Polymerase chain reaction (PCR) for the detection of herpesvirus in tortoises

A polymerase chain reaction (PCR)-based method using a herpesvirus consensus primer was assessed for the identification of herpesviral infections in tortoises. A single band of about 230 bp was detected in PCR products from two out of twenty swabs taken from the oral cavity, three out of three paraffin-embedded tissue sections from the liver (two cases) and oral mucosa (one case), and one out of two fresh tissue samples from the oral mucosa. Nucleotide sequencing of these PCR products indicated that the herpesvirus present in these tortoises might belong to the alphaherpesvirinae. PCR using swabs and biopsy tissues was a sensitive and highly specific method for the diagnosis of herpesviral infections in tortoises.     

Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes

Herpesviruses are an important cause of epidemic disease in tortoises. There are at least two serologically distinct herpesviruses capable of infecting tortoises. Methods for the diagnosis of herpesvirus infections in tortoises include virus isolation and a number of different PCRs. We have compared 11 virus isolates collected from various species in different countries over several years using sequences from three different viral genes. During this study we used four different PCR protocols described for the diagnosis of herpesvirus infections in tortoises. The protocols used included two based on portions of the DNA polymerase gene, one targeting the UL5 homologue, and one targeting the UL39 homologue. Comparison of the methods showed that the tortoise herpesvirus-specific protocols were all serotype specific. Sequences of the obtained amplicons were compared with one another and with sequences of herpesviruses available in GenBank. The sequence alignments showed that the tortoise herpesviruses were most closely related to members of the subfamily Alphaherpesvirinae. They also showed that the tortoise isolates could be clearly divided into two genogroups.     

Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii)

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.     

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.     

沙门菌和志贺菌二重PCR检测方法的建立及应用

根据GenBank提供的沙门菌invA基因序列和志贺菌ipaH基因序列设计引物,建立了二重PCR方法,在同一反应体系同时检测两种致病菌核酸,经二重PCR方法扩增后,在同一泳道同时检出沙门菌284 bp和志贺菌611 bp特异性扩增条带,而普通大肠埃希菌、金黄色葡萄球菌、变形杆菌、阪畸大肠埃希菌、绿脓杆菌、蜡样芽胞杆菌、马链球菌、产单核细胞李斯特菌、鹅大肠埃希菌、猪水肿病大肠埃希菌均未出现这两种条带.对PCR产物进行测序,测序结果与已发表的基因核苷酸序列比较,沙门菌同源性为93%～100%,志贺菌同源性为98%～100%.应用建立的沙门菌和志贺菌二重PCR方法对广西6个试验猴养殖场1 665份猴粪便样品进行检测,检出沙门菌阳性25份,志贺菌阳性90份,阳性检出率分别为1.5%和5.4%.表明建立的沙门菌和志贺菌二重PCR检测方法特异性强、敏感性高,适用于临床粪便样品的快速检测.     

Genetic characterization of a herpesvirus isolate from a superb starling (Lamprotornis superbus) as a psittacid herpesvirus genotype 1

Four genotypes of the psittacid herpesvirus (PsHV) cause Pacheco disease in parrots. Viruses that are serologically cross-reactive to the PsHVs have also been isolated from passerine species. DNA was amplified from a herpesvirus isolated from a superb starling (Lamprotornis superbus) with PsHV-specific primers and polymerase chain reaction. A comparison of the partial sequence of the UL 16 gene from this herpesvirus with sequences from viruses of known PsHV genotypes showed that the herpesvirus from the superb starling was a PsHV genotype 1 virus. This finding expands the range of birds that are known to be susceptible to PsHV genotype 1 infections and suggests that PsHVs should be considered as a differential in passerines with herpesvirus infections.     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

A novel herpesvirus in 3 species of pheasants: mountain peacock pheasant (Polyplectron inopinatum), Malayan peacock pheasant (Polyplectron malacense), and Congo peafowl (Afropavo congensis)

The mountain peacock pheasant (Polyplectron inopinatum), the Malayan peacock pheasant (Polyplectron malacense), and the Congo peafowl (Afropavo congensis) are all listed as vulnerable to extinction under the International Union for Conservation of Nature Red List of Threatened Species. Here the authors report fatal infection with a novel herpesvirus in all 3 species of birds. DNA was extracted from the livers of birds with hepatocellular necrosis and intranuclear eosinophilic inclusions consistent with herpesvirus infection. Based on degenerate herpesvirus primers and polymerase chain reaction, 220- and 519-base pair products of the herpes DNA polymerase and DNA terminase genes, respectively, were amplified. Sequence analysis revealed that all birds were likely infected with the same virus. At the nucleotide level, the pheasant herpesvirus had 92% identity with gallid herpesvirus 3 and 77.7% identity with gallid herpesvirus 2. At the amino acid level, the herpes virus had 93.8% identity with gallid herpesvirus 3 and 89.4% identity with gallid herpesvirus 2. These findings indicate that the closest relative to this novel herpesvirus is gallid herpesvirus 3, a nonpathogenic virus used widely in a vaccine against Marek's disease. In situ hybridization using probes specific to the peacock pheasant herpesvirus DNA polymerase revealed strong intranuclear staining in the necrotic liver lesions of an infected Malayan peacock pheasant but no staining in normal liver from an uninfected bird. The phasianid herpesvirus reported here is a novel member of the genus Mardivirus of the subfamily Alphaherpesvirinae and is distinct from other galliform herpesviruses.     

Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by real-time PCR

Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 (AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.     

Application of immunoperoxidase-based techniques to detect herpesvirus infection in tortoises.

Indirect (IIP) and direct (DIP) immunoperoxidase assays were developed for the serological and histological diagnoses of herpesvirus infection in tortoises, respectively. A mouse monoclonal antibody (MAb HL1546), specific for the heavy chain of tortoise IgY, was used as the secondary antibody in the IIP assay. Rabbit polyclonal antisera raised against 2 sucrose gradient-purified tortoise herpesvirus isolates (HV4295/7R/95 and HV1976) were used as primary antibodies for the detection of herpesvirus antigen either in infected cell cultures or in formalin-fixed, paraffin-embedded tissues. The IIP and DIP assays could detect either the presence of anti-herpesvirus antibody in the plasma of exposed tortoises or the presence of herpesvirus antigen in infected tissues, respectively. Although the IIP test complements the enzyme-linked immunosorbent assay and the serum neutralization test already available for measuring herpesvirus-specific antibody in tortoises, the DIP test is useful for the histological diagnosis of herpesvirus infection in tortoises.     

Detection of chelonid herpesvirus DNA by nonradioactive in situ hybridization in tissues from tortoises suffering from stomatitis-rhinitis complex in Europe and North America

Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatitis-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 gene from a German ChHV isolate was used to generate a digoxigenin-labeled 307-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was detected in paraffin-embedded tissues of five naturally infected tortoises (two Afghan tortoises [Testudo horsfieldii], USA; two Hermann's tortoises [Testudo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many characteristics of alphaherpesvirus but also some characteristics of betaherpesvirus infections. The amino acid sequence of a portion of the ChHV UL5 homolog exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nuclear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia cells and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Importantly, ChHV DNA was detected by ISH in multiple tissues of tortoises originating from different geographic provenances. This indicates a high degree of conservation of the UL5 gene fragment among viruses prevalent in tortoises on different continents. With the described ISH, a molecular biological tool is available for rapid and specific diagnosis of ChHV infections and, more importantly, comparative pathogenetic studies of ChHV isolates from geographically unrelated regions.     

Identification of the porcine cytomegalovirus major capsid protein gene.

A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified. Sequence analysis indicated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues. The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts. Phylogenetic analysis using herpesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse cytomegaloviruses. The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection. Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection. The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving virus-isolation are not useful.     

Ibex-associated malignant catarrhal fever in a bongo antelope (Tragelaphus euryceros).

A 4-yr-old male bongo antelope (Tragelaphus euryceros) died after an acute clinical course involving a febrile illness, anorexia, lethargy, minor oculonasal discharge, and diarrhea. Histologic lesions were compatible with malignant catarrhal fever (MCF). Polymerase chain reaction (PCR) revealed an amplified region of a herpesviral DNA polymerase gene sequence nearly identical to that of a MCF virus previously identified in Nubian ibex (Capra nubiana). The bongo had been housed across from an exhibit containing Nubian ibex that tested positive for MCF viral antibodies by competitive inhibition enzyme-linked immunosorbent assay. Further testing of the zoo's ibex via PCR also revealed viral DNA sequences nearly identical to those found in the bongo's tissues.     

从疑似猪瘟病料中检出牛病毒性腹泻病毒

根据牛病毒性腹泻病毒（ＢＶＤＶ）ＮＡＤＬ株和猪瘟病毒（ＨＣＶ）Ａｌｆｏｒｔ株的核苷酸序列，设计合成了１对ＢＶＤＶ引物和３对ＨＣＶ引物。以从吉林、长春、哲盟３个地区经临床及病理学诊断为猪瘟的病料中提取的ＲＮＡ为模板，采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ），分别以ＢＶＤＶ和ＨＣＶ引物进行扩增。结果，用ＢＶＤＶ引物从哲盟地区的疑似猪瘟病料中扩增出大小约４００ｂｐ的片段，而用３对ＨＣＶ引物在不同的条件下均未从该病料中扩增出相应大小的ＨＣＶ基因片段。此外，用ＨＣＶ的ＰＥ０／ＰＥ４引物从吉林、长春的病料中扩增出了ＨＣＶ的基因片段，但用ＢＶＤＶ引物从这两个地区的病料中均未扩增出ＢＶＤＶ的基因片段。从哲盟疑似猪瘟病料中扩增出的片段经克隆、序列测定及计算机分析证实，该片段的核苷酸序列和氨基酸序列与ＢＶＤＶ的同源性明显高于与ＨＣＶ的同源性。将哲盟病料接种于ＭＤＢＫ传代细胞，出现典型而规律的ＢＶＤＶ样细胞病变。由此证明，从哲盟疑似猪瘟病料中检测出的是ＢＶＤＶ而不是ＨＣＶ     

贝类折光马尔太虫PCR检测方法的建立

根据基因库中折光马尔太虫的基因保守序列,设计了1对特异性引物,通过对PCR扩增条件的优化,研究建立了检测贝类折光马尔太虫的PCR方法。该方法对折光马尔太虫模板进行扩增,得到与试验设计相符的478 bp的特异性扩增带,而对派琴虫、单孢子虫、嗜水气单胞菌、荧光假单胞菌、副溶血弧菌、溶藻弧菌和河弧菌等病原体的扩增,结果全为阴性。敏感性试验结果表明,该技术最低能检测到1 pg的折光马尔太虫DNA。用该PCR对广西沿海的119份牡蛎病料进行检测,折光马尔太虫的阳性率为1.68%,结果提示了中国南方沿海的养殖贝类中存在折光马尔太虫的感染,建立的PCR方法可以用于贝类折光马尔太虫的临床快速检测。     

多重PCR方法检测锦鲤疱疹病毒基因

根据对已报道的PCR检测方法灵敏性评价,以常用KHV病毒PCR检测的目的基因KHVSphI片段(AY568590)、KHV5/9(AF411803)和KHVTK基因(AJ535112)作为靶基因,设计并选择3对特异性引物建立的多重PCR检测体系用于KHV病毒多基因的检测。本研究建立的多重PCR体系具有较高的特异性,能够特异性扩增出KHVSphI片段290bp、KHV5/9片段484bp和KHVTK基因片段409bp,对锦鲤和鲤鱼的另外一种病毒性病原鲤春毒血症病毒检测结果为阴性。多重KHV病毒PCR体系检测KHVSphI、KHV5/9和KHVTK基因片段单一模板的检测下限分别为:10fg、100fg和100fg,在相同模板浓度的情况下,KHVSphI、KHV5/9和KHVTK基因片段同时被检出的检测下限为100fg。对KHV病毒感染组织的检测结果表明,多重KHV病毒PCR检测结果与常规PCR检测结果基本吻合,在多重PCR检测体系中KHVTK基因片段检测的灵敏度高于检验检疫行业标准方法。结果表明,多重KHV病毒PCR检测方法能够快速、准确和灵敏地检测KHV病毒基因。     

应用多重PCR检测鉴别对虾白斑综合征病毒和桃拉病毒

研究建立了一种可同时检测对虾白斑综合征病毒(WSSV)和桃拉病毒(TSV)的多重聚合酶链式反应(多重PCR)技术。根据WSSV和TSV基因序列，设计合成了2对分别与WSSV和TSV某段基因序列互补的引物，用这2对引物对同一样品中的WSSV DNA和TSV RNA模板进行多重PCR扩增。结果均同时得到了2条特异的与实验设计相符的306bp(WSSV)和231bp(TSV)多重PCR扩增带，而对其他对虾病病原的PCR扩增结果均为阴性；敏感性测定结果表明，该多重PCR技术能检出10pg的WSSV DNA和1pg的TSV RNA模板。     

应用多重聚合酶链式反应检测鸡毒支原体、鸡滑液囊支原体和禽衣阿华支原体的研究

根据鸡毒支原体 (MG)、禽衣阿华支原体 (MI)、鸡滑液囊支原体 (MS)的基因文库 ,设计了 3对分别与MG、MI、MS某段基因序列互补的引物。用这 3对引物对同一样品中的MG、MI、MSDNA模板进行多重聚合酶链式反应 (PCR)扩增 ,结果均同时得到了 3条特异性的大小与实验设计相符的 732bp (MG)、 2 99bp (MI)、 2 0 7bp (MS)多重的PCR扩增带 ,而对其他 6种禽病病原的PCR扩增结果均为阴性 ;敏感性测定结果能同时检出 1pg的MG、MI和MSDNA模板