Swine Testicular Cells Research Articles

Reproductive failure in wild boars associated to porcine parvovirus infection and in vivo and in vitro characterization of the causal isolate

Investigations were made to identify the causal agent of an acute outbreak of abortions in a domesticated herd of wild boar. Only porcine parvovirus (PPV) was isolated from samples of organs from the still-born sucklings and mummified aborted fetuses. The isolated virus hemagglutinated erythrocytes of guinea pig, murine, rat, and chicken. Identity of the virus, designated the BQ strain, was confirmed by the production of a specific cytopathic effect on susceptible cells and by the results from ELISA, PCR, immunofluorescence assay, and electron microscopy. PPV BQ strain was adapted to growth in a swine testicular cell line. When inoculated into healthy sows, PPV BQ caused the same reproductive disorder observed in the affected herd.

Nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus

There is little information on the role of nitric oxide ( NO) in innate immunity to respiratory coronavirus (CoV) infections. We examined NO levels by Greiss assay in bronchoalveolar lavage (BAL) of pigs infected with either porcine respiratory coronavirus (PRCV) or porcine reproductive and respiratory syndrome virus (PRRSV), a member of Nidovirales, like CoV. The antiviral effects of NO on these two viruses were tested in an in vitro system using a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We detected a large increase in NO levels in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with increased NO. Moreover, NO levels in cell culture medium of PRRSV-infected alveolar macrophages (AMs) did not differ from that of mock-infected AMs. Antiviral assays showed that NO significantly inhibited PRCV replication in swine testicular (ST) cells, whereas PRRSV was not susceptible to NO based on the conditions tested. Our study suggests that unlike PRRSV which induces apoptosis in AMs, respiratory CoVs such as PRCV that infect pulmonary epithelial cells and cause cytolysis, induce NO production in the respiratory tract. Thus, NO may play a role in innate immunity to respiratory CoV infections by inhibiting viral replication.

Isolation and characterization of the first Chinese strain of porcine Teschovirus-8

Investigations were carried out to identify the causal agent of acute diarrhea, respiratory distress, and death of pigs on a swine farm in Jilin Province, northern China. Only porcine Teschovirus (PTV, designated as PTV-8 Jilin/2003) was isolated from samples of organs. The presence of PTV was confirmed by the production of a specific cytopathic effect on susceptible cells and by the results of the immunoperoxidase monolayer assay (IPMA), polymerase chain reaction, and electron microscopy. Other pathogenic agents causing diarrhea, respiratory distress, and death (including porcine rotavirus, transmissible gastroenteritis virus of swine, porcine epidemic diarrhea virus, classical swine fever virus, pseudorabies virus, porcine circovirus, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, Mycoplasma, Leptospira, Streptococcus, Listeria, and Brucella species) were excluded as possible causal agents because they were not associated consistently with the disease of the pigs. PTV-8 Jilin/2003 was adapted to grow in swine primary kidney (PK-15) cells and in a swine testicular cell line (ST cells). When inoculated into healthy pigs, PTV-8 Jilin/2003 caused the same symptoms as those observed in the affected herd. It is concluded that PTV-8 Jilin/2003 was the causal agent of this disease.

Sequence analysis of the ORF 7 region of transmissible gastroenteritis viruses isolated in Korea

Three (KT2, 133, and DAE) transmissible gastroenteritis viruses (TGEVs) were isolated from pigs suspected of having TGE in Korea. One, KT2 (KT2-L), was passaged 128 times (KT2-H) in swine testicular cells. The open reading frame 7 (ORF 7) gene from each of the four TGEVs (KT2-L, KT2-H, 133, and DAE), which is located at the 3' end of the TGEV genome, was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified PCR products were cloned, sequenced, and compared with published sequences of non-Korean TGEV strains. Differences in replication and cytopathic effect (CPE) between the KT2-L and KT2-H strains in swine testicular cells were investigated. Korean TGEV field strains had 94.8-99.6% nucleotide and 92.1-98.7% amino acid sequence similarity with each other, and 87.8-100.0% nucleotide and 84.2-100.0% amino acid sequence similarity with non-Korean TGEV strains. Compared to the original KT2-L strain, the KT2-H strain differed by 2.2 and 3.9% in nucleotide and amino acid sequences, respectively. Specifically, the KT2-H had six nucleotide and two amino acid deletions compared to the original KT2-L strain. In phylogenetic analysis of the ORF 7 gene, Korean TGEV strains were clustered into two groups. One group (KT2-L, KT2-H, 133) was related to TGEV strains isolated in Japan. Another Korean TGEV isolate (DAE) was related to a strain from China and one from the USA. The Korean TGEV isolates appear to have evolved from a separate lineage of TGEV strain. Differences in growth rate and CPE between the KT2-L and KT2-H strains were discovered in swine testicular cells (STCs). The KT2-H strain exhibited a higher replication rate than KT2-L and produced a CPE distinctly different from that of the KT2-L strain.

Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene

Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.

Effect of conditions of three dimensional clinostating on testicular cell machinery

Our scope was to study the effects of on ground random positioning machine rotation on swine testicular cells in culture. Cells of the 2 n karyotype line, from trypsinized swine testes, were submitted to modeled microgravity ( μ G ) using a 3D RPM from 15 min to 24 h. The cultured cells were then fixed and submitted to immunohistochemistry using antibodies to steroid dehydrogenases, heat shock proteins and the sodium pump ( Na + / K + ATPase). The results revealed that, after 15 min at modeled μ G , all the cells showed damages at cytoskeletal level. The immunoreactions for 3 β HSD , 17 β HSD and Na + / K + ATPase were almost abolished. After 24 h of treatment, the presence of the enzymes was restored, and small heat shock proteins were strongly immunostainable. On the other hand, in the 1 × G cultures, the expression of HSPs was very weak. We conclude that modeled μ G by random positioning of cells affects testicular cells in culture for a short time, while the normal activity of the cells is restored after 24 h.

Analysis of early region 4 of porcine adenovirus type 3

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1–p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to ∼4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to ∼7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.

Heterologous gene expression from transmissible gastroenteritis virus replicon particles.

We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600-10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected approximately 36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident approximately 18 h postinfection. Leader-containing GFP transcripts containing the ORF 3B and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines.

Analysis of early region 1 of porcine adenovirus type 3.

To identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1B(small), or E1B(large) protein. Sera against E1A, E1B(small), and E1B(large) immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1B(small), DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1B(small) and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1B(small), and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1B(large) coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1B(small) is not essential for replication of PAV-3, and (c) E1B(large) is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene.

Transmissible Gastroenteritis Virus Induces Apoptosis in Swine Testicular Cell Lines but not in Intestinal Enterocytes

Evidence of apoptosis caused by infection with the Purdue strain of transmissible gastroenteritis virus (TGEV) was sought in vitro (in infected swine testicular [ST] cells) and in vivo (in the intestinal tissues of infected piglets). The methods used were (1) DNA electrophoresis for detection of DNA fragmentation, and (2) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick and labelling (TUNEL). DNA “laddering” was detected in TGEV-infected ST cells only. Numerous signs of apoptosis were detected in TGEV-infected ST cells by TUNEL assay, the positive (dark brown) staining reaction being present in the majority of cell nuclei, without background staining. No such staining was seen in TGEV-infected enterocytes at various times after inoculation of piglets. Thus, it would appear that apoptosis does not occur in the enterocytes of piglets infected with TGEV.

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.

A Rapid Virus Neutralization Assay for Newcastle Disease Virus with the Swine Testicular Continuous Cell Line

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.

The S gene of canine coronavirus, strain UCD-1, is more closely related to the S gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus

To gain insight into the genetic relationships among six canine coronavirus (CCV) strains, the variable region of the spike (S) protein gene was sequenced. The CCV strains were: two ATCC reference strains, the Insavc-1 vaccine strain, the National Veterinary Services Laboratories (Ames, IA) challenge strain, and two California field isolates (UCD-1 and UCD-2) from the 1970s. All six strains, downstream of the nucleocapsid (N) protein gene, had sufficient size for an ORF 7b, and thus, none were transmissible gastroenteritis virus (TGEV)-like since TGEV lacks ORF 7b. By sequence analysis of the variable domain at the 5′ end of the S gene, five of the six CCV strains had a high degree of identity with feline infectious peritonitis virus (FIPV). However, one CCV field isolate (UCD-1) was different and had a high degree of identity with the 5′ end of the TGEV S gene. This suggests that RNA recombination occurred at this site between antigenically related coronaviruses. The low passage field isolates, UCD-1 and UCD-2, varied in their initial infectivity for swine testicular cells suggesting that sequence differences in the variable domain of the S gene may account for biological variation among CCVs.

Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.

Development of porcine adenovirus-3 as an expression vector.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

Persistent infection promotes cross-species transmissibility of mouse hepatitis virus.

Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.

Complete Development of the Porcine Coccidium Isospora suis Biester, 1934 in Cell Cultures

Development from inoculated sporozoites to unsporulated oocysts of Isospora suis Biester, 1934 is described in a swine testicular (ST) cell line. Sporozoites penetrated ST cells within 1 hr postinoculation (PI). Development was initially by endodyogeny to produce binucleate type I meronts and type I merozoites. Division by endodyogeny continued during the 13-day observation period and type I merozoites were the developmental stages most abundant at observation periods >3 days PI. Mutinucleate type II meronts and type II merozoites were first observed 7 days PI. Gamonts and oocysts were present 12 days PI. Oocysts did not sporulate in vitro. The ultrastructural features of stages were similar to those that occur in the pig host.

Adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine.

A virulent bovine coronavirus isolate (newborn calf diarrheal) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). The same cells have been used to produce modified live porcine rotavirus and coronavirus vaccines that are federally licensed and sold worldwide. Growth of the bovine coronavirus resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Virus yields were relatively high in the ST cells and active replication was confirmed by immune electron microscopy and immunofluorescence. Adaptation of bovine coronavirus to a diploid swine cell line has not been previously reported. Different cell culture passage levels of bovine coronavirus were evaluated by oral inoculation of clean-catch, colostrum-deprived calves. A passage level of bovine coronavirus was identified that multiplied in the calf without the clinical signs of disease associated with virulent passages. The modified live bovine coronavirus vaccine remained safe and efficacious even after 5-backpassages in calves. Further efficacy studies have shown that the modified live bovine coronavirus vaccine significantly protected calves from highly virulent challenges with either winter dysentery or newborn calf diarrheal coronavirus isolates.

Biosynthesis and morphogenesis of group C rotavirus in swine testicular cells.

Polypeptide synthesis and morphogenesis of a group C rotavirus (AmC-1) adapted to a continuous swine testicular cell line was examined. SDS-PAGE analysis of 35S methionine labeled infected cell lysates revealed 9 viral polypeptides (122, 98, 79, 78, 43, 41, 35, 24, and 20 kD). Viral polypeptide synthesis appeared to be maximal at 7-10h post infection. Purified group C virus grown in the presence of trypsin was found to contain seven structural polypeptides (122, 98, 79, 53, 43, 41, and 30 kD) by protein blotting and five polypeptides (98, 79, 78, 43, and 41 kD) by immunoprecipitation with a hyperimmune rabbit antisera. Tunicamycin treatment, Concanavalin A binding, protein blotting, endo-H treatment and 2,6H-mannose labeling suggested that group C rotavirus contains one structural glycoprotein (41 kD) with a corresponding precursor mol. wt. of 37 kD and one not previously identified nonstructural glycoprotein (24 kD) with a corresponding precursor mol. wt. of < or = 20 kD. Electron microscopy of infected swine testicular cells revealed an assembly process for group C rotavirus similar to group A, with single-shelled particles budding through the rough endoplasmic reticulum with concomitant acquisition of a transient membrane.

Group C rotavirus requires sialic acid for erythrocyte and cell receptor binding.

Current immunological and biochemical information regarding the hemagglutinin and virus-cell interactions of rotavirus is obtained exclusively from studies with group A rotaviruses. In this study, I report that the immunologically and genetically distinct group C rotavirus also possesses a hemagglutinin. The viral hemagglutinin was identified on a cultivable porcine group C rotavirus strain (strain AmC-1) by using agglutinated human and guinea pig erythrocytes. Neuraminidase treatment of fresh human erythrocytes or blocking with glycophorin A or fetuin prevented hemagglutination. Infection of swine testicular cells with group C AmC-1 virus was also prevented by glycophorin A, fetuin, and neuraminidase treatment, suggesting that sialic acid constitutes an essential part of the cell receptor.