Ronald D. Wesley

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 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.

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.

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

Abstract 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.

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

Abstract 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 ′AA TT 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.

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.

Increased litter survival rates, reduced clinical illness and better lactogenic immunity against TGEV in gilts that were primed as neonates with porcine respiratory coronavirus (PRCV)

Abstract Establishing immunological memory in female piglets at a young age with PRCV was effective in inducing a secondary immune response to a limiting dose of virulent TGEV given orally 13–18 days prior to farrowing. Subsequently, because of passive antibody transfer, the offspring of these primed gilts were more efficient in surviving a lethal TGEV challenge. An average survival rate of 89% occurred in 6 litters of piglets from primed gilts that were boosted with 2.8×106 plaque forming units (PFU) of TGEV whereas 76% of the piglets survived in three litters that suckled primed gilts boosted with 3.0×105 PFU of TGEV. Non-primed gilts given identical pre-farrowing doses of TGEV had litter survival rates of 63 and 55%, respectively. Moreover, both groups of litters from primed gilts suffered less clinical illness (as measured by the extent of weight loss post-challenge) than control litters. Priming of the piglets as neonates and boosting the pregnant gilts produced an anamnestic systemic immune response and correspondingly higher milk titers in the primed gilts compared to control animals. Thus, priming piglets with PRCV was beneficial in providing resistance to TGEV and could be incorporated into a vaccine strategy that yields better protection against TGEV.

Immunization of pregnant gilts with PRCV induces lactogenic immunity for protection of nursing piglets from challenge with TGEV

Abstract The level of passive protection against transmissible gastroenteritis virus (TGEV) was evaluated by experimentally infecting 12 pregnant gilts with different doses of porcine respiratory coronavirus (PRCV) and challenging their litters at 4 days of age. An overall survival rate of 70% was found for piglets nursing the 12 PRCV-infected gilts, compared to a 16% survival rate for piglets of nine uninfected control gilts. Six of the PRCV-infected gilts had adequate levels of immunity to resist infection with TGEV following the challenge of their litters. These six completely immuned gilts also solidly protected their litters from TGEV as shown by a 96% piglet survival rate through weaning at 3 weeks of age. The results suggest that respiratory infection with PRCV induces a substantial degree of protective lactogenic immunity against TGEV.

Restriction Enzyme Analysis in the Epidemiology of Listeria Monocytogenes

Listeria monocytogenes is a foodborne bacterial pathogen which is transmitted to humans via consumption of dairy products and vegetables. The purpose of this study was to examine the genetic diversity of L. monocytogenes by restriction enzyme analysis and to localize the hemolysin gene by hybridization with a synthetic oligomer specific for the β-hemolysin (β- listeriolysin) toxin gene. Reference strains of serotypes 1A (1/2A), IB (1/2B), 1C (1/2C), 3A, 3B, 3C, 4A, 4B, 4AB, 4C, 4D, and 4E and 39 isolates obtained from food and clinical specimens were serotyped and genomic DNA analyzed for restriction enzyme patterns. Three groups of isolates were examined: two groups were derived from environmental samples and food products, and unrelated sporadic clinical cases. The third set consisted of isolates recovered during the recent listeriosis outbreak in Los Angeles County, California. Isolates were identified as serogroup 1A (factor 1) or as serotype 4B (factor 6). Reference strains exhibited a distinct electro- phoretic pattern after Hha 1 digestion. Isolates assigned to the same serotype exhibited different restriction patterns after Hha 1 digestion. However, when restriction fragments were blotted using the Southern method and probed with a synthetic oligonucleotide specific for the β-hemolysin gene, hybridization occurred with a limited number of restriction fragments. Ten strains recovered from Mexican-style soft cheese incriminated in the California listeriosis outbreak were antigenically identified as serogroup 4B (factor 6). All 10 strains exhibited identical restriction patterns after cleavage with Hha I. Only a single restriction fragment (0.9 kb) hybridized with the β-hemolysin gene probe. This suggests a common contaminating source of Listeria for the cheese samples examined.