Persistence of murine norovirus, bovine rotavirus, and hepatitis A virus on stainless steel surfaces, in spring water, and on blueberries.

The viability of murine norovirus (MNV-1), bovine rotavirus (boRV), and hepatitis A virus (HAV) was evaluated at 21 °C, 4 °C, and -20 °C on stainless steel surfaces, in bottled water, and on blueberries for up to 21 days. After 14 days of incubation at 21 °C on stainless steel, a viability loss >4 log for MNV-1, >8 log for boRV, and >1 log for HAV was observed. Losses were observed for MNV-1 (>1 log) and HAV (>2 log) incubated in water at 21 °C for 21 days. No significant loss was detected for MNV-1 and HAV at 4 °C and -20 °C and for boRV at 21 °C, 4 °C, and -20 °C. On blueberries incubated at 4 °C and -20 °C, they all maintained their infectivity. After 7 days at 21 °C, a loss >2 log, a loss of 3 log, and no loss were observed for boRV, MNV-1, and HAV, respectively. After RNase pretreatment, the detection of extracted RNA from infectious and noninfectious samples suggested the protection of RNA inside the capsid. Even though they all are enteric viruses, their persistence varied with temperature and the nature of the commodity. It is therefore important to use more than one viral surrogate, during inactivation treatments or implementation of control measures.

Association of age and gender with Torque teno virus detection in stools from diarrheic and non-diarrheic people.

BACKGROUND: Torque teno virus (TTV) is a small virus belongs to Anelloviridea family. TTV is a disease orphan virus but it has often been associated with a variety of pathologies and co-infections. TTV was recently identified as an infectious agent that could potentially be involved in cases of acute enteritis. OBJECTIVES: To ascertain the presence of TTV in stools from diarrheic and not diarrheic people, and to investigate an association between infection, and patient age and gender. STUDY DESIGN: Stool samples from people exhibiting signs of enteritis (954) and from non-diarrheic individuals (76) were collected in the former Chinook Health Region (CHR) in Southwestern Alberta, Canada from May 2008 to April 2009. Viral genetic material was extracted, and detection and quantification of TTV were carried out by real-time PCR. The presence of other viral and bacterial enteric pathogens was also investigated. RESULTS: More (P<0.001) diarrheic people (38.8%) tested positive for TTV DNA than non-diarrheic individuals (18.4%). Furthermore, viral load was greater (P<0.001) in stools from diarrheic (2.0×10(7)copies/g) than non-diarrheic (2.0×10(3)copies/g) people. TTV DNA was detected most often in diarrheic individuals that were 0-5 (57.3%) and greater than 81 (59.0%) years of age. Combined across age, the prevalence of TTV was higher among men than women (P=0.003). Co-infections with other enteric pathogens were observed. CONCLUSIONS: This study revealed a significant association between TTV prevalence and viral load, and enteritis. Also, TTV prevalence was significantly higher in the very young and elderly suggesting that immunological status is important.

Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR.

Although uncommon in North America, Hepatitis E virus (HEV) has been identified in some industrialized countries in patients without a history of travel to HEV-endemic countries. Its presence is ubiquitous worldwide in swine populations. Zoonotic transmission of swine HEV to non human primates has been achieved experimentally and transmission of HEV after ingestion of contaminated raw or undercooked meat is well documented. In Canada, so far, no HEV outbreak has been documented but HEV genotype 3 strains have been identified in sera and faecal samples of swine origin. The objective of the present study was to determine the viral load of HEV in liver, loin, bladder, hepatic lymph node, bile, tonsil, plasma and faeces samples of 43 pigs at slaughter. Feline calicivirus (FCV) was used as sample process control to validate the RNA extraction process, as a confirmation of the absence of sample inhibitors and as an amplification control. Using FCV/HEV multiplex TaqMan RT-qPCR system, HEV RNA was detected in 14 out of the 43 animals tested. HEV was detected in lymph nodes (11/43), bladder (10/43), liver (9/43), bile (8/43), faeces (6/43), tonsils (3/43), plasma (1/43) samples from infected animals. No HEV-positive loin samples were observed. Viral loads of 10(3) to 10(7) copies/g were estimated in positive liver and bile samples.

Comparison of commercial viral genomic extraction kits for the molecular detection of foodborne viruses.

When genetic material is extracted from viruses responsible for food illnesses, two broad types of possibilities are offered: conventional methods, which are well established but usually long and exacting to perform, or commercial kits, which are faster and easy to use but much more expensive. Thus, it is important to evaluate some performance parameters such as the analytical sensitivity to be able to select the optimal technique for each situation. The principal objective of this study was to establish and compare the analytical sensitivities of three commercial genetic material extraction methods (TRIzol reagent, FTA cards, and QIAGEN kits) along with three selected viruses, adenovirus, hepatitis A virus, and rotavirus. Viral detection was carried out using a standard PCR technique for adenovirus and reverse transcription PCR for rotavirus and hepatitis A virus. The results obtained showed that with the QIAGEN kit, the sensitivity was 2 logs lower than with the two other methods for all three viruses studied. Nevertheless, despite their lower analytical sensitivities, the other two extraction methods should not be overlooked and ought to be considered when evaluating the most efficient approach suitable for a specific commodity, since food-related outbreaks may be traced to a wide variety of food types.

Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter.

Hepatitis E virus has recently been recognized as having zoonotic potential and could be transmitted from pig to human. Pigs are identified as a potential animal reservoir and HEV is highly prevalent in the swine population around the world. In this study, the presence of HEV was investigated in 51 subjects reared on a simulated commercial farm setting from the age of 2 weeks up to slaughter. Samples were collected on four occasions: at 2, 8, and 18 weeks and between 22-29 weeks of age. Anti-HEV IgG in plasma samples, presence of HEV RNA in plasma samples and feces were monitored. At 2 weeks of age, HEV RNA was detected in feces of 6 subjects (11.8%) but not in their plasma. At 8 weeks, HEV was detected in feces of 27 subjects (52.9%) and in plasma of one subject. At 18 weeks, HEV was detected in feces of 44 subjects (86.2%) and in plasma of 24 subjects (47.1%). At slaughter time (22-29 weeks), HEV was present in plasma of 6 subjects (11.8%) and in stools of 21 subjects (41.2%). Spread of the virus inside the population was evaluated by comparison of means (paired t-test, P<0.05) of anti-HEV IgG ELISA results from the 4 bleedings. Significant differences were noted between the results of populations at 8 and 18 weeks and also between 18 and 22 to 29 weeks indicating an immune response to the virus. Based on the comparison of a 304 nucleotides sequence of the 5' ORF 2 gene, all amplified fragments clustered in genotype 3a.

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus.

Three novel real-time TaqMan RT-PCR assays targeting the 5'-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5'-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5'-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5'-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.

Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material.

Using different primer and probe sets, RT-PCR, NASBA and TaqMan RT-PCR molecular methods were compared to detect NoV GII in 13 clinical stool samples. The RT-PCR results observed by gel electrophoresis (Ando, Kageyama and Anderson amplification and probe systems), dot blot hybridization (Ando and Kageyama) and real-time TaqMan assay (Ando and Kageyama) were shown to be consistent and reproducible for the detection of NoV GII. Whereas, the NASBA assay using Ando primers showed some reproducibility discrepancies. Detection limits of the NoV GII/Kageyama system were found to be equal or significantly higher than the Ando system. Real-time TaqMan RT-PCR assay showed similar detection limits to that of NASBA with the Kageyama amplification and detection system, while it was 1log less sensitive than the Ando system. In a clinical context, RT-PCR, NASBA and real-time TaqMan RT-PCR methods using undiluted samples were all suitable for the detection of NoV GII, however the NASBA assay provided less consistent signals. The NoV GII Kageyama real-time TaqMan RT-PCR assay was reliable with a high analytical sensitivity and has shown the capability of detecting one genomic equivalent copy.