Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes

A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Serotyping and antigenic comparison of some animal rotaviruses isolated in China.

Eight strains of rotaviruses isolated from diarrheal animals (4 from calves and 4 from piglets) in China were compared by serotyping with reference animal rotavirus strains (bovine NCDV, porcine OSU and simian SA-11 and human rotavirus Wa strain). Two-way cross neutralization test showed no antigenic difference between all 4 local strains of bovine rotavirus (BRV007, BRV014, HN-7 and BRV6555) and reference NCDV, so they belonged to rotavirus serotype 6 (bovine rotavirus serotype 1 or NCDV-serotype). Meanwhile, the four strains of Chinese porcine rotavirus could be determined into 2 different serotypes. One (Li99) was neutralised to a high titer with the antiserum against reference OSU virus and probably related to OSU (serotype 5 or porcine serotype 1). The other three strains (Lin71, Nan86 and Jiang150) were antigenically obviously different from Li99 and did not react with the antiserum against OSU. They were tentatively considered as porcine rotavirus serotype 2. All the strains of bovine and porcine rotavirus did not cross-neutralise with simian SA-11 and human Wa strain. There was also no antigenic relationship between bovine rotaviruses and porcine rotaviruses.     

The use of biotin-labeled cDNA probes for the detection of infectious bursal disease viruses

A cDNA library was prepared from the double-stranded RNA genome of the infectious bursal disease virus (IBDV) strain ST-C. The cDNA molecules were annealed into the plasmid pUC9 and used to transform Escherichia coli strain JM107. A cDNA clone that contained IBDV-specific nucleotide sequences was selected and designated STC-1. Radiolabeled probes were prepared from STC-1 and hybridized to genome segment A of ST-C in a northern blot hybridization assay. The STC-1 cDNA was 448 base pairs in length, and its nucleotide sequence indicated that it is located near the VP-2/VP-4 junction in IBDV genome segment A. Biotin-labeled probes were prepared from STC-1 and used in a dot-blot hybridization assay to detect IBDV. Under relatively low stringency conditions of hybridization, the biotinylated probes detected four subtypes of IBDV serotype 1 and a serotype 2 IBDV isolate.     

Differential detection of infectious bursal disease virus serotypes, using cDNA probes to VP2 coding region.

Two nonoverlapping clones, pOH405 and pOH632, containing cDNA inserts in the VP2 coding region of genome segment A were selected from a cDNA library prepared from the double-stranded RNA genome of the OH strain of infectious bursal disease virus (IBDV) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with IBDV strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with IBDV serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-IBDV double-stranded RNA, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 IBDV double-stranded RNA, was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for IBDV serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 IBDV serotypes by nucleic acid hybridization.     

Genetic heterogeneity of wild-type G4P[6] porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd

Group A rotavirus (RV-A) with short electropherotype was identified by ss-PAGE in a neonatal diarrhea outbreak at a Brazilian pig farm where the sows were regularly vaccinated with a commercial vaccine containing OSU (G5P[7]) and Gottfried (G4P[6]) porcine RV-A (PoRV-A) strains. The ss-PAGE positive stool samples (n=20) were characterized as P[6] genotype by multiplex-nested-RT-PCR assay. The nucleotide analysis of the VP4 gene (VP8*) state that the viruses clustered in P[6] lineages that are also shared by RV-A strains identified in human hosts. Nucleotide analysis of the VP7 gene identified different lineages in G4 including a new lineage tentatively designated IX. The immunological pressure induced by commercial vaccine with a rotavirus containing a G4P[6] genotype of porcine origin (Gottfried strain) might have allowed the selection of PoRV-A strains with characteristics found in RV-A strains isolated of human hosts, such as P[6]-Ie and If, and promoted the selection or emergence of RV-A strains with a new lineage of the G4 genotype. The characterization of PoRV-A strains with unusual genotypes described in this study highlight the importance of surveys on the relationship between human and animal rotavirus strains.     

Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec.

Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates.     

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.     

A群猪轮状病毒JS株vp7基因序列分析

根据已发表的猪轮状病毒OSU毒株vp7基因核苷酸序列ORF两端保守区序列,设计一对特异引物,以猪轮状病毒JS毒株反转录cDNA为模板,通过PCR方法扩增出长约1000 bp目的片段。将其进行T-A克隆、序列测定和分析。结果表明,vp7基因全长1062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与已知的15个毒株vp7全长基因的核苷酸及推导的氨基酸序列比较,同源性分别为74.5%～78.5%和75.2%～83.1%,核苷酸系统发育进化树结果表明,JS毒株与轮状病毒G9型参考毒株ICB2185、O-1亲缘关系较近,分为一个群,表明JS毒株血清型为G9型。目前,我国尚未见猪及其它动物轮状病毒G9型流行株的报道。     

Development of hybridization probes on the basis of recombinant DNA to identify porcine parvoviruses and rotaviruses

Porcine parvovirus (PPV) and porcine rotavirus (PRV) infections can be diagnosed, and the diagnosis greatly contributes to their control. For the diagnosis of PPV and PRV of pigs recombinant DNA were obtained which could be used as hybridization probes. Homology between PPV-RNA and cloned cDNA was confirmed, and the ability of plasmid probes to detect PRV was demonstrated. Hybridization with preparations of both PPV-infected cells and purified PPV gave positive reproducible results. The results are useful as an experience for the development of a sandwich hybridization system for PPV detection.     

Efficacy of an orally administered modified-live porcine-origin rotavirus vaccine against postweaning diarrhea in pigs

A commercially available, porcine-origin rotavirus vaccine was evaluated for efficacy against postweaning diarrhea due to rotavirus infection in pigs. Weight gains were compared at 5 intervals after weaning. Visual scoring was used to evaluate fecal consistency. Rectal swab specimens were cultured for hemolytic E coli and evaluated for rotaviral antigen by use of enzyme-linked immunosorbent assay. Milk from dams and sera from pigs and dams were evaluated for rotavirus-neutralizing antibodies by use of a plaque-reduction test. Significant differences between vaccinates and controls were not found in the determinants evaluated. Selected rotavirus-positive fecal and rectal swab specimens were examined for double-stranded (ds) RNA by use of direct electropherotyping, and the results were compared with the dsRNA pattern of rotavirus propagated from the vaccine. Only electropherotypes typical of field strain virus were found in the fecal and rectal swab specimens evaluated. Sera from guinea pigs and from a gnotobiotic pig immunized against the field strain rotavirus neutralized Ohio State University strain rotavirus (homologous to the vaccine rotavirus strain), but did not neutralize the Gottfried strain of rotavirus. This indicated that, although the dsRNA electropherotypes of the field and vaccine strains of the virus were different, the serotypes were similar, if not identical.     

Detection of bovine viral diarrhea virus by spot hybridization with probes prepared from cloned cDNA sequences

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3' end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virus-specific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10-100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by 10-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪轮状病毒GD1株VP7基因的克隆及序列分析

参考GenBank中发表的PRV OSU毒株VP7基因核苷酸序列ORF两端保守区序列,设计1对特异引物,以PRV GD1株反转录cDNA为模板,通过PCR方法扩增出长约1 kb的基因片段,并克隆到pMD18-T载体中进行序列测定。测序结果表明:VP7基因全长1 062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与国内外已知的13个毒株VP7全长基因的核苷酸及推导的氨基酸序列比较,相似性分别为77.4%～100%和78.6%～99.7%;核苷酸系统发育进化树结果表明,GD1毒株与轮状病毒A2,JP3-6毒株亲缘关系较近,为一个进化群。     

Serotypes and intimin types of intestinal and faecal strains of eae+ Escherichia coli from weaned pigs

Enteropathogenic Escherichia coli (EPEC) that are known to cause severe diarrhoea in children and young rabbits are well characterized, but there are few reports on the serotypes and intimin (eae) types of EPEC in weaned pigs. Based on detection of the eae gene by PCR and by DNA-hybridisation with LEE specific gene probes, 20 intestinal and 17 faecal eae(+) strains from diarrhoeal (164) and non-diarrhoeal (57) weaned pigs from 13 Hungarian farms, representing 12.8% of diarrhoeal and 14.0% of non-diarrhoeal pigs, were identified. The dominant serotype was O123:H11 (40%) among intestinal, and O108:H9 (23%) among faecal strains. The majority (85%) of the intestinal strains possessed eae-beta and 10% carried eae-gamma gene. In contrast, significantly (p<0.025) fewer faecal strains (53%) harboured the eae-beta gene, and 23% were eae-gamma positive. In vitro adhesion tests of intestinal and faecal eae(+) strains indicated adhesion of 20/37 of the strains to PK15 (porcine kidney) cells while only 3/37 strains adhered to HeLa cells. The ultrastructure of intimate bacterial attachment of representative porcine eae(+) strains to PK15 cells showed no pedestal formation, in contrast to the human EPEC (O127:H5, eae-alpha) strain. In conclusion, the data do not demonstrate a significant role for the eae(+)E. coli in porcine post-weaning diarrhoea, but provide new information on a dominant porcine serotype (O123:H11, eae-beta), and on differences of serotypes and intimin types of porcine eae(+) strains according to their site of isolation. Furthermore there was an indication that the PK15 cell line could be used as a model to study in vitro adherence of eae(+)E. coli of some human and porcine origin.     

A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定

To prepare the monoclonal antibodies against VP7 protein of group A porcine rotavirus (PoRV) serotype G11,VP7 gene of PoRV serotype G11 was cloned into the vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli competent cells DH5α and induced by IPTG. Hybridomas were produced by fusing SP2/0 cells with spleen cells from mouse immunized with GST-VP7 recombinant protein. One hybridomas secreting MAb against VP7 protein was identified by indirect ELISA. Western blotting analysis showed that the MAb could recognize the recombinant VP7 protein and authentic VP7 protein of PoRV，and the specific immunoflurescence was detected in PoRV infected MA104 cells by indirect immunoflurescence assay.The result of MTT method showed that the MAb had the neutralizing activity. The subtype of the MAb was IgG2b. The results showed that VP7 protein was successfully expressed in E.coli， one MAb was preparated and identified，and it could be used for further study of prevention，diagnosis and treatment of porcine rotavirus disease.     

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.     

Relative prevalence of typical and atypical strains among rotaviruses from diarrheic pigs in conventional swine herds

Polyacrylamide gel electrophoresis was conducted on genomic RNA extracted from rotaviruses detected in diarrheic pigs from conventional swine herds. Ninety samples contained sufficient virus for RNA band visualization and genome classification. Genome profiles were characteristic of typical group A rotaviruses in 67.8% of the 90 samples, of group B rotaviruses in 10.0%, and of group C rotaviruses in 11.1%. In 11.1% of the samples, the presence of more than 11 bands suggested concurrent infection with more than 1 strain of rotavirus. In infections among nursing pigs, 76.4% were group A rotaviruses, 7.4% were group B, 7.4% were group C, and 8.8% were coinfections. In infections among weaned pigs, 40.9% were group A, 18.2% were group B, 22.7% were group C, and 18.2% were coinfections. Coelectrophoresis with prototype OSU and Gottfried strains revealed a great diversity in electropherotype among field strains of rotavirus.