The interferon sensitivity of selected porcine viruses.

The objective of this study was to compare the sensitivity of 11 porcine viruses to the antiviral effects of porcine interferon-alpha in serum from piglets which had been infected 19 h previously with transmissible gastroenteritis virus, and of porcine interferon-beta prepared in PK-15 cells by induction with polyinosinic:polycytidylic acid, in yield reduction assays in pig kidney cells which were treated with interferon before virus challenge, and both before and after virus challenge. The most sensitive virus to both types of interferon was vesicular stomatitis. A porcine isolate of bovine herpesvirus type 1, hemagglutinating encephalomyelitis virus and porcine enterovirus types 1 and 2 were also highly sensitive to interferon-alpha. There was little reduction in the yield of porcine parvovirus or porcine rotavirus, while swinepox, swine influenza and transmissible gastroenteritis viruses were intermediate in their sensitivity to interferon-alpha. In addition to vesicular stomatitis virus, porcine adenovirus type 3, swine influenza, hemagglutinating encephalomyelitis and porcine rotavirus were highly sensitive to interferon-beta, while swinepox, bovine herpesvirus type 1, porcine parvovirus, transmissible gastroenteritis and porcine enteroviruses were less sensitive than the above viruses to interferon-beta, although all showed significant reductions in virus yield.     

Transmissible gastroenteritis virus: plaques and a plaque neutralization test.

A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.     

Laboratory evaluation of selected disinfectants as virucidal agents against porcine parvovirus, pseudorabies virus, and transmissible gastroenteritis virus

Of a variety of disinfectants evaluated, only sodium hypochlorite and sodium hydroxide inactivated porcine parvovirus (PPV) after a 5-minute incubation period. After the same incubation time, pseudorabies and transmissible gastroenteritis viruses were inactivated by all of the disinfectants tested. When the incubation time was increased to 20 minutes, 2% glutaraldehyde and a double-strength concentration of a commercial formaldehyde preparation also inactivated PPV. Formaldehyde vapor and ultraviolet radiations inactivated PPV also, but relatively long exposure times were required.     

PEDV、TGEV、PoRV和PKV多重RT-PCR检测方法的建立及初步应用

为快速鉴别诊断猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)和猪嵴病毒(PKV),根据PEDV的M基因、TGEV的N基因、PoRV的VP6基因和PKV的3D基因序列设计4对特异性引物,通过PCR扩增目的片段并构建重组质粒,建立了一种可同时检测4种病毒的RT-PCR诊断方法,该方法可特异性扩增这4种病毒的相应片段,而对猪瘟病毒(CSFV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)均无扩增,最低检出量分别为1.33×10^4、1.33×10^3、1.33×10^4、1.33×10^5copies/μL。应用该方法对临床55份猪腹泻样品进行检测,结果检测出14份PEDV、1份PoRV和27份PKV,未检出TGEV,其中PEDV和PKV混合感染9份。上述结果表明,建立的多重RT-PCR检测方法快速、特异、敏感,可用于以上4种腹泻病毒的临床检测和流行病学调查。     

PEDV、TGEV和PRoV多重RT-PCR检测方法的建立及应用

为建立猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）及猪轮状病毒（PRoV）的快速鉴别检测方法,本试验针对PEDV、TGEV、PRoV的基因组序列设计3对特异性引物PEDV-N、TGEV-M和PRoV-VP6,分别扩增PEDV N基因、TGEV M基因和PRoV VP6基因。经优化反应条件,成功建立了能同时检测并区分PEDV、TGEV、PRoV的多重RT-PCR方法。该方法可特异扩增PEDV、TGEV、PRoV相应的基因片段,而与猪瘟病毒（CSFV）、猪口蹄疫病毒（FMDV）、猪伪狂犬病病毒（PRV）、猪细小病毒（PPV）、猪圆环病毒2型（PCV2）均无交叉反应;对PEDV、TGEV、PRoV基因重组质粒标准品的检出限分别为1.41×10³、1.41×10²和1.41×10³拷贝/μL;在相同条件下重复试验可获得一致的结果。应用该方法对临床采集的190份腹泻病料进行检测,结果PEDV阳性42份,阳性率22.11%;TGEV阳性58份,阳性率30.53%;PRoV阳性34份,阳性率17.89%,且存在不同病毒混合感染的现象。结果表明,所建立的多重RT-PCR方法具有特异性强、敏感性高、重复性好的优点,可用于PEDV、TGEV和PRoV的临床检测和流行病学调查。     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

Viral susceptibility of a cell line derived from the pig oviduct.

Seventeen of 24 RNA viruses and eight of nine DNA viruses replicated in a cell line derived from a pig fallopian tube. The following RNA viruses grew poorly in it: the virus of transmissible gastroenteritis of pig and the swine-influenza, Sendai and bovine para-influenza type 3 viruses. Among other RNA viruses an untyped swine para-myxovirus and some picornaviruses, rhabdoviruses and togaviruses attained high titers and produced an extensive cytopathic effect. Among the DNA viruses a porcine adeno, equine rhinopneumonitis, infectious bovine rhinotraceheitis, pseudorabies and porcine cytomegalo viruses replicated in pig fallopian tube cells as well as in other cells generally used to grow them.     

猪流行性腹泻病毒、传染性胃肠炎病毒、A群轮状病毒和嵴病毒多重RTPCR检测方法的建立及临床应用

本研究建立了可同时检测猪流行性腹泻病毒（Porcineepidemicdiarrheavirus,PEDV）、传染性胃肠炎病毒（TransmissiblegastroenteritisVirus,TGEV）、A群轮状病毒（GroupArotavirus,GARV）和猪嵴病毒（Porcinekobu—virus）的多重RT—PCR方法。检测中,建立的多重RT—PCR方法能够检测到500Pg的TGEV、PEDV、GARV和猪嵴病毒等量混合RNA模板,与常规的单一RT—PCR检测结果基本相同（检测TGEV、PEDV、GARV和猪嵴病毒的灵敏性分别为1000A、1000／6、93．33％和96．67％,特异性均为i00％）。结果表明,建立的多重RTPCR方法敏感性和特异性良好,可作为临床上猪病毒性腹泻病因快速、高效的诊断工具。应用该方法对2010—2012年华中地区190份腹泻仔猪样本进行检测,PEDV、TGEV、GARV和猪嵴病毒的阳性率分别为62．11％、0．53％、7．37％和82．11％。混合感染方面,PEDV和猪嵴病毒混合感染率为47．89％,PEDV和GARV混合感染率为4．74％,GARV和猪嵴病毒混合感染率为7．37％,PEDV、GARV和猪嵴病毒混合感染率为4．74％,未发现TGEV与其它3种病毒的混合感染情况。另外,有27份样本中仅检出PEDV（14．21％）,57份样本只检出猪嵴病毒（30％）。分析表明,我国自2010年底大面积暴发的病毒性腹泻是多病原混合感染造成的,主要病原为PEDV,猪嵴病毒在其中所起作用尚待进一步验证和研究。     

A seroepizootiologic study of five viruses in a swine-evaluation station.

This serologic study was done to gain information on the spread, maintenance, and effect upon performance of five porcine viruses. Blood samples were taken from two groups of 8- to 11-week-old pigs from a large number of Indiana swine herds in a performance-testing station 1 week after entry, 7 weeks after entry (one group only), and at slaughter. The sera were tested by indirect fluorescent antibody tests for antibodies to transmissible gastroenteritis virus (TGEV), swine influenza virus (SIV), hemagglutinating encephalomyelitis virus (HEV), porcine adenovirus (PAV), and pseudorabies virus (PRV). Seroconversions to TGEV, HEV, and PAV occurred in a group of pigs entered in May and slaughtered in August (group 1). In the group that was entered in October and slaughtered in January (group 2), pigs developed antibodies to SIV, HEV, and PAV, but not to TGEV. Only 1 of the 434 pigs tested had antibodies to PRV, and there were no seroconversions to this virus. The only statistically valid effect of infection on performance was found in group 1 pigs, which had seroconverted to TGEV during the first 7 weeks of their stay. These pigs gained 0.077 kg less per day than pigs that did not develop antibodies to TGEV during that period. The pattern of serologic reactions was indicative of a relatively slow spread of these viruses in the groups. We interpret this as supporting the concept that a relatively slow spread of these viruses through large groups of pigs kept under conditions that are less than optimum for virus spread may be an important means of their interepizootic survival.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

2018年广西重要猪源病毒性疫病流行病学调查

为了解2018年广西猪群重要疫病流行情况,试验采集广西各地的病死猪组织样品及病猪腹泻拭子,应用多重实时荧光定量RT-PCR检测猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）,应用多重实时荧光定量PCR检测猪伪狂犬病病毒（PRV）、猪圆环病毒1型（PCV1）、猪圆环病毒2型（PCV2）及猪圆环病毒3型（PCV3）,应用多重RT-PCR检测猪流行性腹泻病毒（PEDV）、猪德尔塔冠状病毒（PDCoV）、猪传染性胃肠炎病毒（TGEV）和猪轮状病毒（PRoV）。结果显示,所检测的694份组织样品中,CSFV、PRRSV、HP-PRRSV、PRV、PCV1、PCV2、PCV3的阳性率分别为11.10%、18.88%、7.20%、5.19%、2.45%、67.00%和5.76%;2种病原混合感染率为41.21%,3种病原混合感染率为4.32%,其中PRRSV和PCV2混合感染率最高。所检测的792份肠内容物及拭子腹泻样品中,PEDV、PDCoV、TGEV、PRoV的阳性率分别为9.72%、5.81%、1.77%和6.31%;2种病原混合感染率为5.30%,其中PEDV和PRoV混合感染率最高。结果表明,当前多种重要病毒性疫病仍在广西猪群发生和流行,并且多重感染普遍存在,应进一步加强监测和防控。     

山东种公猪精液携带主要病原的检测

采用PCR技术,对2007年-2009年山东部分地区的38个猪场和人工授精站的生产公猪精液样品727份进行了猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)和猪细小病毒(PPV)5种主要病原的检测。结果检出CSFV、PRRSV、PRV、PCV-2和PPV的阳性数和阳性率分别为18份(2.48%)、27份(3.71%)、7份(0.96%)、33份(4.54%)、9份(1.24%),有7份样品为2种以上病原混合感染,其中以PRRSV+PCV-2混合感染最多。结果表明精液传播病毒仍是当前母猪繁殖障碍的重要原因之一,应重点加强对种公猪的疫病净化和公猪精液管理,从源头控制传染源。     

Transmissible gastroenteritis: demonstration of the virus from field specimens by means of cell culture and pig inoculation.

Isolation of transmissible gastroenteritis virus was attempted from segments of jejunum collected from piglets submitted for diagnosis of transmissible gastroenteritis. The virus was isolated more frequently in susceptible piglets than in pig kidney or pig thyroid cells. Practically, both cell systems were equally capable of demonstrating the virus when the tissue suspensions were sonicated. The pig thyroid cells prepared with glands collected from minimal disease pigs were preferred to the pig kidney cells for initial virus isolation because of their ability to respond to transmissible gastroenteritis virus with a progressive cytopathic effect. However, the pig thyroid cells, prepared from pool of glands collected in abattoirs, were often contaminated with parvoviruses and could not be used for diagnostic work. Controlled ultrasound treatments of the inoculum increased the frequency of virus isolation in both cell systems.     

Immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine.

Immune electron microscopy (IEM) was developed as a diagnostic aid for detecting and identifying transmissible gastroenteritis virus and rotavirus (reovirus-like agent) in fecal and intestinal contents from cases of gastroenteritis in young pigs. Variables involved in use of direct IEM and its sensitivity were determined. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous convalescent antiserum, but not in control samples containing preexposure serum or antibody directed against a heterologous virus. At least a ten fold enhancement of the sensitivity of direct IEM for virus detection was accomplished using indirect IEM employing rabbit anti-porcine IgG to further aggregate virus-antibody complexes. The technique was used to investigate the size and morphology of the porcine rotavirus. Particles ranged from 55 to 70 nm in diameter and had capsomere structures. Morphologically, the porcine rotavirus resembled the calf and human rotaviruses. By IEM, employing specific antiserums for each virus, porcine rotavirus was found to be antigenically related to these 2 viruses, but not to the reovirus type 3.     

广西德保县黑猪主要疫病病原学和血清学检测

为了解德保黑猪主要疫病流行状况和免疫效果,2018—2020年釆集166个德保黑猪饲养场点的300份病死猪样品进行非洲猪瘟(ASF)、猪瘟(CSF)、口蹄疫(FMD)、猪繁殖与呼吸综合征(PRRS)、猪伪狂犬病(PR)、猪圆环病毒病(PCV-2)、猪支原体病(PPLO)、猪传染性胸膜肺炎(PCP)、猪链球菌病(SS)、...     

Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.

A virologic survey was conducted to determine the frequency of transmissible gastroenteritis (TGE) virus infection in farm-raised sows. Pharyngeal swab specimens collected in an abattoir were examined for TGE virus by inoculation onto swine-testes cell culures. The virus was detected in 61 (3%) of a sample of 2,058 Iowa sows after slaughter. All TGE viral isolates, given orally to 2- or 3-day-old pigs, caused acute gastroenteritis and in some cases death. All pigs that recovered from illness had serum antibody to TGE virus.     

The prevalence of antibodies to influenza virus and respiratory coronavirus in fattening pigs in Spain

The presence of antibodies to two influenza viruses of the type A (H1N1 and H3N2) and to a porcine respiratory coronavirus was investigated in a study lasting a year. 735 blood serum samples were collected from 79 closed pig fattening farms in the province Segovia (Spain). Hemagglutination inhibition was used with influenza viruses. The percentage of positive results was 78.5% and 62.5% respectively for the serotypes H1N1 and H3N2. A clear reduction in the spread of antibodies was observed in the autumn. The ELISA technique was used with the porcine respiratory coronavirus. As antigen we used the antigenically related transmissible porcine gastroenteritis virus. Using this technique 87% of the sera were positive. Some of these sera with representative ELISA values were confirmed by means of serum neutralisation and radioimmune precipitation of the viral proteins. The incidence of these antibodies remained unchanged over the whole year of the investigation.     

猪6种常见病毒多重PCR检测方法的建立及应用

旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)和猪巨细胞病毒(PCMV)的多重PCR检测方法。参考相关文献及序列比对结果设计6对特异性引物,建立了可同时检测以上6种病毒的多重PCR方法并应用此方法对临床病例进行了检测。结果表明,所建立的多重PCR方法灵敏度高,对6种病毒的最低核酸检测量分别为12.8pg(PRRSV)、46pg(CSFV)、16pg(PRV)、23.5pg(PCV-2)、72pg(PPV)、8.6pg(PCMV),对猪流感病毒(SIV)、猪乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)无特异性扩增。应用该方法对临床145份样品进行检测,总阳性率为83.45%,2种以上病毒混合感染阳性率为69.66%。说明所建立的多重PCR检测方法敏感,可用于猪群中上述6种猪病病原的单一感染或混合感染的鉴别诊断。     

Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome.

A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.