Miller Strain Research Articles

Porcine ex-vivo intestinal mucus has age-dependent blocking activity against transmissible gastroenteritis virus

Transmissible gastroenteritis virus (TGEV) causes high mortality in young piglets (< 3 days of age). With aging, the susceptibility/morbidity/mortality rates drop. We previously hypothesized that the age-related changes in the intestinal mucus could be responsible for this resistance. Hence, this study investigated the effect of porcine intestinal mucus from 3-day and 3-week-old pigs on the free mobility of the virulent TGEV Miller strain, and on the infection in swine testicle (ST) cells. Single particle tracking (SPT) revealed that TGEV had significantly higher diffusion coefficients in 3-day mucus compared to 3-week mucus. TGEV and charged and uncharged control nanoparticles diffused freely in 3-day mucus but were hindered by 3-week mucus in the diffusion model; TGEV mimicked the diffusion behavior of negatively charged carboxylated particles. Inoculation of ST cells with TGEV in the presence of 3-week mucus resulted in a significantly lower average number of infected cells (30.9 ± 11.9/5 fields) compared with 3-day mucus (84.6 ± 16.4/5 fields). These results show that 3-week mucus has a significant TGEV-blocking activity compared to 3-day mucus in free diffusion and infection of the underlying susceptible cells. Additionally, a label-free proteomics analysis revealed an increased expression of mucin 13, known for negatively regulating the tight junctions in intestinal epithelium, in 3-day-old pigs. In 3-week-old pigs, a higher expression of mucin 2, a type of secreted mucin which is known for inhibiting coronavirus infection, was observed. Concludingly, this study demonstrated a protective effect of 3-week mucus against viral infections.

A Study on Low Cycle Fatigue Life Assessment of Notched Specimens Made of 316 LN Austenitic Stainless Steel

Abstract The local strains obtained from the best-known analytical approximations, namely, Neuber's rule, equivalent strain energy density method, and linear rule, were compared with those resulting from finite element analysis. It was found that apart from Neuber's rule with the elastic stress concentration factor Kt, all the aforementioned analytical methods underestimate the local strains for all notch root radius, strain amplitude levels, at room temperature and 550 °C. Neuber's rule with Kt slightly overestimates the maximum strains for lower notch root radius, namely, 1.25 mm, at high temperature. Based on the analytically and numerically obtained notch root strains, the fatigue lives were estimated using the Coffin–Manson–Basquin equation. Besides, a numerical assessment of fatigue lives was made based on Brown–Miller and maximum shear strain multi-axial fatigue life criteria. It was found that all these methods provide inaccurate fatigue life results for all notch root radius, strain amplitude level, and under both temperatures conditions. Therefore, a new method was suggested, for which only the applied strain amplitude is needed to calculate the fatigue life of notched components. It was revealed that the suggested method provides a good fatigue life prediction at a higher temperature loading state.

Establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses

A stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. Because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. As subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. First, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain RVA/pig-tc/BEL/RV277/1977/G1P[7] and different other rotavirus genotypes (fecal samples containing G5P[7], G5P[13], G9P[23], G4P[6]). Next, the TGEV Purdue strain infected both ileocytes and colonocytes whereas the Miller strain only infected ileocytes. Last, the PEDV CV777 Vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. The infectivity of the CV777 Vero adapted strain was higher when the cells were cultured without fetal bovine serum and the CV777 fecal suspension only infected the ileocytes cultured without fetal bovine serum. In conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses.

Reverse transcription-PCR assays for the differentiation of various US porcine epidemic diarrhea virus strains

Concurrently, several porcine epidemic diarrhea virus (PEDV) variants are circulating in US swine farms, including the original US and the spike insertion-deletion (S-INDEL) strains. In this study, reverse transcription (RT)-PCR assays for the detection and differentiation of different US PEDV variants were developed based on the differences in the S1 domain of the spike (S) gene. This assay successfully differentiated three PEDV strains: PC22A (the original US virulent), Iowa106 (S-INDEL), and PC177 (S-197DEL) that was derived from cell culture adaptation and has a 197 amino acid-deletion in the S1 domain. The assays did not amplify the porcine deltacoronavirus OH-FD22 strain or transmissible gastroenteritis virus Miller strain. It is the first report on the development of RT-PCR assays allowing the detection and differentiation of all major types of US PEDV variants.

Low‐cycle fatigue of 316L stainless steel under proportional and nonproportional loadings

AbstractThis paper discusses low‐cycle fatigue characteristics of 316L stainless steel under proportional and nonproportional loadings. Tension–torsion multiaxial low‐cycle fatigue tests were performed using five strain paths. Additional hardening was observed under nonproportional loadings and was more significant in tests with larger nonproportionality. Mises equivalent strain, Smith–Watson–Topper, Fatemi–Socie, Kandil–Brown–Miller and nonproportional strain parameters were applied to the experimental data to evaluate the multiaxial low‐cycle fatigue damage. The applicability of the damage laws to practical design was discussed.

Complete genomic sequence of the coronavirus transmissible gastroenteritis virus SHXB isolated in China.

A strain of transmissible gastroenteritis virus (TGEV), SHXB, was isolated in Shanghai, China. The complete genome of strain SHXB was sequenced, and its sequence was compared those of other TGEV strains in the GenBank database. The comparison showed that there were no insertions or deletions in the 5′ and 3′- non-translated regions, in the nonstructural genes ORF1, ORF3, and ORF7, or in the genes encoding the structural proteins envelope (E), membrane (M) and nucleoprotein (N). A phenomenon in common with other strains was that nucleotide (nt) 655 of the spike (S) gene was G, and a common change in nt 1753 of the S gene was a T-to-G mutation that caused a serine-to-alanine mutation at amino acid 585, which is in the region of the main major antigenic sites A and B of the TGEV S protein. A 6-nt deletion was also found at nt 1123-1128 in all Purdue strains except the strain Virulent Purdue. Phylogenetic analysis showed that TGEV SHXB was closely related to the Purdue strains and shared a common ancestor with the Miller strains as well as strain PRCV-ISU-1.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-014-2080-9) contains supplementary material, which is available to authorized users.

Complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus

Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506–706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522–744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.

Experimental Infection of Piglets with Transmissible Gastroenteritis Virus: a Comparison of Three Strains (Korean, Purdue and Miller)

Eighty-four colostrum-deprived piglets aged 1 day were inoculated with either a Korean strain of transmissible gastroenteritis virus (TGEV) or one of two American strains (Purdue and Miller). The purpose was to compare, by morphometric analysis and in-situ hybridization, the Korean strain with the two American strains in respect of pathogenicity and viral distribution over a period of 72 h. The progression of infection in pigs infected with the Korean strain was much slower than that in pigs infected with either of the two American strains. At 6 h post-inoculation (hpi), the mean score of the villous height/crypt depth (VH/CD) ratio of pigs inoculated with the Purdue or Miller strain was significantly less than that of pigs inoculated with the Korean strain or uninfected control pigs. At 12–72 hpi, however, the VH/CD ratio of all infected pigs was significantly less than that of control pigs. In-situ hybridization showed significant differences between the Korean and American strains in terms of the amount of TGEV nucleic acid at 6, 12 and 60 hpi. TGEV nucleic acid was detected in the duodenum, jejunum and ileum of pigs inoculated with the Purdue or Miller strain but only in the jejunum and ileum of those inoculated with the Korean strain. The results suggested that the Korean strain was less virulent than the two American strains.

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets.

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.

Downstream Ribosomal Entry for Translation of Coronavirus TGEV Gene 3b

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5′-terminal on mRNA 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3–1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

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.

Field isolates of transmissible gastroenteritis virus differ at the molecular level from the Miller and Purdue virulent and attenuated strains and from porcine respiratory coronaviruses.

The diversity in selected regions of the transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) genomes was analyzed among known TGEV and PRCV strains and field isolates. The N-terminal half of the spike (S) glycoprotein gene and open reading frames (ORF) 3, 3-1 and 4 were amplified by reverse transcriptase reaction and polymerase chain reaction (RT/PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. Reference TGEV strains (Miller and Purdue) and a PRCV strain (ISU-1), and TGEV and PRCV field isolates were analyzed. Based on the size of the ORF 3, 3-1 and 4 RT/PCR products, TGEV and PRCV strains could be quickly and easily differentiated into three groups designated TGEV Miller, Purdue types and PRCV. By RFLP analysis of the N-terminal region of the S glycoprotein gene and ORFs 3, 3-1 and 4, TGEV and PRCV strains were differentiated into five groups using the restriction enzyme Sau3AI. Sequence analysis of a PCR product in the ORFs 3, 3-1 and 4 from virulent and attenuated Miller strains demonstrated additional differences in that region which have been correlated with a change in virulence of TGEV isolates.

Active and passive immune responses to transmissible gastroenteritis virus (TGEV) in swine inoculated with recombinant baculovirus-expressed TGEV spike glycoprotein vaccines

Abstract Objective Baculovirus-expressed transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein vaccines were inoculated parenterally in swine to determine whether such vaccines could induce serum and whey virus-neutralizing (VN) antibodies and protective lactogenic immunity for TGEV-challenge-exposed pigs. Animals and Procedures 3 recombinant baculoviruses that expressed full or partial length TGEV Miller strain S glycoproteins were inoculated SC in 17 conventionally raised 11-day-old TGEV-seronegative pigs to determine whether the recombinant S glycoproteins would elicit serum VN antibodies. Eleven TGEV-seronegative pregnant sows were inoculated SC or intramammarily with subunit vaccines (R2-2 or R3-5) or control proteins. Pigs born to 9 of the 11 sows were challenge exposed at 4 to 5 days of age with the virulent Miller strain, and passive immunity was assessed. Serum and whey antibody responses to TGEV were analyzed by VN and ELISA testing. Results Recombinant S glycoproteins (R2-2 or R3-5) containing the 4 major antigenic sites induced similar VN antibody titers to TGEV in serum and colostrum, but low (some sows) or no VN antibody titer was detected in milk. Subcutaneous inoculation of sows with R2-2 or R3-5 elicited IgG, but not IgA antibodies to TGEV in colostrum. Morbidity was 100%, and mortality ranged from 20 to 80% in TGEV challenge-exposed pigs nursing sows inoculated SC or intramammarily with TGEV S glycoprotein vaccines. Conclusions and Clinical Relevance Parenterally administered TGEV S glycoprotein vaccines elicit VN antibodies to TGEV in serum and colostrum that do not fully provide active or passive immunity in swine. (Am J Vet Res 1997;58:242–250)

Cloning and sequencing of a 8.4-kb region from the 3′-end of a Taiwanese virulent isolate of the coronavirus transmissible gastroenteritis virus

The nucleotide sequence (8396 nucleotides) was determined, from the 3′-end of the putative polymerase gene to the poly(A) tail, for a Taiwanese virulent isolate, TFI, of transmissible gastroenteritis virus (TGEV). The TFI nucleotide sequence had very high identity to the British virulent field isolate FS772/70 (98.3%), the attenuated Purdue 115 (96.7%) and from the S gene to ORF-4 gene region, to the low passaged virulent Miller (98.3%) strains of TGEV. Comparison of the TFI S protein sequence with those determined from other TGEV strains and those of the TGEV variant, porcine respiratory coronavirus, isolated from Europe and North America showed that they had changed very little over a period of 4 decades. The two extra amino acids found to be present in the spike proteins of the virulent FS772/70 and Miller strains when compared to the avirulent Purdue strain were found to be present in the TFI strain. The genomic organisation of the TFI strain was the same as that of the other TGEV viruses.

The use of arms PCR and RFLP analysis in identifying genetic profiles of virulent, attenuated or vaccine strains of TGEV and PRCV.

The use of ARMS (amplification refractory mutation system) PCR coupled with RFLP (restriction fragment length polymorphism) analysis has been used to identify a unique genetic marker on the Ambico oral vaccine strain. This method was also used to characterize the genetic profiles of a number of other TGEV strains. This procedure takes advantage of the nucleotide differences between the Ambico strain, and the Miller and Purdue strains. Within the S gene there are three nucleotide differences between the Ambico strain and the published Purdue sequence. There are additional nucleotide differences in the structural and non-structural gene sequences, but we have chosen to focus on the differences contained within the S gene. The Ambico strain has a closer sequence homology to the Purdue strain than to the Miller strain. The Ambico and Purdue strains contain a six nucleotide deletion at position 1122 that is not present in the Miller published sequence or the ISU-1 strain of PRCV (based on our PCR experiments). We have designed a 5' oligo whose sequence is homologous to a region located 80 nucleotides upstream of the TGEV and PRCV S gene initiation codons to be used in conjunction with either of two 3' oligos whose sequences are identical with the exception of the last six nucleotides of their 3' ends. When utilized with the appropriate PCR conditions, these oligos can differentiate between PRCV, Miller and Purdue prototype virus strains. These PCR products were then subjected to RFLP analysis using four separate restriction enzymes (BstE II, Alw26 I, Dra III, or MspA1 I). We have used this procedure to analyze six TGEV vaccine strains, intestinal derived virulent viruses, cell cultured viruses at different cell passage numbers, and field isolates of TGEV or PRCV.

Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains. Correlation with antigenicity and pathogenicity.

Transmissible gastroenteritis virus (TGEV) causes an economically important enteric disease of swine. Differences in the pathogenicity, antigenicity and tissue tropism have been observed among porcine coronaviruses. Although porcine respiratory coronavirus (PRCV) is antigenically similar but not identical to TGEV isolates, these respiratory coronaviruses differ markedly in pathogenicity and tissue tropism compared to TGEV isolates. Using a reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay, TGEV and PRCV isolates were assigned to several distinct groups. By RFLP analysis of the 5' region of the S gene, TGEV strains were differentiated into 4 groups using the restriction enzyme Sau3AI. A fifth Sau3AI group contained the PRCV isolates. These 5 groups correlated with antigenic groups previously defined using monoclonal antibodies in our laboratory. Several restriction enzymes could be used to differentiate the TGEV strains into Miller and Purdue types. Analysis of a PCR amplified product in the 3 and 3-1 genes indicated the RT/PCR-RFLP assay results for TGEV Miller strains could be correlated with lower virulence created by passage in cell culture.

Competition ELISA, using monoclonal antibodies to the transmissible gastroenteritis virus (TGEV) S protein, for serologic differentiation of pigs infected with TGEV or porcine respiratory coronavirus

SUMMARY Monoclonal antibodies (MAB) to subsite A (25C9) and subsite D (44C11) of the S protein of transmissible gastroenteritis virus (tgev) were used in a blocking elisa on fixed tgev-infected swine testis cells to differentiate sera from pigs experimentally inoculated with either tgev or porcine respiratory coronavirus (prcv). Serum samples were obtained from pigs at various intervals from postinoculation day (pid) 0 through at least PID 22 to 40. Eleven-day-old pigs, seronegative for tgev-neutralizing antibodies at the time of inoculation, were inoculated orally and nasally with either the virulent Miller (M5C) strain or the attenuated Purdue (P115) strain of tgev, or with the ISU-1 strain of prcv. Gastroenteritis was observed in 100% of the M5C-tgev-inoculated pigs; but clinical signs of disease were not observed in either the PI 15-tgev- or prcv-inoculated pigs. Virus-neutralization (vn) antibody titer in sera was determined by use of a plaque-reduction assay. Blocking ELISA antibody titer for subsites A and D was determined from the serum dilution that produced 50% reduction in the absorbance values when it competed with biotinylated MAB 25C9 and 44C11, respectively. In sera from the inoculated pigs, the VN antibody titer began to increase by pid 7 and reached maximum by PID 15 to 16. For pigs inoculated with tgev M5C, subsite A and subsite D blocking antibody titers in the serum paralleled the vn antibody titer, began to increase after pid 7, and reached maximum by pid 15 to 16. The blocking antibody titer to sub-sites A and D began to increase in the PI 115- tgev-inoculated pigs after pid 15 to 16 and reached maximum by PID 22 to 26. Blocking antibody titer to subsite A in prcv-inoculated pigs behaved similarly to blocking antibody titer to subsite A in the M5C-tgev-inocuIated pigs, reaching maximum by PID 15 to 16; however, blocking antibody titer was not detected for subsite D up to PID 24 (the latest time point examined) in sera from the prcv-inoculated pigs. Serum antibody responses and clinical signs of disease were monitored in pigs initially inoculated with either M5C-tgev or -prcv and challenge-exposed with M5C-tgev on PID 24. Clinical signs of gastroenteritis were not observed in the M5C-tgev-inoculated pigs after challenge-exposure with M5C-tgev. Low increases in vn antibody titer and in sub-site A or D blocking antibody titer were detected in the M5C-tgev-inoculated and challenge-exposed pigs. Of the 12 pigs initially inoculated with prcv then challenge-exposed with M5C-tgev, 5 pigs developed diarrhea, the vn and subsite A antibody blocking titers began to increase by postchallenge-cxposure day (PCD) 2 and reached maximal titer by PCD 9, increasing approximately 100-fold above the prechallenge-exposure titer. Subsite D antibody-blocking titer began to appear after PCD 9 and, by PCD 12, had reached nearly the same level as that for the primary response to the M5C-tgev inoculation.

Detection of transmissible gastroenteritis virus using cDNA probes

SummaryFive cDNA probes prepared from molecular clones representing genomic RNA sequences of the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were used in a dot blot hybridization assay to detect TGEV in cell culture and fecal specimens. Two clones (pA2 and pB4) represent nucleotide base pairs at the 3′ terminus of the Miller TGEV genome. The other three clones represent various portions of the 5′ end of the E 2 gene, which codes for the major surface glycoprotein of TGEV. Each of the32P-labeled cDNA probes hybridized to the virulent Miller, attenuated Purdue and four field strains of TGEV. The probes detected 200 to 2000 pg of TGEV RNA extracted from density gradient purified virions and did not hybridize RNA from mock-infected cell cultures, porcine rotavirus or antigenically unrelated coronaviruses. The pB4 and Hpa-1600 probes detected TGEV RNA sequences in 79 and 88%, respectively of 34 field samples identified as TGEV positive by the immunofluorescence assay and electron microscopy (EM). The pD24 clone, which is able to differentiate TGEV from the antigenically related coronaviruses, also compared favorably with conventional methods of EM and immunofluorescence for the detection of TGEV in fecal specimens.

Differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cDNA probes specific for the 5' region of the S glycoprotein gene

Two cDNA clones prepared from the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were identified, and their nucleotide sequences were determined. The clones were nonoverlapping and located in the 5' region of the S glycoprotein gene. Their nucleotide and predicted amino acid sequences were compared with published sequences of the attenuated Purdue strain of TGEV and feline infectious peritonitis virus (FIPV). TGEV clone pE21 contained 381 bp of the S glycoprotein gene and had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV and over 87% nucleotide and amino acid sequence homology with FIPV. TGEV clone pD24 contained 267 bp of the S glycoprotein gene. It had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV but only 54% nucleotide sequence homology and 24% amino acid sequence homology with FIPV. A probe prepared from pD24 could differentiate TGEV from porcine respiratory coronavirus and other antigenically related coronaviruses, FIPV, feline enteric coronavirus, and canine coronavirus in a dot blot hybridization assay.

Genetic basis for the pathogenesis of transmissible gastroenteritis virus

Intracellular RNAs of an avirulent small-plaque (SP) transmissible gastroenteritis virus variant and the parent virulent Miller strain of transmissible gastroenteritis virus were compared. Northern RNA blotting showed that the Miller strain contained eight intracellular RNA species. RNAs 1, 2(S), 5, 6(M), 7(N), and 8 were similar in size for both viruses; however, the SP variant lacked subgenomic RNAs 3 and 4. Instead, the SP virus contained an altered RNA species (delta 4) that was slightly smaller than RNA 4. S1 nuclease protection experiments showed a deletion of approximately 450 nucleotides in the SP genome downstream of the peplomer S gene. Sequencing of cDNA clones confirmed that SP virus contained a 462-nucleotide deletion, eliminating the transcriptional recognition sequences for both RNAs 3 and 4. These RNAs encode open reading frames A and B, respectively. An alternative consensus recognition sequence was not readily apparent for the delta 4 RNA species of SP virus. Since open reading frame A is missing in SP virus, it is not essential for a productive infection. The status of the potential protein encoded by open reading frame B is not clear, because it may be missing or just truncated. Nevertheless, these genes appear to be the contributing entities for transmissible gastroenteritis virus virulence, SP morphology, tissue tropism, and/or persistence in swine leukocytes.