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Infantile gastroenteritis associated with excretion of Pestivirus antigens
Abstract
Faeces from children under 2 years old who had gastroenteritis that could not be attributed to recognised enteric pathogens were examined with a monoclonal-antibody-based immunoassay for Pestivirus antigens. Such antigens were detected in 30 of 128 episodes of gastroenteritis. Children without diarrhoeal disease and children infected with rotaviruses had little evidence of Pestivirus infection (faeces positive in 1 of 28 and 1 of 31, respectively). The diarrhoeal disease in children excreting Pestivirus antigens resembled that in other children except that it was more commonly associated with signs and symptoms of respiratory inflammation.
... It is a flavivirus that causes the most prevalent infectious disease of cattle, the bovine diarrheal disease [27]. It can be transmitted vertically and produce malformations and microcephaly in calves [27][28][29][30][31][32][33][34]. Its horizontal transmission is done by body fluids, including milk, saliva and fomites [27][28][29][30][31][32][33][34]. ...
... It can be transmitted vertically and produce malformations and microcephaly in calves [27][28][29][30][31][32][33][34]. Its horizontal transmission is done by body fluids, including milk, saliva and fomites [27][28][29][30][31][32][33][34]. ...
... Some studies have confirmed its presence in outbreaks of diarrhea in children [33]. Nevertheless, BVDV has been seen as of little relevance to human pathology [14]. ...
While there is no doubt about the participation of Zika virus in microcephaly, its epidemiology is not entirely clear and doubts remain about the intervention of other factors. In studies on the epidemiology of dengue, the infestation by Aedes aegypti peridomiciliary and the population density are the main determinants for viral spread. However, in Rio Grande do Norte state (RN), the counties that have confirmed cases of microcephaly overlapped the river basins regions surrounded by agriculture and livestock. In addition, the prevalence of microcephaly at the end of the first year of the epidemic was higher in small towns than in larger ones, elements that seem to contradict what is known about the epidemic by other arboviruses. Methods: 234 cases of microcephaly were analyzed from three states and 144 counties. Results: An exponential trend of higher prevalence of microcephaly in the smaller cities (r ² =0,7121) was found.The correlation coefficients (R) between the Prevalence of microcephaly and the variables that measured the density of animals in the territory ranged from moderate to strong. Discussion: Concerning microcephaly, studies in progress point to the possibility of association between the Zika Virus and the BVDV, a virus known to produce birth defects in farm animals but perceived as innocuous in humans. Conclusions: The overlap of cases of microcephaly in river basins, their higher prevalence in smaller cities, the strength of the correlation coefficient, render necessary new etiological and pathophysiological studies.
4.
ABBREVIATIONS
BVDV Bovine diarrhea virus
CE Ceará state
IBGE Instituto Brasileiro of Geography and Statistics
IPESQ Instituto de Pesquisa da Paraíba
PB Paraíba state
RN Rio Grande do Norte state
UFRJ Rio de Janeiro Federal University
ZKV Zika Virus.
... Pestivirus is ass RNA( + ) virus which is 50 nm in diameter, pleomorphic, probably icosahedral, enveloped, and difficult to identify by EM. Pestivirus is widely recognized as a cause of highly economically important diseases in animals, but has never been demonstrated in or to cause disease in humans, either directly or indirectly (21,22). ...
... Recently, a monoclonal antibodybased EIA was developed and used to demonstrate pestivirus antigens in 23% of the stools of children <2 yr of age with gastroenteritis that was not attributable to recognized enteric pathogens (22). Similar methods have been used to detect pestivirus-associated gastroenteritis and pestivirus exposure in the United States, Bangladesh, and Peru (1,2 ...
... Only rabbits appear to support virus replication (Fernelius et al. 1969). Reports of antibodies to pestiviruses in human sera (Giangaspero et al. 1988; Wilks et al. 1989), of pestivirus antigens in human stools (Yolken et al. 1989) and human leukocyte (Giangaspero et al. 1997) require further investigations. There are indications of a possible impact of infection in humans such as microencephalic infants and gastroenteritis associated with respiratory illness in children (Yolken et al. 1989; Giangaspero et al. 1997). ...
... Reports of antibodies to pestiviruses in human sera (Giangaspero et al. 1988; Wilks et al. 1989), of pestivirus antigens in human stools (Yolken et al. 1989) and human leukocyte (Giangaspero et al. 1997) require further investigations. There are indications of a possible impact of infection in humans such as microencephalic infants and gastroenteritis associated with respiratory illness in children (Yolken et al. 1989; Giangaspero et al. 1997). Among free-living ruminants, pestiviruses have been recovered from roe deer (Capreolus capreolus) (Baradel et al. 1988), fallow deer (Dama dama) (Frölich 1995), dromedary camel (Camelus dromedarius) (Hegazy et al. 1996); African buffalo (Syncerus caffer) (Hamblin & Hedger 1979); giraffe (Giraffa camelopardalis) (Soine et al. 1992) and wildebeest (Connochaetes spp.) (Doyle & Heuschele 1983) but in some cases, the contribution of the virus to the cause of death was uncertain. ...
Thesis (MSc (Veterinary Science))--University of Pretoria, 2001. Includes bibliographical references.
... The presence of BVDV was confirmed for instance in the measles, mumps, rubella, and polio vaccines administered to children (Giangaspero et al., 2001;Sasaki et al., 1996). Although pestiviruses are not considered zoonotic, their antigens were found in the feces of children showing signs of gastroenteritis and respiratory disorders (Yolken et al., 1989). Furthermore, BVDV was isolated from human leukocytes in patients in Belgium (Giangaspero et al., 1997). ...
In this report, we describe the detection of bovine viral diarrhea virus (BVDV) contamination in commercial animal-derived sera and vaccines against animal viral pathogens on the market in Poland. Antibodies against BVDV were detected in 4/45 sera samples (8.9%) using an ELISA test. The presence of BVDV antigen using ELISA was found using ELISA in 3/45 serum samples (6.6%) and 18/172 vaccine samples (10.5%). An RT-PCR was conducted using primers targeting two genome regions, the five prime untranslated region (5’UTR) and N-terminal protease (N pro ). BVDV RNA was detected in 33/45 (73.3%) of sera, and 11/172 samples (6.4%) of collected vaccines, of which one vaccine did not declare BVDV strain in its composition. A single serum showed the presence of an infectious virus and only one was contaminated with all 3 species of BVDV. The most frequent species in sera was BVDV-3 (75.5%), whereas in vaccines only BVDV-1 was identified. Sequence analysis showed that the tested commercial sera and one vaccine were contaminated by six genotypes of BVDV: -1a, -1b, -1c, -1d, -2a, and -3. Identification of BVDV and its genetic material in animal-derived products is important due to the possibility of pestivirus transmission as well as the chance of falsifying the results of a diagnostic test. It also demonstrates the necessity of rigorous monitoring of the bioproducts used in the laboratory and industry level.
... A number of other viruses, including coronavirus 82 ( Figure 45.12), torovirus, 83 picobirnavirus 84,85 and pestivirus, 86 have been detected in stool specimens of patients with acute gastroenteritis, but their signifi cance as aetiological agents of diarrhoea remains to be established. ...
... Similarly, BVDV antibodies were found in 40% of sera collected from twins incongruous for schizophrenia [20]. Another study showed the presence of BVDV antigen in 23.6% of faecal samples collected from children suffering from gastroenteritis [21]. Recently, unpublished data from proteomics analysis of brain tissue samples from foetuses bearing microcephaly during the 2015 Zika outbreak in Brazil was reported to suggest the presence of peptide(s) from the polyprotein of a BVDV-like virus, although they were also related to peptides from host ubiquitin [22,23]. ...
Bovine viral diarrhoea virus (BVDV) is a cattle pathogen that has previously been reported to be present in bovine raw materials used in the manufacture of biological products for human use. Seven lots of trivalent measles, mumps and rubella (MMR) vaccine and 1 lot of measles vaccine from the same manufacturer, together with 17 lots of foetal bovine serum (FBS) from different vendors, 4 lots of horse serum, 2 lots of bovine trypsin and 5 lots of porcine trypsin were analysed for BVDV using recently developed techniques, including PCR assays for BVDV detection, a qRT-PCR and immunofluorescence-based virus replication assays, and deep sequencing to identify and genotype BVDV genomes. All FBS lots and one lot of bovine-derived trypsin were PCR-positive for the presence of BVDV genome; in contrast all vaccine lots and the other samples were negative. qRT-PCR based virus replication assay and immunofluorescence-based infection assay detected no infectious BVDV in the PCR-positive samples. Complete BVDV genomes were generated from FBS samples by deep sequencing, and all were BVDV type 1. These data confirmed that BVDV nucleic acid may be present in bovine-derived raw materials, but no infectious virus or genomic RNA was detected in the final vaccine products.
... This virus infects mainly domestic and wild ruminants and pigs, easily determining fetal infection and causing abortion or malformations at the central nervous system characterized by demyelination and cerebellar hypoplasia. In addition, there are some reported cases in humans where it showed strong tropism to nervous tissue [12][13][14][15]. In 2016, researchers from the Paraiba Research Institute (IPESQ) and the Federal University of Rio de Janeiro (UFRJ) pointed to the possibility that ZIKV could function as a facilitator of infection by the BVDV [11]. ...
A retrospective study was conducted on available data of Zika virus associated microcephaly epidemics
occurred in Brazil during 2015 and 2016. Prevalence by live births was associated with the distribution of farm animals in the territory, demographic density and environmental aspects. The correlation coefficient (r) between the prevalence of microcephaly by live births and the variables that measured the density of farm animals in the territory ranged from moderate to strong in all the Regions of Brazil. The prevalence of microcephaly at the end of the first year of the epidemic was exponentially higher in small counties than in larger ones, elements that contradict the epidemiology of other arboviruses, influenced by vector infestation and by demographic density, introducing a surprising rurality to the epidemiology of microcephaly. These findings add epidemiological elements which show a relation with livestock in the epidemiology of microcephaly in Brazil, and suggest the involvement of potential cofactors related with the rural environment and animal farming as the bovine viral diarrhoea virus (BVDV), which was also found in fetal tissues and amniotic fluids of fetuses with microcephaly, or other unidentified zoonotic agents in the ZIKV epidemiology and the associated microcephaly.
... Similarly, serological and antigenic evidence of BVDV in humans has been reported [20]. The virus has been demonstrated in viraemic patients [21], and supposed to be related with gastro enteric or neurological neonatal pathologies [22][23][24] or post-infective nevritis [25], but these observations have been reported in experimental or exceptional conditions and does not correspond to common evidence in normal conditions, and for this reason diseases not listed as zoonoses in the framework of disease categorization and prioritization. ...
With the aim to present advances in risk assessment of animal diseases, recent methods have been considered in a brief review. Various initiatives relating to the risk assessment of animal diseases for categorization and prioritization have been undertaken with the objective to provide decision-makers with elements of priority for the application of optimal prevention and control measures. Theoretically, a technical approach should be harmonized and internationally recognized. However, methodologies remain complex, and different fields of applications (for example animal species, production systems) multiply variants. Local dimension play an essential role for the definition of a final result, often not necessarily comparable with results obtained when considering different geographical realities. Furthermore, other elements, as political or cultural aspects, may influence final decisions taken by competent authorities. Nevertheless, further efforts will be needed to harmonize procedural tools. In conclusion, despite methodological limits, the application of categorization and prioritization protocols represents a precious support for the competent authorities in relation to the various aspects of animal health management, from legislation, surveillance, or control measures.
... Nie stwierdzono, aby wirusy BVD-MD by³y niebezpieczne dla zdrowia ludzi, jednak z badañ laboratoryjnych wynika, ¿e wirus ³atwo namna¿a siê w hodowlach komórek ludzkich (15). Badania przeprowadzone w szpitalu Johna Hopkinsa w Baltimore (USA) wykaza³y, ¿e antygeny pestiwirusowe obecne by³y u ponad 20% dzieci poni¿ej drugiego roku ¿ycia cho-ruj¹cych z objawami zapalenia ¿o³¹dka i jelit, u których wykluczono zaka¿enie rotawirusami (42). Ryzyko zaka¿enia pestiwirusami poprzez szczepionki jest tym wiêksze, ¿e patogen wprowadzany jest bezporednio do organizmu zwierzêcia z pominiêciem naturalnych barier ochronnych. ...
The paper presents the current state of knowledge on the prevalence and risk of a new type of bovine viral diarrhea virus known as type 3 (BVDV-3). The first discovered atypical pestivirus was the isolate D32/00-'HOBI, detected in fetal calf serum (FCS) originating from Brazil. The isolates CH-KaHO/cont, SVA/cont-08 and IZSPLVJT-To are further examples confirming the presence of BVDV type 3 in the FCS. This new species of pestiviruses (BVDV-3) is a problem not only for research laboratories using bovine serum, but also for cattle breeders. Natural infections with this virus have been reported in Brazil, Thailand and Italy, which may suggest that the new species of pestivirus is also present in European cattle. The methods used for routine diagnosis of BVDV infection are ineffective in detecting atypical pestiviruses, which may pose a risk of false negative results. It may also influence the safety of vaccines and biological products produced on the basis of contaminated batches of fetal bovine serum.
... To our knowledge, there is no evidence to substantiate the contamination of human vaccines with infectious pestiviruses. Although it has not been established that Pestivirus infections cause specific symptoms in humans, infantile gastroenteritis associated with the excretion of pestiviral antigens and microencephaly in infants who were born from mothers seropositive for the virus have been reported (Yolken et al., 1989;Potts et al., 1987). Thus, our results stress the importance of improving the tests applied to products from the biological industry, especially in those for human use. ...
This study presents a phylogenetic analysis of 115 bovine pestiviruses. A sequence data set from the 5′ untranslated genomic region was analyzed with maximum parsimony, bootstrapping and parsimony jackknifing. We tested for the proposed classifications of the group and analyzed the evolution of the symptoms associated with Pestivirus infections in bovines. Based on the historical framework provided by our phylogenetic trees, we also characterized the extent and importance of contamination caused in biologicals by the virus. Our phylogenetic analyses showed that the previously defined genotypes are monophyletic, except for genotype 1a. Based on our cladograms, we propose the existence of more than 12 monophyletic groups within the species BVDV 1. The mapping of clinical symptoms suggests that the emergence of some genotypes could have been driven by a change in the pathogenic process. Enteric Problems appear to be ancestral, while Reproductive and Respiratory Problems arise with the emergence of genotypes 1b, 1d and the herein-proposed genotype Arg 1. The distribution of contaminant strains on the cladograms shows that pestiviral contamination is a common process, and also suggests that a contaminated product might be a vehicle for virus dispersion. Implications for virus evolution, virus taxonomy, veterinary medicine and biotechnology are discussed.
... This includes antilocapridae (e.g., pronghorn), bovidae (e.g., cattle, sheep, goats), camelidae (e.g., camels, llamas), cervidae (e.g., deer, moose), giraffidae (e.g., giraffe), suidae (e.g., pigs), and traguilidae (e.g., mouse deer; Carbrey et al., 1976;Grøndahl et al., 2003;Topliff et al., 2009;Krametter-Froetscher et al., 2010;. Infectious virus, viral nucleic acids, or viral antigens of BVDV or a closely related pestivirus have been detected in peripheral white blood cells of 2 people (Giangaspero et al., 1993), in an intestinal biopsy from a person exhibiting Crohn's disease (Van Kruiningen et al., 2007), and in the feces of 30 children with gastroenteritis (Yolken et al., 1989). However, BVDV was not definitely diagnosed as a disease in any of these human patients. ...
Bovine viral diarrhea virus (BVDV) is a pestivirus that is enzootic in most cattle populations throughout the world. This virus is present throughout the body of persistently infected (PI) cattle. Previous research has not assessed the cooking temperature at which BVDV in meat from PI cattle can be inactivated. Therefore, muscle tissue from 6 PI cattle was harvested, refrigerated, frozen, and heated to various internal temperatures. The concentration of virus present was determined by virus isolation. Average cell culture infective doses (50% endpoint; CCID(50)) of BVDV per gram of frozen, uncooked meat from PI cattle were 10(5.85) CCID(50)/g of whole cuts and 10(6.02) CCID(50)/g of ground meat. The virus in whole and ground meat was consistently inactivated when cooked to temperatures greater than or equal to 75°C. A second objective of this research was to thoroughly reassess if Vero cells were permissive to BVDV infection in our laboratory to provide further indication of whether primates, including humans, might be susceptible to BVDV. Vero cells were not permissive to infection with any of 43 different strains of BVDV that readily replicated in Madin Darby bovine kidney cells. In conclusion, this bovine pathogen, which is not considered to be a human pathogen, can be inactivated by cooking ground or whole cuts of meat to 75°C or higher. Care should be taken to ensure that susceptible hosts such as pigs are not fed improperly cooked meat, meat by-products, or waste food originating from PI cattle.
... Human cells may essentially be susceptible to BVDV infection because serum antibodies against pestivirus have been detected in humans who had no contact with potentially infected animals (12,38). Pestiviruses adapted to human cell cultures may be harmful because serious BVDV infections in humans have frequently been suggested (27,39). The presence of BDV, a pathogen to sheep and goats, in the Ch1Es cell line was first confirmed in this report. ...
We examined 20 cell lines of various animal origins for the presence of pestivirus contamination by the reverse transcription-polymerase chain reaction (RT-PCR), and found 15 (75%) cell lines were positive. The RT-PCR products of the 5' untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. Stem-loop structures at three variable regions in the 5' UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with genotypes I, II, or III of bovine diarrhea virus (BVDV). Cell lines of canine, feline, and primate origin were contaminated with genotype II of BVDV. Cell line Ch1Es of caprine origin was contaminated with border disease virus (BDV).
... In particular, HRV, subgenus F (types 40 and 41) of ADV, astroviruses and caliciviruses are recognized as the most prevalent viruses implicated in the aetiology of childhood gastroenteritis in developed countries [3]. Recently other viral agents such as picobirnavinus (PBV) and pestivirus have been reported to be associated with diarrhea [7,11]. The assignment of a casual role to an isolated pathogen is complicated because often multiple enteric pathogens are isolated from the same patient or because the rate of isolation of the potential pathogen is high also from asymptomatic controls [1]. ...
Polyacrylamide gel electrophoresis of nucleic acid extracted from stool samples of diarrhoeic children revealed in 3 out of 690 (0.43%) specimens two electrophoretic bands with a migration pattern characteristic of picobirnavirus ds-RNA. In none of the 92 control children were similar bands detected. No other potential enteric pathogens were found in the patients with picobirnavirus infection.
... However, our findings do not imply that the vaccines giving a positive reaction by PCR were all contaminated with infectious pestivirus. Although it has not been established that BVDV infection causes specific symptoms in man, infantile gastroenteritis associated with excretion of pestivirus antigens 24 and microcephaly in infants born to mothers seropositive for pestiviruses have been reported. 25 Serum antibodies against BVDV have been detected in humans who had had no contact with potentially infected animals. ...
PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.
... In a study conducted in the U.S.A., in mothers with microcephalic infants, two sera were positive for antibodies against the NADL strain of BVDV (22). Pestivirus antigen was detected in 23.6% fecal samples from children under two years old affected by gastroenteritis often associated with respiratory illness, where no pathogen could be identified (30). Samples were collected from the population of the White Mountain Indian reservation, Ariz., U.S.A. Similar preliminary studies in children from urban areas (Baltimore, Md., U.S.A., and Houston, Tex., U.S.A., and Dhaka, Bangladesh) showed pestivirus-associated gastroenteritis, suggesting transmission without animal involvement. ...
The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.
... Furthermore, they can cross the host-species barrier, infecting different species from among the cloven-hoofed ungulates (Paton et al., 1992;Edwards et al., 1995;Wensvoort and Terpstra, 1988). While pestivirus infections in humans have often been suspected (Yolken et al., 1989;Wilks et al., 1989;Yolken et al., 1993), a pestivirus strain was isolated recently from a human buffy coat sample (Giangaspero et al., 1993). Such cross-infections may weaken the definition of pestivirus species by the host. ...
A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5'-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5'-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes.
Background
The variation in the pathogen type as well as the spatial heterogeneity of predictors make the generality of any associations with pathogen discovery debatable. Our previous work confirmed that the association of a group of predictors differed across different types of RNA viruses, yet there have been no previous comparisons of the specific predictors for RNA virus discovery in different regions. The aim of the current study was to close the gap by investigating whether predictors of discovery rates within three regions—the United States, China, and Africa—differ from one another and from those at the global level.
Methods
Based on a comprehensive list of human-infective RNA viruses, we collated published data on first discovery of each species in each region. We used a Poisson boosted regression tree (BRT) model to examine the relationship between virus discovery and 33 predictors representing climate, socio-economics, land use, and biodiversity across each region separately. The discovery probability in three regions in 2010–2019 was mapped using the fitted models and historical predictors.
Results
The numbers of human-infective virus species discovered in the United States, China, and Africa up to 2019 were 95, 80, and 107 respectively, with China lagging behind the other two regions. In each region, discoveries were clustered in hotspots. BRT modelling suggested that in all three regions RNA virus discovery was better predicted by land use and socio-economic variables than climatic variables and biodiversity, although the relative importance of these predictors varied by region. Map of virus discovery probability in 2010–2019 indicated several new hotspots outside historical high-risk areas. Most new virus species since 2010 in each region (6/6 in the United States, 19/19 in China, 12/19 in Africa) were discovered in high-risk areas as predicted by our model.
Conclusions
The drivers of spatiotemporal variation in virus discovery rates vary in different regions of the world. Within regions virus discovery is driven mainly by land-use and socio-economic variables; climate and biodiversity variables are consistently less important predictors than at a global scale. Potential new discovery hotspots in 2010–2019 are identified. Results from the study could guide active surveillance for new human-infective viruses in local high-risk areas.
Funding
FFZ is funded by the Darwin Trust of Edinburgh ( https://darwintrust.bio.ed.ac.uk/ ). MEJW has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 874735 (VEO) ( https://www.veo-europe.eu/ ).
Viruses interact with the gastro-intestinal tract in a number of ways. Some viruses such as hepatitis A virus and the enteroviruses use the intestine as a portal of entry and rarely, if ever, produce diarrhoeal disease. Others cause diarrhoeal disease only when the immune system is compromised, for example, HIV and cytomegalovirus (HHV-5). Human papillomaviruses and Kaposi’s sarcoma associated herpesvirus (HHV-8) can affect the gastro-intestinal tract causing local tumours. On stool electron microscopy, bacteriophages can be seen (Fig. 1) which can be mistaken for other viruses. Bacteriophages are viruses that infect bacteria and are involved only indirectly in human disease, for example, acting as vectors for toxin genes (e.g. shiga toxins 1 and 2 in Escherichia coli 0157). However, here we will concentrate on the virology and laboratory diagnosis of the enteric viruses that are primary pathogens causing diarrhoeal disease (Table 1). The relative importance of viruses and the various enteric viruses depends upon the patient’s age and their state of immunity. Undoubtedly, viruses are the most important causes of diarrhoeal disease in infants and young children whether HIV-infected or not [1].
IntroductionPestivirus of Chamois and Border DiseaseBovine Viral DiarrhoeaClassical Swine FeverReferences
Contamination of bovine-derived bioproducts with pestiviruses, in particular with bovine viral diarrhea virus, creates a risk of infection for animals and even for humans. Owing to the worldwide spread of pestivirus infections in cattle population, many biological products based on materials derived from cattle or from their tissues are contaminated with this pathogen or contain the viral RNA. Contamination of fetal bovine serum (FBS) by BVDV is an intra-laboratory problem that hinders the diagnostics of infections caused by related viruses, such as classical swine fever virus (CSFV) or hepatitis C (HCV) in humans. Furthermore, vaccines, hormones or other bioproducts used in animals and humans may be contaminated with the virus. The article describes numerous examples of transmission of virulent pestiviruses to susceptible populations through the use of contaminated animal bioproducts.
Bovine Viral Diarrhea virus species of the genus Pestivirus, important pathogens affecting zootechnics worldwide, have been reported as adventitious contaminants of biological products for veterinary and human use. The Bovine Viral Diarrhea virus 1 species showed potential for an emerging zoonosis. According to World Organization for Animal Health (Office International des Épizooties: OIE), Bovine Viral Diarrhea is a notifiable disease of importance to international trade. Recently, Bovine Viral Diarrhea virus 3 tentative species have been isolated from Brazil and Thailand. The virus has been diffused from South America to other countries probably through the commercialization of contaminated fetal bovine serum.
Background/Aims Infectious agents have long been suspected of playing a role in the initiation of Crohn's disease. The objective of this study was to search for likely microbial agents in diseased tissues using immunocytochemical techniques. Methods Intestines and mesenteric lymph node specimens of 21 patients from two French families with a high frequency of Crohn's disease and from Connecticut were studied. The microbial agents searched for included Bacteroides vulgatus, Borrelia burgdorferi, Escherichia coil, Listeria monocytogenes, Streptococcus spp., bovine viral diarrhea virus, influenza A virus, measles virus, parainfluenza virus, and respiratory syncytial virus. Results Seventy-five percent of the patients with Crohn's disease (12 of 16) were positively labeled with the antibody to Listeria. Macrophages and giant cells immunolabeled for this antigen were distributed underneath ulcers, along fissures, around abscesses, within the lamina propria, in granulomas, and in the germinal centers of mesenteric lymph nodes. In addition, 57% (12 of 21) of the cases contained the E. coli antigen, and 44% (7 of 16) contained the streptococcal antigen. The immunolabeling for the latter two agents also occurred within macrophages and giant cells, distributed in a pattern similar to that of Listeria antigen. Conclusions The results suggest that Listeria spp., E. coli, and streptococci, but not measles virus, play a role in the pathogenesis of Crohn's disease.
A procedure is described for the detection of hepatitis C virus (HCV) RNA in blood by means of the polymerase chain reaction (PCR) in which the reverse transcription step and two rounds of amplification are carried out in a single tube. This results in fewer manipulations, reduced risk of contamination, and economy of time. The procedures are generally applicable to other assays based on the PCR. We describe the preparation (from 100 μL serum) of test samples that remain stable for at least 6 days under specified conditions and an assay that employs nested primer pairs homologous to conserved sequences in the 5′ noncoding region. The method was tested on 107 sera from the United States and Japan. Correlation with first-generation anti-HCV was 77%. Two sets of nested primer pairs homologous to sequences in the 5′ noncoding region and one set based on structural region sequences showed differences in their reactivities with serum HCV RNA. The recommended single tube procedure specified a primer for reverse transcription that was conserved in all reported HCV genomes but absent from pestivirus genomic sequences. The effects of preanalytical factors on the detection of HCV RNA were studied. Qualitatively, there was no change in the HCV RNA-positivity of sera that were exposed to room temperature for 24 hours. Quantitative studies showed a decrease in titer in some specimens. Three cycles of freeze-thawing had no detectable effects on the titers of HCV RNA. © 1992 Wiley-Liss, Inc.
Classical, live viral vaccines have been developed by adapting viruses by serial passages in animals, tissue or cell cultures
during which multiple mutations in the viral genome have accumulated. The majority of vaccines in use today were developed
in this way and a number of similar investigational vaccines are currently in development. The principal advantage of live
vaccines is that they mimic natural infection and induce durable immunity, including cytotoxic T cell responses that are not
generated by soluble proteins and inactivated vaccines. Recent studies of gene activation following a live vaccine (yellow
fever 17D) have shed light on the role of innate immune responses in provoking strong, polyfunctional adaptive immunity following
the administration of live vaccines. The principal disadvantage of live vaccines is that, occasionally, infection caused by
the live vaccine causes adverse events resembling the parental (virulent) virus. Such events can be due to reversions in critical
attenuating mutations or to host-specific susceptibility factors. The attenuating mutations in live vaccines increase the
inapparent: apparent infection ratio compared to the parental virus, but overt infection (adverse events) while far less frequent
than in natural infection can still occur. This problem is inconsequential for infections that are typically mild or self-limited,
such as measles, mumps, rubella, and varicella, but can be devastating for infections that are frequently lethal such as yellow
fever or in individuals who are immunocompromised. Despite these issues, live vaccines have had dramatic benefits in reducing
the incidence of the most important infections of humankind, and in one case (smallpox) a live vaccine helped eradicate a
viral disease.
Pestivirus bovine viral diarrhea virus 2 (BVDV-2) strains from 61 isolates from cattle and sheep, and from some adventitious contaminants of biologicals, have been assessed using the palindromic nucleotide substitutions (PNS) method at three variable loci (V1, V2 and V3) located delin the 5' untranslated region (UTR) of genomic RNA. This genotyping procedure is new, simple and practical. Two characteristics of the base pairings common to BVDV-2 species, a C-G or U-A pairing at the V1 locus, and a G*U pairing at the V2 locus, were observed in isolates tested. The PNS method showed six genotypes: BVDV-2a, BVDV-2b, BVDV-2c, BVDV- 2d, BVDV-2e and BVDV-2f. Twenty-five strains showed the BVDV-2a genotype specific combination of three base pairings (A-U in position 1 and C-G or U*G in position 18 in V1 and U-A or U*G in position 4 in V2). Ten strains were identified by a single C-G pairing in position 4 from the bottom of the V2 stem region, characteristic to genotype BVDV-2b. Three strains were assigned to genotype BVDV-2c, due to their recognition by a G*U pairing at the bottom of the V1 stem region. A U-A pairing, characteristic of the BVDV-2d genotype when found in position 18 of the V1 stem region, was observed in fourteen strains. Genotype BVDV- 2e, present in only six South American cattle isolates, was characterized by G-C pairing in position 12, by U-A pairing in position 16 and G_G or G-_A bulges in position 18 in the V1 region. One strain from Argentina was classified as genotype BVDV-2f, showing: A-U pairing in position 9 and 12, U-A in position 16 and G_A bulge in position 18 in V1 region. Two strains were not characterized due to incomplete sequence of V1 locus.
The Parsonage-Turner syndrome, a rare form of neuralgic amyotrophy of unknown aetiology, was diagnosed in a patient involved in an outbreak of bovine viral diarrhoea virus (BVDV). The patient, suffering from inflammation of the right shoulder with a permanent atrophy, developed anti-BVDV antibody titres which remained very high during the four following years of monitoring.
Consensus Statements of the American College of Veterinary Internal Medicine (ACVIM) provide the veterinary community with up-to-date information on the pathophysiology, diagnosis, and treatment of clinically important animal diseases. The ACVIM Board of Regents oversees selection of relevant topics, identification of panel members with the expertise to draft the statements, and other aspects of assuring the integrity of the process. The statements are derived from evidence-based medicine whenever possible and the panel offers interpretive comments when such evidence is inadequate or contradictory. A draft is prepared by the panel, followed by solicitation of input by the ACVIM membership, which may be incorporated into the statement. It is then submitted to the Journal of Veterinary Internal Medicine, where it is edited prior to publication. The authors are solely responsible for the content of the statements.
OBJETIVO Estudio de la incidencia de Gastroenteritis aguda GEA viral (Rotavirus, Adenovirus, Astrovirus, Calicivirus) adquirida en la Comunidad, en niños menores de 4 años. Se estudia también la GEA viral de origen nosocomial, en niños menores de 2 años. Se analiza la distribución de los serotipos circulantes de Rotavirus grupo A. MÉTODOS Estudio prospectivo de 2 años de duración. En el primer años 822 niños atendidos en Urgencias fueron estudiados para obtener datos sobre GEA viral adquirida en la Comunidad (clínica, epidemiología y microbiología. En el segundo año de 801 niños ingresados en la Unidad de lactantes, con el fin de obtener datos sobre la GEA viral nosocomial. En todas las muestras se investigó la presencia en heces de Rotavirus grupo A. Adenovirus, Astrovirus y Calicivirus. En las muestras negativas, se estudió además la presencia de Rotavirus grupo C. RESULTADOS Rotavirus grupo A ha sido el agente más frecuentemente encontrado tanto en la GEA adquirida en la Comunidad como nosocomial: asociándose estos a una mayor gravedad. El segundo en frecuencia fue Galicivirus tipo Norwalk. Astrovirus y Rotavirus del grupo C se encontraron algo menos frecuente. En la GEA nosocomial del segundo periodo, se encontró mayoritariamente Rotavirus del grupo A. En ambas fases, los serotipos G1 y G2 fueron los predominantes, si bien de forma alternante. CONCLUSIONES Se confirman que Rotavirus del grupo A es el agente más frecuente de GEA en nuestro medio. Por primera vez en nuestro país se estudian los Calicivirus por Técnicas moleculares, siendo el segundo agente en nuestra serie. También por primera vez se aportan datos sobre Rotavirus del grupo C que hacen valorar su búsqueda. También se aportan datos de la distribución de los serotipos del Rotavirus grupo A, por primera vez en la Comunidad de Madrid y se plantea la necesidad de realizar una vigilancia epidemiológica continuada
We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.
Pestiviruses are a group of small enveloped, positive-strand RNA viruses well known to veterinary virologists. Based on their morphology and the polarity of their genome, they originally held generic status in the family Togaviridae. Pestiviruses have never been model viruses like other togoviruses, because they are difficult to work with. In cell cultures virus yield is usually low and virus purification is difficult if not impossible, because the virions are fragile and they seem to be intimately associated with the host cell membranes. However, the availability of modern virological techniques revived interest in pestiviruses in the early to mid 1980s and today there is a much broader understanding of this group of viruses. Although many unsolved questions remain important, details concerning the pathogenesis, genomic organization, and replication of pestiviruses have been established. However, notably among ruminant pestiviruses, cytopathogenic (cp) variants can arise during persistent infections with ncp viruses and they may play an important role in causing disease in their host animals. The name of the group is derived from the infectious agent of pestissuum (hog cholera virus, HoCVl). All pestiviruses are antigenically closely related, but no significant relationships with other known viruses have been detected, either at the antigenic or at the molecular level. Their spread is worldwide and, apart from the somewhat indirect evidence of pestivirus infections in humans, they are restricted to pigs and a variety of ruminant species, both domestic and wild living. Of the economic importance regarding domestic animals are: HoCV, bovine viral diarrhea virus (BVDV), and border disease virus (BDV) of sheep. These viruses are under extensive investigation and are the focus of this chapter.
An antigen-capture ELISA was developed for the detection of pestivirus-specific antigens in peripheral blood leucocytes (PBLs), blood clots and tissue samples of immunotolerant cattle persistently infected with virus. The ELISA demonstrated complete agreement with conventional virus isolation procedures undertaken on specimens from a total of 58 carrier animals and 360 uninfected animals. The technique is based on capturing antigen with a high-titred goat polyclonal antiserum and detecting the bound antigen with a combination of 3 broadly-reactive monoclonal antibodies. Increased sensitivity was obtained with the use of an avidin-biotin complex (ABC) amplification method. On average, ELISA optical densities (ODs) for PBL and blood clot samples derived from carrier animals were 1.53 and 0.95, respectively, while uninfected animals had corresponding values of less than 0.15 for all blood samples. Tissue samples from carrier cattle had OD values ranging from an average of 0.95 for liver to 1.77 for spleen, with negative values for all tissues again averaging less than 0.20. Signal-to-noise (S/N) ratios calculated from the ELISA OD readings for carrier cattle showed an average of 15.6 for blood samples and 16.4 for tissues. In contrast, all samples from negative cattle had S/N ratios less than 2.0. The antigen-capture ELISA has been validated on field samples and is suitable for routine diagnostic and certification testing.
Infectious diarrhea, caused by a wide variety of viral, bacterial and parasitic pathogens, is a common reason for morbidity and hospitalization for children in the United States. Overall, rotavirus is the most common cause of acute diarrheal disease in infants. Salmonella, Shigella, and Campylobacter are the most frequently isolated bacterial pathogens, and Giardia and Cryptosporidium are the parasites that most commonly produce acute infectious diarrhea. The mechanisms by which these enteropathogens cause diarrhea are highly variable, and include crypt cell proliferation, cellular invasion, elaboration of enterotoxins or cytotoxins, and enteroadhesion. In infants the incidence of diarrheal disease is higher and the severity of the illness is greater than in older children and adults. An increased rate of exposure to enteropathogens, as a result of fecal-oral contamination, may explain some of the increased incidence of diarrhea in infants. However, age-specific differences in host defense mechanisms may also account for the increased susceptibility to and severity of certain enteric infections in infants.
A monoclonal antibody capture enzyme-linked immunosorbent assay (ELISA) has been developed to detect a pestivirus-specific antigen in leucocytes of sheep persistently infected with border disease virus. A blind trial was conducted to compare the specificity of the ELISA with conventional tissue culture virus isolation on blood samples from 58 sheep, aged 3 to 48 months. There was total agreement between the two tests; 27 sheep were shown to be BDV-infected. The ELISA OD values of the positive samples ranged from 0.12 to 0.86 and were not related to age, strain of virus with which they were infected or presence of serum neutralising antibody. Negative samples had OD values between 0 and 0.02.
Border disease (BD) of sheep is caused by a virus in the genus Pestivirus that results in decreased myelination throughout the CNS when acquired congenitally. Pregnant ewes were inoculated with BD virus at 50 days of gestation, and myelin proteins were quantified in several regions of the CNS during prenatal and postnatal development of infected lambs for comparison with age-matched controls. Newborn field-infected lambs were also examined. Myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were measured by densitometric scanning of western blots. Deficiencies in the myelin proteins were detected as early as 116 days of gestation, and the deficiencies of myelin proteins were most pronounced in the cerebellum at all ages examined. PLP and MBP increased from 10-30% of normal in cerebellar white matter at birth to 40-60% of normal at 6 months, suggesting some catch-up in the amount of compact myelin with development. MAG and CNP were between 70 and 80% of control levels in the cerebellum at birth and at 6 months. Similar results were obtained for the corpus callosum and spinal cord of infected lambs, but the deficiencies of myelin proteins were not as great. A common finding in all regions examined was that MBP and PLP were reduced more than MAG and CNP. This is probably explained by a greater deficit of compact myelin, in which MBP and PLP are localized, than of associated oligodendroglial membranes, in which MAG and CNP are concentrated.(ABSTRACT TRUNCATED AT 250 WORDS)
Infectious agents have long been suspected of playing a role in the initiation of Crohn's disease. The objective of this study was to search for likely microbial agents in diseased tissues using immunocytochemical techniques.
Intestines and mesenteric lymph node specimens of 21 patients from two French families with a high frequency of Crohn's disease and from Connecticut were studied. The microbial agents searched for included Bacteroides vulgatus, Borrelia burgdorferi, Escherichia coli, Listeria monocytogenes, Streptococcus spp., bovine viral diarrhea virus, influenza A virus, measles virus, parainfluenza virus, and respiratory syncytial virus.
Seventy-five percent of the patients with Crohn's disease (12 of 16) were positively labeled with the antibody to Listeria. Macrophages and giant cells immunolabeled for this antigen were distributed underneath ulcers, along fissures, around abscesses, within the lamina propria, in granulomas, and in the germinal centers of mesenteric lymph nodes. In addition, 57% (12 of 21) of the cases contained the E. coli antigen, and 44% (7 of 16) contained the streptococcal antigen. The immunolabeling for the latter two agents also occurred within macrophages and giant cells, distributed in a pattern similar to that of Listeria antigen.
The results suggest that Listeria spp., E. coli, and streptococci, but not measles virus, play a role in the pathogenesis of Crohn's disease.
We examined live virus vaccines against measles, mumps, and rubella for the presence of pestivirus RNA or of pestiviruses by reverse transcription PCR. Pestivirus RNA was detected in two measles-mumps-rubella combined vaccines and in two monovalent vaccines against mumps and rubella. Nucleotide sequence analysis of the PCR products indicated that a modified live vaccine strain used for immunization of cattle against bovine viral diarrhea is not responsible for the contamination of the vaccines.
An epidemiological survey for pestivirus was undertaken in Zambia and Europe, in view of the recent serological findings obtained by previous studies in Europe with humans. Collected sera were tested for anti-bovine viral diarrhea virus (BVDV) specific antibodies by IIF and Western Blotting. Of those individuals tested (n = 1272), 15.3% showed a seropositive reaction to the BVDV. Anti-BVDV antibody prevalence in immuno-depressed patients (e.g. HIV positive) was investigated. A higher prevalence was revealed in HIV patients suffering from chronic diarrhoea and in those having developed AIDS Related Complex (ARC). Our of 212 persons tested for pestivirus isolation, a non cytopathic virus strain was detected in 2 buffy coat samples using IIF with a specific anti-BVDV serum. The isolation could be repeated three times during 31 days in one person. The virus was identified as a pestivirus with radioimmuno-precipitation assays and IIF-flow cytometry. A doublet of 120 kD was identified only in cell lysates, indicating a non-structural protein. In order to rule out cross reactivity 30 sera from Hepatitis C seropositive patients were tested against the isolate by IIF-flow cytometry. No antigen-specific binding could be observed. These findings indicated the occurrence of a pestivirus in man and might suggest a relationship with a pestivirus of animal origin.
Two French families were investigated. In the first a husband, wife, and 4 children had Crohn's disease; in the second 7 of 11 children had the disease. There was no history of Crohn's disease in antecedent generations and no linkage to HLA haplotypes.
Methods included family interviews; review of medical records, radiographs, and pathology slides; serology; selective stool culture; enzyme-linked immunosorbent assay for fecal viral detection; and immunocytochemistry.
In both families multiple cases occurred among siblings in 7-13-month periods. There appeared to be a 4-8-year recurrence of new disease in both families. Radiographs showed a remarkable similarity in the pattern of disease, confined to distal ileum and cecum, in the members of family 1. Examination for pathology showed granulomas in all 8 patients for whom tissues were available. Acid-fast organisms or Campylobacter-like organisms were not found in tissue sections, and immunocytochemistry was negative for mycobacteria and Yersinia. Stool cultures were negative for mycobacteria, Yersinia, and Mycoplasma. Torovirus and coronavirus antigens were not found in stool. Serology was negative for antibodies to Brucella, Yersinia, influenza, and three enteropathogenic viruses of animals.
The circumstances and data suggest that an infectious microorganism is responsible for these clusterings of Crohn's disease.
The ruminant pestiviruses, bovine virus diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens which infect ruminant species worldwide. Although the serological relationships among ruminant pestiviruses require further clarification, there is growing evidence for two antigenic groups, one of which predominates in cattle and one in sheep. The success of pestiviruses stems from the ability of the non-cytopathic (NCP) biotype of the virus to cross the placenta and establish a persistent infection (PI) in the developing foetus. This biotype should be regarded as the 'normal' biotype with the cytopathic (CP) biotype being an abnormal virus that is usually isolated only from PI animals dying from mucosal disease. Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination events that include the insertion of host RNA and/or the duplication of viral RNA sequences. However, the biological mechanism through which CP viruses kill cells remains unknown. Virtually all CP and NCP viruses cause only mild, transient clinical symptoms in healthy adult animals and stimulate a protective immune response. Despite the urgent requirement for a safe, effective vaccine, there is still no commercial vaccine that has been shown to immunize dams so that foetal infection is prevented. In the absence of an effective vaccine, reliable diagnostic techniques are essential to implement effective control measures. There is now a range of monoclonal antibody-based enzyme-linked immunosorbent assays for identifying PI or convalescent animals. These tests are specific, rapid, sensitive and reliable but may themselves become redundant as they are superceded by ever-increasing molecular biology-based techniques.
Is some white matter damage in preterm neonates induced by a human pestivirus? We oVer the hypothesis that some forms of cerebral white matter damage (WMD) in preterm neonates might be caused by transplacental viral infection of the fetus during the first or second trimester of pregnancy. Potts and colleagues 1 speculated on the possible role of a human pes-tivirus (PV) in the aetiology of congenital microcephaly. We suggest PV might be a candidate virus for some form of WMD. We further oVer our view that a virus induced cytokine cascade might place the fetus at double jeopardy—that is, disturb white matter development and lead to preterm birth. Virus related WMD White matter damage is the most important predictor of childhood neuromotor disability among those born preterm. About 50% of infants who have echolucent zones in the periventricular white matter or ventriculomegaly on neonatal cranial ultrasound scans subsequently develop cerebral palsy. 2 The multiple expressions of WMD, both on histological examination and neonatal cranial ultrasound scan, as focal, multifocal, or diVuse WMD, 3 make multiple aetiologies likely. Some forms of WMD might be related to a hypoxic–ischaemic insult, but others might be associated with infection.
The family Togaviridae is described; it contains four genera--Alphavirus, Flavivirus, Pestivirus and Rubivirus--and additional members. The main characteristics of the family are as follows: single-stranded, linear RNA, molecular weight about 4 X 10(6). Virions have isometric nucleocapsids surrounded by a lipoprotein envelope containing host cell lipid and virus-specified polypeptides including one or more glycopeptides. Virions yield infectious RNA. There are at least 80 members; all the alphaviruses and most flaviviruses are arboviruses in the biological sense.
A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.
Neutrophil function and mononuclear cell proliferative responses to mitogens were determined in healthy cattle and in cattle persistently infected with bovine viral diarrhea (BVD) virus. Uptake of [3H]thymidine by resting and mitogen-stimulated peripheral blood mononuclear cells was significantly lower in cattle persistently infected with BVD virus than in healthy cattle. Neutrophils from cattle persistently infected with BVD virus had significantly impaired capability to ingest Staphylococcus aureus, but were normal in respect to random migration under agarose, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Impairment of neutrophil function in cattle persistently infected with BVD virus differs from impairment of neutrophil function reported in healthy cattle mounting an immune response to recent BVD virus infection.
Electron micrographs of intestinal epithelium of infant mice infected with epizootic diarrhea virus have demonstrated intracellular
spherical structures measuring 65 to 75 mµ in diameter which have a complex morphology resembling several virus particles. They have ben interpreted as being the etiologic
agent of this disease. The particles were present in association with, in some cases within, vesicles of the endoplasmic reticulum
of intestinal epithelial cells. They were never seen in the nucleus.
Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.
Solid-phase enzyme immunoassays can be utilized to detect antigens directly in clinical specimens. However, a small number of stools which we tested for human rotavirus by enzyme-linked immunosorbent assay (ELISA) were found to have nonspecific activity in the absence of rotaviral antigen. Similar nonspecific activity was found in eight of eight sera which contained rheumatoid factor. This nonspecific activity was markedly reduced by pretreatment of the specimens with reducing agents, normal goat serum, and anti-human immunoglobulin M (IgM). Thus, it is likely that these specimens contain an IgM antibody capable of reacting nonspecifically with the other components of the assay. Although pretreatment with the mild reducing agent N-acetylcysteine markedly reduced this nonspecific activity, such treatment did not reduce the specific ELISA activity due to rotavirus. Other treatments did produce a reduction in specific activity. Thus pretreatment with N-acetylcysteine offers a practical means to increase the specificity of ELISA systems without reducing their sensitivity.
Group B rotaviruses (GBRs) are fastidious agents which cause enteric disease in humans and a number of other animal species. Detailed study of the role of GBRs in human disease has been hampered by the lack of immunoreagents suitable for large-scale studies. We developed a monoclonal antibody which recognizes a group-reactive antigen contained in a number of strains of GBRs. When utilized in conjunction with a hyperimmune guinea pig antiserum to GBR, this monoclonal antibody can be used in an enzyme immunoassay system to detect a wide range of GBRs. Alternatively, this monoclonal antibody can be combined with sera obtained from GBR-infected animals to devise assays which are largely specific for the homologous strain of GBR. Reactivity was not noted in either system with strains of group A or group C rotaviruses or with other members of the family Reoviridae. These results indicate that GBRs contain both group-reactive and species-specific antigens which are distinct from those found in group A rotaviruses. The availability of well-defined immunoreagents will facilitate detailed studies of GBR infections in humans and animals.
The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus (BVDV) has been molecularly cloned and the nucleotide sequence determined. The cloned sequence was 12,573 nucleotides in length, corresponding to a molecular weight of 4.3 X 10(6), having a base composition of 32.2% A, 25.7% G, 22.1% U, and 20.0% C. However, the sequences at the 5' and 3' termini of the RNA have not been unequivocally established. A single major open reading frame extending the length of the molecule was found in the viral-sense (positive polarity) sequence. This open reading frame was capable of encoding 3988 amino acids, representing 449 kDa of protein.
Both cytopathic and noncytopathic bovine viral diarrhea virus (BVDV) were isolated from 16 of 17 bovine spleens representing 11 herds that had experienced acute BVD and from 12 of 21 bovine spleens from 1 herd affected with chronic BVD. It was concluded that isolation of cytopathic and noncytopathic BVDV from the same spleen probably indicates that an animal with a persistent, noncytopathic BVDV infection was superinfected with a cytopathic BVDV. The prevalence (greater than 70%) of 2 viruses in the spleen of cattle with acute or chronic BVD suggested that persistent infection with noncytopathic BVDV may be an important factor in the pathogenesis of BVD.
Eight healthy cattle that were persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) were inoculated with cell culture fluids that contained noncytopathic or cytopathic BVDV. A severe disease occurred after inoculation with cytopathic BVDV. The clinical signs, lesions, and immune response were consistent with those of clinical BVDV infections.
Bovine fetal lung tissue culture cells were infected with bovine viral diarrhea virus. The cells released substances into the supernatant fluid that suppressed the proliferative response of bovine peripheral blood mononuclear cells which had been stimulated with concanavalin A. Activity could be found in low molecular weight fractions (less than or equal to 2,000 daltons). During the assay, these fractions were required to inhibit the response of leukocytes to concanavalin A. Addition of indomethacin to the infected tissue culture cells inhibited generation of immunosuppressive supernatants; addition of indomethacin to the suppressive supernatants did not block their activity. The possible involvement of prostaglandins in this immunosuppressive activity is discussed.
Rotaviral diarrhea is endemic in most areas of the world, yet community-wide epidemics have not been reported in prospectively
monitored populations. This prospective study of the etiology of diarrhea included biweekly visits to the homes of 10070 of
the population of the White Mountain Apache Indians and began in April 1981. During a three-week period beginning 21 October,
1981, 342 new cases of diarrhea were identified on different parts of the reservation. Rotaviral antigen, detected by an enzyme-linked
immunosorbent assay, was identified in 169 (73%) of the 233 stool samples that were tested. Rotavirus was not detected in
any of the stool samples taken six months before or after the epidemic. During the epidemic, respiratory symptoms were present
in 44 (33%) of 135 rotavirus-positive patients compared with 17 (17%) of 98 rotavirus-negative patients (P < .05). This rapidly spreading epidemic involving all areas of the reservation, in the absence of a common source of exposure
of ill persons, suggests the possibility of respiratory transmission.
Electron microscopy of duodenal mucosa from nine children with acute non-bacterial gastroenteritis revealed virus particles in epithelial cells from six patients. The morphology of the virus particles was identical in each of the six children. The virus belonged to the orbivirus group. No virus particles were observed in duodenal mucosa obtained from three of these children after clinical recovery. This orbivirus is believed to have been an important cause of sporadic gastroenteritis in children in Melbourne during the 3 months of the survey.
Neutralizing antibody in serial serums from calves infected with non-cytopathogenic (NCP) strain No.12 of bovine viral diarrhea-mucosal disease (BVD-MD) virus was comparatively titrated by the semi-micro reverse plaque fromation (RPF) and the END methods. There was a tendency that the neutralizing antibody titer was estimated eightfold higher in the semi-micro RPF method than in the END method. Antigenic relationships among three Japanese strains and two American strains were examined by the cross-neutralization and kinetic neutralization tests using the RPF technique. In cross-neutralization test, differences in antibody titers were not more than twofold among three Japanese strains. However, there were eightfold differences in some combinations of the Japanese and American strains. Kinetic neutralization test also demonstrated that three NCP strains of BVD-MD virus isolated in Japan were closely related. The American strains were antigenically distinct from three Japanese strains used in this study although some antigenic similarity was recognized in some combinations of the strains and the antisera. Moreover, these two American strains appeared to differ one another slightly.
Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed.
The morphology of bovine viral diarrhea virus (BVDV) was studied by electron microscopy. The NADL strain of BVDV was plaque purified 3 times, concentrated by polyethylene glycol precipitation, and purified by centrifugation to equilibrium in continuous potassium tartrate density-gradients. The virus was examined by negative-stain electron microscopy in the presence or absence of specific antiserum. The density of BVDV was between 1.101 g/cm3 and 1.174 g/cm3, with the peak at maximum infectivity at 1.122 g/cm3. Oval to pleomorphic viral particles, 120 ( +/- 30) nm in diameter, were enriched in the peak of maximum infectivity. The detailed structure of virions was revealed: a 5- to 7-microns thick unit membrane-like envelope layer with numerous projecting knobs, 4 to 5 nm in diameter, surrounding an interior core-like structure. Viral particles measuring 120 ( +/- 30) nm were found in large aggregates in the presence of specific antiserum.
Electron micrographs of intestinal epithelium of infant mice infected with epizootic diarrhea virus have demonstrated intracellular spherical structures measuring 65 to 75 m(micro) in diameter which have a complex morphology resembling several virus particles. They have ben interpreted as being the etiologic agent of this disease. The particles were present in association with, in some cases within, vesicles of the endoplasmic reticulum of intestinal epithelial cells. They were never seen in the nucleus.