = 0.6128) during 2016-2018. AIV outbreak in 2010 (y) along the Atlantic Coast was also correlated with humpback whale stranding (2016-2018) (x) as y = 0.1387x + 6.8184 (R2 = 0.3966). Since AIV outbreak was linearly (R2 = 0.9967) correlated with the minimum sunspot number, it was postulated that the unusual mortality events of humpback whale stranding might be caused by an infected mutant virus in the Arctic. Consequently, the humpback whales were stranded along major CO2 producing Atlantic Coast States toward the winter habitat of the West Indies during the CO2 emissions and the minimal sunspot number with strong UV radiation. The stranded dead whales should be burned as soon as possible to prevent further deadly viral interspecies transmission of AIV by the coastal animals. Since CO2 emissions were increased in 2017 and the sunspot number was minimal at the end of 2018, serious numbers of whales are expected to be stranded at the Gulf of Maine, States of North Carolina, New York, and Virginia from November 2018 till April 2019. To save humpback whales from the unusual mortality event along the Atlantic Coast, the reduction of CO2 emissions is suggested by replacement of fossil fuels combustion plants with nuclear power plants along the Atlantic Coast of the USA.展开更多
[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus （AIV） H9N2 subtype in Madin- Darby canine kidney （MDCK） cells. [Method] Three AIV H9 subt...[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus （AIV） H9N2 subtype in Madin- Darby canine kidney （MDCK） cells. [Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively. Then, DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells. The cytopathic effect （CPE） was observed once every 24 h, and the HA titer of the supematant was measured by HA assay. [Result] When the trypsin concentration was 10 -20 μg/ml in DMEM, the HA titer of virus culture reached 7 log2 （1：128）. Almost all cells were cytopathic after 96 h post inoculation with 1：1 000 or 1：10 000 dilution of AIV culture, and the virus titer reached a peak after 72 -96 h. [ Conclusion] The optimal concentration of trypsin is 10 -20 pg/ml for proliferation of AIV H9N2 subtype in MDCK cells.展开更多
Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including tra...Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.展开更多
基金Fundamental Research Program of Shanxi Province,China(202103021224156)National Natural Science Foundation of China(32202788)+5 种基金Special Research Fund of Shanxi Agricultural University for High-level Talents,China(2021XG004)Science and Technology Innovation Program of Shanxi Agricultural University,China(2021BQ78)special fund for Science and Technology Innovation Teams of Shanxi Province,China(202304051001041)?Shanxi Province Excellent Doctoral Work Award-Scientific Research Project,China(SXBYKY2021005,SXBYKY2021063,SXBYKY2022014)the Fund for Shanxi“1331 Project”,China(20211331-13)earmarked fund for Modern Agro-industry Technology Research System of Shanxi Province,China.
文摘The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.
基金Supported by a Sub-project of 973 Program of China(2005CB523001)~~
文摘[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.
文摘[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.
基金Supported by subproject of Major State Basic Research Development Program of China (2005CB523001)~~
文摘[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.
基金supported by funds provided by South China Agricultural University and Guangzhou work team project(No 2011A020102009)
文摘This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.
基金supported by subproject of National Program on Key Basic Research Project (973 Program )(2005CB523001)
文摘A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.
基金supported by the National Basic Research Program of China (973 program: 2005CB523006)
文摘Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.
基金funded by an internal project of the Reference Laboratory for Veterinary Quality Control on Poultry Production,Animal Health Research Institutesupported in part by the Swedish Research Council VR(Grant Numbers 2016-02596 and 2018-02569)to MMN
文摘Background:Avian influenza viruses(AIVs)have been identified from more than 100 different species of wild birds around the globe.Wild migratory birds can act as potential spreaders for AIVs to domestic birds between different countries.Egypt is situated on important migratory flyways for wild birds between different continents.While much is known about circulation of zoonotic potential H5N1 and H9N2 AIVs in domestic poultry in Egypt,little is known about the pivotal role of migratory birds in the maintenance and transmission of the viruses in Egypt.Methods:Targeted AIV surveillance has been conducted in 2017 in different wetlands areas in Northern and Eastern Egypt.Results:AIV of subtype H5 was detected in two bird species.In addition,a novel reassortant strain of the H6N2 subtype was identified which reveals the continuous risk of new influenza virus(es)introduction into Egypt.This novel virus possesses a reassortant pattern originating from different AIV gene pools.Conclusions:Intervention control strategies should be performed to minimize the possible contact of domestic birds with wild birds to lower the risk of virus transmission at this interface.In addition,constant monitoring of AIVs in migratory birds is essential in the early detection of influenza virus introduction into Egypt.
文摘Low pathogenic Avian Influenza (AI) virus has the ability to evolve to high pathogenic viruses resulting in significant economic losses in the poultry sector. This study aims at assessing the impact of H9N2 viral passaging in broilers and its relatedness to pathogenicity and amino acid (a.a) sequences of the hemagglutinin (HA) cleavage site and neuraminidase (NA) stalk. The original H9N2 AI virus (P0) was used to challenge ten-21 days old broilers. Individual recovery of H9N2 virus from homogenates of trachea, lungs and airsacs was attempted in 9 days old chicken embryos, as a conclusion of the first passage (P1). Tracheal isolates of H9N2 were passaged for a second (P2) and a third (P3) time in broilers, followed by a similar embryonic recovery procedure. The a.a. sequence of a part of HA1 cleavage site and Neuraminidase stalk were compared among the differently passaged viruses;an assessement of the relatedness of the determined a.a. sequences to the pathogenicity in broilers, based on frequency of mortality, morbidity signs, gross and microscopic lesions at 3 days post challenge with the P1, P2, and P3-H9N2, is concluded. An increase in certain morbidity signs and specific lesions was observed in P2- and P3-H9N2 challenged broilers compared to birds challenged with P1-H9N2. A conserved R-S-S-R amino acid sequence at the HA1 cleavage site was observed in the differently passaged H9N2, associated with a variability in the NA stalk-a.a sequences. The passaging of the low pathogenic H9N2 virus in broilers leads to a trend of increase in pathogenicity, manifested in higher frequency of morbidity signs, and of specific gross and microscopic lesions of the examined organs. This passaging was associated with a conserved a.a. sequence of the hemaglutinin cleavage site and a variability in the sequence of the neuraminidase stalk. A detailed study of the potential of the detected variability in the neuraminidase stalk of H9N2 in induction of a higher pathogenicity in broilers will be the subject of future investigations.
基金supported by National Bai Qian Wan Talents Engineering Foudation (Grant No. 9452006-03 )Guangxi Science Technology Bureau (GKG- 0719004-3A)Guangxi Husbandry and Fisheries Bureau.
文摘Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.
基金This work was supported by grants from Natural Sciences and Engineering Research Council of Canada Discovery Grant(Number 386466 to KCW and 06538 to JTW)the Human Frontier Science Program(Grant Number RGP0062/2016).
文摘Background:Flying birds,especially those that hover,need to meet high energetic demands.Birds that meet this demand through nectarivory face the added challenges of maintaining homeostasis in the face of spikes in blood sugar associated with nectar meals,as well as transporting that sugar to energetically demanding tissues.Nectarivory has evolved many times in birds and we hypothesized that the challenges of this dietary strategy would exert selective pressure on key aspects of metabolic physiology.Specifically,we hypothesized we would find convergent or parallel amino acid substitutions among different nectarivorous lineages in a protein important to sensing,regulating,and transporting glucose,glucose transporter 2(GLUT2).Methods:Genetic sequences for GLUT2 were obtained from ten pairs of nectarivorous and non-nectarivorous sister taxa.We performed PCR amplification of the intracellular C-terminal domain of GLUT2 and adjacent protein domains due to the role of this region in determination of transport rate,substrate specificity and glucosensing.Results:Our findings have ruled out the C-terminal regulatory region of GLUT2 as a target for selection by sugar-rich diet among avian nectarivores,though selection among hummingbirds,the oldest avian nectarivores,cannot be discounted.Conclusion:Our results indicate future studies should examine down-stream targets of GLUT2-mediated glucosensing and insulin secretion,such as insulin receptors and their targets,as potential sites of selection by nectarivory in birds.
文摘Influenza type A, is an avian disease with a complicated ecology and transmission routes in verity of avian and mammalian species. The present study aimed to demonstrate the characteristic, clinical and experimental features as well as pathogenecity of Avian Influenza Virus H5N2 through a laboratory-based experiment in western Iran. A post-mortem examination of experimentally chickens was undertaken in 2007. Overall 25 local native chickens including 15 layers and 10 roosters suspected with AI infection as well as 50 experimental chickens were studied. The virus was isolated from the embryonated specific pathogen-free （SPF） chicken eggs. There was an embryo mortality rate of 71% within 48 hours post inoculation （PI）. Hemagglutinin （HA） inhibition titres against AIV subtype H5N2 in the layers ranged from 4.20 to 4.75 （acute） and 6.21 to 7.82 （convalescent）. Accumulated mucous in trachea of the dissected birds, congested lungs, atrophied bursa, haemorrhagic cecal tonsils and inflamed thymus were the main clinical symptoms. Thickened and infected air sacs, pre hepatitis and enteritis signs were also observed, in experimental birds, the eyes＇ colour became red and the eyelashes were almost double in diameters after being infected. The AI virus found in the present study was classified as a highly pathogenic avian influenza.
文摘Humpback whales are migratory, spending summers in cooler, high-latitude waters and mating and calving in tropical and subtropical waters in 14 identified district population segments. It may be possible that the coastal areas are infected with low pathogenic avian influenza (LPAI) during the release of infected humpback whale feces. Therefore, humpback whales can be an effective reservoir of the avian influenza virus (AIV) from the Poles to the Continents to spread AIV to coastal animals. Strong ultraviolet (UV) exposure amidst CO2 emission increase and minimal sunspot number might cause mutations of aquatic virus and humpback whale in the Antarctic and the Arctic. LPAI or highly pathogenic avian influenza (HPAI) is expressed in the Continents under appropriate environmental factors. Since penguins are birds while humpback whales are marine mammals, the humpback whales infected by the mutant virus might cause interspecies transmission to a new host with evolutionary changes. The migration pattern is seasonally similar between migratory bird and humpback whale except: 1) different species of bird versus whale, 2) different landing area of land versus coast, 3) similar infection means of bird feces versus humpback whale feces. The contribution of AIV transmission by whales was several times larger than that by migratory birds. Therefore, the routes of humpback whales should be considered to prevent AIV outbreaks in addition to the flyways of migratory birds. Humpback whale stranding (y) along the Atlantic Coast of the USA was correlated with CO2 emissions (x) to have y = 0.3515x + 18.595 (R2 = 0.4069) during 1992-2016 while y = 0.0652x + 4.5847, (R2 = 0.6128) during 2016-2018. AIV outbreak in 2010 (y) along the Atlantic Coast was also correlated with humpback whale stranding (2016-2018) (x) as y = 0.1387x + 6.8184 (R2 = 0.3966). Since AIV outbreak was linearly (R2 = 0.9967) correlated with the minimum sunspot number, it was postulated that the unusual mortality events of humpback whale stranding might be caused by an infected mutant virus in the Arctic. Consequently, the humpback whales were stranded along major CO2 producing Atlantic Coast States toward the winter habitat of the West Indies during the CO2 emissions and the minimal sunspot number with strong UV radiation. The stranded dead whales should be burned as soon as possible to prevent further deadly viral interspecies transmission of AIV by the coastal animals. Since CO2 emissions were increased in 2017 and the sunspot number was minimal at the end of 2018, serious numbers of whales are expected to be stranded at the Gulf of Maine, States of North Carolina, New York, and Virginia from November 2018 till April 2019. To save humpback whales from the unusual mortality event along the Atlantic Coast, the reduction of CO2 emissions is suggested by replacement of fossil fuels combustion plants with nuclear power plants along the Atlantic Coast of the USA.
基金funded by the Beijing Academy of Agriculture and Forestry Sciences (2010A007)
文摘[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus （AIV） H9N2 subtype in Madin- Darby canine kidney （MDCK） cells. [Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively. Then, DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells. The cytopathic effect （CPE） was observed once every 24 h, and the HA titer of the supematant was measured by HA assay. [Result] When the trypsin concentration was 10 -20 μg/ml in DMEM, the HA titer of virus culture reached 7 log2 （1：128）. Almost all cells were cytopathic after 96 h post inoculation with 1：1 000 or 1：10 000 dilution of AIV culture, and the virus titer reached a peak after 72 -96 h. [ Conclusion] The optimal concentration of trypsin is 10 -20 pg/ml for proliferation of AIV H9N2 subtype in MDCK cells.
文摘Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.
