Comparison of a commercial enzyme-linked immunosorbent assay with hemagglutination inhibition assay for serodiagnosis of swine influenza virus (H1N1) infection.

A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.     

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.     

Expression of myeloperoxidase in swine influenza virus (SIV)-infected neutrophils in lungs from pigs experimentally infected with SIV subtype H1N2

The expression of myeloperoxidase (MPO) was examined in the swine influenza virus (SIV)-infected neutrophils in the lungs of pigs experimentally infected with swine influenza virus (SIV) subtype H1N2 by immunohistochemistry. Five pigs each from the infected and non-infected group were euthanized 1, 3, 5, 7, and 10?days post-inoculation (dpi). Immunohistochemical reactivity was mainly seen in neutrophils. The score for pulmonary histopathological lesions correlated with the score for MPO immunohistochemical reactivity (r ( s )?=?0.962, P?相似文献   

H9N2亚型猪流感病毒血凝素基因的表达及LAT诊断方法的研究

根据H9N2亚型猪流感病毒血凝素基因序列设计并合成引物,从本室分离保存的H9N2亚型猪流感病毒中扩增出血凝素基因,并将其克隆进pMD18-T载体,限制性酶切及序列测定后,进一步将其信号肽缺失并亚克隆到pGEX-KG中,使其在E.coli BL21（DE3）中与GST融合表达。SDS-PAGE显示表达的融合蛋白的相对分子质量约为90 000。Western印迹表明表达的蛋白质具有免疫学活性。表达产物位于包涵体中,包涵体经变性、复性处理,利用复性产物作为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,建立了H9亚型猪流感病毒抗体的乳胶凝集试验的检测方法,结果表明,应用HA重组蛋白作为诊断抗原具有特异性强、敏感性高、重复性好的特点,可用于H9亚型猪流感抗体水平监测、流行病学调查和现地疫病普查。     

An immunoperoxidase monolayer assay for the detection of antibodies against swine influenza virus.

An immunoperoxidase monolayer assay (IPMA) has been developed to detect antibodies against swine influenza A virus (SIV) in pig sera. The test was evaluated by using sequential sera from pigs experimentally infected with H1N1 subtype of SIV. Two hundred field serum samples that had been examined by the hemagglutination inhibition (HI) test were also tested. Antibodies specific to SIV were detected as early as 3 days postinoculation (dpi) in the IPMA test as compared with 7 dpi by the HI test. Unlike HI, no serum treatment was required in the IPMA test. Regardless of the virus used in the test, IPMA detected antibodies to both H1N1 and H3N2 subtypes of SIV whereas HI detects antibodies against either H1N1 or H3N2, depending upon the virus used in the test. Results of this study indicate that IPMA is a useful test for screening of pig sera for SIV antibodies.     

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a na?ve swine population.     

具有HI活性的H1亚型流感病毒特异性单克隆抗体的制备与应用

为了制备具有HI活性的HI亚型流感病毒特异性单克隆抗体(MAb),本研究以H1N1亚型猪流感病毒(SIV)株A/Swine/Guangdong/718/01(H1N1)为免疫原,免疫BALB/c小鼠,经常规细胞融合后,血凝抑制(HI)方法进行检测,融合细胞经稀释克隆纯化后,获得11株能稳定分泌抗血凝素特异性HI MAb的杂交瘤细胞株。鉴定表明,所获MAb与其他具有血凝活性的病毒以及其他14个HA亚型的流感病毒均不具有HI交叉反应,表明这11株MAb均具有良好的流感病毒亚型特异性。其中A6F、2BBF和2BB与其他H1亚型流感病毒分离株的HI试验证实我国不同地区分离株之间的抗原性存在一定差异。11株MAb对H1SIV抗原的HI试验结果显示其HI效价有明显差异。叠加实验表明这些MAb分别识别HA抗原的不同表位。间接免疫荧光试验表明,2BBF、8HB、1DH、7FC和2BB均可与2009年流行H1N1病毒A/California/04/2009HA抗原发生特异性反应。这些MAb特异性的研制为H1亚型流感病毒的疫情病原学快速诊断以及病毒抗原性变异的相关研究提供了物质基础。     

上海地区猪群中H1N1甲型流感抗体检测

对2009年H1N1甲型流感流行前后的上海地区养殖场户410份猪血清样品,分别采用血凝抑制试验（hemagglutination inhibition,HI）和酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）进行检测H1N1甲型流感病毒和猪流感病毒（Swine in？uenza virus,SIV）。检测结果表明,除2007年外,2008~2010年猪血清中均存在不同水平的HI抗体,阳性率呈显著上升趋势,且抗体水平与猪群饲养周期及饲养密度正相关,而与猪流感病毒的流行无相关性。     

Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain

This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain.     

猪流感病毒HA基因的原核表达及其ELISA检测方法的建立

根据H1N1亚型猪流感病毒血凝素基因序列设计并合成特异性引物,从本室保存的H1N1亚型猪流感病毒中扩增信号肽和跨膜区缺失的血凝素基因,并将其克隆到原核表达载体pET-28a中,在大肠埃希菌中诱导表达.对表达蛋白进行SDS-PAGE和Western blot的结果表明,HA基因在大肠埃希菌中获得表达,产物具有免疫学活性,表达蛋白分子质量约为55 ku.表达产物经过纯化作为包被抗原建立了检测H1亚型猪流感抗体的间接ELISA方法.该方法具有较高的特异性和敏感性,重复性良好.应用该方法对2006年2 584份临床猪血清进行检测,结果显示多个地区检测猪流感抗体出现阳性,平均为20.5%.在6月、7月和12月分别出现抗体水平高峰,分别高达25.4%、25.0%和35.5%.     

Seroprevalence and risk factors of swine influenza in Spain

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI(95%): 89.1-98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI(95%): 60.2-64.3%) were seropositive. A total of 1622 animals (75.4%; CI(95%): 73.6-77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI(95%): 87.7-98.1%) had H1N1 seropositive animals; 63 (64.3%; CI(95%): 54.6-73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI(95%): 87.7-98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI(95%): 83.7-95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.     

猪流感病毒H1、H3、N1、N2亚型分型 RT-PCR方法的建立

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。     

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine

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.     

Comparison of three serological assays to determine the cross-reactivity of antibodies from eight genetically diverse U.S. Swine influenza viruses.

Swine influenza virus is an economically important pathogen to the U.S. swine industry. New influenza subtypes and isolates within subtypes with different genetic and antigenic makeup have recently emerged in U.S. swineherds. As a result of the emergence of these new viruses, diagnosticians' ability to accurately diagnose influenza infection in pigs and develop appropriate vaccine strategies has become increasingly difficult. The current study compares the ability of subtype-specific commercial enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HI), and serum neutralization (SN) assays to detect antibodies elicited by multiple isolates within different subtypes of influenza virus. Pigs were infected with genetically and antigenically different isolates of the 3 major circulating subtypes within populations of swine (H1N1, H1N2, and H3N2). Serum was collected when all pigs within a group collectively reached HI reciprocal titers >or=160 against that group's homologous challenge virus. The antibody cross-reactivity of the sera between isolates was determined using ELISA, HI, and SN assays. In addition, the correlation between the 3 assays was determined. The assays differed in their ability to detect antibodies produced by the viruses used in the study. The results provide important information to diagnostic laboratories, veterinarians, and swine producers on the ability of 3 common serological assays used in identifying infection with influenza in pigs.     

Prime-boost immunization with HA/C3d DNA followed by a recombinant pseudorabies virus boost enhanced protective immunity against H3N2 swine influenza virus in mice

DNA and recombinant virus vaccines against swine influenza virus (SIV) have been pursued with promising results, but induce poor immunogenicity. This study evaluated the effects of a vaccine regimen in mice including priming with three DNA vaccines expressing soluble HA (sHA), complete HA (tmHA), or sHA fused with three copies murine C3d (sHA-mC3d3) and boosting with recombinant pseudorabies virus expressing HA (rPRV-HA). Immune responses were monitored by ELISA, HI assays, and virus neutralization. Protective efficacy was evaluated by virus isolation from lungs, distribution in tissues, and pathology following challenge with H3N2 SIV. Priming with sHA-mC3d3 and boosting with rPRV-HA induced higher levels of HA-specific antibodies and yielded the most effective protection. This finding implied that priming with a DNA vaccine expressing C3d fused with antigen and boosting with a recombinant vector vaccine is an effective way to induce protective humoral immunity and prevent some infectious diseases.     

华南地区猪群猪流感病毒病原学和血清学调查

为了解华南地区猪群中猪流感病毒(SIV)的流行及其遗传变异情况,本研究从2016年~2017年广东、广西等地猪群236份猪肺脏病料组织和143份鼻拭子样品中分离鉴定得到3株SIV,全基因组测序和遗传演化分析结果显示,3个分离株均属于H1N1亚型欧亚类禽分支SIV,并且均与pdm09分支病毒株发生了重组。HA蛋白分子特征分析结果显示,A/Swine/Guangxi/NK/2016 HA蛋白第23位糖基化位点发生了缺失。3265份血清样品抗体监测结果显示,欧亚类禽H1N1、pdm09 H1N1和H3N2 SIV的血清抗体阳性率分别为27.53%、20.98%和34.85%。另外,0.64%的(21份)血清样品为H9N2亚型流感病毒抗体阳性,并且猪群中不同亚型和不同分支SIV之间混合感染的情况非常普遍。猪群中流感病毒血清抗体监测结果显示,EA H1N1、pdm09和H3N2亚型SIV HI抗体滴度最高均可达到1:1280,而H9N2亚型HI抗体滴度最高为1:160,表明H9N2 AIV虽然可以感染猪,但对猪还不适应。各月份的血清抗体阳性率分析显示,SIV的流行具有季节性,冬季(11月、12月和1月份)的流行最为严重。本研究可为华南地区猪群SI防控及疫苗株的筛选提供参考依据。     

Immune responses and protective efficacy of a recombinant swinepox virus co-expressing HA1 genes of H3N2 and H1N1 swine influenza virus in mice and pigs

The recombinant swine poxvirus rSPV/H3-2A-H1 co-expressing HA1 genes of H3N2 and H1N1 subtype SIV has been constructed and identified. Inoculations of rSPV/H3-2A-H1 yielded ELISA and neutralization antibodies against SIV H1N1 and H3N2, and elicited potent H1N1 and H3N2 SIV-specific INF-γ response from T-lymphocytes in mice and pigs in this study. Complete protection against SIV H1N1 or H3N2 challenge in pigs was observed.     

Fusion of C3d with hemagglutinin enhances protective immunity against swine influenza virus

H1N1 and H3N2 are the dominant subtypes causing swine influenza in China and other countries. It is important to develop effective vaccines against both H1N1 and H3N2 subtypes of swine influenza virus (SIV). We examined the effects of a DNA vaccine expressing an influenza HA fused to three copies of murine complement C3d in mice. Plasmids encoding soluble HA (sHA), complete HA (tmHA), or a soluble fused form of HA (sHA-mC3d3) were constructed from the H3N2 subtype of SIV. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assays, and virus neutralization tests. Analysis of antibody titers indicated that immunization with HA-mC3d3 resulted in higher titers of anti-HA antibodies and higher antibody affinities, compared with serum from mice immunized with sHA or tmHA. Furthermore, the C3d fusion increased the Th2-biased immune response, by inducing IL-4 production. Splenocytes from mice immunized with sHA-mC3d3 produced about three-fold more IL-4 than did splenocytes from mice immunized with sHA or tmHA. Seven days post-challenge with homologous virus (H3N2), no virus was isolated from the mice immunized with HA-expressing plasmids. However, 10 days post-challenge with heterologous virus (H1N1), only mice immunized with sHA-mC3d3 had no virus or microscopic lesions in the kidneys and cerebrum. In conclusion, C3d enhanced antibody responses to hemagglutinin and protective immunity against SIV of different subtypes.     

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.     

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against borth rVP2 and rNS1 could be detected for this period of time.