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1.
根据WHO推荐的检测A型猪流感病毒的引物和探针序列,建立了A型猪流感病毒实时荧光定量PCR检测方法。使用含有选定检测序列的重组质粒标准品绘制标准曲线,检测结果表明,该方法的敏感性可达100拷贝/25μL反应体系,显示出良好的敏感性和重复性,临床上已用于实验室鼻拭子样品的检测,并成为一种快速定量检测A型猪流感病毒的方法。  相似文献   

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4.
Since the first detection of human H3N2 influenza virus in Taiwanese pigs in 1970, infection of pigs with wholly human viruses has been known to occur in other parts of the world. These viruses, referred to as human‐like H3N2 viruses, have been known to cause clinical and subclinical infections of swine populations. Due to the paucity and complete unavailability of information on transmission of influenza viruses from other species, especially humans, to swine in Nigeria and Ghana, respectively, this study was designed to investigate the presence and prevalence of a human strain of influenza A (H3N2) in swine populations at three locations in two cities within these two West African countries in January and February, 2014. Using stratified random technique, nasal swab specimens were collected from seventy‐five (75) pigs at two locations in Ibadan, Nigeria and from fifty (50) pigs in Kumasi, Ghana. These specimens were tested directly by a sensitive Quantitative Solid Phase Antigen‐detection Sandwich ELISA using anti‐A/Brisbane/10/2007 haemagglutinin monoclonal antibody. Influenza virus A/Brisbane/10/2007 (H3N2) was detected among pigs at the three study locations, with an aggregate prevalence of 4.0% for the two locations in Ibadan, Nigeria and also 4.0% for Kumasi, Ghana. Transmission of influenza viruses from other species to swine portends serious sinister prospects for genetic reassortment and evolvement of novel viruses. We therefore recommend that further studies should be carried out to investigate the presence of other circulating human and avian influenza viruses in swine populations in West Africa and also determine the extent of genetic reassortment of strains circulating among these pigs. This would provide an early warning system for detection of novel influenza viruses, which could have pandemic potentials.  相似文献   

5.
Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.  相似文献   

6.
Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.  相似文献   

7.
A midwestern producer reported high incidence of conjunctivitis and keratoconjunctivitis in a herd of crossbred finishing swine. Complete necropsy was performed on 3 pigs with bilateral mucopurulent conjunctivitis and chemosis; other gross lesions were not seen. Mycoplasma sp was isolated from conjunctival swab specimens obtained from 1 pig; small numbers of streptococci and coagulase-negative staphylococci were isolated from conjunctival swab specimens from all 3 pigs. Neither swine influenza virus nor pseudorabies virus was isolated from conjunctival swab specimens. Histologically, the 3 pigs had chronic lymphoplasmacytic conjunctivitis with lymphofollicular hyperplasia and foci of epithelial and goblet cell hyperplasia. Ultrastructural examination of conjunctival specimens from the 3 pigs revealed large numbers of Mycoplasma-like organisms adhered to superficial conjunctival cells. Mycoplasma-like organisms also were seen in membrane-bound vacuoles in superficial conjunctival cells. Bacteria (including chlamydiae) or viruses were not seen ultrastructurally. The lymphoproliferative nature of the conjunctival lesion and the evidence of adhered and intracellular organisms suggested an etiologic role for a Mycoplasma-like organism in the disease in these pigs.  相似文献   

8.
The susceptibilities of culture cells to twelve avian influenza virus strains were determined with ten established cell lines including MDCK and ESK cells and three primary culture cells. The established cell lines derived from embryonic swine kidney (ESK) and chicken kidney (CK) primary culture cells were more sensitive to the avian influenza viruses than the other eleven cells. The ESK cell had a particularly higher infective titer than the MDCK cell with and without trypsin supplement in culture medium, and dispersion of the infective titers was narrower than that of the MDCK cell. The ESK cell is a suitable candidate for routine work on avian influenza viruses in laboratories.  相似文献   

9.
Pharyngeal swab samples were collected at a central Iowa abattoir from 6,010 market-weight slaughter hogs from September 1, 1979 to August 31, 1980. The swab samples were examined for cytopathic viruses by inoculation of monolayer cultures of continuous line of swine testicular cells. Of the 6,010 swab samples tested, transmissible gastroenteritis virus was isolated from 91 (1,51%), pseudorabies virus was isolated from 431 (7.17%), and porcine enterovirus was isolated from 21 (0.35%). Although all 3 viruses were identified throughout the year, transmissible gastroenteritis and pseudorabies viruses were found more frequently during the winter and early spring. In contrast, porcine enterovirus was detected more frequently during the spring and summer.  相似文献   

10.
To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.  相似文献   

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12.
猪流感是猪常见的呼吸道传染病,临床以高热、呼吸困难、咳嗽和衰竭、迅速康复或死亡为特征。猪流感不仅给养猪业造成巨大损失,也严重威胁着人类健康。本研究从发病猪场中分离到1株H1N1亚型猪流感病毒,序列分析结果显示,分离毒株属于欧洲类禽猪流感H1N1亚型病毒。将分离毒株分别接种到MDCK与ST细胞,观察病毒的生长特性,结果显示分离的猪流感病毒在ST细胞中复制能力较强。采用RT-PCR技术分别扩增8个基因片段,克隆到流感病毒反向遗传系统,成功拯救出猪流感病毒毒株,测序结果显示拯救的猪流感病毒与亲本毒序列一致。本研究成功分离的猪流感病毒,以及建立的反向遗传技术为研究欧洲类禽猪流感病毒跨种传播的机制以及研发新型猪流感疫苗株奠定了基础。  相似文献   

13.
用荧光定量RT-PCR方法检测猪瘟病毒   总被引：4，自引：1，他引：4  
为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。  相似文献   

14.
Like other influenza A viruses, equine influenza virus undergoes antigenic drift. It is therefore essential that surveillance is carried out to ensure that recommended strains for inclusion in vaccines are kept up to date. Here we report antigenic and genetic characterisation carried out on equine influenza virus strains isolated in North America and Europe over a 2-year period from 2008 to 2009. Nasopharyngeal swabs were taken from equines showing acute clinical signs and submitted to diagnostic laboratories for testing and virus isolation in eggs. The sequence of the HA1 portion of the viral haemagglutinin was determined for each strain. Where possible, sequence was determined directly from swab material as well as from virus isolated in eggs. In Europe, 20 viruses were isolated from 15 sporadic outbreaks and 5 viruses were isolated from North America. All of the European and North American viruses were characterised as members of the Florida sublineage, with similarity to A/eq/Lincolnshire/1/07 (clade 1) or A/eq/Richmond/1/07 (clade 2). Antigenic characterisation by haemagglutination inhibition assay indicated that the two clades could be readily distinguished and there were also at least seven amino acid differences between them. The selection of vaccine strains for 2010 by the expert surveillance panel have taken these differences into account and it is now recommended that representatives of both Florida clade 1 and clade 2 are included in vaccines.  相似文献   

15.
Ip HS  Dusek RJ  Heisey DM 《Avian diseases》2012,56(1):114-119
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16.
猪流感病毒RT-PCR快速检测方法的建立   总被引：3，自引：2，他引：3  
根据猪流感病毒M基因序列设计了一对扩增M基因684 bp片段的特异性引物,建立了RT-PCR快速诊断猪流感的方法,采用该方法检测出H1、H3、H5和H9 4个亚型猪流感病毒标准参考株为阳性,猪副黏病毒、圆环病毒2型和猪繁殖与呼吸综合征病毒检测结果呈阴性.RT-PCR最少可检测到1 000EID50的病毒量核酸,可直接从猪流感病毒感染小鼠的组织样品中检测到病毒.  相似文献   

17.
Several cell lines including Vero and MDCK cells were tested for susceptibility to the Miami and Prague strains of equine influenza virus to find cell cultures suited for study of these viruses. The viruses readily multiplied with cytopathic effects in these cell cultures. Of the cells tested, ESK cells derived from swine embryo kidney were the most susceptible to the viruses. Based on these results an infectivity assay of the viruses was worked out using ESK cell cultures prepared in microplates. The method is not only simple enough for routine use, but is also practically as sensitive as the egg inoculation method. The method was further adapted to a neutralization test.  相似文献   

18.
In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.  相似文献   

19.
根据A型流感病毒基质蛋白（matrix protein,M）基因保守序列设计并合成特异性引物和TaqMan BHQ探针,建立快速检测A型流感病毒的实时荧光定量RT-PCR方法。通过RT-PCR方法克隆A型流感病毒M基因靶序列并将其连入pMD-18T载体,制备阳性标准品,优化反应条件,以10倍系列稀释的标准品绘制标准曲线,其相关系数为0.998。检测结果显示,该方法的灵敏度可达10 copies/μL或1 EID50病毒且特异性良好,除A型流感病毒外,对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪圆环病毒2型、猪输血传播病毒检测结果均为阴性。该方法重复性好,批内和批间变异系数均小于3%。对40份实验感染样品的检测结果表明,该方法与病毒分离结果一致,比常规RT-PCR检测方法灵敏度更高,特异性更强。  相似文献   

20.
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   

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