Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA 104 cell line

Avian rotaviruses were isolated from turkeys with enteritis using MA 104 cell line. MA 104 cells were suitable for primary isolation and propagation of avian rotaviruses. Trypsin appeared essential for the enhancement of infectivity and the occurrence of cytopathic effect (CPE). Serum neutralization (SN), electron microscopy (EM), and analysis of genomic RNA were done to identify and confirm the identity of rotaviruses. Electrophoretic migration patterns of genomic RNA from avian rotaviruses were examined, and they were compared with those from mammalian rotaviruses. The migration patterns differed between these groups.     

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

Characterization of viral polypeptides from avian rotavirus

The synthesis of avian rotavirus (AvRV-1) polypeptides in MA 104 cells was investigated. Extracts of cells labeled with either [35S]methionine or [3H]mannose were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was detected first at 6 hours postinfection. Ten major viral polypeptides were detected at this time. The presence of five viral structural proteins (100K, 90K, 88K, 45K, and 37K) were demonstrated in AvRV-1-infected cells by immunoprecipitation analysis. One structural polypeptide (37K) was identified as a glycoprotein. Two viral polypeptides (30K and 28K) were identified as nonstructural glycoproteins. By tunicamycin inhibition of glycosylation, a 32K polypeptide was identified. This 32K polypeptide later was proven to be the precursor of 37K structural glycoprotein by immunoprecipitation analysis, beta-N-acetylglucosaminidase H (Endo H) treatment, and peptide mapping analysis. Avian rotavirus contained high-mannose oligosaccharide content in the glycoproteins similar to the glycoproteins of mammalian rotaviruses.     

Isolation and serial propagation of turkey rotaviruses in a fetal rhesus monkey kidney (MA104) cell line

Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.     

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。     

Propagation of avian rotavirus in primary chick kidney cell and MA104 cell cultures

The Ch2 strain of avian rotavirus was propagated in primary chick kidney cell (CKC) and MA104 cell cultures in the presence of trypsin. Cultures were evaluated for the presence of rotavirus by an indirect fluorescent antibody (IFA) assay and by an indirect enzyme-linked immunosorbent assay (ELISA). After two passages, the viral titer was significantly higher in CKC than MA104 cell cultures. Also, the IFA assay was more sensitive than the indirect ELISA for detecting rotavirus-positive cultures.     

猪轮状病毒Rotavirus A pig/China/NMTL/2009/G9P[23]株的分离与鉴定

A群轮状病毒(GAR)是引起人类和多种动物腹泻的主要病原体,病原的分离与鉴定是轮状病毒(RV)流行病学、病原学等研究的基础.本研究采用接种MA 104细胞的方法,从内蒙古某猪场患病猪腹泻粪便中分离一株病毒,经3次蚀斑克隆纯化后,采用电镜观察、PCR鉴定和序列测定进行分析,结果表明该分离株为猪轮状病毒(PoRV).致病性试验表明该分离株能够引起仔猪急性腹泻,RT-PCR扩增其VP4、VP6和VP7的基因节段并进行序列测定,按照A群RV的最新分类方法,确定该分离株的VP6、VP7和VP4基因型分别为I5型、G9型和P[23]型.因此,将该分离株命名为Rotavirus A pig/China/NMTL/2009/Q9P[23].     

Production and characterization of monoclonal antibodies against an avian group A rotavirus.

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.     

The isolation, propagation and characterization of tissue-cultured equine rotaviruses

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.     

Isolation, identification and characterization of group A rotavirus from a chicken: the inner capsid protein sequence shows only a distant phylogenetic relationship to most other avian group A rotaviruses

Rotavirus particles were identified in the intestinal content of a 35-day-old stunted chicken. The virus was isolated, RNA pattern was analysed and the viral genome segment 6 was sequenced. In particular, the sequence data showed a very close similarity to the chicken rotavirus isolate Ch-1 (99.2% amino acid homology), this is distantly related to all known avian rotaviruses and supports the existence of different VP6 types amongst avian group A rotaviruses.     

Physical, chemical, and serological characterization of avian rotaviruses

Physical, chemical, and serological characterization of rotavirus isolates from turkeys was done. Cesium chloride (CsCl)-gradient isopycnic centrifugation of infected cell cultures revealed the presence of rotavirus particles of three different densities. They were double-shelled, single-shelled, and core particles. The double-shelled particles had a buoyant density (in CsCl) of 1.34 g/cml3, and that of single-shelled particles in CsCl was 1.36 g/cm3. The buoyant density of core particles in CsCl was greater than 1.40 g/cm3. These rotavirus isolates were not inactivated by chloroform and were relatively stable at pH 3.0. Their replication was not affected by 5-bromo-2'-deoxyuridine. Avian rotaviruses were not completely inactivated by heat treatment of 56 C for 8 hr. All six avian rotavirus isolates examined were antigenically related to each other. However, there was no antigenic relationship between mammalian rotaviruses and the avian rotavirus isolates examined.     

Detection of a mammalian-like group A rotavirus in diarrhoeic chicken

The present investigation describes detection of a mammalian-like electropherogroup A rotavirus in chicken with diarrhoea. This also records the first detection of a rotavirus in an avian species from India. During the investigation 75 diarrhoeic faecal samples collected from adult chicken were screened for the presence of group A rotavirus antigen by sandwich ELISA. All three samples positive for rotavirus antigen revealed 11 bands of RNA in polyacrylamide gel electrophoresis (PAGE). In contrast to avian group A rotavirus, segment 5 was found to migrate closer to 6 as is the case with mammalian group A rotaviruses. Segments 7, 8 and 9 were found to migrate as a tight triplet, which is characteristic of group A rotavirus.     

A study of cytopathic rotavirus strains isolated from calves with acute enteritis

Nine cytopathic bovine rotavirus strains were isolated in MA-104 cell cultures from fecal specimens of dairy calves suffering from diarrhea. Isolation of the virus was accomplished from three outbreaks which occurred on dairy farms located in Central and Southern Italy. Fecal suspensions were treated with a high concentration (1000 micrograms/ml) of trypsin, and inoculated into MA-104 cell cultures grown out in Eagle's minimum essential medium (MEM) containing 5 micrograms/ml of the enzyme. Cytopathic effects (CPE), characterized by intracytoplasmic inclusion bodies of different sizes and shapes, were observed on the 1st passage with five of the strains and on the 2nd (2 strains) or the 3rd (2 strains) passage for the others. The presence of trypsin and the use of MA-104 cells appeared to be essential for the occurrence of CPE, inasmuch as no CPE was detected when trypsin was omitted in the MA-104 cell system. Replication failed to occur when primary bovine embryo kidney cell cultures with or without trypsin were used. Electron microscopy revealed the presence of particles with a typical rotavirus morphology. In MA-104 cells, the titre of virus reached its maximum 48 hr after inoculation. Small, clear-cut plaques were produced by the isolates in MA-104 cells under the overlay of MEM containing carboxymethyl cellulose, trypsin and DEAE-dextran. The nine rotavirus strains were antigenically related, whereas the relationship to either the Nebraska or the Compton rotaviruses was quite weak.     

Identification and characterization of a plaque forming avian rotavirus isolated from a wild bird in Japan.

From fresh faeces of a wild bird (Melanitta fusca), a virus that showed granular cytopathic effects (CPE) on chicken kidney cell (CKC) cultures was isolated. By indirect immunofluorescence analyses (IFA), this isolate reacted with an antiserum against a bovine rotavirus. The isolate produced clear plaques on CKC by conventional techniques, without trypsin. Three virus plaques were selected by plaque size (small, medium, and large) and cloned by three successive plaque cloning. In the SDS-PAGE analyses, dsRNA bands showed a typical profile of avian rotavirus and quite different from that of avian reovirus. With dsRNA patterns, IFA results, CPE, and a morphological property, the clones were identified as avian rotaviruses of group A rotavirus. The clones killed chicken embryos, when they were inoculated to yolk sac.     

猪轮状病毒的分离鉴定及部分特性研究

从吉林省某猪场发生腹泻的猪群粪便中,用MA-104细胞培养,分离到一株猪轮状病毒,盲传至4代,感染细胞出现明显病变,经电镜观察,见有典型的轮状病毒颗粒.其RNA电泳型为4:2:3:2型,属A群轮状病毒,同时对其蚀斑特性和血凝特性进行了初步研究,这株病毒被命名为JL94株.     

Rapid coagglutination test for detection of rotaviruses in turkeys

Avian rotaviruses present in fecal samples were readily detected using a staphylococcal protein-A coagglutination test on a white porcelain plate. Staphylococci, which produced large amounts of protein-A, were coated with rabbit anti-avian rotavirus serum. The antibody-coated staphylococci were agglutinated specifically by rotavirus present in the fecal sample. The macroscopic agglutination reaction occurred within a few minutes. A total of 40 fecal samples were tested by the coagglutination test. The sensitivity and specificity of the coagglutination test were compared with those of electron microscopy, enzyme-linked immunosorbent assay, and tissue-culture virus-isolation methods. Of the 31 fecal samples positive for rotavirus on electron microscopy, 27 (87%) were positive on coagglutination test. Of the nine electron-microscopy-negative samples, seven (78%) were also negative on coagglutination test. It was concluded that the staphylococcal protein-A coagglutination test can be used as a simple, rapid screening test for avian rotavirus.     

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].