Bovine Polyomavirus Research Articles

Investigation of Human and Animal Viruses in Water Matrices from a Rural Area in Southeastern Region of Brazil and Their Potential Use as Microbial Source-Tracking Markers.

This study assessed the sources of contamination of water matrices in a rural area using detection of a host-specific virus (human adenovirus [HAdV], porcine adenovirus [PAdV] and bovine polyomaviruses [BoPyV]) as potential microbial source-tracking tool, and rotavirus A [RVA], given its epidemiological importance in Brazil. From July 2017 to June 2018, 92 samples were collected from eight points (P1-P8) of surface and raw waters in southeastern region of Brazil. Fifty-five (59.8%) were positive for HAdV, 41 (44.5%) for RVA, 10 (10.9%) for PAdV and four (4.3%) for BoPyV. HAdV and RVA were detected at all sites, and over the entire sampling period, PAdV was detected at a porcine breeding area and at Guarda River site, presenting high concentrations up to 2.6 × 109 genome copies per liter [GC/L], and viral concentrations ranging from 9.6 × 101 to 7.1 × 107, while BoPyV (1.5 × 104 GC/L-9.2 × 105 GC/L) was only detected in samples from the bovine breeding areas. The combination of human and animal virus circulation presents a potential impact in the environment due to raw sewage discharge from regional communities, as well as potential hazard to human and animal health.

Assessment of Microbiological Quality of Fresh Vegetables and Oysters Produced in Buenos Aires Province, Argentina.

Fresh vegetables and shellfish are prone to microbial contamination through irrigation or breeding with sewage-polluted waters, as well as by infected food handlers. In this work, we studied the presence of human and bovine polyomaviruses and human norovirus in fresh lettuces, strawberries and oysters produced in Buenos Aires province, Argentina. In oysters, we also investigated F-specific RNA bacteriophages, indicator Escherichia coli (E. coli) and pathogen bacteria of concern (Salmonella spp., Vibrio spp.). Within vegetables, we found viral contamination of human origin given the presence of human-associated polyomaviruses -MCPyV, HPyV6, JCPyV, and SV40- in lettuce and strawberry samples (16 and 10%, respectively), probably coming from irrigation waters and food handling. Among oysters, human (MCPyV, 4.2%) and bovine (BPyV1, 8.4%) polyomaviruses were detected even with low counts of E. coli. Bacteriophages (n = 3) and Salmonella spp. (n = 1) were also found, while Vibrio spp. was not detected. These results may indicate that the contamination in oysters comes from human and animal excreta, probably present in breeding waters. Norovirus was not detected in any food sample. To our knowledge, this is the first description of SV40 in lettuces and MCPyV and BPyV1 in oysters. The detection of different viral contaminants encourages further studies to evaluate the need for including viral indicators in microbiological standards. The identification of possible sources and routes of contamination using viral markers during routine microbiological controls, such as the polyomaviruses used in this work, would be useful to focus attention on the most hazardous stages of the food production chain.

Assessment of the Incidence of Human Adenovirus in Surface Waters of Southwest Greece: Vouraikos River as a Case Study.

The purpose of this study is to assess the overall impact of different anthropogenic activities in the Vouraikos River basin (southwestern Greece, Natura 2000 area). Virological quality of river water samples was investigated. Positive samples for human adenoviruses were found occasionally, while porcine adenoviruses and bovine polyoma viruses were not detected. It is the first time that virological data are collected in the study area.

Microbial Source Tracking Analysis Using Viral Indicators in Santa Lucía and Uruguay Rivers, Uruguay.

The aim of this study was to determine the origin (human, bovine or porcine) and the concentration of the fecal sources of contamination in waters from Santa Lucía basin and Uruguay River in Uruguay by using host-specific viral markers (adenoviruses and polyomaviruses) as microbial source tracking (MST). Between June 2015 and May 2016, monthly collections of surface water samples were performed in six sites in Santa Lucía basin and four sites in Uruguay River (n = 120 samples). Viral concentration was carried out using an absorption-elution method. Detection and quantification of human and porcine adenovirus (HAdV and PAdV, respectively) and human and bovine polyomavirus (HPyV and BoPyV, respectively) were performed by quantitative PCR (qPCR). To evaluate the infectivity of circulating HAdV, an integrated cell culture-qPCR (ICC-qPCR) was used. A logistic regression analysis was carried out to estimate the influence of environmental variables on the virus presence in surface waters. Overall, HAdV was the prevalent (18%; 21/120) followed by BoPyV (11%; 13/120) and HPyV (3%; 3/120), whereas PAdV was not detected in this study. The mean concentration ranged from 1.5 × 104 genomic copies/L (gc/L) for HAdV to 1.8 × 102 gc/L for HPyV. Infective HAdVs were observed in two out of ten analyzed samples. A significant effect of environmental temperature (p = 0.001) and river (p = 0.012) on the presence of human viruses was found. These results suggest that fecal contamination could affect the water quality of these rivers, showing deficiencies in the procedure of sewage discharge from regional cities, livestock and dairy farms.

Viral metagenomics reveals significant viruses in the genital tract of apparently healthy dairy cows.

The virome in genital tract secretion samples collected from 80 dairycattle in Shanghai, China, was characterized. Viruses detected included members of the families Papillomaviridae, Polyomaviridae, Hepeviridae, Parvoviridae, Astroviridae, Picornaviridae, and Picobirnaviridae. A member of a new species within the genus Dyoxipapillomavirus and six circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viral genomes were fully sequencedand phylogenetically analyzed. The prevalence of bovine polyomaviruses 1 and 2 was measured by PCR to be 10% (8/80) and 6.25% (5/80), respectively.PCR screening also indicated that the novel papillomavirus ujs-21015 and bovine herpesvirus 6 were present in three and two out of the 80 samples, respectively.

Screening human cell lines for viral infections applying RNA-Seq data analysis.

Monitoring viral infections of cell cultures is largely neglected although the viruses may have an impact on the physiology of cells and may constitute a biohazard regarding laboratory safety and safety of bioactive agents produced by cell cultures. PCR, immunological assays, and enzyme activity tests represent common methods to detect virus infections. We have screened more than 300 Cancer Cell Line Encyclopedia RNA sequencing and 60 whole exome sequencing human cell lines data sets for specific viral sequences and general viral nucleotide and protein sequence assessment applying the Taxonomer bioinformatics tool developed by IDbyDNA. The results were compared with our previous findings from virus specific PCR analyses. Both, the results obtained from the direct alignment method and the Taxonomer alignment method revealed a complete concordance with the PCR results: twenty cell lines were found to be infected with five virus species. Taxonomer further uncovered a bovine polyomavirus infection in the breast cancer cell line SK-BR-3 most likely introduced by contaminated fetal bovine serum. RNA-Seq data sets were more sensitive for virus detection although a significant proportion of cell lines revealed low numbers of virus specific alignments attributable to low level nucleotide contamination during RNA preparation or sequencing procedure. Low quality reads leading to Taxonomer false positive results can be eliminated by trimming the sequence data before analysis. One further important result is that no viruses were detected that had never been shown to occur in cell cultures. The results prove that the currently applied testing of cell cultures is adequate for the detection of contamination and for the risk assessment of cell cultures. The results emphasize that next generation sequencing is an efficient tool to determine the viral infection status of human cells.

Viral tools for detection of fecal contamination and microbial source tracking in wastewater from food industries and domestic sewage

Alternative indicators may be more suitable than thermotolerant coliform bacteria to assess enteric virus pollution in environmental waters and their removal from wastewaters. In this study, F-specific RNA bacteriophages (F-RNAPh) showed to be potential viral indicators of fecal contamination when they were quantified from domestic and food-industrial effluents containing human, chicken, swine or bovine wastes. In addition, they showed to be resistant to the primary and secondary treatments of the wastewater treatment plants. The viable F-RNAPh count showed correlation with viable thermotolerant coliforms but also with human polyomaviruses (HPyV) quantified by a new molecular method. In domestic effluents, F-RNAPh and HPyV indicators significantly correlated with a human viral pathogen, norovirus, while the bacterial indicator did not, being then better predictors of the behavior of enteric pathogenic viruses. In addition, we assessed human, bovine and fowl microbial source tracking markers, based on the molecular detections of human polyomavirus, bovine polyomavirus, and fowl adenovirus, respectively. The techniques implemented extend the range of viruses detected, since they target different viral types simultaneously. These markers could be applied when multiple source pollution is suspected, contributing to making decisions on public health interventions.

Assessment of the Virological Quality of Marine and Running Surface Waters in NW Greece: A Case Study.

The virological quality of surface marine and running water samples collected from Igoumenitsa gulf and Kalamas river (NW Greece) was assessed from October 2012 to September 2013. Sampling sites were exposed to different land and/or anthropogenic effects. Seawater samples were collected monthly from five sampling stations (new harbor, old harbor, wastewater treatment plant outlet, protected Natura area, Drepano beach). Viral targets included human adenoviruses (hAdVs), as index human viruses, while noroviruses (NoVs) and hepatitis A virus (HAV) were also studied. Kalamas river samples were collected seasonally, from three sampling stations (Soulopoulo, Dam, Sagiada-estuaries), while viral targets included also porcine adenoviruses (pAdVs) and bovine polyoma viruses (bPyVs), as additional index viruses. All water samples were analyzed for standard bacterial indicators, as well. Physicochemical and meteorological data were also collected. Based on the standard bacterial indices, both sea and river water samples did not exceed the limits set according to Directive 2006/7/EU. However, positive samples for hAdVs were found occasionally in all sampling sites in Igoumenitsa gulf (23.3%, 14/60) showing fecal contamination of human origin. Moreover, HAV was detected once, in the sampling site of the old port (at 510 GC/L). Most of the Kalamas water samples were found positive for hAdVs (58.3%, 7/12), while human noroviruses GI (NoVGI) (8.3%, 1/12) and GII (NoVGII) (16.7%, 2/12) were also detected. HAV, pAdVs, and bovine polyomaviruses (bPyVs) were not detected in any of the analyzed samples. No statistically significant correlations were found between classic bacterial indicators and viral targets, nor between viruses and meteorological data. Overall, the present study contributed to the collection of useful data for the biomonitoring of the region, and the assessment of the overall impact of anthropogenic activities. It provided also valuable information for the evaluation of the risk of waterborne viral infections and the protection of public health. It was the first virological study in the area and one of the few in Greece.

Hydrologic, land cover, and seasonal patterns of waterborne pathogens in Great Lakes tributaries

Great Lakes tributaries are known to deliver waterborne pathogens from a host of sources. To examine the hydrologic, land cover, and seasonal patterns of waterborne pathogens (i.e. protozoa (2), pathogenic bacteria (4) human viruses, (8) and bovine viruses (8)) eight rivers were monitored in the Great Lakes Basin over 29 months from February 2011 to June 2013. Sampling locations represented a wide variety of land cover classes from urban to agriculture to forest. A custom automated pathogen sampler was deployed at eight sampling locations which provided unattended, flow-weighted, large-volume (120–1630 L) sampling. Human and bovine viruses and pathogenic bacteria were detected by real-time qPCR in 16%, 14%, and 1.4% of 290 samples collected while protozoa were never detected. The most frequently detected pathogens were: bovine polyomavirus (11%), and human adenovirus C, D, F (9%). Human and bovine viruses were present in 16.9% and 14.8% of runoff-event samples (n = 189) resulting from precipitation and snowmelt, and 13.9% and 12.9% of low-flow samples (n = 101), respectively, indicating multiple delivery mechanisms could be influential. Data indicated human and bovine virus prevalence was different depending on land cover within the watershed. Occurrence, concentration, and flux of human viruses were greatest in samples from the three sampling locations with greater than 25% urban influence than those with less than 25% urban influence. Similarly, occurrence, concentration, and flux of bovine viruses were greatest in samples from the two sampling locations with greater than 50 cattle/km2 than those with less than 50 cattle/km2. In seasonal analysis, human and bovine viruses occurred more frequently in spring and winter seasons than during the fall and summer. Concentration, occurrence, and flux in the context of hydrologic condition, seasonality, and land use must be considered for each watershed individually to develop effective watershed management strategies for pathogen reduction.

Detection and genome characterization of bovine polyomaviruses in beef muscle and ground beef samples from Germany

Polyomaviruses are small, non-enveloped, circular double-stranded DNA viruses. Some polyomaviruses can induce tumors and cancer under certain circumstances. The bovine polyomaviruses (BPyV) 1–3 have been only scarcely analyzed so far. It was hypothesized that the consumption of beef meat containing polyomaviruses could contribute to the development of cancer in humans. In order to assess the distribution of the BPyV genome in meat from Germany, 101 beef muscle samples and 10 ground beef samples were analyzed here. A specific sample preparation method combined with or without rolling circle amplification (RCA), and BPyV-specific PCRs were developed and applied. BPyV-1 DNA was detected in 1/101 (1%) samples from beef meat and in 2/10 (20%) ground beef samples. BPyV-2 DNA was detected in 3/10 (30%) ground beef samples, whereas BPyV-3 was not detected in the samples. Application of RCA did not increase the detection rate in ground beef samples. Sequence analysis of the PCR products indicated the presence of BPyV-1, BPyV-2a and BPyV-2b. The whole genome of a BPyV-1 strain from ground beef meat showed 97.8% sequence identity to the BPyV-1 reference strain and that of a BPyV-2a strain from ground beef meet showed 99.9% sequence identity to strain 2aS11. It can be concluded that BPyV genomes can be frequently detected in ground beef samples, although higher sample numbers should be investigated in future to confirm this finding. Further studies should focus on the infectivity, tumorigenicity and heat resistance of the contained viruses in order to assess the risk of cancer induction through consumption of BPyVs present in beef products.

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains.

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1%, 18/64), followed by that of porcine (15.4%, 6/39) and bovine (5.1%, 2/39) origins. Hepatitis E virus (5%, 1/20) and noroviruses GII (14.3%, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3%, 9/108), followed by that of porcine, 4.5% (4/89) and bovine, 1.1% (1/89) origins. Norovirus GII (3.6%, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.

Complete Genome Sequence of Murine Pneumotropic Virus (Polyomaviridae) Clone pKV(37-1).

The murine pneumotropic virus genome encoded by the pKV(37-1) clone was sequenced to completion. The regulatory region harbored a mutation not previously reported. The protein coding regions (large and small T antigens, viral proteins 1 to 3) showed multiple regions of high amino acid identity to the human, simian, and bovine polyomaviruses.

Exploring the virome of cattle with non-suppurative encephalitis of unknown etiology by metagenomics

Non-suppurative encephalitis is one of the most frequent pathological diagnosis in cattle with neurological disease, but there is a gap in the knowledge on disease-associated pathogens. In order to identify viruses that are associated with non-suppurative encephalitis in cattle, we used a viral metagenomics approach on a sample set of 16 neurologically-diseased cows. We detected six virus candidates: parainfluenza virus 5 (PIV-5), bovine astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), bovine polyomavirus 2 (BPyV-2 SF), ovine herpesvirus 2 (OvHV-2), bovine herpesvirus 6 (BHV-6) and a novel bovine betaretrovirus termed BoRV-CH15. In a case-control study using PCR, BoAstV-CH13 (p=0.046), BoPV-2 SF (p=0.005) and BoHV-6 (p=4.3E−05) were statistically associated with the disease. These data expand our knowledge on encephalitis-associated pathogens in cattle and point to the value of NGS in resolving complex infection scenarios in a clinical disease setting.

Complete Genome Sequence of Bovine Polyomavirus Type 1 from Aborted Cattle, Isolated in Belgium in 2014.

The complete and fully annotated genome sequence of a bovine polyomavirus type 1 (BPyV/BEL/1/2014) from aborted cattle was assembled from a metagenomics data set. The 4,697-bp circular dsDNA genome contains 6 protein-coding genes. Bovine polyomavirus is unlikely to be causally related to the abortion cases.

Genome Sequence of Bovine Polyomavirus 1 Detected in a Salers Cow (Bos taurus) from Catalonia, Spain.

We identified a variant of the first bovine polyomavirus (BPyV1; family Polyomaviridae) in a lymph node of a Salers cow. As the 2 previously published genome sequences of this virus originated from fetal bovine serum and ground beef, respectively, this is the first BPyV1 genome that could be traced back to an individual.

Human and Bovine Viruses and Bacteria at Three Great Lakes Beaches: Environmental Variable Associations and Health Risk.

Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65-87% for pathogenic bacteria, and 13-35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 2 × 10(-5), 8 × 10(-6), and 3 × 10(-7) [corrected] for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.

Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters.

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.

Hamburger polyomaviruses.

Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.

Presence of Human and Animal Viruses in Surface Waters in Vojvodina Province of Serbia.

For the first time in Serbia, a small surveillance study was conducted in order to estimate the presence and frequency of occurrence of selected human [adenoviruses (HAdV), noroviruses, (NoV GI, NoV GII) and hepatitis A virus (HAV)], animal [porcine adenovirus (PAdV) and bovine polyomavirus (BPyV)] and zoonotic [hepatitis E virus (HEV)] viruses in selected surface waters. In total, 60 surface water samples were collected in two sampling occasions at 30 locations, with each sampling time being separated by 1-5months. In addition, six sewage effluent samples were collected at one sampling site per each of the three tested town sewage systems, in two sampling occasions with 2months intervals, before their discharge into the surface waters. The most prevalent virus found was HAdV which was detected in 43.33% samples. NoV GII was found in 40% samples. NoV GI was found in 10% samples, and PAdV, BPyV and HEV were detected in 5 (8.33%), 4 (6.67%) and 2 (3.33%) samples, respectively. HAV was not found in any of analysed surface waters or urban sewage samples. The obtained results confirm the presence of pathogenic enteric viruses of both human and animal origin in surface waters in Serbia indicating the existence of diverse contamination sources.

What is for dinner? Viral metagenomics of US store bought beef, pork, and chicken

We describe here the metagenomics-derived viral sequences detected in beef, pork, and chicken purchased from stores in San Francisco. In beef we detected four previously reported viruses (two parvoviruses belonging to different genera, an anellovirus, and one circovirus-like virus) and one novel bovine polyomavirus species (BPyV2-SF) whose closest relatives infect primates. Detection of porcine hokovirus in beef indicated that this parvovirus can infect both ungulate species. In pork we detected four known parvoviruses from three genera, an anellovirus, and pig circovirus 2. Chicken meat contained numerous gyrovirus sequences including those of chicken anemia virus and of a novel gyrovirus species (GyV7-SF). Our results provide an initial characterization of some of the viruses commonly found in US store-bought meats which included a diverse group of parvoviruses and viral families with small circular DNA genomes. Whether any of these viruses can infect humans will require testing human sera for specific antibodies.