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An immunohistochemical investigation of porcine epidemic diarrhoea.

A sudden outbreak of epidemic diarrhoea of piglets occurred in Japan, the principal features being watery diarrhoea, dehydration and high mortality in newborn animals. The microscopical lesions were villous atrophy in the small intestine, the villous enterocytes being vacuolated and cuboidal in shape. The villus-crypt ratio was severely reduced, varying from 1:1 to 3:1. Transmission electron microscopy showed numerous coronaviruses within the cytoplasm of enterocytes and among microvilli. Specific antigens of porcine epidemic diarrhoea (PED) virus were detected in the cytoplasm of enterocytes by the streptavidin-biotin (SAB) technique. Infected cells, which were most abundant in the villous epithelia of the jejunum and ileum, were present in small numbers in the large intestine, the crypt epithelia, the lamina propria and Peyer's patches. The study suggests that the SAB technique is useful for the diagnosis of PED.

Animals↗

Specific cytotoxic lymphocyte response in swine against structural proteins of transmissible gastro-enteritis virus: a study using lymphoblastoid cell line and recombinant vaccinia virus.

To determine the specificity, if any, of cellular cytotoxicity against transmissible gastro-enteritis virus (TGEV) infected cells, we developed a test using B lymphoblasts from a MHC histocompatible (d/d haplotype) cell line (L14), as stimulating and target cells. These cells were previously infected with recombinant vaccinia virus including different TGEV structural genes, either the spike (vS), membrane (vM) or nucleoprotein gene (vN). Lymphocytes from a TGEV immunized (d/d) swine developed a cytotoxic activity after secondary in vitro stimulation in the presence of vS, vM or vN infected L14 cells. The cytotoxic activity was induced and directed against the homologous vS and vM infected cells but no cytotoxic activity occurred at all against vN infected cells. While vM infected cells induced a cytotoxic activity against vM infected cells only, vS infected cells stimulated a cross-reactive cytotoxic activity against vM and vN infected cells in addition to that against vS infected cells. This latter cytotoxicity may be due to an increase in a non-specific background of Natural Killer or lymphocyte activated killer activity, which is seen also after coculture with wild type vaccinia virus (vW) infected cells. Thus these results are of practical importance in two respects. First, lymphoïd B cell lines represent an excellent tool for determining which viral antigens are recognized by cytotoxic lymphocytes and second, they indicate the need to incorporate the M and S genes into a TGEV vaccine to induce cellular immunity against TGEV.

Animals↗

25- to 30-nm virus particle associated with a hospital outbreak of acute gastroenteritis with evidence for airborne transmission.

Between November 1 and 22, 1985, an outbreak of acute, nonbacterial gastroenteritis occurred in a 600-bed hospital in Toronto, Ontario, Canada. Illness in 635 of 2,379 (27%) staff was characterized by fatigue, nausea, diarrhea, and vomiting and had a median duration of 24-48 hours. The finding of virus-like particles measuring 25-30 nm in six stool specimens and low rates of seroresponse to Norwalk virus (3/39) and Snow Mountain agent (1/6) suggest that a Norwalk-like virus was responsible for the outbreak. The outbreak was of abrupt onset and high incidence, affecting 79 people in a single day. No common food or water exposure could be identified. The attack rate was greatest (69%) for staff who had worked in the Emergency Room. Of 100 patients and their companions who visited the Emergency Room on November 11-12 for unrelated problems, 33 (33%) developed gastroenteritis 24-48 hours after their visit, versus 0 of 18 who visited the Emergency Room on November 8 (p less than 0.001). An analysis of housekeepers who worked at least once during the period from November 9-13, which included those who became ill during the period of November 9-14, showed that the risk of becoming ill was four times greater for those who visited or walked through the Emergency Room than for those who did not (p = 0.028). These data are consistent with the possibility of the airborne spread of a virus.

Adult↗

Further studies on human enteric coronaviruses.

Comparisons were made between human enteric coronaviruses and the enteric coronaviruses of pigs and calves by negative staining. Examination of human intestinal organ culture fluids at various time intervals after inoculation with the human enteric coronavirus showed increasing numbers of particles in the fluids. Thin sections of the columnar epithelial cells of these explants showed a number of features consistent with the replication of known human and animal coronaviruses. Virus particles found in thin sections had a mean diameter of 68 nm. In addition, a structure was found in thin sections which has not been described previously. This structure may represent the viral nucleocapsid.

Animals↗

Water and non-water-related risk factors for gastroenteritis among bathers exposed to sewage-contaminated marine waters.

All previously published epidemiological studies of the health effects of bathing in marine waters contaminated with domestic sewage contain three major methodological weaknesses in study design: (1) failure to control for the substantial amount of temporal and spatial variation in indicator organism densities shown to occur within just a few hours at marine water bathing locations; (2) failure to relate indicator organism density directly to the individual bather; and (3) failure to rigorously control for non-water-related risk factors on previously reported associations between bathing in marine waters and illness among such bathers. We report the results of two intervention follow-up studies specifically designed to address these methodological weaknesses. We restricted study outcome to bathing-associated gastroenteritis since this is the illness most consistently reported to be associated with bathing in marine waters, and upon which both current US Marine Water Quality Criteria and other standards used worldwide are based. Our results show that faecal streptococci was the only indicator organism to predict the occurrence of gastroenteritis among bathers, and this occurred at only one of the three water quality sampling depths used in our study. The consumption of three different foods known or suspected to act as vectors in the transmission of gastroenteritis, as well as one non-food, non-water-related risk factor for gastroenteritis were found to significantly increase the risk of gastroenteritis among bathers. Multiple logistic regression modelling showed that these non-water-related risk factors confounded the relationship between exposure to marine waters of varying faecal streptococci densities and the occurrence of gastroenteritis among bathers to a moderate degree. Moreover, these analyses showed that the risk of gastroenteritis to the individual bather caused by these non-water-related risk factors, approximated the risk of gastroenteritis among bathers exposed to waters containing relatively high faecal streptococci densities. The implications of these findings with regard to the validity of present marine water quality criteria and on the need for, and design of, future epidemiological studies of bathing water associated illness are discussed.

Adult↗

Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs.

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1-5 ml dose, 10(5.8)-10(6.0) TCID(50)/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load. This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population.

Animals↗

Possible factors influencing the immunoglobulin G concentration in swine colostrum.

The immunoglobulin (Ig) G concentration in swine colostrum was determined by the single radial immunodiffusion method, using 157 samples collected from farm-raised sows in the Yamaguchi Prefecture of Japan during 1976 and 1977. The mean IgG value was 53.03 mg/ml, and the maximum and minimum values were 101.39 mg/ml and 11.74 mg/ml, respectively. The amount of IgG varied greatly among sows. To clarify the possible factors influencing the amount of IgG in colostrum, the following items were surveyed: season, district, breed, age of sows, number of parturitions, udder section from which samples were collected, kind of feed, vaccinations of swine erysipelas live-organism vaccine, hog cholera live-virus vaccine, Japanese encephalitis live-virus vaccine, tramsmissible gastroenteritis liver-virus vaccine, type of farming, and number of sows raised on a farm. Relationships between the amount of IgG in colostrum and each of these 13 items were analyzed. Seemingly, strong correlations with the amounts if IgG in colostrum were found with five items (district, number of parturitions, kind of feed, type of farming, and number of sows). To the contrary, five items (age, udder section, and vaccinations of swine erysipelas live-organism vaccine, hog cholera live-virus vaccine, and Japanese encephalitis live-virus vaccine) had poor correlations. Other items had moderate correlations. The multiple correlation coefficient obtained was 0.5499.

Age Factors↗

Persistence of caliciviruses on environmental surfaces and their transfer to food.

The noroviruses (NoV) are a common cause of human gastroenteritis whose transmission by foodborne routes is well documented. Fecally contaminated surfaces are likely to contribute to this foodborne transmission and to the propagation of viral disease outbreaks. The purpose of this study was to (i) investigate the stability of NoV on various food preparation surfaces; and (ii) evaluate the degree of virus transfer from these surfaces to a model-ready-to-eat (RTE) food. For the virus persistence experiments, stainless steel, formica and ceramic coupons were artificially contaminated with Norwalk virus (NV), the prototype genogroup I NoV; NV RNA; or feline calicivirus (FCV) F9 (a NoV surrogate), stored at ambient temperature for up to 7 d, and periodically assayed for detection. In the transfer experiments, stainless steel coupons were inoculated with NV or FCV F9 and allowed to dry for 10, 30 and 60 min, after which lettuce leaves were exposed to the surface of the coupons at various contact pressures (10, 100, and 1000 g/9 cm2). Virus recovery was evaluated by RT-PCR (for NV and NV RNA) or by plaque assay (for FCV F9) using Crandell Reese Feline Kidney (CRFK) cells. NV and FCV were detected on all three surfaces for up to 7 d post-inoculation; for FCV, there was an approximate 6 to 7-log10 drop in virus titer over the 7 d evaluation period. By contrast, when stainless steel was inoculated with purified NV RNA, RT-PCR detection was not possible beyond 24 h. Transfer of both NV and FCV from stainless steel surfaces to lettuce occurred with relative ease. This study confirms lengthy NoV persistence on common food preparation surfaces and their ease of transfer, confirming a potential role for environmental contamination in the propagation of viral gastroenteritis.

Calicivirus, Feline↗

Pathogenic rotaviruses isolated from pigs and calves.

Rotavirus is commonly isolated from diarrhoeic calves and pigs. Bacterium-free faecal filtrates containing rotavirus from five different outbreaks of disease in calves all caused diarrhoea and clinical illness in gnotobiotic calves and five different isolates from pigs were inoculated into gnotobiotic pigs with similar results. The author was unsuccessful in finding an avirulent strain although one of the calf isolates was from a non-diarrhoeic calf. The laboratory strain of calf virus retained its virulence after being passaged seven times in gnotobiotic calves, which included sucrose density gradient purification on two occasions. The calf tissue culture-adapted virus retained its virulence. Rotavirus isolates from humans, calves, pigs and foals were infectious to pigs. Although sharing a common antigen the viruses were separable according to host infectivity, virulence and neutralizing antigens. In both calves and pigs the main lesion was loss of the epithelial cell of the small intestine and stunting of villi. Passive protection of the calf and pig was poor. Circulating antibody was not protective and although high levels of clolostral antibody in the gut lumen at the time of infection protected calves clinically, the antibody level secreted in milk declined 10-fold 48 hours after parturition. Frequently other viruses are found together with rotavirus in cases of diarrhoea. Their role is being investigated.

Animals↗

Tandem placement of a coronavirus promoter results in enhanced mRNA synthesis from the downstream-most initiation site.

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3'-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

Animals↗

Coronavirus genome structure and replication.

In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. Their lengths range from 27,317 nt for HCoV-229E to 31,357 nt for the murine hepatitis virus-A59, establishing the coronavirus genome as the largest known among RNA viruses. The basic organization of the coronavirus genome is shared with other members of the Nidovirus order (the torovirus genus, also in the family Coronaviridae, and members of the family Arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mRNA synthesis (transcription) (an estimated 14-16 end products for coronaviruses) are encoded within the 5'-proximal two-thirds of the genome on gene 1 and the (mostly) structural proteins are encoded within the 3'-proximal one-third of the genome (8-9 genes for coronaviruses). Genes for the major structural proteins in all coronaviruses occur in the 5' to 3' order as S, E, M, and N. The precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. This chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (DI) RNA molecules], the minimum sequence requirements for autonomous replication of an RNA replicon, and the importance of gene order in genome replication.

Animals↗