Molecular Characterization of HE, M, and E Genes of Winter Dysentery Bovine Coronavirus Circulated in Korea During 2002–2003

The different bovine coronavirus (BCoV) strains or isolates exhibited various degrees of substitutions, resulting in altered antigenicity and pathogenicity of the virus. In the previous our study, we demonstrated that the spike glycoprotein gene of Korean winter dysentery (WD) BCoV had a genetic property of both enteric (EBCV) and respiratory BCoV (RBCV) and were significantly distinct from the ancestral enteric strains. In the present study, therefore, we analyzed the other structure genes, the hemagglutinin/esterase (HE) protein, the transmembrane (M) protein and the small membrane (E) protein to characterize 10 WD BCoV circulated in Korea during 2002–2003 and compared the nucleotide and deduced amino acid sequences with the other known BCoV. Phylogenetic analysis indicated that the HE gene among BCoV could be divided into three groups. The first group included only RBCV, while the second group contained calf diarrhea BCoV, RBCV, WD and EBCV, respectively. The third group possessed only all Korean WD strains which were more homologous to each other and were sharply distinct from the other known BCoV, suggesting Korean WD strains had evolutionary distinct pathway. In contrast, the relative conservation of the M and E proteins of BCoV including Korean WD strains and the other coronaviruses suggested that structural constraints on these proteins are rigid, resulting in more limited evolution of these proteins. In addition, BCoV and human coronavirus HCV-OC43 contained four potential O-glycosylation sites in the M gene. However, the M gene sequence of both BCoV and HCV-OC43 might not contain a signal peptide, suggesting the M protein might be unlikely to be exposed to the O-glycosylation machinery in vivo. Nucleotide sequence data reported is available in the GenBank database under the accession no. HE, M and E genes: KWD1; AH014866, KWD2; AH014867; KWD3; AH014868, KWD4; AH014869, KWD5; AH014870, KWD6; AH014871, KWD7; AH014872, KWD8; AH014873, KWD9; AH014874, and KWD10; AH014875.     

Nucleotide and Predicted Amino Acid Sequences of All Genes Encoded by the 3′ Genomic Portion (9.5 kb) of Respiratory Bovine Coronaviruses and Comparisons Among Respiratory and Enteric Coronaviruses

The 3′-ends of the genomes (9538 bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 3′-end of the genome (9545) of the wild-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specific nucleotide and amino acid differences were disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specific nucleotide and amino acid changes were located within the S and E genes, and the 32 kDa open reading frame. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antibody responses of cattle with respiratory coronavirus infections during pathogenesis of shipping fever pneumonia are lower with antigens of enteric strains than with those of a respiratory strain

The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from <0.0001 to 0.0483. The IN and HAI antibody responses against the two EBCV strains did not differ significantly, but the lowest titers were detected with EBCV strain L9-81.     

Antibody Responses of Cattle with Respiratory Coronavirus Infections during Pathogenesis of Shipping Fever Pneumonia Are Lower with Antigens of Enteric Strains than with Those of a Respiratory Strain

The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from <0.0001 to 0.0483. The IN and HAI antibody responses against the two EBCV strains did not differ significantly, but the lowest titers were detected with EBCV strain L9-81.     

Temperature-sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses

Numerous respiratory bovine coronaviruses (RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses (EBCV). The RBCV strains had a receptor-destroying enzyme function mediated by acetylesterase (AE) activity of the haemagglutinin-esterase (HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 (OK) and LSU-94LSS-051 (LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HE proteins in COS-7 cellular extracts and in purified virus preparations were assayed at room temperature, 37 degrees C and 39 degrees C by two different assays. One assay used p-nitrophenyl acetate (PNPA) as substrate and detected serine-esterase activity; the second assay monitored AE function with bovine submaxillary mucin (BSM) as substrate. The PNPA tests confirmed that HE proteins of EBCV and RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AE activity of purified RBCV OK and LSU particles showed lower AE activity at 39 degrees C than at 37 degrees C, whereas the purified EBCV LY particles retained full AE activity at both 37 degrees C and 39 degrees C. Transiently expressed RBCV HE exhibited a marked reduction of AE activity after 40 min of assay time at 37 degrees C. In contrast, the AE activity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HE proteins specified by the RBCV strains OK and LSU contained specific amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectivelyreplicate in respiratory tissues and that HE may play a role in this tissue tropism.     

Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation

The genetic diversity of 2 pairs (AH65 and AH187) of wild type bovine coronaviruses (BCoV) sequenced directly from nasal (respiratory) and rectal (enteric) swabs of two feedlot calves with respiratory and enteric symptoms [Hasoksuz, M., Sreevatsan, S., Cho, K.O., Hoet, A.E., Saif, L.J., 2002b. Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res. 84 (1-2), 101-109.]. was analyzed. Sequence analysis of the complete genomes revealed differences at 123 and 149 nucleotides (nt) throughout the entire genome between the respiratory and enteric strains for samples AH65 and AH187, respectively, indicating the presence of intra-host BCoV quasispecies. In addition, significant numbers of sequence ambiguities were found in the genomes of some BCoV-R and BCoV-E strains, suggesting intra-isolate quasispecies. The tissue culture (TC) passaged counterparts of AH65 respiratory BCoV (AH65-R-TC) and enteric BCoV (AH65-E-TC) were also sequenced after 14 and 15 passages and 1 plaque purification in human rectal tumor cells (HRT-18), respectively. Compared to the parental wild type strains, tissue culture passage generated 104 nt changes in the AH65-E-TC isolate but only 8 nt changes in the AH65-R-TC isolate. Particularly noteworthy, the majority of nucleotide changes in the AH65-E-TC isolate occurred at the identical positions as the mutations occurring in the AH65-R strain from the same animal. These data suggest that BCoV evolves through quasispecies development, and that enteric BCoV isolates are more prone to genetic changes and may mutate to resemble respiratory BCoV strains after tissue culture passage.     

Detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons

Bovine coronavirus (BCoV) is an etiological agent associated with winter dysentery (WD), prevalent in adult cattle during the winter. Although we previously detected, isolated, and characterized BCoV strains from adult cattle with WD (WD-BCoV strains) during the winter in South Korea, the precise epidemiology, as well as the causative agent of diarrhea in adult cattle in the warmer seasons, has not been examined. We examined 184 diarrheic fecal specimens collected from 75 herds of adult cattle from seven provinces during the spring (warm), autumn (warm), and summer (hot) seasons. Bovine coronavirus-positive reactions were detected for 107 (58.2%) diarrheic fecal samples (in 47/75 herds). Of these 107 positive samples, 90 fecal samples from 33 herds tested positive for BCoV alone and 17 fecal samples from 14 herds also tested positive for other pathogens. Biological comparisons between the 9 BCoV strains isolated in this study and the 10 previously isolated WD-BCoV strains revealed that there was no receptor-destroying enzyme (RDE) activity against mouse erythrocytes in the 9 BCoV strains but the 10 WD-BCoV strains had high RDE activity. Phylogenetic analysis of the spike (S) and hemagglutinin/esterase (HE) proteins revealed that all the Korean BCoVs clustered together regardless of season and were distinct from the other known BCoVs, suggesting a distinct evolutionary pathway for the Korean BCoVs. These and previous results revealed a high prevalence and widespread geographical distribution of BCoV, suggesting that this virus is endemic in adult cattle with diarrhea in all seasons in South Korea.     

Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates.

Seven hybridoma cell lines producing monoclonal antibodies (MAbs) to the hemagglutinin-esterase (HE) glycoprotein of bovine coronavirus (BCV) were obtained from BALB/c mice that were immunized with an enriched peplomeric fraction of the winter dysentery (WD)-associated strain BCQ.2590. The specificities of these MAbs to either the dimeric (140-kDa) or the monomeric (65-kDa) form of the HE glycoprotein were determined by Western immunoblotting experiments with purified virus and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts. Four of these anti-HE MAbs inhibited the hemagglutinating activity of the virus and three weakly neutralized its infectivity in vitro. In addition, competition binding assays allowed for the definition of two independent antigenic domains (domains A and D) and two overlapping antigenic domains (domains B and C) for the HE glycoprotein of the WD-associated strain; epitopes located within antigenic domain A were not associated with hemagglutination inhibition (HAI) and virus neutralization activities. In HAI tests, the four anti-HA MAbs defined two distinct antigenic subgroups among 24 BCV field isolates that have been associated with either typical outbreaks of WD or neonatal calf diarrhea (NCD) in Quebec dairy herds from 1986 to 1996. The Quebec WD-associated strains of BCV, as well as some of the NCD-associated strains isolated since 1991, fell within a subgroup distinct from that of the prototype Mebus strain.     

Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates

It is unclear whether respiratory and enteric bovine coronavirus (BoCV) strains are distinctive in biological, antigenic and genetic characteristics. In the present study, we analyzed the nucleotide and amino acid sequence of the S1 subunit of the S glycoprotein, including the cleavage site, of both respiratory (n=5) and enteric (n=3) BoCV isolates including two paired isolates from the same feedlot animals and compared them with the prototype Mebus and two enteric and one respiratory BoCV strains from Quebec. A total of 75 polymorphic nucleotides were identified in the S1 subunit of the spike glycoprotein of BoCV isolates compared with the Mebus strain. These polymorphisms led to 42 amino acid changes at 38 distinct sites. The amino acid changes were distributed throughout the S1 subunit with clustering around residues 40-118, 146-179, and 458-531. Among these variations, only 19 amino acid substitutions altered the charge, hydrophobicity and surface probability of the protein. Based on phylogenetic analysis, our respiratory and enteric isolates clustered into two major groups with two subgroups. Although, there were only a few amino acid changes between the respiratory and enteric paired isolates, the other two respiratory isolates, one isolated from the same farm as a paired strain and the other from a different farm, showed more sequence diversity. Amino acid alterations in residues 113, 115, 118, 146, 148, 501, 510 and 531 of respiratory isolates conferred significant changes in the predicted secondary structure compared with the prototype winter dysentery (WD) and the calf diarrhea (CD) strains of BoCV. In conclusion, the data suggests that respiratory strains of BoCV may differ genetically from the classical calf enteric and adult WD strains.     

Dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves

Summary Although winter dysentery (WD), which is caused by the bovine coronavirus (BCoV) is characterized by the sudden onset of diarrhea in many adult cattle in a herd, the pathogenesis of the WD-BCoV is not completely understood. In this study, colostrum-deprived calves were experimentally infected with a Korean WD-BCoV strain and examined for viremia, enteric and nasal virus shedding as well as for viral antigen expression and virus-associated lesions in the small and large intestines and the upper and lower respiratory tract from 1 to 8 days after an oral infection. The WD-BCoV-inoculated calves showed gradual villous atrophy in the small intestine and a gradual increase in the crypt depth of the large intestine. The WD-BCoV-infected animals showed epithelial damage in nasal turbinates, trachea and lungs, and interstitial pneumonia. The WD-BCoV antigen was detected in the epithelium of the small and large intestines, nasal turbinates, trachea and lungs. WD-BCoV RNA was detected in the serum from post-inoculation day 3. These results show that the WD-BCoV has dual tropism and induces pathological changes in both the digestive and respiratory tracts of calves. To our knowledge, this is the first detailed report of dual enteric and respiratory tropisms of WD-BCoV in calves. Comprehensive studies of the dual tissue pathogenesis of the BCoV might contribute to an increased understanding of similar pneumoenteric CoV infections in humans.     

Antigenic variation among bovine enteric coronaviruses (BECV) and bovine respiratory coronaviruses (BRCV) detected using monoclonal antibodies

Summary.  Bovine coronavirus (BCV) causes neonatal calf diarrhea (CD) and is associated with winter dysentery (WD) in adult dairy cattle. It can also be isolated from the respiratory tracts of cattle entering feedlots. Monoclonal antibodies (MAbs) specific for the hemagglutinin esterase (HE) and spike (S) surface proteins of 2 bovine enteric coronavirus (BECV) strains and two bovine respiratory coronavirus (BRCV) strains were tested against 6 BECV strains and 6 recently isolated BRCV strains, in order to characterize the antigenicity of BCV strains with varied tissue tropisms. All MAbs had high immunofluorescence (IF) titers against BECV and BRCV strains, indicative of conserved cross-reactive epitopes. In hemagglutination inhibition (HI) tests, the S-MAbs were more broadly reactive than HE-MAbs. The BRCV and CD MAbs were more broadly reactive in HI than the WD MAbs. The HA activity of the Mebus vaccine CD strain was not inhibited by any of the MAbs tested. The HI activity of BRCV strain R6 was unique among the 6 BRCV isolates. In virus neutralization assays, MAbs to the BRCV strain R4 neutralized all 6 BECV strains tested. Antigenic variation exists among both BECV and BRCV strains, but it cannot be attributed soley to the clinical origin of the strain. Received February 9, 1998/Accepted July 22, 1999     

Molecular analysis of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43

Summary. Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.     

Analysis of the genome sequence of an alpaca coronavirus

Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3' end. This virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs) characteristic of Group 2 bovine coronavirus (BCoV). Phylogenetic analysis showed that the ACoV genome is clustered closely (>99.5% identity) with two BCoV strains, ENT and LUN, and was also closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (>96%), and porcine hemagglutinating encephalomyelitis virus (>93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT, respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same ancestor as BCoV ENT and LUN.     

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.     

Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups

Bovine coronaviruses (BCoVs) isolated in Japan consist of four genetic groups, as determined by phylogenetic analysis using the polymorphic region (aa 456–592) of the S glycoprotein gene. Japanese field isolates of BCoV, reference Kakegawa strain, and vaccine strain 66/H were analyzed for their antigenic properties by indirect immunofluorescence and neutralization testing. There were no significant differences observed among these BCoVs in direct immunofluorescence tests. However, antigenic differences were observed between BCoVs in the neutralization tests, although there was no clear indication of a distinct serotype. A monoclonal antibody, 4H4, against the Kakegawa strain belonging to group 1 lacked significant neutralizing activity for viruses of groups 2, 3, and 4. Therefore, we speculate that the genetic differences between these groups may have altered their antigenicity. Analysis of mutant viruses resistant to neutralization by 4H4 revealed that the antigenic site of the Kakegawa strain maps to amino acid position 284 of the S glycoprotein. This site is not homologous to a known antigenic site (aa 528) of the Quebec strain belonging to group 1, and it is not located in the conformational domain comprising domain I (aa 351–403) and domain II (aa 517–621). This amino acid constitutes a neutralization epitope of BCoV, which is distinct from aa 528 of the Quebec strain. These results indicate antigenic evolution of BCoV between the genetic groups circulating in Japan.     

Sequencing, phylogenetic analysis, and potential recombination events of infectious bronchitis viruses isolated in Korea

The S2 glycoprotein and membrane (M) protein genes and S1 glycoprotein and nucleocapsid (N) genes of 11 Korean infectious bronchitis virus (IBV) isolates were amplified by RT-PCR, cloned, and sequenced. The resultant nucleotide sequences were compared with the published sequences for non-Korean IBV strains. Korean IBV isolates formed two independent subclusters within the phylogenetic tree based on S2 glycoprotein gene sequences. However, four and two different clusters were formed in the phylogenetic tree based on S1 glycoprotein and M gene sequences, respectively. In particular, Korean IBV K446-01 and K203-02 strains appeared to be the result of recombination between an indigenous Korean IBV strains and a vaccine strain (Massachusetts serotype) currently used in Korea. The recent IBV isolate, K026-10, formed a new subgroup that was closely related to traditional Korean IBV group in a phylogenetic tree based on the S1 and S2 genes, but it was grouped into the traditional Korean IBV cluster in a phylogenetic tree based on the M and N genes. Our data show that field IBVs in Korea are continuing to evolve and that vaccine strains might actually play a critical role in the appearance of new IBV strains via recombination in the field.     

Genomic and antigenic variations of the HE glycoprotein of bovine coronaviruses associated with neonatal calf diarrhea and winter dysentery

Summary.  In this study, we attempted to define differences in the hemagglutinin-esterase (HE) glycoprotein between 11 bovine coronaviruses (BCV) recent (post-1991) and past (pre-1991) isolates from neonatal calf diarrhoea (NCD) and winter dysentery (WD) syndromes as a basis for strain differentiation related to the clinical source of the isolates. The five WD-associated BCV isolates studied could be distinguished from past NCD-isolates by their hemagglutinating (HA) properties at 4 ° and 37 °C, their receptor-destroying enzyme (RDE) activities with rat erythrocytes and lack of reactivity of these NCD isolates to four HA inhibiting (HAI) monoclonal antibodies (MAbs) directed against the HE glycoprotein of the reference WD-associated BCQ.2590 Quebec strain. Although minor or no differences could be demonstrated by comparing biological properties of the HE of WD-isolates to those of recent NCD-isolates, past NCD isolates lacked reactivity with the WD HAI MAbs, whereas recent NCD isolates displayed two distinct reactivity patterns. Attempts to define sequence differences in the HE genes of the WD and NCD strains revealed high nucleotide (NT) identities with only scattered amino acid differences, seemingly unrelated to the clinical origin of the isolates or HAI MAb reactivities. Accepted January 9, 2001 Received September 26, 2000     

Antibody responses to respiratory coronavirus infections of cattle during shipping fever pathogenesis

Summary.  Antibody responses against respiratory bovine coronavirus (RBCV) infections were monitored in cattle from the onset of a naturally occurring severe shipping fever (SF) epizootic to complete recovery of affected cattle or fatal outcomes. The infection with RBCV was detected in nasal secretions of 86 cattle, and 81 of them developed acute respiratory tract disease, including fatal pneumonia. Cattle nasally shedding RBCV at the beginning of the epizootic experienced characteristic primary immune responses with specific antibodies for hemagglutinin-esterase (HE) and spike (S) glycoproteins. Virus shedding in nasal secretions of the majority of the cattle ceased between days 7 and 14 with the appearance of HE- and S-specific antibodies. Nasal samples and lung tissues from 9 of the 10 fatal cases had high titers of RBCV, but these cattle had only IgM responses to RBCV infections. Cattle remaining negative in RBCV isolation tests entered this epizootic with antibodies against HE and S. Protection against respiratory tract disease was apparently associated with high level of opsonic and virus-neutralizing IgG2. The HE and S glycoproteins were recognized earliest by the bovine immune system while the N protein induced antibody responses during the later stage of initial infection and the early stage of reinfection. The membrane (M) glycoprotein was the least immunogenic of the major viral structural proteins. Received December 13, 2000/Accepted May 11, 2000     

Detection of a porcine rotavirus strain with VP4, VP7 and NSP4 genes of different animal origins

A new rotavirus strain, sh0902, was detected in diarrheic piglets on a farm in Shanghai, China, and its genotype was characterized as G1P[7]. Analysis of the VP4, VP7 and NSP4 genes demonstrated VP4 homology to bovine and swine rotavirus strains; the nucleotide (nt) and amino acid (aa) identities were 99.7% and 99.5%, respectively. The VP7 gene was highly homologous to that of a giant panda rotavirus strain, with 98.5% similarity at the nt level and 99% similarity at the aa level. The nucleotide sequence of the NSP4 gene displayed high homology to human rotavirus strain R479, with 99.7% identity at the nt level and 99.3% identity at the aa level. This is the first report of an unusual porcine rotavirus strain with VP4, VP7 and NSP4 genes that are highly homologous to bovine, swine, giant panda and human strains isolated at geographically distant sites (South Korea, China and India). Our data indicate that rotaviruses have circulated among humans and animals and undergone genome reassortment.