Swine Testicular Research Articles

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

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.

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.

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.

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.

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.

Evidence for a Porcine Respiratory Coronavirus, Antigenically Similar to Transmissible Gastroenteritis Virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.

Identification of a 17000 molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells.

Pulse labelling of cells with [35S]methionine at different times after infection followed by SDS-PAGE was used to resolve and to identify polypeptides designated as specific to transmissible gastroenteritis virus (TGEV)-infected swine testicular (ST) cell cultures. The major TGEV structural proteins, with apparent molecular weights of 200,000 (200K), 47K and 30K were detected in radiolabelled cell extracts by 6 h postinfection. Additionally, a 17K major polypeptide was present in infected cells but not in mock-infected control cultures. Labelling with [3H]glucosamine revealed only the 200K and 30K proteins to be glycosylated. TGEV-primed porcine lymphocytes, secondarily stimulated in vitro with sucrose gradient-purified virus, produced antibody only to the two glycoproteins (gp) indicating that the 17K polypeptide is not a surface feature of the virion. Two pigs were infected oronasally with the virulent Miller strain of TGEV and their sera were analysed by immunoprecipitation. At 25 days postinfection convalescent sera responded strongly to gp30 and gp200 and there was a weak initial response to the 17K polypeptide. Serum immunoglobulins at 60 days postinfection reacted strongly to the 17K protein while the antibody response to gp30 was significantly reduced and that to gp200 was slightly reduced.

Attachment of Salmonella to mammalian cells in vitro.

The attachment of Salmonella typhimurium strain PHL67342 to several mammalian tissue culture cell lines was investigated. Strain PHL67342 failed to attach in significant numbers to the Buffalo green monkey (BGM), swine testicular (ST), and HeLa cell lines. Significant attachment was observed with the Henle intestinal cell line. Log-phase cells of strain PHL67342 attached in greatest numbers to the Henle cells after 45 min of incubation at 37 degrees C. Attachment to the Henle cells was not affected by D-mannose or D-galactose, but was markedly inhibited by high concentrations of alpha-methyl-D-mannoside. Also, Salmonella lipopolysaccharide had no effect on the attachment of strain PHL67342 to the Henle cells. Fimbriae were not detected on the bacterial cells used in the adherence experiments. These results suggest that some bacterial factor(s) other than fimbriae and lipopolysaccharide mediate the attachment of strain PHL67342 to the Henle cells.