Interactions Between Infectious Foodborne Viruses and Bacterial Biofilms Formed on Different Food Contact Surfaces.

Bacterial biofilms contribute to contamination, spoilage, persistence, and hygiene failure in the food industry, but relatively little is known about the behavior of foodborne viruses evolving in the complex communities that make up biofilm. The aim of this study was to evaluate the association between enteric viruses and biofilms on food contact surfaces. Formed biofilms of mono- and multispecies cultures were prepared on glass, stainless steel, and polystyrene coupons and 105 pfu/ml of murine norovirus, rotavirus, and hepatitis A virus were added and incubated for 15 min, 90 min, and 24 h. The data obtained clearly demonstrate that the presence of biofilms generally influences the adhesion of enteric viruses to different surfaces. Many significant increases in attachment rates were observed, particularly with rotavirus whose rate of viral infectious particles increased 7000 times in the presence of Pseudomonas fluorescens on polystyrene after 24 h of incubation and with hepatitis A virus, which seems to have an affinity for the biofilms formed by lactic acid bacteria. Murine norovirus seems to be the least influenced by the presence of biofilms with few significant increases. However, the different factors surrounding this association are unknown and seem to vary according to the viruses, the environmental conditions, and the composition of the biofilm.

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.

Impact of seasonal temperature transition, alkalinity and other abiotic factors on the persistence of viruses in swine and dairy manures.

Animal manures are a valued source of nutrients for crop production. They frequently do, however, contain zoonotic pathogens including a wide range of viruses. Ideally, manures would be treated prior to land application, reducing the burden of zoonotic viruses, and thus the potential for transmission to adjacent water resources or crops intended for human or animal consumption. In the present study, manure was obtained from four dairy and three swine farms. The manure was incubated anaerobically in the laboratory for 28 weeks at temperatures ranging from 4 to 25 °C, and multiple physical and chemical parameters were monitored. The abundance of various DNA and RNA viruses was measured throughout the incubation by amplifying virus-specific gene targets. A combination of statistical analyses were applied to identify whether the viruses are significantly impacted by temperature transition or affected by other abiotic factors. Temperature had no effect on the persistence of any of the viruses studied. An increase in pH of the manures during the incubation was significantly (P < 0.05) associated with decreased persistence, suggesting that pH manipulation during storage could reduce the abundance of viruses.

An efficient recovery method for enteric viral particles from agricultural soils.

Enteric viruses have been recognized as the leading cause of non-bacterial gastroenteritis and hepatitis outbreaks around the world. Understanding their prevalence and persistence in the environment is important for the effective control of these infections. The aim of this study was to develop an efficient recovery procedure for viral infectious particles from agricultural soils. Samples (25 g) of soil (black earth soil, loamy soil, and sandy soil) were spiked with murine norovirus (MNV) and feline calicivirus (FCV), mixed with five different buffers and viral genetic material was extracted by 3 commercial kits. The combination consisted by the modified Eagle's medium buffer followed by Dynabeads nucleic acid extraction kit, when the detection is conducted by molecular biology, has been identified as being the most effective procedure to preserve the viral particle infectivity and also to remove PCR inhibitors. The recovery percentages of infectious MNV for the 3 types of soils were 54.3%, 54.4%, and 56.9%. In contrast, the titres of the FCV varied depending on the type of soil, and the recovery percentages were 47.8% in the black soil, 15.6% in the loamy soil, and 17.7% in the sandy soil. Also, the results presented in this study highlight the importance of using an internal process control such as artificial inoculation with MNV at known concentrations during detection by molecular methods, in order to avoid the occurrence of false negative reactions.

Evaluation of survival of murine norovirus-1 during sauerkraut fermentation and storage under standard and low-sodium conditions.

Sodium reduction strategies have raised a few concerns in regards to possible outbreaks in unpasteurised raw fermented vegetables. Among potential outbreak agents, foodborne viruses are recognized as an important cause of food-borne illnesses. As most of them are acid-resistant, evaluation of the efficacy of lactic fermentation in inactivating enteric viruses must be considered to ensure the safety of these foods. In particular with the sodium reduction trend which could impair adequate fermentation in vegetables, we have challenged sauerkraut fermentation at a final concentration of 4 log TCID50/mL with the murine norovirus (MNV-1). Three sodium chloride concentrations (1.0%, 1.5%, 2.0%) were evaluated in spontaneous and starter fermentation of sauerkraut and were followed during fermentation and over a storage phase of 90 days. Detection of MNV-1 genetic material was carried out by real-time RT-PCR and the infectivity on cell culture. Real-time RT-PCR results showed that viral RNA was still detected after 90 day in sauerkraut under all the different conditions. Furthermore, MNV-1 viral particles were able to infect RAW cells after 90 days of storage with a non-significant viral charge reduction. Sodium reduction has a significant impact on the fermentation processing of sauerkraut but no influence on the destruction of norovirus particles or on their survival.

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.

Survey of Canadian retail pork chops and pork livers for detection of hepatitis E virus, norovirus, and rotavirus using real time RT-PCR.

Over the past 15 years, hepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV) have been hypothesized to be potentially zoonotic; swine and pork have been suggested as possible human infection sources for all 3 viruses. Our objective was to estimate HEV, NoV, and RV prevalence and load on Canadian retail pork chops and livers. Using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) sampling platform, pork livers (n=283) and chops (n=599) were collected, processed, and assayed for the 3 viruses by four collaborating federal laboratories using validated real time reverse transcriptase polymerase chain reactions (qRT-PCR). Follow-up qRT-PCR estimating viral load in genomic copies/g was followed by nested classical RT-PCR and isolate sequencing of a partial segment of the ORF2 gene. Local alignments were performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation); a phylogenetic tree was created. Twenty-five livers and 6 chops were classified 'positive' (thresholds for viral RNA detected in both replicates of the assay) or 'suspect' (thresholds detected in one of two replicates) for HEV. Follow-up qRT-PCR detected HEV on 16 livers, 0 chops, and nested classical RT-PCR, on 14 livers and 0 chops. Initial qRT-PCR classified 12 chops 'suspect' for NoV. Follow-up qRT-PCR detected viral RNA on only one sample with thresholds greater than 40 in both replicates. No amplicon was yielded, and therefore no isolate was sequenced from this sample. Partial ORF2 genes from 14 HEV isolates were sequenced, and compared via sequence identity and phylogenetic analysis with selected human case isolates listed in NCBI-GenBank. Overall, HEV prevalence on retail pork was comparable with other published reports.

Presence, viral load and characterization of Torque teno sus viruses in liver and pork chop samples at retail.

Torque teno viruses (TTV) are widespread in humans, swine as well as in several other animal species. In market ready swine, the reported prevalence ranges between 11% and 100%. Through a national retail sampling plan from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program, 283 and 599 liver and pork chop samples, respectively, were collected over a 12-month period from commercial establishments in 5 selected geographical regions of Canada to assess the presence of Torque teno sus viruses (TTSuVs) in these products. TTSuVs were detected in 97.9% of pork chops with viral loads ranging between 1×10(4) and 9.9×10(5) genomic copies (gc)/g and 98.6% of liver samples with viral loads ranging from 1×10(5) to 9.9×10(6) gc/g. A selection of 20 positive samples (10 pork chop and 10 liver) from the 5 geographical regions were further tested for the production, of a 305bp fragment for TTSuV1 and a 253bp fragment for TTSuV2 in the non-coding region. TTSuV1 was present in all 10 liver and 10 pork chops samples while TTSuV2 was detected in 10 liver and 9 pork chop samples. Two different TTSuV1 sequences were simultaneously detected from 5 of 20 samples and 2 different TTSuV2 sequences were detected from 6 of 19 samples. The omnipresence of TTSuVs in commercial pork samples may allow its use as a viral indicator to monitor the effectiveness of cleaning and disinfecting process in slaughtering, cutting, slicing and packaging facilities.

Torque Teno virus in children who underwent orthotopic liver transplantation: new insights about a common pathogen.

BACKGROUND: Torque Teno virus (TTV) is a ubiquitous infectious agent. Transplant recipients are at risk of hepatitis E virus (HEV) infection and could be vulnerable to TTV-associated adverse effects. The aim of this study was to evaluate the influence of immunosuppression and HEV infection on TTV replication and liver injury in pediatric patients after orthotopic liver transplantation (OLT). METHODS: Pediatric recipients of liver transplants were classified into the following 2 groups: (1) those with normal serum aminotransferases levels and (2) those with persistently increased serum aminotransferases levels and histological features of chronic hepatitis of unknown etiology. The TTV load was assessed in 342 serum samples by use of TaqMan real-time polymerase chain reaction, along with TTV genogroups and coinfection with HEV. RESULTS: TTV DNA was detected in 96% of tested serum samples. Viral load was significantly lower in patients with features of chronic hepatitis, of whom 78% had liver fibrosis scores of ≥2. Viral load decreased during posttransplantation follow-up. Viral load and genogroups were influenced by immunosuppression. Lower viral load was observed in patients coinfected with HEV. CONCLUSIONS: TTV infection is widespread, and its replication is closely related to immune status and viral coinfection. High TTV viremia is not associated with hepatitis after OLT, but, conversely, liver inflammatory activity impairs TTV replication.

Real-time PCR study of the infection dynamics of Torque teno sus viruses in naturally infected pigs from nursery to slaughterhouse.

Although torque teno viruses are frequently detected in humans and animals the pathogenicity and virulence of these viruses are currently unknown, and little information on the dynamics of infection is available. In this study, 24 pigs were monitored from 2 weeks of age to slaughter to determine the natural infection dynamics of Torque teno sus virus (TTSuV) species 1 (TTSuV1) and species 2 (TTSuV2). Plasma and fecal material were collected at 2, 8 and 18 weeks of age and between 22 and 29 weeks of age, while organs and tissues were collected at slaughter. Both viruses were found to disseminate very quickly within the herd. At 8 weeks, TTSuV2 and TTSuV1 were observed at rates of 88% and 58%, respectively, and peak excretion occurred at 18 weeks. Furthermore, TTSuV DNA was detected in the plasma of between 96% and 100% of the animals, and viremia was maintained at a viral load between 5 × 10(4) and 5 × 10(6)copies/mL until slaughter. All tested organs and tissues contained TTSuVs in amounts ranging from 10(3) to 10(8)copies/g; the highest concentration of viruses was found in the liver. This study did not demonstrate any association with disease, despite the high prevalence of TTSuVs.

Detection of human food-borne and zoonotic viruses on irrigated, field-grown strawberries.

This study evaluated the presence of pathogenic human and zoonotic viruses on irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus, and swine hepatitis E virus genogroup 3 were detected on strawberries, and irrigation water is suspected as the contamination origin.

Simultaneous recovery of bacteria and viruses from contaminated water and spinach by a filtration method.

Water and leafy vegetables eaten fresh are increasingly reported as being involved in food-borne illness cases. The pathogenic agents responsible for these infections are mainly bacteria and viruses and are present in very small quantities on the contaminated food matrices. Laboratory techniques used to isolate or detect the contaminating agent differ enormously according to the type of microorganisms, generating time and economical losses. The purpose of this study was to optimize a single method which allows at the same time the recovery and concentration of these two main types of pathogenic organisms. Water and spinach samples were artificially contaminated with the feline calicivirus (FCV), rotavirus, hepatitis A virus (HAV), Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella Typhimurium. The principle behind the recovery technique is based on the use of a positively charged membrane which adsorbs both viruses and bacteria present in the water or in the rinse from the vegetables. Using conventional microbiology, PCR and RT-PCR, this filtration technique allowed a detection level superior to 10² CFU/g for S. Typhimurium, E. coli, L. monocytogenes and C. jejuni and to 10¹ PFU/g for FCV, HAV and rotavirus. This combined method can also be applied to other bacterial and viral species for the identification of the responsible agent for food-borne illnesses.

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.

Characterization of novel porcine sapoviruses.

Sapoviruses are common caliciviruses known to cause enteric diseases in humans and animals. SaVs are genetically highly heterogeneous and are presently classified in five genogroups that are further subdivided in a number of genotypes. In recent years, a number of novel animal SaV strains, mostly of swine origin, have been partially characterized and proposed to represent novel genogroups or genotypes. We previously reported the detection and partial characterization of a wide range of variable and novel SaV strains of uncertain taxonomic status in Canadian swine. We now report on further genomic characterization of two novel strains to clarify their taxonomic relationship to other swine and human SaVs. Detailed analysis of different regions of their genomes, including determination of their complete capsid sequence, did not permit clear taxonomic assignment according to current criteria. This situation appears reminiscent of that of a number of SaV strains of swine origin and calls for a classification update for this calicivirus genus. We also report the detection of swine GIII SaVs for the first time in Canada.

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.

The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses.

Many food and waterborne outbreaks of infectious disease are caused by viruses. While numerous methods exist and are being developed to test food and water for the presence of enteric viruses, there is no standard control for the comparison of different methods. Potential control viruses should be well characterized, share the physical characteristics of the enterically infecting viruses and not normally be associated with foods. Here, the feline calicivirus (FCV) is proposed as a sample process control for methods aimed at the extraction and detection of RNA viruses in food and water. FCV is shown to be useful as a control for the extraction of hepatitis A virus (HAV) from water using filtration technology and from strawberries using the Pathatrix system. The FCV standard provides a valuable quality control tool when testing potentially contaminated food samples.

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.