A V Parwani

Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines

Abstract Heterotypic passive immunity to IND (P[5]G6) bovine rotavirus (BRV) was evaluated. Three groups of calves (n = 5 per group) were fed 1% pooled colostrum supplements (birth to 7 days of age) from BRV seropositive cows vaccinated with recombinant SA11(P[2]G3) rotavirus-like particles (VLPs), recombinant SA11 rotavirus core-like particles (CLPs), or inactivated SA11 rotavirus (SA11). Control calves (n = 5 per group) received either pooled colostrum from unvaccinated (BRV field exposure seropositive) control cows, or no colostrum. IgG1 antibody titers to IND BRV for the pooled colostrum were: 1048576 (VLP); 1048576 (CLP); 262144 (SA11); and 16384 (control colostrum). Elevated titers of BRV neutralizing (VN) antibodies were present in VLP colostrum (98 000), and SA11 colostrum (25 000), but not in CLP colostrum (1400), compared to colostrum from nonvaccinates (2081). Calves were orally inoculated with virulent IND BRV at 2 days of age and challenged at post-inoculation day (PID) 21. Calves were monitored daily for diarrhea and faecal BRV shedding through PID 10 and post-challenge day (PCD) 10. After colostrum feeding, the IgG1 antibody titers were highest in serum and faeces of calves fed VLP and CLP colostrum, but VN and IgA antibodies were highest in calves fed VLP colostrum. After BRV inoculation, calves fed colostrum from vaccinated cows had significantly fewer days of BRV-associated diarrhea and BRV shedding than control calves. All calves fed VLP colostrum were protected from diarrhea after BRV inoculation; two calves shed BRV. In the CLP colostrum group, one calf developed BRV-associated diarrhea and all calves shed virus. In the SA11 colostrum group, three calves developed BRV-associated diarrhea and four calves shed virus. BRV-associated diarrhea and shedding occurred in 9 of 10 control calves. Active IgM antibody responses occurred in faeces and/or serum of most calves after BRV inoculation. However, the highest active antibody responses (IgM and IgG1 in serum, and IgM, IgG1 or IgA in faeces) after BRV inoculation were in calves fed control or no colostrum, in association with clinical diarrhea in most of these calves. After challenge at PID 21, BRV-associated diarrhea and shedding were of short duration or absent, in all groups. These results demonstrate the efficacy of colostrum from VLP vaccinated cows to provide heterologous, passive protection against BRV diarrhea and shedding in calves. In comparison, calves fed CLP or SA11 colostrum were only partially protected against BRV diarrhea or shedding.

The characterization of VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses from field samples using RT-PCR and RFLP analysis

SummaryCharacterization of the VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses (BRV) from field samples was performed using RT-PCR and RFLP analysis. After RT-PCR amplification of the full length VP7 genes and partial length VP4 genes (nucleotides 1 to 1096), four enzymes,EcoRV,NlaIV,BamHI andHpaII were used for digestion analysis. For VP7, four RFLP profiles were observed after analysis of the digests: they were designated as G6, G6s (subtype, showed about 86% nucleotide and 90% amino acid identity to reference G6 strains), G8 and G10. For VP4, three RFLP profiles were observed: designated as P[1], P[5] and P[11].The G typing analysis of 86 BRV fecal samples from 5 states, representing at least 11 different herds revealed that 60.5% (52/86) were G6, which included G6s (9/52); 19.8% (17/86) were G10; 7% (6/86) were G8; 10.4% (9/86) were G6 and G10 mixtures including two G6s samples; and 2.3% (2/86) were G6 and G6s mixtures. The P typing analysis of the same 86 fecal samples revealed that 64% (55/86) were P[5]; 28% (24/86) were P[11]; 1.2% (1/86) were P[1] and 6 samples (7%) were mixtures of either P[11] or P[5]. When the same samples were analyzed according to G and P type specificity, all possible combinations of G and P types existed in the field. The G6P[5] type was most prevalent and accounted for 46.7% (41/86) of the samples; 12.8% (11/86) were G10P[11]; 7% (6/86) were G10P[5] and an equal number were G6sP[11]. The G6P[11] (n=2), G8P[1] (n=1), G8P[5] (n=1) and G8P[11] (n=3) combinations were also observed. The following mixed BRV infections were observed in the field samples; G6sP[5+11] (n=1), G8P[5+11] (n=1), G6+G10P[5] (n=1) G6+G10P[5+11] (n=2), G6+G6sP[11] (n=1), G6+G6sP[1+11] (n=1), G6s+G10P[11] (n=1) and G6s+G10P[5+11] (n=1). Information on the G and P types and G/P combinations in the field samples should be useful for understanding the epidemiology of BRV and designing vaccination strategies to control BRV in the field.

Serial propagation of porcine enteric calicivirus in a continuous cell line. Effect of medium supplementation with intestinal contents or enzymes

SummaryA porcine enteric calicivirus (PEC) was adapted to serial propagation in a continuous porcine kidney cell line (LLC-PK) by inclusion of an intestinal contents preparation (ICP), derived from uninfected gnotobiotic pigs, in the maintenance medium. Infected cells were detected by immunofluorescent staining of cell monolayers. Attempts to propagate PEC in the presence of various intestinal enzymes of porcine origin, bacterial protease, bovine ICP, avian ICP or without medium supplementation were unsuccessful.

Molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (TGEV) and porcine respiratory coronavirus (PRCV) field isolates co-circulating in a swine herd

Summary. TGEV replicates in intestinal enterocytes and causes diarrhea in young pigs. PRCV, a spike (S) gene deletion mutant of TGEV with an altered respiratory tissue tropism, causes mild or subclinical respiratory infections. Comparisons of TGEV and PRCV strains suggest that tropism and pathogenicity are influenced by the S gene and ORF3, respectively. Recently, outbreaks of TGE of reduced virulence were reported in the field. We investigated a similar suspect TGEV outbreak of reduced virulence in nursery pigs from a swine herd in the Midwest. A TGEV strain (BW021898B) was isolated in swine testicular cells from gut contents of a diarrheic pig and three PRCV strains (BW126, BW154, BW155) were isolated from nasal swabs from normal TGEV-seronegative sentinel pigs in contact with the diarrheic pigs. Sequence analysis of the TGEV isolate in the partial S gene and ORF3/3a and ORF3-1/3b revealed high homology with enteropathogenic TGEV strains. Gnotobiotic pig inoculation and histopathological results revealed that this TGEV isolate retained virulence even though in the field outbreak the diarrheal disease was of reduced severity. Sequence analysis of the S gene deletion region of the three PRCV isolates revealed identical deletions between nt 105–752, which differ from deletions previously reported among PRCV strains. The three PRCV isolates had variable sequence changes in ORF 3/3a and ORF 3-1/3b, affecting the ORF size and amino acid sequence. Thus, sequence analysis and pathogenicity studies indicate that this TGEV isolate resembles other enteropathogenic TGEV strains. Therefore, the reduced severity of TGE observed in this herd may be due to the ongoing PRCV infections, which induce antibodies cross-reactive with TGEV and result in decreased disease severity. The results outlined in this study highlight the need to monitor the molecular epidemiology of TGEV/PRCV strains with sensitive differential diagnostic assays, followed by sequence analysis of the critical regions to identify changes and pathogenicity studies to confirm the disease potential of the TGEV isolates.

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

Abstract The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30–200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P <0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6–100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were ∼2–10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

A Survey of G6 and G10 Serotypes of Group a Bovine Rotaviruses from Diarrheic Beef and Dairy Calves using Monoclonal Antibodies in ELISA

Group A bovine rotaviruses (BRV) have been identified worldwide as a major cause of diarrhea in the young of many species, including humans. Group A rotaviruses are classified into serotypes on the basis of the outer capsid proteins, VP7 (G types) and VP4 (P types). To date, there are 14 G types of group A rotaviruses, with G1, G6, G8, and G10 described for BRV isolates. In this study, G6- and G lo-specific monoclonal antibodies (MAbs) were used in an enzyme-linked immunosorbent assay (ELISA) for the G typing of BRV-positive stool samples from diarrheic beef and dairy calves from South Dakota, Ohio, Michigan, Nebraska, and Washington, USA, and Ontario, Canada. ELISA plates were coated using a broadly reactive VP7 MAb (Common 60) or with G6- or G10-specific MAbs. BRV-positive fecal samples were diluted and added to duplicate wells, followed by the addition of polyclonal guinea pig anti-group A rotavirus serum as the secondary antibody. Several reference G6 and G10 BRV strains as well as other G types previously reported in cattle (G1, G2, G3, G8) and BRV-negative samples were included as G type specificity and negative controls. From a total of 308 field samples analyzed, 79% (244/308) tested positive by the broadly reactive VP7 MAb; of these, 54% (131/244) were G6 positive, 14% (35/244) were G10 positive, 4% (9/244) were both G6 and G10 positive, and 28% (69/244) were G6 and G10 negative. The negative samples may represent additional or undefined serotypes. The 89 samples from South Dakota were further subdivided into samples from beef (n = 43) or dairy (n = 46) herds. G6 was more prevalent in beef herd samples (67%) than in dairy herd samples (47.5%). In addition, dairy herds had higher percentages of G10-positive samples (17.5%) G6-G10 double positives (10%), and untypable samples (25%) than did beef herds, in which the prevalence of G10 positive samples was 5.5%, G6-G10 double positives was 5.5%, and untypable samples was 22%. Application of the serotype ELISA for the analysis of additional BRV samples will provide further epidemiologic data on the distribution of BRV serotypes in beef or dairy cattle, an important consideration for the development of improved BRV vaccines.

Comparative sequence analysis of the VP7 genes of G6, G8 and G10 bovine group A rotaviruses and further characterization of G6 subtypes

Summary. We previously reported the relatively high prevalence (15%) of bovine G6 subtypes (G6s) in the field using RT-PCR and restriction fragment length polymorphism (RFLP) analysis (Chang et al., Arch. Virol. 141: 1727–39). In the present study, we report the nucleotide and antigenic characterization of a G6s strain (C-8336). We also sequenced the VP7 genes of four additional bovine rotavirus (BRV) strains: another G6s (MC27), G6 (IND), G8 (C-8008) and G10 (2292B) and compared these with other bovine and human rotavirus strains. The C-8336 and MC27 strains were confirmed as P[11]G6s by RT-PCR and RFLP analysis. The VP7 genes of the C-8336 and MC27 strains showed high homology to each other (∼98%) and with other bovine G6s strains (greater than 95% homology in nucleotide and amino acid sequence with KN-4{P[11]G6s}) and also showed lower, but substantial sequence homology with human G6s strains and prototype G6 BRV (79–87% in nucleotide and 88–91% in amino acid). Serologic analysis of the cell culture adapted C-8336 strain showed that it was neutralized by a G6 monoclonal antibody (MAb IC3) to similar titers as the reference NCDV and IND G6 strains. In two-way cross-neutralization tests, strain C-8336 showed 4- to 16-fold differences in antibody titers with NCDV and IND G6 BRV. Moreover polyclonal antiserum against strain C-8336 neutralized the NCDV and IND strains weakly. Genetic variability was also observed among G8 and G10 bovine and human group A rotaviruses: the VP7 genes of the bovine C-8008 (P[5]G8) and 2292 B (P[11]G10) strains showed from 10 to 17% nucleotide divergence with those of Cody I801 (P[1]G8, bovine), A5 (P[1]G8, bovine), 69 M (P[10]G8, human) and Hal 1166 (P[14]G8, human), and I321(P[11]G10, human) and MC35 (P[14]G10, human) rotaviruses, respectively. The divergence of VP7 genes among bovine and human G6, G8 and G10 strains appears related to host species origin and their combination with VP4 (P type). The data presented in this report confirms the genetic variability among homotypic bovine and human strains and highlights the importance of continued monitoring of BRV G and P types circulating in the field for the future development and monitoring of effective vaccines.

Biochemical characterization of porcine enteric calicivirus : analysis of structural and nonstructural viral proteins

SummaryIn this report, the molecular weight and antigenicity of the proteins of a porcine enteric calicivirus (PEC) were characterized. The PEC virions were purified from intestinal contents of infected pigs and from infected cell culture lysates. The average buoyant density of the purified virus was 1.37 gm/cm3 in cesium chloride. One major structural protein with a molecular weight of approximately 58k was found in the gut and cell culture-passaged PEC using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Using immunoblotting techniques only one immunoreactive protein (58 k) ws identified. The PEC and a prototype calicivirus, feline calicivirus (FCV) were propagated in pig kidney and feline kidney (Crandell) cell lines, respectively and intrinsically labeled using [35S]methionine at various times post-inoculation (PI). SDS-PAGE of the radiolabeled proteins indicated the presence of the major structural protein (58 k) and one probable nonstructural protein (28 k) synthesized in the PEC-infected cell lysates by 12 h PI. Other minor protein bands were also evident by 24 h PI (32 k and 82 k). Only the 58 k major protein was detected by radioimmunoprecipitation (RIP) analysis using hyperimmune anti-PEC serum. SDS-PAGE and RIP analysis of FCV-infected cell lysates using hyperimmune anti-FCV serum identified a single major protein of approximately 64 k. No antigenic relationship between PEC and FCV proteins was detected by RIP analysis. The single major structural protein of PEC, the morphological appearance and size of the virus, and its average density of 1.37 gm/cm3 in cesium chloride are consistent with properties of other members of the familyCaliciviridae.

Comparative nucleotide and deduced amino acid sequence analysis of VP7 gene of the NCDV Cody (I-801) strain of group A bovine rotavirus

SummaryThe prevalent G serotypes of group A bovine rotavirus (BRV) reported are G6, G10, and less commonly, G8. Neonatal Calf Diarrhea Virus (NCDV), Lincoln and Cody strains were first isolated from diarrheic calves in Nebraska. The NCDV Lincoln strain is the currently used U.S. vaccine strain and has a G6 serotype. In this study, the complete nucleotide sequence of the VP7 gene of NCDV Cody (I-801 strain) was determined using the primer extension method. The VP7 gene nucleotide sequence homologies between Cody I-801 and established G8 rotaviruses, A5 (Thailand BRV), 678 (UK BRV), B37 (human RV) and 69M (human RV) were 84.7%, 86.4%, 84.7% and 85.9%, respectively. The deduced VP7 amino acid sequence of Cody I-801 was similar to that of A5, 678, B37 and 69M (93.6%, 95.7%, 92.6% and 95.1%, respectively). The VP7 gene nucleic acid sequence homologies between NCDV Cody (I-801) and NCDV Lincoln or B223 (G10) was 76.2% and the deduced VP7 amino acid sequence homologies between Cody I-801 and NCDV Lincoln or B223 were 82.5% and 81.3%, respectively. Thus, our sequence data suggests that the VP7 gene of Cody I-801 strain of BRV is genetically most similar to G8 rotaviruses and unrelated to the NCDV Lincoln G6 rotavirus strain.

Serotypic Analysis of Group a Bovine Rotavirus Field Specimens Using G1-, G2-, and G3-Specific Monoclonal Antibodies in ELISA

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