In this study, we report the characterization of a novel calicivirus (CV), the Tulane virus (TV), which was isolated from stool samples of captive juvenile rhesus macaques (Macaca mulatta) of the Tulane National Primate Research Center. The complete genome of TV contains 6,714 nucleotides plus a poly(A) tail and is organized into three open reading frames (ORFs) that encode the nonstructural (NS) polyprotein (ORF1); the capsid protein (ORF2), with an estimated molecular mass of 57.9 kDa; and a possible minor structural protein (ORF3), with an isoelectric point (pI) of 10.0 and a calculated molecular mass of 22.8 kDa. The NS polyprotein revealed all typical CV amino acid motifs, including GXXGXGKT (NTPase), EYXEX (Vpg), GDCG (protease), and GLPSG and YGDD (polymerase). Phylogenetic trees constructed for the NS polyprotein, NTPase, protease, polymerase, and capsid protein sequences consistently placed the TV on a branch rooted with Norovirus, but with distances equal to those between other genera. The TV can be cultured in a monkey kidney cell line (LLC-MK2) with the appearance of typical cytopathic effect. TV exhibits a typical CV morphology, with a diameter of 36 nm, and has a buoyant density of 1.37 g/ml. According to these physicochemical and genetic characteristics, TV represents a new CV genus for which we propose the name "Recovirus" (rhesus enteric CV). Although the pathogenicity of TV in rhesus macaques remains to be elucidated, the likelihood of TV causing intestinal infection and the availability of a tissue culture system make this virus a valuable surrogate for human CVs.
Chronic enterocolitis is the leading cause of morbidity in colonies of captive rhesus macaques (Macaca mulatta). This study's aim was to identify the common enteric pathogens frequently associated with chronic enterocolitis in normal, immunocompetent rhesus monkeys and to elucidate the influence of this clinical syndrome on the host immune system. We analyzed the fecal specimens from 100 rhesus macaques with or without clinical symptoms of chronic diarrhea. Retrospective analysis revealed an increased incidence of Campylobacter spp. (Campylobacter coli and Campylobacter jejuni), Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni in samples collected from animals with chronic diarrhea (P < 0.05). The presence of additional enteric pathogens, such as Escherichia coli, carrying the eaeA intimin or Stx2c Shiga toxin virulence genes, Balantidium coli, Giardia lamblia, Enterocytozoon bieneusi, and Trichuris trichiura was found in all animals regardless of whether diarrhea was present. In addition, the upregulation of interleukin-1␣ (IL-1␣), IL-3, and tumor necrosis factor alpha cytokine genes, accompanied by an increased presence of activated (CD4 ؉ CD69 ؉ ) T lymphocytes was found in gut-associated lymphoid tissues collected from animals with chronic enterocolitis and diarrhea in comparison with clinically healthy controls (P < 0.05). These data indicate that chronic enterocolitis and diarrhea are associated, in part, with a variety of enteric pathogens and highlight the importance of defining the microbiological status of nonhuman primates used for infectious disease studies. The data also suggest that chronic colitis in rhesus macaques may have potential as a model of inflammatory bowel disease in humans.
Background and AimsGluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity.MethodsUsing ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity.ResultsWhen fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse.ConclusionsGluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.
Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.
BackgroundBased on clinical, histopathological and serological similarities to human celiac disease (CD), we recently established the rhesus macaque model of gluten sensitivity. In this study, we further characterized this condition based on presence of anti-tissue transglutaminase 2 (TG2) antibodies, increased intestinal permeability and transepithelial transport of a proteolytically resistant, immunotoxic, 33-residue peptide from α2-gliadin in the distal duodenum of gluten-sensitive macaques.Methodology/Principal FindingsSix rhesus macaques were selected for study from a pool of 500, including two healthy controls and four gluten-sensitive animals with elevated anti-gliadin or anti-TG2 antibodies as well as history of non-infectious chronic diarrhea. Pediatric endoscope-guided pinch biopsies were collected from each animal's distal duodenum following administration of a gluten-containing diet (GD) and again after remission by gluten-free diet (GFD). Control biopsies always showed normal villous architecture, whereas gluten-sensitive animals on GD exhibited histopathology ranging from mild lymphocytic infiltration to villous atrophy, typical of human CD. Immunofluorescent microscopic analysis of biopsies revealed IgG+ and IgA+ plasma-like cells producing antibodies that colocalized with TG2 in gluten-sensitive macaques only. Following instillation in vivo, the Cy-3-labeled 33-residue gluten peptide colocalized with the brush border protein villin in all animals. In a substantially enteropathic macaque with “leaky” duodenum, the peptide penetrated beneath the epithelium into the lamina propria.Conclusions/SignificanceThe rhesus macaque model of gluten sensitivity not only resembles the histopathology of CD but it also may provide a model for studying intestinal permeability in states of epithelial integrity and disrepair.
Abstract. Transmissible gastroenteritis virus (TGEV), a coronavirus, replicates in intestinal enterocytes and causes diarrhea in young pigs. Porcine respiratory coronavirus (PRCV), a spike (S) gene natural deletion mutant of TGEV, has a respiratory tissue tropism and causes mild or subclinical respiratory infections. Conventional antigen-based diagnostic tests fail to differentiate TGEV and PRCV, and a blocking ELISA test to serologically differentiate TGEV/PRCV-infected pigs is conducted on convalescent serum retrospectively after disease outbreaks. A reverse transcription (RT)-nested polymerase chain reaction (PCR) with primers targeted to the S gene deletion region to differentiate TGEV/PRCV was developed. The specificity of the RT-nested PCR was confirmed with reference and recent field strains of TGEV/PRCV, and its sensitivity was analyzed by testing nasal and fecal samples collected from pigs at various days postinoculation (DPI) with TGEV or PRCV. Specific PCR products for TGEV/PRCV were detected only with the homologous reference or field coronaviruses and for 10-14 DPI of pigs with TGEV (feces) or PRCV (nasal samples). The RT-nested PCR assay was more sensitive than antigen-based assays on the basis of duration of virus detection in experimentally infected pigs and was directly applicable to nasal as well as fecal specimens from the field.Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family and is enveloped with a positive-stranded RNA genome. 3,7 Porcine respiratory coronavirus (PRCV) represents a natural deletion mutant of TGEV that appeared in 1983-1984 in Europe and in 1988 in the US. 3 Coronaviruses have 3 major structural proteins: the spike (S), the integral membrane glycoprotein, and the nucleocapsid protein. 3 TGEV replicates primarily in small intestinal enterocytes, whereas PRCV replicates predominantly in the respiratory tract. 3,7 According to sequence comparisons of PRCV and TGEV, PRCV has a large deletion in the 5Ј region of the S gene and minor deletions in genes 3 and 3-1. 3,11 These deletions are thought to influence the viral tissue tropism and virulence. The deletion size in the S gene ranges from 621 to 681 bp depending on the origin of the strain. 11 Recently, strains of TGEV with reduced enteropathogenicity were reported in the field. 6 A similar suspect TGEV outbreak of reduced virulence (mild diarrhea and intestinal lesions, slow disease spread among pigs) in nursery pigs from a swine herd in the US Midwest was investigated. Diagnosis of TGEV in these pigs was sporadic and inconsistent and presumably complicated by the presence of antibodies to PRCV confirmed by a blocking differential ELISA test on sera from a number of pigs in this herd (L. J. Saif and P. Lewis, unpublished). However, this latter test showed inconsistent results for TGEV/PRCV differentiation with serially collected samples from the same pigs within the herd (inconsistent individual immune status), and some pigs in the Received for publication May 20, 1999. herd tested only PRCV...
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