Molecular characterization of a new PToV strain. Evolutionary implications.

Toroviruses are emergent viruses, belonging to the Nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. Thus far, only five variants of porcine torovirus (PToV) have been identified. In this report we describe the identification and partial characterization of a new strain of porcine torovirus (PToV-BRES) that was detected by RT-PCR in a swine faecal specimen from a farm in Brescia (Italy). The complete genes coding for the nucleocapsid (N), hemagglutinin-esterase (HE) and membrane (M) proteins were amplified, and sequence analysis showed that PToV-BRES is a new PToV strain that, based on the HE gene sequence, is phylogenetically related to P4 strain, that was up to now the only member of a distinct PToV lineage. The nucleocapsid protein from PToV-BRES was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an ELISA method to detect antibodies against PToV. This assay was evaluated using a serum collection including 45 samples from three commercial farms from Spain. High antibody prevalence against PToV was observed in the three farms, both in adult animals and in piglets, which could suggest that PToV might be endemic in Spanish porcine population. The ELISA method developed in this work could be useful in future epidemiological surveys about toroviruses.

Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

Rapid and sensitive detection of porcine torovirus by a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP).

Porcine torovirus (PToV) is associated with swine gastroenteritis, but its pathogenesis is uncertain because there is limited information regarding PToV due to its difficulty to adapt in vitro. This study has developed a rapid one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of PToV. A set of four primers specific to six regions within the PToV's highly conserved fragment of the M gene was designed for use with the RT-LAMP assay. The RT-LAMP assay was sensitive with a detection limit of 1 × 10(1)copies/µL, which was 100-fold higher than reverse-transcription PCR. No cross-reaction was observed with other similar viruses. A total of 175 clinical specimens were collected from the Sichuan province, and PToV was detected by the established RT-LAMP assay with a positive rate of 39.2% (69/175). This study developed the first rapid, sensitive, simple, cost-effective and accurate method for the detection of PToV. The results show that the RT-LAMP assay is highly feasible in clinical settings.

Detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle.

The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (< or = 6 months old), 27 young adults (52 years), 125 adults (> or = 2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV. Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves (P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent.

Enteric and nasal shedding of bovine torovirus (Breda virus) in feedlot cattle.

OBJECTIVE: To assess fecal and nasal shedding patterns of bovine torovirus (BoTV) in cattle at time of arrival and periodically throughout the first 21 days after arrival at a feedlot. ANIMALS: 57 steers. PROCEDURE: Fecal and nasal-swab samples collected on days 0, 4, 14, and 21 after arrival were tested for BoTV, using ELISA. A subset of samples from calves testing positive and negative for BoTV was analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR). Paired serum samples were collected on days 0 and 21 and tested for BoTV antibodies, using a hemagglutination inhibition assay. RESULTS: Overall rate of fecal shedding of BoTV was 21 of 57 (37%) by ELISA and 40 of 42 (95%) by RT-PCR with peak shedding on day 4. Diarrhea was more common in calves shedding BoTV than those not shedding the virus (odds ratio, 1.72). Overall rate of nasal shedding of BoTV was 15 of 57 (26%) by ELISA and 42 of 42 (100%) by RT-PCR, with peak shedding on day 0. Specificity of the RT-PCR product was confirmed by sequence analysis. Approximately 93% of the calves seroconverted to BoTV (> 4-fold increase in titer). Differences were not detected between calves shedding BoTV and nonshedders in relation to disease and treatments, perhaps because of the low number of cattle in the study. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirmed BoTV infections in feedlot cattle, including BoTV antigen and viral RNA in nasal secretions, and the shedding pattern during the first 21 days after arrival in a feedlot.

Association of enteric shedding of bovine torovirus (Breda virus) and other enteropathogens with diarrhea in veal calves.

OBJECTIVE: To determine the prevalence, fecal shedding pattern, and association of bovine torovirus (BoTV) with diarrhea in veal calves at time of arrival and periodically throughout the first 35 days after their arrival on a veal farm. ANIMALS: 62 veal calves. PROCEDURE: Fecal samples collected on days 0, 4, 14, and 35 after arrival were tested for BoTV by use of ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Paired serum samples obtained from blood collected on days 0 and 35 were analyzed for BoTV antibodies with a hemagglutination inhibition assay. Fecal samples were also screened for other enteric pathogens, including rotavirus, coronavirus, and Cryptosporidium spp. RESULTS: Fecal shedding of BoTV was detected in 15 of 62 (24%) calves by use of ELISA and RT-PCR assay, with peak shedding on day 4. A significant independent association between BoTV shedding and diarrhea was observed. In addition, calves shedding > or = 2 enteric pathogens were more likely to have diarrhea than calves shedding < or = 1 pathogen. Calves that were seronegative or had low antibody titers against BoTV (< or = 1:10 hemagglutination inhibition units) at arrival seroconverted to BoTV (> 4-fold increase in titer); these calves were more likely to shed virus than calves that were seropositive against BoTV at arrival. CONCLUSIONS AND CLINICAL RELEVANCE: Shedding of BoTV was strongly associated with diarrhea in neonatal veal calves during the first week after arrival at the farm. These data provide evidence that BoTV is an important pathogen of neonatal veal calves.

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses.

The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.

Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses.

Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 x 10(2) BToV and 2.17 x 10(3) PToV copies/reaction (correlation coefficiency=0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 x 10(5) and 2.54 x 10(4) (BToV) and 2.17 x 10(7) and 2.17 x 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n=121) and piglets (n=86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P<0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.

Detection of porcine torovirus by real time RT-PCR in piglets from a Spanish farm.

Toroviruses are enteric viruses belonging to the Nidovirales order that infect different animal species and humans. The lack of "in vitro" culture systems for toroviruses, except for the prototype Berne virus or BEV, isolated originally from an infected horse, has hampered their study and the development of diagnostic assays. This report describes a real time RT-PCR method to detect porcine torovirus (PToV) RNA in clinical fecal samples using primers corresponding to the gene coding for the nucleocapsid protein which are conserved in all PToV strains known to date. This method can be used to determine viral loads allowing quantitation within a range between 10(1) and 10(8) genomic units per reaction tube. The assay was evaluated with 48 rectal swabs from piglets from a Spanish farm. Nineteen out of 48 animals were shedding virus at the time of sample collection, indicating a high incidence of PToV infection in this farm. This is the first report showing the presence of PToV in Spain. The real time RT-PCR assay described in this report provides a rapid, highly sensitive, specific and reliable detection and quantitation method enabling future PToV epidemiological studies.

Human torovirus: a new virus associated with neonatal necrotizing enterocolitis.

AIM: Toroviruses have been associated with gastroenteritis in both animals and humans. The aim of this study was to examine the fecal excretion of torovirus in infants with necrotizing enterocolitis (NEC). METHODS: We reviewed all infants with NEC admitted to our tertiary care NICU over a 5-y period who had stool specimens sent for microbial culture and virology. Infants in the NICU during the same period with diagnoses other than NEC served as controls. RESULTS: Forty-four infants with NEC stages I-III were identified, and pathogenic organisms were identified in 27 (61%). Toroviruses were identified in stool cultures in 48% of patients with NEC, and 17% of the non-NEC controls (p<0.001). There was no significant difference in illness severity or mortality between the torovirus-positive and -negative infants with NEC. CONCLUSION: Torovirus should be added to the list of infectious agents associated with NEC in newborn infants. The exact role torovirus plays in the etiology and progression of NEC warrants further investigation.

[Emergent riboviruses implicated in gastroenteritis]. / Ribovirus emergentes implicados en las gastroenteritis.

Viral agents are one of the main causes of acute diarrhea, particularly in infants and young children. Astrovirus, coronavirus, torovirus, and picobirnavirus are increasingly being identified as causative agents of gastroenteritis. Astroviruses have been detected in the stools of between 1.2% and 20% of children with diarrhea requiring medical care in a variety of geographical areas. Outbreaks have been described in schools, day care settings and pediatric wards. Children younger than 3 years old are the most frequently affected. In temperate climates incidence is greater in winter whereas in tropical areas infection occurs throughout the year. Transmission is mainly through the fecaloral route. At least seven serotypes of human astroviruses have been recognized and serotype 1 is more common than the other serotypes. Astroviruses are often shed in stools during long periods and can be detected by electron microscopy. An enzymeimmunoassay technique that detects the astrovirus group antigen has been widely used in epidemiological studies. Nucleic acid hybridization and polymerase chain reactionbased techniques have also been used. Enteric coronaviruses have most frequently been associated with gastrointestinal disease in neonates and children younger than 12 years old. The role of toroviruses and picobirnaviruses as causative agents of gastroenteritis is still emerging. Further epidemiological studies to determine the frequency of these viruses in the community and to identify their mechanisms of transmission are needed, as are further studies to elucidate the pathophysiology of diseases due to these agents.