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Biomedical subjects

R D Wesley

Publications and source records attributed to R D Wesley.

At least 37 records · Page 2Linked to original sources

Seroconversion of pigs in contact with dogs exposed to canine coronavirus.

In order to determine if canine coronavirus (CCV) could be transmitted to pigs, two dogs were inoculated orally with virulent CCV. After 24 h, the dogs were moved to an isolation room that contained three three-day-old pigs. A wire mesh fence, allowing close contact between the animals, separated the dogs from the pigs. The dogs and pigs were observed for 14 days for clinical signs of disease. Samples of blood were obtained from dogs and pigs immediately before the dogs were inoculated with virus and 14 and 28 days later. The dogs developed mild clinical signs of an infection, but the pigs remained normal throughout the observation period. The dogs shed CCV for eight days after exposure. All three pigs developed neutralizing antibodies against CCV and transmissible gastroenteritis virus by 14 days after they were exposed to the dogs.

Animals↗

Efficacy of antiserum produced in goats and pigs to passively protect piglets against virulent transmissible gastroenteritis virus.

The protective effect of sera produced in swine and goats exposed to virulent transmissible gastroenteritis virus (TGEV) or modified-live TGEV was tested in hysterectomy-derived, colostrum-deprived three-day-old pigs. Pigs were given serum with their daily ration of milk, and their immunity to virulent TGEV was determined. The pigs were observed for ten days for clinical signs of TGEV infection. One of nine pigs receiving goat serum was protected whereas all three pigs receiving three doses of swine serum per day were protected. Because virus was not isolated from the goats after oral/intranasal vaccination, it is suggested the virus did not replicate in either the respiratory or digestive tract of the goat.

Animals↗

Detection of transmissible gastroenteritis virus using cDNA probes.

Five 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 E2 gene, which codes for the major surface glycoprotein of TGEV. Each of the 32P-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 immunofluoresence for the detection of TGEV in fecal specimens.

Animals↗

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.

Amino Acid Sequence↗

Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.

Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA.

Animals↗

Genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus.

The genome and transcriptional pattern of a newly identified respiratory variant of transmissible gastroenteritis virus were analyzed and compared with those of classical enterotropic transmissible gastroenteritis virus. The transcriptional patterns of the two viruses indicated that differences occurred in RNAs 1 and 2(S) and that RNA 3 was absent in the porcine respiratory coronavirus (PRCV) variant. The smaller RNA 2(S) of PRCV was due to a 681-nucleotide (nt) deletion after base 62 of the PRCV peplomer or spike (S) gene. The PRCV S gene still retained information for the 16-amino-acid signal peptide and the first 6 amino acid residues at the N terminus of the mature S protein, but the adjacent 227 residues were deleted. Two additional deletions (3 and 5 nt) were detected in the PRCV genome downstream of the S gene. The 3-nt deletion occurred in a noncoding region; however, the 5-nt deletion shortened the potential open reading frame A polypeptide from 72 to 53 amino acid residues. Significantly, a C-to-T substitution was detected in the last base position of the transcription recognition sequence upstream of open reading frame A, which rendered RNA 3 nondetectable in PRCV-infected cell cultures.

Base Sequence↗

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.

Animals↗

Nucleotide sequence of the E2-peplomer protein gene and partial nucleotide sequence of the upstream polymerase gene of transmissible gas gastroenteritis virus (Miller strain).

The E2-peplomer protein gene of the virulent Miller strain of transmissible gastroenteritis virus (TGEV) was sequenced from cDNA clones and compared to the E2 gene sequence of the avirulent Purdue strain. Sequence comparisons indicate that most amino acid differences occur in the N-terminal half of the E2-peplomer which represents the most exposed region of the protein. In addition, analysis of an incompletely sequenced open reading frame (ORF) to the immediate 5' side of the E2 gene indicates extensive sequence homology with the infectious bronchitis virus (IBV) F2 gene which is thought to encode a RNA polymerase.

Amino Acid Sequence↗

Characterization of Listeria monocytogenes isolates by Southern blot hybridization.

A synthetic deoxyribonucleotide probe for virulent Listeria monocytogenes, designated ADO7, was evaluated for its ability to identify restriction fragments of L. monocytogenes with nucleic acid sequences homologous with the beta-hemolysin gene by Southern blot hybridization of clinical and food isolates. The synthetic probe hybridized with three restriction fragments (approximately 1.1, 0.86, and 0.76 kb) of the serotype 1/2A isolates. Southern blot hybridization of the serogroup 4B isolates indicated that the nucleic acid sequences homologous with the beta-hemolysin gene probe were limited to a single restriction fragment of approximately 1 kb.

Animals↗

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.

Animals↗

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.

Animals↗

Nucleotide sequence of coronavirus TGEV genomic RNA: evidence for 3 mRNA species between the peplomer and matrix protein genes.

The region of the TGEV genome between the E1-matrix protein gene and the E2-peplomer protein gene has been sequenced from a cDNA clone. The consensus recognition sequence, 5'TTAA CTAAAC was found upstream from 3 large open reading frames. In coronaviruses these homologous recognition sequences are involved in the initiation of transcription suggesting that there are 3 mRNA species in this region of the TGEV genome. Northern blot analysis and nuclease S1 mapping confirmed the presence of 3 mRNA species between mRNA 3 encoding the E2-peplomer protein and mRNA 6 encoding the E1-matrix protein. The 5' regions of these 3 mRNAs encode potential polypeptides of predicted molecular weight; 7859, 27744 and 9287, respectively. The potential translation product of ORF B (27744 Da) is considerably larger than previously reported and could be difficult to distinguish by size from the E1-matrix protein.

Animals↗

Lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus.

Monoclonal antibodies (Mabs) specific for the E1 and E2 surface glycoproteins of the transmissible gastroenteritis virus (TGEV) of swine were examined either alone or in combination to evaluate their potential value in protecting neonatal pigs against a lethal dose of TGEV. Cesarean-delivered colostrum-deprived (CDCD) piglets were given one pre-challenge dose of Mab and an equal dose of the same Mab at each successive feeding after challenge. In vivo challenge results demonstrated that neither Mabs given individually nor combinations of the Mabs were able to protect neonatal pigs against a lethal dose of TGEV. However, in parallel experiments, polyclonal antibodies from immune colostrum or serum were protective.

Animals↗

Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies.

Monoclonal antibodies (MAB) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared, using their virus-neutralizing (VN) activity in the presence or absence of guinea pig, rabbit, or swine complement. The MAB against the peplomer protein had similar VN titers for TGEV in the presence or absence of complement from any source. The MAB against the matrix protein had VN activity for TGEV only in the presence of complement, whereas MAB specific for the nucleocapsid protein did not possess VN activity in the presence or absence of complement. Serum from sows containing antibodies against TGEV peplomer and matrix proteins had slightly higher VN titers in the presence of complement than in the absence of complement. High concentrations of complement from swine serum (128 U) had little effect on the infectivity of TGEV for swine testes cells, whereas 32 U of complement from rabbit serum and 64 U of complement from guinea pig serum were able to neutralize virulent and attenuated TGEV in the absence of known antibodies for TGEV. Complement (less than or equal to 8 U) from any source did not decrease the infectivity of TGEV by greater than 0.5 log10 units.

Animals↗

Complement-dependent neutralization of transmissible gastroenteritis virus by monoclonal antibodies.

Monoclonal antibodies (MAb) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared as to their virus neutralizing activity in the presence or absence of guinea pig complement. MAbs to the peplomer protein had neutralizing activity for TGEV with or without complement and the titers were similar in either case. MAbs to the matrix protein had neutralizing activity for TGEV only in the presence of complement. Antibodies to the nucleocapsid protein were without neutralizing activity with or without complement. High concentrations of guinea pig complement, but not swine complement, had neutralizing activity for TGEV even in the absence of any known TGEV antibodies.

Animals↗

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.

Animals↗

Immune response in sows given transmissible gastroenteritis virus or canine coronavirus.

Twelve pregnant sows were inoculated oral-nasally 8 weeks before farrowing with attenuated transmissible gastroenteritis virus (TGEV), tissue culture-adapted canine coronavirus (CCV), or fluids from mock-infected culture (controls). A 2nd dose of the same inoculum, one-half oral-nasally and one-half intramammarily, was given 6 weeks later. Neutralizing antibodies for TGEV and CCV were demonstrated in sera, colostrum, and milk whey from the virus-vaccinated sows. Homologous geometric mean neutralizing titers were generally 4-fold higher than were heterologous titers. After challenge exposure of the nursing pigs with virulent TGEV, average morbidity and mortality for the pigs were 81% and 34% (mortality range = 11% to 63%), respectively, in the TGEV-vaccinated group; 83% and 39% (mortality range = 15% to 83%), respectively, in the CCV-vaccinated group; and 97% and 84% (mortality range = 78% to 100%), respectively, in the controls. Seemingly, sera from swine exposed to CCV could test serologically positive for TGEV-neutralizing antibody, and TGEV and CCV share at least 1 common neutralizing determinant that may be involved in protection.

Animal Population Groups↗

In vitro and in vivo association of African swine fever virus with swine erythrocytes.

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.

African Swine Fever↗