Comparative study of rotavirus strains of bovine and rabbit origin

Newborn calves were susceptible to infection with a rotavirus strain isolated from rabbits and, conversely, rabbits 4–5 weeks of age became ill and some died when inoculated with the rotavirus strain of bovine origin. However, the latter isolate appeared to be more virulent than the rabbit isolate in either calves or rabbits. Moreover, in reciprocal cross protection tests, carried out in calves and in rabbits, the rabbit rotavirus antiserum afforded only weak protection to challenge infection with either the homologous or the heterologous virus. By contrast, the protective level of the bovine rotavirus antiserum was relatively high.     

The stability of porcine rotavirus in feces

Rotaviruses are known as major causal agents of diarrhea in humans and animals. They affect young animals in intensive rearing and cause great economic losses. This study evaluated the infectivity of porcine rotavirus maintained for 32 months at approximately 10 degrees C in the original stool specimens. Thirty stool specimens of 1-4-week-old piglets from breeding farms located in the southwest of the State of Parana were selected for this study. They were randomly chosen from stool samples positive for rotavirus RNA by polyacrylamide gel electrophoresis (PAGE) at the time of collection. The thirty stool samples maintained for 32 months were re-tested by PAGE and 11 out of 30 were still positive showing physical integrity of the eleven segments of viral RNA. In order to demonstrate the maintenance of viral infectivity processed fecal homogenates were inoculated in MA-104 cell cultures. After an average of three blind passages 5 out of 11 samples demonstrated cytopathic effect similar to that of a simian rotavirus (SA-11) used as positive control. To confirm these findings an immunofluorescence test was performed and typical cytoplasmatic granular fluorescence was observed. Electron microscopy of stool samples showed that most of the virus particles were single-shelled and some were found to be in advanced state of degradation. The viral nucleic acid extracted from six fecal specimens out of those that showed physical integrity of rotavirus RNA by PAGE were also amplified when submitted to RT-PCR demonstrating stability of viral RNA. We therefore concluded that porcine rotavirus infectivity is maintained for a long period of time in stool specimens at low temperature.     

规模化奶牛场犊牛腹泻的病原检测与分析报告

为了调查某规模化奶牛场犊牛腹泻的发病原因,试验采用血清抗体检测、细菌分离培养、寄生虫卵镜检和病毒核酸检测方法对腹泻犊牛的血样和粪便进行了检查。结果表明,该奶牛场牛病毒性腹泻/黏膜病病毒抗体阳性率为65.00%,牛轮状病毒抗体阳性率25.00%,牛冠状病毒抗体阳性率15.00%;分离到1 株结肠弯曲杆菌和1 株空肠弯曲杆菌;所检腹泻犊牛粪便和血液样品中,4 份粪便样品和1 份血液样品检出牛冠状病毒,2 份粪便样品检出轮状病毒,1 份粪便样品和1 份血液样品检出牛病毒性腹泻/黏膜病病毒,同一份粪便样品中同时检出牛病毒性腹泻/黏膜病病毒和牛冠状病毒;通过镜检,球虫卵囊检出率77.78%,在1 份粪样中发现蛔虫卵,2 份粪样中发现其他线虫卵。该牛场流行性腹泻的原因可能为夏季高温潮湿引起犊牛免疫力下降导致的多病原体感染。     

犬轮状病毒荧光定量RT—PCR检测方法的建立

目的:建立轮状病毒快速准确的检测方法,为研发诊断试剂盒和疫苗奠定基础。方法:用Primer5软件设计VP7引物,从采集的腹泻犬粪便样品抽提病毒RNA,用反转录聚合酶链式反应(RT-PCR)技术,将RNA反转录为cDNA,并进行PCR扩增,对扩增产物进行非变性聚丙烯酰胺凝胶电泳和测序,与代表株的VP7进行同源性比较。结果:从腹泻病犬粪便样品中抽提到了轮状病毒RNA,反转录后扩增产物为51bp的基因片段,经核苷酸序列分析,其PCR序列与犬轮状病毒VP7基因编码源序列的同源性为86.3%。结论:正确克隆了轮状病毒主要保护性抗VP7基因中抗原表位区,建立了犬轮状病毒实时定量诊断方法,为研制实时定量诊断试剂盒奠定了基础。     

Recurrent rotavirus diarrhoea outbreaks in a stud farm, in Italy

A total of 47 stool samples were collected at the same stud farm from young foals with rotavirus diarrhoea and from their stud mares. Illness involved foals during three consecutive winter seasons. Infection in the farm appeared firstly in January-February 2008. After vanishing in the warm seasons, cases reappeared in March 2009 and 2010. Determination of the rotavirus G- and P-types was carried out using nested RT-PCR in samples collected in 2009 and 2010. A total of 19 of 47 samples resulted positive for rotavirus. The G type was determined in 19/47 samples, whereas the P genotype was determined in 17/47 samples. All equine strains presented a G14 VP7 in combination with a P[12] VP4, suggesting persistence of the same viral strain in the stud farm, during at least two consecutive winter periods. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 revealed that the virus had a close relationship between strains recently isolated in the rest of Europe.     

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: Comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen.

A sandwich ELISA procedure using the chosen monoclonal as “capture and detecting” antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.     

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.     

Evaluation of killed and modified live porcine rotavirus vaccines in cesarean derived colostrum deprived pigs

Twenty-eight cesarean derived, colostrum deprived (CDCD) piglets were used to evaluate the efficacy of killed and modified live rotavirus (MLV) vaccines against challenge with virulent A-1 and A-2 rotaviruses. Two killed rotavirus vaccines were evaluated: an experimental vaccine and a commercially available vaccine. Efficacy parameters included: average daily weight gains, rotavirus shedding in feces, morbidity incidence and duration, and rotavirus serum antibody conversion post-vaccination and post-challenge. Piglets vaccinated orally/intramuscularly with the modified live vaccine were completely protected from A-1 and A-2 virulent rotavirus challenge. Nonvaccinated control piglets and piglets receiving killed rotavirus vaccines developed diarrhea, shed virus and exhibited reduced weight gains post-challenge. Only the MLV rotavirus vaccine was able to prevent virus shedding in feces after virulent challenge. Both controls and pigs which received killed vaccines intraperitoneally, orally or intramuscularly shed virus in the feces for 7 days post-challenge and virus peak titers approached 10(7) fluorescent antibody infectious dose (FAID)50/g feces. These studies clearly reflected the inability of killed rotavirus vaccines to induce active local immunity to rotaviral diarrhea in piglets.     

Neonatal calf diarrhea induced by rotavirus

This presentation summarizes the results of a comprehensive study on rotaviruses isolated in Italy from calves and rabbits affected by neonatal diarrhea. The results clearly indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. The evidence of differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by three rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits, respectively. When rotavirus strains of bovine, simian, porcine and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. On the other hand, a reciprocal antigenic correlation was found between bovine and rabbit isolates. Finally, it was proven that feeding newborn calves with colostrum of their dams, previously vaccinated with an inactivated rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.     

猪A群轮状病毒BJ株的分离与鉴定

轮状病毒是引起幼龄动物和儿童病毒性腹泻的主要病原,轮状病毒的分离鉴定为该病的流行病学调查和分子生物学特性研究奠定了基础。本研究采集北京某猪场腹泻发病仔猪肠内容物,将其接种MA104细胞,分离得到一株能产生明显细胞病变的病毒。对分离毒株进行胶体金、实时荧光定量RT-PCR和免疫荧光等方法鉴定,并对分离毒株的VP4、VP6和VP7基因进行测序及序列分析。结果表明,分离毒株为猪A群轮状病毒。按照A群轮状病毒的最新分类方法,确定该分离株VP4、VP6和VP7基因的基因型分别为P[13]型、I5型和G11型。因此,将该分离株命名为Rotavirus A pig/China/BJ/2015/G11P[13]。     

Isolation and Identification of Porcine Group A Rotavirus BJ Strain

Rotavirus infections are a major cause of viral diarrheas in young animals and children. Isolation and identification of rotavirus make a contribution to epidemiological survey and molecular biology study.A strain of porcine rotavirus was isolated in MA104 cell cultures from the intestinal contents of piglets with diarrhea in Beijing.The virus was identified to be porcine rotavirus by immunochromatography strip test, Real-time RT-PCR, immunofluorescence test and sequencing analysis.According to the sequence analysis, the virus was classified as group A porcine rotavirus, the genotype of VP4, VP6 and VP7 genes belonged to P[13], I5 and G11, respectively.The virus was designated Rotavirus A pig/China/BJ/2015/G11P[13].     

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.     

The effect of dexamethasone-induced immunosuppression on the development of faecal antibody and recovery from and resistance to rotavirus infection.

Rotavirus-naive and rotavirus-immune gnotobiotic calves were treated with high doses of dexamethasone (DX) to suppress the immune system. Calves were then infected with a virulent rotavirus inoculum, J-160, to investigate the role of immune responses both in recovery from primary rotavirus infection and in resistance to secondary rotavirus infection. Treatment of calves with DX markedly suppressed in vitro responsiveness of peripheral blood lymphocytes to mitogens within 48 h of the start of DX treatment. Suppression was similar in rotavirus-naive and rotavirus-immune calves. In contrast, the effect of DX treatment on specific antibody responses differed depending on when DX treatment started in relation to rotavirus infection. When DX treatment commenced prior to primary rotavirus infection both systemic and local specific antibody responses were inhibited. These calves, in which mitogen and antibody responses were suppressed, exhibited greater clinical signs than did control calves after infection with virulent rotavirus, but virus excretion was affected in only one of the two calves. When DX treatment was started after primary rotavirus infection but before secondary infection, systemic and local antibody responses to the primary infection and to the challenge infection were not affected. These calves resisted challenge with virulent virus as did DX-untreated rotavirus-immune calves, even though mitogen responses were suppressed. We conclude that in a primary rotavirus infection, virus excretion ceased when both antibody and mitogen responses were suppressed. Resistance to secondary rotavirus infection occurred when mitogen responsiveness was suppressed, but when antibody levels were normal. Thus, no evidence was obtained that fully functional cell-mediated immune mechanisms are essential for resistance to rotavirus infection. Evidence was provided for the ability of parenteral treatment with DX to suppress mucosal as well as systemic antibody responses.     

Rotavirus infection in foals

Fecal samples from 86 foals with diarrhea were examined by electron microscopy during a 2.5 year period. Of these, 26 (30%) were positive for rotavirus. All of the cases were found in epizootic areas. The disease was produced in an experimental foal by inoculation via stomach tube of a bacteria-free fecal filtrate containing rotavirus. Examination of postmortem tissues from the duodenum and jejunum of 2 naturally infected foals and an experimentally infected foal revealed replicating virus in the intestinal epithelial cells. A limited survey of complement-fixing antibody to rotavirus in horses from Kentucky, Virginia, and France indicated that all horses had antibody to the virus. The sole exception was 1 foal from which blood samples were collected prior to suckling. These results were presumptive evidence that rotavirus is a major cause of diarrhea in foals, and the presence of antibody in horses from diverse areas is evidence for the ubiquitousness of this infection.     

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Paraná, Brazil. Fecal (n=66) and serum (n=182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1]+P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission.     

Rotavirus associated diarrhoea in nursing piglets and detection of antibody against rotavirus in colostrum, milk and serum

A blocking method of ELISA for the detection and quantification of antibody against porcine rotavirus in serum, colostrum and milk has been compared with a plaque reduction test. The results obtained with the two techniques correlated (Fig. 1). Antibody against rotavirus was demonstrated in 384 serum samples representing 25 swine farms, indicating a widely spread and dense distribution of the infection with porcine rotavirus among Danish swine. The antibody contents in milk samples from 7 gilts and sows from 2 farms with a previously diagnosed problem with rotavirus associated diarrhoe showed a rapid decline during the first few days of the lactation periods (Fig. 1). An increase in the contents of rotavirus specific antibody was observed from day 15 in the milk samples from one of the gilts. The excretion of rotavirus with feces from the 7 litters of piglets were followed through the suckling period. Rotavirus war found in all litters but one and the virus was excreted in periods ranging from 4 to 9 days.     

Design and evaluation of primer pairs for efficient detection of avian rotavirus

The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing and allows the identification of putative reassortant strains. Reagents and methods for genotyping the virus need constant modification because of the reassortant nature of the virus. This study was aimed at designing and evaluating new oligonucleotide degenerate primer pairs that provide increased sensitivity and specificity for detecting avian rotavirus. Gene-specific primer pairs were designed by analysing different rotavirus strains isolated during the last decade by downloading them from the GenBank. The alignments were generated using clustal analysis from the BioEdit program. Degenerate nucleotides were included due to the reassortant nature of rotavirus. The consensus sequences were aligned using the BioEdit program and then treated with the Fast PCR software to derive the primers. The derived primer sequences were submitted for a BLAST search to ensure alignment was exclusive to the desired target genes. The designed primers had specific bands and were efficient in detecting rotavirus in faecal samples than previously published primers. Thus, a successful surveillance of rotaviruses requires that primer pairs be updated regularly in order to detect the emergence of novel or “unusual types”, which have occurred by genetic drift causing nucleotide changes at the primer binding sites that result in typing failures. We recommend the use of the proposed primers in molecular surveillance studies for efficient detection of avian rotavirus.     

禽轮状病毒的分离鉴定及部分特性研究

从北京某自然腹泻的鸡场 中成功地分离到一伯禽轮状病毒，并经鸡胚单层肝细胞和ＭＡ１０４细胞进行了传代培养，对其生物学特性作了部分研究，从而为我国禽轮状毒的毒株鉴定、诊断及疫苗等系列研究提供了一定依据。