Experimental infection of recent field isolates of feline herpesvirus type 1

Two field isolates of feline herpesvirus type 1 (FHV-1) designated as 00-015 and 00-035, were obtained from cats diagnosed as feline viral rhinotracheitis (FVR) in Japan. To analyze the character of recent FHV-1, these two isolates and our laboratory strain C7301 were inoculated experimentally to specific-pathogen-free cats. Although all cats showed typical FVR symptoms, more severe clinical symptoms were observed on cats infected with the isolates 00-015 and 00-035 compared with those of C7301-infected cats. Severe ocular lesions including conjunctivitis were found in the cats infected with the isolates, indicating that the recent FHV-1 has a potential to induce severe FVR symptoms including ocular lesions.     

绵羊肺腺瘤病毒中国NM株gag基因3''''端951 bp段的分子克隆与序列分析

究参照Genebank中已发表的绵羊肺腺瘤病毒的全基因序列,设计合成一对引物,对JSRV中国NM株的gag基因3'端中主要编码核衣壳(NC)蛋白的基因段进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现一条约951 bp的特异条带,将其回收后克隆人pMD-18T载体中,并进行序列测定.结果表明,与南非代表株(基因序列号NC-001494)的gag基因序列比较,核苷酸同源性为95.4%,推导出的氨基酸同源性为95%.与美国代表株(基因序列号AF105220)的gag基因序列比较,核苷酸同源性为91.3%,氨基酸同源性为91%.这是我国首次报道的绵羊肺腺瘤病毒的gag基因的一段序列,为我国科研工作者进行更深入的研究奠定基础.     

Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7-10 DNA fragments ranging in size from 40 to 373kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.     

鸭瘟病毒UL47基因克隆及其分子特性分析

通过测定本实验室构建的鸭瘟病毒(Duck plague virus,DPV)DNA基因文库中重组质粒的DNA序列,结合NCBI的ORF Finder和Blast工具分析得到了该病毒UL47基因的ORF。采用PCR扩增出了UL47基因并将其克隆到pMD18-T载体上,经PCR和酶切鉴定以及进一步的核酸斑点杂交试验证实该基因即为DPV UL47基因。利用生物信息学软件ProtScale、SignalP3.0、Scansite、TMpred、Prosite、DNAStar以及在线的EMBOSS等分析了UL47基因的分子特性。结果显示,该基因大小为2 367bp,编码788aa,而且与GenBank上多种α疱疹病毒同源蛋白的核酸和氨基酸序列具有较高的同源性。系统进化树分析表明,DPV UL47与禽类疱疹病毒(α-疱疹病毒)的进化关系最近。密码子偏爱性结果显示,DPV UL47编码相同氨基酸的不同密码子使用频率差异较大,UL47基因密码子使用模式更接近真核生物。研究结果为进一步开展DPV UL47基因功能研究奠定了基础。     

Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates.

Sixteen isolates of transmissible gastroenteritis virus and one isolate of porcine respiratory coronavirus were characterized using RT-PCR amplification of 4 antigenic subsites in the site A epitope on the TGEV spike gene. The PCR products were digested with restriction enzymes Sau3AI and SspI and the sizes of the fragments were determined. Three different digestion patterns were observed with each enzyme. The recognition site for Sau3AI was missing in 1 isolate, was present in 13 isolates and 3 isolates had 2 sites. PCR-products with a single site had 3 different fragment sizes and the other isolates produced 2 fragments with different sizes. The SspI recognition site was not present in 5 isolates and 12 isolates had a single site that produced 2 fragments of different sizes. Based on the restriction fragment sizes, the 17 isolates were separated into 7 groups. Direct sequencing of the 455 bp nested set fragments demonstrated greater than 96% sequence homology among the 16 isolates and 100% homology in the 4 antigenic subsites in the conserved site A epitope. The groups are discussed in relation to their sequence homology and virulence. In vitro procedures have been developed to identify several porcine enteric coronavirus isolates at the strain level.     

伪狂犬病病毒Bartha-K61株UL49基因的克隆和序列分析

根据已经发表的伪狂犬病病毒(PRV)国内Ea株UL49基因序列,设计并合成了1对引物,通过PCR方法扩增到了PRV Bartha-K61株UL49基因的编码区,并克隆到pMD18-T载体中.重组质粒pMD-UL49经XhoI酶切和PCR鉴定后,进行了序列测定和分析.结果表明,重组质粒pMD-UL49含有PRV Bartha-K61株UL49基因的编码区.同源性分析显示,PRV Bartha-K61株UL49基因序列与国外Kaplan株、国内Ea株相应基因的核苷酸同源性分别为98.9 %和94.1 %,推导的氨基酸序列同源性分别为96.7 %和87.2 %.     

伪狂犬病病毒Ea株UL31基因克隆、序列分析及其在大肠杆菌中的表达

以伪狂犬病病毒Ea株基因组DNA为模板,通过PCR扩增含UL31基因的1 000bp片段,扩增产物克隆于pMD18-T中,双脱氧末端终止法序列测定.通过OM1GA2.0软件包分析发现,Ea株UL31基因编码271个氨基酸,蛋白质分子量为30.38 kD,与Ka株UL31基因核苷酸与氨基酸序列同源性均在98%以上.将α-疱疹病毒亚科9个不同成员UL31同源基因编码的氨基酸序列进行多重比对分析,发现4个保守的功能性结构域.将该片段插入原核表达载体pGEX-KG中GST下游,构建的原核表达质粒pGEX-UL31在大肠杆菌BL32(DE3)中获得了高效表达,SDS-PAGE结果显示,表达的融合蛋白质分子量为56 kD.     

Genomic diversity among equine herpesvirus-4 field isolates

Infection with equine herpesvirus-4 (EHV-4) is a major cause of respiratory tract disease, equine rhinopneumonitis, in horses. Although the full sequence of EHV-4 has been reported, genomic differences among EHV-4 field isolates have not yet been characterized. In this study, the genomic diversity between 23 Japanese EHV-4 isolates was analyzed by digestion with restriction endonucleases (BamHI, BgIII, EcoRI, SacI, and SalI) and polymerase chain reaction (PCR). The restriction endonuclease digestion patterns of the EHV-4 field isolates showed distinct differences which included mobility shifts of some fragments as well as loss and/or gain of fragments. Two EHV-4 genes containing repeat sequences, ORFs 24 and 71, were amplified by PCR and the amplified fragments were compared among the field isolates. The sizes of the amplified fragments varied among epizootiologically unrelated isolates, while the fragments of related isolates had the same size. The observed genomic diversity among EHV-4 field isolates may be a useful tool for epidemiological study of equine rhinopneumonitis by EHV-4 infection.     

PCR用于鸭瘟病毒诊断的研究

根据鸭瘟病毒UL6和UL7基因序列,设计合成了一对引物,以2株疫苗株、1株强毒株和1株山东分离株DNA为模板,进行PCR扩增,得到预期690bp的目的片段.将扩增的目的片段克隆到pMD18-T载体,经Amp平板筛选,HindⅢ、BamHⅠ双酶切鉴定,获得阳性重组质粒.对重组质粒进行序列测定,与参考序列比较,山东分离株与参考序列的同源性为99.7%,其余3株DPV与参考序列的同源性均为100%.应用PCR可检测人工感染和自然感染鸭瘟的组织中的鸭瘟病毒,表明PCR检测鸭瘟病毒具有很高的特异性、敏感性,该法能够用于鸭瘟急性及亚临床感染的检测与诊断.     

An etiological investigation of domestic cats with conjunctivitis and upper respiratory tract disease in Japan

Chlamydophila felis (C. felis), feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV) were detected in 39 (59.1%), 11 (16.7%) and 14 (21.2%) cats respectively, from 66 domestic cats presented with conjunctivitis and upper respiratory tract disease (URTD) in 9 prefectures of Japan. Dual and multiple infections were found in 7 (10.6%) cats with both C. felis and FHV-1, 10 (15.2%) cats with both C. felis and FCV, and 1 (1.5%) cat with all three agents. C. felis was isolated from 11 (28.2%) of 39 PCR positive cats. Antigenic difference was found in a 96 kDa protein of our isolates and Fe/145 strain isolated in USA. In conclusion, C. felis is the most common agent of feline conjunctivitis and URTD, and the coinfection with C. felis, FHV-1 and FCV are also common in cats in Japan.     

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.     

Biological and molecular characterization of chicken anemia virus isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.     

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.     

绵羊肺腺瘤病毒NM株前病毒gag基因的克隆与序列分析

参照GenBank中已发表的绵羊肺腺瘤病毒(JSRV)的全基因序列，设计合成3对引物，对JSRVNM株的gag基因分3段进行PCR扩增，经琼脂糖凝胶电泳分析，分别呈3条531、888和949 bp的特异条带，将其分别克隆人pMD-18T载体中，进行序列测定并拼接序列，得到完整的gag基因序列。分析结果表明，与南非代表株(基因序列号NC-001494)的gag基因序列比较，核苷酸同源性为89．0%，推导出的氨基酸同源性为90％。与美国代表株(基因序列号AF105220)的gag基因序列比较，核苷酸同源性为86．3％，氨基酸同源性为87％。     

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis

Infectious laryngotracheitis is a dramatic disease of the upper respiratory tract in poultry caused by a herpesvirus. In this study we investigated the characteristics of western European field isolates of infectious laryngotracheitis virus (ILTV) to gain more information on their diversity. The examined 104 isolates, collected from acute outbreaks during the last 35 years, originated from eight different countries: Switzerland (48), Germany (21), Sweden (14), the United Kingdom (9), Italy (5), Belgium (4), Austria (2), and Norway (1). Two vaccines, a chicken embryo origin product and a tissue culture origin product, were included in the survey. Polymerase chain reaction (PCR) was performed to amplify a 2.1-kb DNA fragment of ILTV using primers generated for the thymidine kinase (TK) gene. After digestion of the resulting PCR products by restriction endonuclease HaeIII, restriction fragment length polymorphism analysis was carried out. PCR amplicons of three field isolates and both vaccine strains were selected for sequencing. Here 98 field isolates showed the same cleavage pattern and were identical to both vaccine strains (clone 1). They differed from five Swiss isolates with identical cleavage pattern (clone 2) and one Swedish isolate (clone 3). The present study demonstrated that at least three clones of ILTV have been circulating in western Europe during the last 35 years. The 104 isolates analyzed showed a high genetic similarity regarding the TK gene, and a large majority of the field isolates (98/104) were genetically related to the vaccine strains.     

Provirus variants of bovine leukemia virus in naturally infected cattle from Argentina and Japan

A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.     

9株鸡毒支原体29 Ku多肽基因的克隆与序列分析

根据已发表的鸡毒支原体(MG)S6株29Ku多肽基因序列设计了1对引物,以9株(广西分离株5株、标准株4株)DNA为模板进行PCR扩增,均得到802bp的特异性片段,将9株MG PCR产物纯化后克隆到pMD18-T载体上,得到重组质粒.重组质粒经PCR法和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定了9株29 Ku多肽基因序列,并在基因库中S6标准株的29 Ku多肽基因序列进行分析比较.结果表明,5株分离株与5株标准株29Ku多肽基因核苷酸序列同源性分别为94.4%～99.9%,推导的氨基酸同源性分别为89.7%～99.2%.从各毒株的进化分析表明,5个分离株与标准强毒株S6、A5969、K1501和PG31强毒株间遗传距离较近,而5个分离株与标准株F疫苗株间遗传距离则较远.     

A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains

In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.     

Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).     

Diarrheal condition in dogs associated with viruses antigenically related to feline herpesvirus

Viruses with properties consistent with herpesvirus were isolated from dogs with diarrhea. The viruses were shown to be antigenically related to feline herpesvirus-1 (FHV-1) by virus neutralization tests. It was also observed that a canine herpesvirus (CHV) prototype, D004, and two field isolates from fatal CHV infections in 2-week-old and 6-week-old puppies were neutralized at a low level by antiserum to FHV-1. Reciprocal neutralization tests with CHV antiserum against FHV-1 were negative. These results indicated that viruses related to FHV-1 can infect the dog and that there appears to be uni-directional virus neutralization of CHV by FHV-1 antibody.