Integrated hepatitis e virus monitoring in central Argentina: a six-year analysis of clinical surveillance and wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has gained prominence worldwide as a powerful tool in public health. This study aimed to monitor the circulation of Hepatitis E Virus (HEV) from wastewater samples collected during a six-year period and compare these results with clinical surveillance in the central region of Argentina. From 2017 to 2022, 1008 raw wastewater samples were analyzed, including four wastewater treatment plants from four cities (n=319), and 7 local neighborhood collector sewers in Córdoba city (n=689). Serum and/or stool samples from patients suspected of HEV infection were also analyzed (n=48). HEV molecular detection and viral load quantification were performed by real time RT-qPCR, and genetic characterization by two RT-Nested PCRs (targeting partial ORF-1 and ORF-2 genomic regions), sequencing and phylogenetic analysis. Fifty-three (5.3%) wastewater samples were RNA-HEV positive by real time RT-qPCR, with variations according to the location and year (0.0% - 21.6%). Out of these, ORF-2 genomic region was amplified in 20 samples (37.7%) and ORF-1 partial region in 12 (22.6%), and eighteen sequences were obtained. Throughout the study period, two (4.2%) HEV confirmed infections were reported, and one sequence was obtained. Phylogenetic analyses for both genomic regions showed that all the isolates were genotype HEV-3 clade abchijklm. Our study detected HEV in wastewater over a six-year period, despite a low number of clinical cases, emphasizing WBE as a valuable tool that complements clinical surveillance, by detecting pathogens' presence; identifying their transmission, circulation dynamics and excretion hotspots; and revealing changes in their genomic diversity.

Circulation of picobirnavirus in Neotropical free-ranging mammals.

Picobirnavirus (PBV) is a family of non-enveloped double-stranded RNA viruses with bisegmented genomes. Segment 1 encodes the capsid protein and segment 2 encodes RNA-dependent RNA polymerase. They exhibit high genomic heterogeneity and infect a wide range of vertebrate hosts, including humans. The objective of this study was to expand our knowledge of the circulation of PBV in free-living animals from two regions (Brazil and Argentina) of the Atlantic Forest. Fecal samples were analyzed from free-living animals: tapir, brocket deer, peccary, and different species of rodents and marsupials. A total of 133 samples were collected and analyzed by RT-PCR, of which 44 (33.08%) were PBV-positive. Nine amplicons were sequenced, five species from Argentina and four from Brazil, and phylogenetic analysis was performed. The nucleotide and amino acid identities of the PBV strains detected in animals from Argentina and Brazil were between 66.3% and 82.5% and between 55.3% and 74.2%, respectively. The analysed strains presented conserved nucleotide blocks without distinction of the host species. The phylogenetic tree showed that PBV strains from Atlantic Forest animals belonging to genogroup I were grouped into different clusters, without defining groups according to host species (human or animal) or the geographical area of detection. This is the first study on PBV in free-living animals in the Atlantic Forest. Our analysis suggested that PBV strains can infect different animal species, leading to PBV transmission between animals and humans. This reinforces the hypothesis of previous crossover points in the ecology and evolution of heterologous PBV strains.

Hepatitis A virus monitoring in wastewater: A complementary tool to clinical surveillance.

Monitoring wastewater is an effective tool for tracking information on trends of enteric viral dissemination. This study aimed to perform molecular detection and genetic characterization of HAV in wastewater and to correlate the results with those obtained from clinical surveillance. Wastewater samples (n=811) of the second most populous city in Argentina were collected from the main wastewater treatment plant (BG-WWTP, n=261), and at 7 local neighborhood collector sewers (LNCS, n=550) during 2017-2022. Clinical samples of acute hepatitis A cases (HA, n=54) were also analyzed. HAV molecular detection was performed by real time RT-PCR, and genetic characterization by RT-Nested PCR, sequencing and phylogenetic analysis. RNA-HAV was detected in sewage samples throughout the entire period studied, and detection frequencies varied according to the location and year (2.9% - 56.5%). In BG-WWTP, 23% of the samples were RNA-HAV+. The highest detection rates were in 2017 (30.0%), 2018 (41.7%) and 2022 (56.5%), which coincides with the highest number of HA cases reported. Twenty-eight (28) sequences were obtained (from clinical and sewage samples), and all were genotype IA. Two monophyletic clusters were identified: one that grouped clinical and wastewater samples from 2017-2018, and another with specimens from 2022, evidencing that environmental surveillance might constitute a replica of viral circulation in the population. These findings evidence that WBE, in a centralized and decentralized sewage monitoring, might be an effective strategy to track HAV circulation trends over time, contributing to the knowledge of HAV in the new post-vaccination epidemiological scenarios in Argentina and in Latin America.

A Preliminary Study of SARS-CoV-2's Permanence and Potential Infective Capacity in Mineromedicinal Waters of Copahue, Neuquén, Argentina.

Copahue Thermal Center is characterized by the presence of mineromedicinal acidic waters with high temperatures, therapeutic peloids, and relevant consortia of extremophiles species, distributed in small natural pools which cannot be disinfected. The objective of this research was to investigate the survival of SARS-CoV-2 in Copahue's waters and its remaining infective capacity. In a first assay, a decrease of more than 50% of the initially viral load compared to the initially inoculated positive sample was detected for all the water samples analyzed. After that, two of the Copahue springs, which are used as an immersion bath in closed environments without going through any disinfection treatment, was selected to determine the viral viability. VERO cell infections were performed, with no cytopathic effect detected, but a strikingly high resistance of the virus, detecting its genome by real time PCR, during the seven days of study under laboratory conditions. SARS-CoV-2 survival in acid media was reaffirmed, which is a peculiarity for a covered virus. A decrease in the detectable viral load of the positive sample was found as the infection time passed, becoming completely negative in the subsequent blind passages. More research is needed to further study the feasibility of SARS-CoV-2 in mineromedicinal waters, especially natural acidic waters that cannot disinfected, in order to expand information about the risk to populations that are exposed to them.

Wastewater based epidemiology as a silent sentinel of the trend of SARS-CoV-2 circulation in the community in central Argentina.

Monitoring wastewater for the traces of viruses allows effective surveillance of entire communities, including symptomatic and asymptomatic infected individuals, providing information on whether a specific pathogen is circulating in a population. In the context of the COVID-19 pandemic, 261 wastewater samples from six communities of the province of Córdoba, Argentina were analyzed. From mid-May 2020 to the end of August 2021, raw sewage samples were collected from the central network pipe that enters into the Wastewater Treatment Plants (WWTP) in Córdoba city and five communities in the Punilla Valley. SARS-CoV-2 was concentrated by using the polyethylene glycol-6000 precipitation method. Viral genomes were extracted from concentrated samples, and N- and E-SARS-CoV-2 genes were detected by using real time RT-PCR. Wastewater samples that resulted positive for SARS-CoV-2 genome detection were subjected to viral variants of concern (VOCs) identification by real time RT-PCR. Overall, just by using the identification of the N gene or E gene, the rates of viral genome detection were 43.4% (86/198) and 51.5% (102/198) respectively, and by using both methodologies (positivity criterion: detection of N and / or E gene), the detection rate was 71.2% (141/198). Thereby, the optimal strategy to study the SARS-CoV-2 genome in wastewater would be the use of the combined detection of both genes. Detection of SARS-CoV-2 variants in wastewater reflected their circulation in the community, showing no VOCs detection in the first COVID-19 wave and their co-circulation with Gamma, Alpha and Delta VOCs during 2021. Therefore, SARS-CoV-2 Wastewater Based Epidemiology (WBE) described the introduction, permanence and/or the co-circulation of viral variants in the community. In geographical areas with a stable population, SARS-CoV-2 WBE could be used as an early warning sign of new COVID-19 cases, whereas in localities with a low number of inhabitants and high tourist influx, WBE may only be useful to reflect the circulation of the virus in the community. Overall, the monitoring of SARS-CoV-2 in wastewater can become a silent sentinel of the trend of viral circulation in the community, providing supplementary information for clinical surveillance to support public health measures.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

Monitoring of SARS-CoV-2 RNA in wastewater as an epidemiological surveillance tool in Mendoza, Argentina.

Wastewater-based epidemiology (WBE) is an emerging tool that gives temporal and spatial information on a population's health status. Here, we report the epidemiological dynamics of a population of ~1.2 million residents in the metropolitan region of Mendoza province, Argentina, within the period July 2020 to January 2021. We combined the use of WBE of two wastewater treatment plants with epidemiological surveillance of the corresponding populations. We applied two viral concentration methods (polyethylene glycol precipitation and aluminum-based adsorption-flocculation) and RNA isolation methods in each wastewater sample to increase the possibility of detection and quantification of nucleocapsid markers (N1 and N2) of SARS-CoV-2 by RT-qPCR. Overall, our results allowed us to trace the rise, exponential growth, plateau, and fall of SARS-CoV-2 infections for 26 weeks. Individual analysis for each wastewater treatment plant showed a positive correlation between the viral load of SARS-CoV-2 genetic markers and COVID-19 cases that were diagnosed per week. Our findings indicate that WBE is a useful epidemiological indicator to anticipate the increase in COVID-19 cases and monitor the advance of the pandemic and different waves of infections.

Environmental Surveillance of Human Enteroviruses in Córdoba City, Argentina: Prevalence and Detection of Serotypes from 2009 to 2014.

Environmental surveillance is an effective approach to investigate the circulation of human enteroviruses (EVs) in the population. EVs excreted by patients who present diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 6-year (2009-2014) surveillance study was conducted to detect non-polio enteroviruses (NPEVs) in the urban sewage of Cordoba city, Argentina. Echovirus 6 (E-6) was the most prevalent (28%), followed by E-14 (17%), E-16 (14%), Coxsackievirus (CV) A9 (11%), E-20 (9%), and CVA24 (6%). Other minority serotypes (E-7, E-13, E-21, E-25, and CVB4) were found, which together represented 14% of the total. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.

Searching variables to assess recreational water quality: the presence of infectious human enterovirus and its correlation with the main variables of water pollution by multivariate statistical approach in Córdoba, Argentina.

Surface waters are used by local populations for different purposes, such as recreational activities, water source for human and animal consumption, and irrigation among others, which lead to the need for management strategies on water health and associated risks. During this study, we investigated physicochemical parameters, fecal coliform bacteria, and infectious human enterovirus detection to determine the water quality in different beaches (categorized as an urban area, non-urban areas, and an intermediate position) from San Roque Dam, in Argentina. Multivariate techniques were applied. Principal component analysis allowed identification of subgroup of variables responsible for the water quality. A cluster analysis and multivariate analysis of variance showed the urban beach as the highest pollution area. The following variables (measured at the urban beach) would be enough to describe the quality of the aquatic body: nitrites, fecal coliforms, total phosphorous, and infectious human enterovirus. The infectious human enterovirus was an independent variable detected in 69.1% of the samples showing a steady frequency of detection during the whole period studied and could identify human fecal contaminations as a source of water pollution. The selected variables would contribute to water quality regarding the risk for human health using San Roque dam waters for recreational propose.

Environmental Surveillance of Enteroviruses in Central Argentina: First Detection and Evolutionary Analyses of E14.

Environmental surveillance is an effective approach to investigate the circulation of human enteroviruses in the population. Enteroviruses E14, CVA9, E-6, E16, E20, E25, E13, and CVA24 were detected in sewage and a watercourse in central Argentina. E14 was the most frequent serotype and was found for the first time in environmental samples in our region. Phylogenetic and coalescence analyses showed at least two recent introduction events.

Maintenance of picobirnavirus (PBV) infection in an adult orangutan (Pongo pygmaeus) and genetic diversity of excreted viral strains during a three-year period.

The present work provide data about the maintenance of picobirnavirus (PBV) infection during adulthood in a mammalian host. For this purpose PBV infection was studied in an adult orangutan (Pongo pygmaeus) by PAGE/SS, RT-PCR and nucleotide sequencing. PBV infection in the animal was asymptomatic and was characterized by interspaced silent and high/ low active viral excretion periods. The PBV strains excreted by the studied individual were identified as genogroup I and revealed a nucleotide identity among them of 64-81%. The results obtained allowed to arrive to a deeper understanding of the natural history of PBV infection, which seems to be characterized by new-born, juvenile and adult asymptomatic hosts which persistently excrete closely related strains in their feces. Consequently, picobirnaviruses could be considered frequent inhabitants of the gastrointestinal tract, leaving the question open about the molecular mechanisms governing persistent and asymptomatic coexistence within the host and the potential host suitability to maintain this relationship.

Animal picobirnavirus.

Picobirnavirus (PBV) is a small, non-enveloped, bisegmented double-stranded RNA (dsRNA) virus of vertebrate hosts. The name 'Picobirnavirus' derives from the prefix 'pico' (latin for 'small') in reference to the small virion size, plus the prefix 'bi' (latin for 'two') and the word 'RNA' to indicate the nature of the viral genome. The serendipitous discovery of PBV dates back to 1988 from Brazil, when human fecal samples collected during the acute gastroenteritis outbreaks were subjected for routine rotavirus surveillance by polyacrylamide gel electrophoresis (PAGE) and silver straining (S/S). The PAGE gels after silver staining showed a typical 'two RNA band' pattern, and it was identified as Picobirnavirus. Likewise, the feces of wild black-footed pigmy rice rats (Oryzomys nigripes) subjected for PAGE assay by the same research group in Brazil reported the presence of PBV (Pereira et al., J Gen Virol 69:2749-2754, 1988). PBVs have been detected in faeces of humans and wide range of animal species with or without diarrhoea, worldwide. The probable role of PBV as either a 'primary diarrhoeal agent' in 'immunocompetent children'; or a 'potential pathogen' in 'immunocompromised individuals' or an 'innocuous virus' in the intestine remains elusive and needs to be investigated despite the numerous reports of the presence of PBV in fecal samples of various species of domestic mammals, wild animals, birds and snakes; our current knowledge of their biology, etiology, pathogenicity or their transmission characteristics remains subtle. This review aims to analyse the veterinary and zoonotic aspects of animal Picobirnavirus infections since its discovery.

Evaluation of concentration efficiency of the Pseudomonas aeruginosa phage PP7 in various water matrixes by different methods.

Enteric viruses monitoring in surface waters requires the concentration of viruses before detection assays. The aim of this study was to evaluate different methods in terms of recovery efficiencies of bacteriophage PP7 of Pseudomonas aeruginosa, measured by real-time PCR, using it as a viral control process in water analysis. Different nucleic acid extraction methods (silica-guanidinium thiocyanate, a commercial kit (Qiagen Viral RNA Kit) and phenol-chloroform with alcohol precipitation) exhibited very low recovery efficiencies (0.08-4.18 %), being the most efficient the commercial kit used for subsequent experiments. To evaluate the efficiency of three concentration methods, PBS (as model for clean water) and water samples from rivers were seeded to reach high (HC, 10(6) pfu ml(-1)) and low concentrations (LC, 10(4) pfu ml(-1)) of PP7. Tangential ultrafiltration proved to be more efficient (50.36 ± 12.91, 17.21 ± 9.22 and 12.58 ± 2.35 % for HC in PBS and two river samples, respectively) than adsorption-elution with negatively charged membranes (1.00 ± 1.34, 2.79 ± 2.62 and 0.05 ± 0.08 % for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95 ± 7.43, 4.01 ± 1.12 and 3.91 ± 0.54 %, for HC in PBS and two river samples, respectively), being 3.2-50.4 times more efficient than the others for PBS and 2.7-252 times for river samples. Efficiencies also depended on the initial virus concentration and aqueous matrixes composition. In consequence, the incorporation of an internal standard like PP7 along the process is useful as a control of the water concentration procedure, the nucleic acid extraction, the presence of inhibitors and the variability of the recovery among replicas, and for the calculation of the sample limit of detection. Thus, the use of a process control, as presented here, is crucial for the accurate quantification of viral contamination.

Genetic and antigenic evolution profiles of G1 rotaviruses in córdoba, Argentina, during a 27-year period (1980-2006).

Rotavirus G1 strains represent the most common genotype that causes diarrhea in humans and has been incorporated into both, monovalent and multivalent, rotavirus licensed vaccines. The aim of this study was to determine the evolution profile of G1 rotaviruses in Córdoba, Argentina, over a 27-year period (1980-2006). Intragenotype diversity, represented by lineages within rotavirus circulating strains, was observed. Phylogenetic analysis of the VP7-gene of G1 rotavirus clinical strains showed the circulation of G1 lineage IV and V strains in the 1980s, and co-circulation of lineage I and II strains in the 1990s and 2000-2006. The distribution of G1 in lineages could be linked to multiple nucleotide substitutions distributed across lineages that did not correlate with the emergence of G1 antigenic variants. Moreover, temporal lineage distribution was not linked to significant changes in G1 prevalence. Therefore, the continuous and dominant circulation of G1 over time could not be related to the emergence of antigenic variants in the community. Continuous rotavirus surveillance is necessary to understand rotavirus evolution and to measure how genetic and antigenic changes might affect the effectiveness of vaccines in the future.

Establishment and maintenance of persistent infection by picobirnavirus in greater rhea (Rhea Americana).

A study aimed to determine the infection model that picobirnavirus (PBV) established in birds was conducted in a farm of greater rheas in Córdoba, Argentina. Analysis of stools collected during a longitudinal study involving seven birds provided evidence that PBV is acquired very early in life and establishes a persistent infection in the host, which is characterized by intermingled periods of high, low and silent viral activity. Genomic analysis indicated that the rheas excreted virus with nucleotide sequence identity between 90.5-100 % and that more than one PBV strain with different electropherotype profiles could be involve in the infection. This report provides the first evidence of persistent infection of PBV in birds. The natural history of PBV infection has begun to be understood, and it appears that asymptomatic PBV-infected mammals and birds could persistently excrete the virus in stool samples, contributing to wide circulation of the virus in the environment.

Picobirnavirus infections: viral persistence and zoonotic potential.

Picobirnaviruses (PBVs) are small, non-enveloped, bisegmented double-stranded RNA genomic viruses of vertebrate hosts. Since their discovery in the late 1980s in clinical specimens from outbreaks of acute gastroenteritis in children, significant efforts have been made to investigate the role of PBV in diarrheic diseases. PBV has been detected in sporadic episodes of diarrhea as sole pathogen or coinfection as well as in outbreaks of acute gastroenteritis and in immunocompromised patients with diarrhea. However, PBV is frequently detected in non-diarrheic healthy hosts, and prolonged shedding has been observed in some individuals. Of interest, similar patterns of PBV infection have also been observed in pigs and other animal hosts. The increasing amount of PBV sequence data gathered from molecular epidemiological studies has evidenced a great sequence diversity of PBVs in various hosts and environmental samples. Importantly, evidence has been found for genetic relatedness between human and animal PBV strains, suggesting extant crossing points in the ecology and evolution of heterologous PBV strains. At present, no cell culture and animal model exists for PBVs. Well-structured epidemiological studies are still the only alternative to demonstrate the potential etiological role of PBVs in acute gastroenteritis or other diseases. This review aims to analyze the public health aspects of PBV infection, especially its possible association with zoonosis.

Genetic and evolutionary characterization of norovirus from sewage and surface waters in Córdoba City, Argentina.

Noroviruses (NoVs) are among the most common viral agents that cause gastroenteritis in humans of all ages worldwide. They are excreted in the feces and introduced into environmental waters as raw or treated sewage. In this work, sewage and water samples collected from the Suquía River in the city of Córdoba, Argentina, were evaluated for the presence of NoV. Phylogenetic analysis demonstrated that the main genotype detected was GII.4, belonging to the widely-distributed 2006b variant, followed by strains related to the putative recombinant GII.g virus. Detected NoVs were more phylogenetically related with recent viruses from other countries than with previous local sequences, suggesting a rapid and wide spread of viral strains that prevents a geographically structured phylogeny. A Bayesian coalescent analysis demonstrated that variants isolated in this work have a most recent common ancestor placed in 2007-2008 with estimated substitution rates of 3.7-5.8×10(-3)s/s/y. Environmental samples showed a mixture of both viral types, pointing up to the co-circulation and the risk of mixed infections and recombination. This is the first report on the detection and characterization of NoV in sewage and river water in Argentina.

Genogroup I picobirnavirus in diarrhoeic foals: can the horse serve as a natural reservoir for human infection?

Picobirnaviruses (PBV) are small, non-enveloped viruses with a bisegmented double-stranded RNA genome. In this study a PBV strain, PBV/Horse/India/BG-Eq-3/2010, was identified in the faeces of a 10 month old weaned female foal with diarrhoea in January 2010 from Kolkata, India. Surprisingly, sequence comparison and phylogenetic analysis of a short stretch of the RNA dependent RNA polymerase gene revealed close genetic relatedness (> 98% nucleotide identity) to a human genogroup I PBV strain (Hu/GPBV1) detected earlier from the same part of India. Our observations together with earlier findings on genetic relatedness between human and animal PBV warrant further studies on zoonotic potential.

Evidence of closely related picobirnavirus strains circulating in humans and pigs in Argentina.

BACKGROUND: On the basis of the published literature, it is still difficult to draw conclusions as to whether picobirnavirus (PBV) circulation is influenced by host species restriction. OBJECTIVE: To provide data regarding the genetic relatedness between porcine and human PBV strains present in Argentina as a means of defining the host range and epidemiology of these viruses. METHODS: Fecal specimens (n = 74) collected from kidney transplant patients (n = 55) and piglets (n = 19) were analyzed by RT-PCR using primers designed to amplify the porcine PBV genomic segment 2. Amplified sequences were further examined phylogenetically. RESULTS: By RT-PCR amplification 14 of 74 samples rendered amplicons of the expected 282 base pair size (8 detected from humans and 6 from pigs). Eleven amplicons (5 from humans and 6 from pigs) were selected for sequencing and subjected to phylogenetic analysis. The eleven amplicons revealed similarities between human and porcine viral sequences that ranged between 94.7 and 100% in identity. Phylogenetic analysis identified these 11 strains as PBV genogroup I-related strains and showed that they grouped as a single separate clade distinct from other PBV strains detected in humans and porcine from other countries. CONCLUSIONS: The present study suggests that closely related PBV strains infect both pigs and humans in Argentina and that the epidemiology of PBVs is not species restricted.

Picobirnavirus causes persistent infection in pigs.

A study aimed to further understand the biology of porcine picobirnaviruses (PBV) was conducted between November 2003 and January 2008, on a farm located in the outskirts of Córdoba City, Argentina. PBV prevalence was examined by polyacrylamide gel electrophoresis and silver staining (PAGE S/S) on a total of 265 samples collected from pigs divided into four groups, according to age and physiological status. PBV detection rate was highest in the group of sows sampled within the lactogenic period (38.02%; p<0.05), followed by pregnant sows (15.09%), piglets aged 2-5 months of age (18.42%) and adult (> or =50 weeks) male pigs (0%). In addition, 103 samples collected in 3 follow-up studies were analyzed by PAGE S/S and reverse transcription followed by PCR (RT-PCR). Two of these studies followed female pigs from weaning up to slaughter and a third one from weaning up to 4 pregnancy periods. The results provide evidence that PBV establishes a persistent infection in the host with periods of silence intermingled with periods of low and high viral excretion. High PBV excretion levels were detected by PAGE S/S and were conditioned by age (primary infection) and host physiological status. Low PBV excretion levels were detected by RT-PCR throughout the entire study period. Sequence analysis of selected amplicons indicated that the virus excreted through the follow-up study was the same. These results suggest that porcine PBV is maintained in nature by transmission from infected asymptomatic individuals to susceptible ones.