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1.
猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立   总被引：2，自引：3，他引：2  
对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。  相似文献   

2.
The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.  相似文献   

3.
Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.  相似文献   

4.
Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.  相似文献   

5.
The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian‐like H1N1 and two reassortant H1N2 and H3N2 viruses with human‐origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross‐reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.  相似文献   

6.
本研究对2011年分离自吉林省猪群的3株流感病毒进行了遗传进化分析。结果表明欧亚类禽H1N1猪流感病毒和古典H1N1猪流感病毒在吉林省猪群中共同流行,因此加强猪流感流行病学调查具有重要意义。  相似文献   

7.
猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立   总被引：1，自引：0，他引：1  
根据GenBank中H1N1和H3N2亚型猪流感病毒(SIV)血凝素(hemagglutinin,HA)、神经氨酸酶(neuraminidase,NA)和M基因保守序列,分别设计合成了5对特异性引物,利用RT-PCR技术对SIV的型和亚型进行鉴定。结果表明,该方法的型RT-PCR可以检测出104 EID50病毒量所提取的RNA;H1、H3、N1和N2的亚型RT-PCR均可以检测出104 EID50病毒量所提取的RNA。除每对特异性引物所对应的亚型外,对其他亚型及猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的检测均为阴性,应用该方法对临床样品进行检测,其结果与病毒分离结果符合率为100%。结果表明,该方法特异性好、敏感性高,有望成为SIV的一种特异、敏感、快速的分型检测方法,为猪流感分子流行病学的调查奠定了良好的基础。  相似文献   

8.
中国类禽型H1N1亚型猪流感病毒的发现和遗传分析   总被引：1，自引：0，他引：1  
采用禽流感病毒通用引物,对2006年发现的1株H1N1亚型的类禽型猪流感病毒的全基因组进行了测序,并进行了遗传学分析。序列分析表明它的8个片段与欧洲的类禽型猪流感病毒A/swine/Ile et Vilaine/1455/99(H1N1)病毒和A/swine/Cotes d'Armor/1488/99(H1N1)病毒的相应基因具有高度的同源性,同源性可达97%~99%,表明类禽型猪流感病毒已在中国出现。其血凝素基因的190E→D和225G→E的突变使得其结合NeuAc-a2,6Gal受体的能力高于NeuAca2,3Gal受体。欧洲的类禽型猪流感病毒可以直接感染人,并且可导致人的肺炎和死亡。中国类禽型猪流感病毒的发现及其的NeuAca2,6Gal受体结合特性使其成为一个潜在可感染人的病毒。  相似文献   

9.
Sun H  Jiao P  Jia B  Xu C  Wei L  Shan F  Luo K  Xin C  Zhang K  Liao M 《Veterinary microbiology》2011,152(3-4):258-265
In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.  相似文献   

10.
We investigated influenza interspecies transmission in two commercial swine farms in Thailand. Sera from swine-exposed workers (n=78), age-matched non-swine-exposed healthy people (n=60) and swine populations in both farms (n=85) were studied. Hemagglutination-inhibition (HI) assay was performed on Thai swine H1 viruses (swH1N1 and swH1N2) isolated from both farms. Thai human H1N1 (huH1N1) and pandemic H1N1 2009 (pH1N1) were also used as test antigens. The hemagglutinin (HA) 1 genes of swH1N1 and swH1N2 viruses were sequenced and shown to be genetically distinct from the Thai huH1N1 and pH1N1 viruses. Evidence of pig-to-human influenza virus transmission was found in farm workers with increased odds of elevated antibody titers to both swH1N1 (OR 42.63, 95% CI, 14.65-124) and swH1N2 (OR 58, 95% CI, 13.12-256.3) viruses. No evidence of human-to-pig influenza virus transmission was detected in this study.  相似文献   

11.
旨在了解河南省猪流感病毒的流行情况及其遗传进化和基因组特征。2018年4月，从河南省某一出现疑似流感症状猪群中采集鼻拭子样品150份用于分离病毒，对分离病毒的全基因组进行序列测定和分析。同时感染6周龄BALB/c小鼠，研究其对小鼠的致病性。结果显示，获得1株H1N1亚型病毒[命名为A/swine/Henan/NY20/2018（H1N1）]。遗传进化表明，其HA和NA基因属于欧亚类禽H1N1分支，PB2、PB1、PA、NP和M基因属于2009甲型H1N1分支，NS基因属于经典H1N1分支。HA蛋白的裂解位点序列为PSIQSR↓GL，具有低致病性流感病毒的分子特征，在小鼠肺和鼻甲有效复制并能引起肺组织病理学变化。本研究分离到1株3源重排H1N1亚型病毒，对小鼠呈现一定致病力，提示应进一步加强对SIV的监测。  相似文献   

12.
In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 106 EID50 of GSGD/1/96, 103 EID50 of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 106 EID50 of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.  相似文献   

13.
Several highly pathogenic H5N1 avian influenza viruses were isolated from swine populations in Fujian Province, China, since 2001. Because it is thought that H5N1 infection in pigs might result in virus adaptation to humans, we surveyed swine populations in Fujian Province in 2004 and 2007 for serological evidence of the infection. Twenty‐five pig farms covering all nine administrative districts of Fujian Province were sampled and a total of 1407 serum specimens were collected. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection and only a few cases of H9 infection. The negative results for H5 infection were further verified by micro‐neutralization tests. By contrast, H1 influenza virus infections were prevalent in swine in both surveys according to the results of enzyme‐linked immunosorbent assay (ELISA). The H3 infection rate was reduced dramatically in 2007 compared with 2004, when examined by HI and ELISA. In summary, the results imply that the swine populations in Fujian Province had not been affected greatly by the H5N1 avian influenza virus, given that there is no serological evidence that H5N1 influenza virus has infected the pig populations. The reported isolates represent only sporadic cases.  相似文献   

14.
对南京市首例甲型H1N1(2009)病毒进行细胞分离,获得一株具有较高血凝活性的病毒,命名为A/Nanjing/1/2009。在全基因组测序的基础上,对分离株的血凝素基因(haemagglutinin,HA)的遗传特征进行了详细研究。分离株HA蛋白不具有多碱基HA裂解位点,具有低致病性流感病毒特点。与参考株A/California/04/2009相比,分离株A/Nanjing/1/2009HA蛋白的有5个氨基酸发生了突变,其中一个位于Ca抗原位点208位氨基酸(R→K),这一突变虽然还不会影响抗原性的改变,但预示了新甲型H1N1(2009)抗原漂移的启动。分离株有5个潜在糖基化位点,这与近年来古典猪H1N1和北美三源重配猪H1病毒完全一致,保留了古典猪H1病毒的特点。与禽H1病毒相比,分离株HA蛋白受体结合位点上的190(E→D)和225(G→D)位点发生突变,这可能成为新甲型H1N1(2009)在人际间传播的一个重要分子基础。此外,其它受体结合位点上相关氨基酸同时具有人和猪流感病毒的特点。本研究对南京市早期流行的甲型H1N1(2009)流感病毒的HA蛋白的分子遗传特征进行了详细研究,对进一步监测病原变异具有重要指导意义。  相似文献   

15.
In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 108/EID50 /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log2). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.  相似文献   

16.
The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.  相似文献   

17.
Novel swine influenza virus subtype H3N1 in Italy   总被引：2，自引：0，他引：2  
To date, three subtypes of swine influenza viruses, H1N1, H1N2, and H3N2 have been isolated in Italy. In 2006, a novel swine influenza virus subtype (H3N1) was isolated from coughing pigs. RT-PCR performed on lung tissues, experimental infection in pigs with the novel isolate, and cloning the virus by plaque assay confirmed this unique H and N combination. The novel isolate was also antigenically and genetically characterized. Genetic and phylogenetic analysis showed that the complete HA gene of the H3N1 strain has the highest nucleotide identity to three Italian H3N2 strains, one isolated in 2001 and two in 2004, whereas the full length NA sequence is closely related to three H1N1 subtype viruses isolated in Italy in 2004. The remaining genes are also closely related to respective genes found in H1N1 and H3N2 SIVs currently circulating in Italy. This suggests that the novel SIV could be a reassortant between the H3N2 and H1N1 SIVs circulating in Italy.  相似文献   

18.
OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.  相似文献   

19.
From May to September 2013, monthly samples were collected from swine in a Vietnamese slaughterhouse for influenza virus isolation and serological testing. A(H1N1)pdm09 viruses and a novel H3N2 originating from reassortment between A(H1N1)pdm09 and novel viruses of the North American triple reassortant lineage were isolated. Serological results showed low seroprevalence for the novel H3N2 virus and higher seroprevalence for A(H1N1)pdm09 viruses. In addition, serology suggested that other swine influenza viruses are also circulating in Vietnamese swine.  相似文献   

20.
《Veterinary microbiology》2015,175(2-4):224-231
During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.  相似文献   

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