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.

Environmental surveillance of norovirus in Argentina revealed distinct viral diversity patterns, seasonality and spatio-temporal diffusion processes.

Norovirus (NoV) contamination was evaluated in five rivers of Argentina between 2005 and 2011. NoV was present in all sampled rivers, with distinct NoV patterns in waters impacted by different-sized communities. In rivers affected by medium-sized populations (Salta and Córdoba cities) only one or two genotypes were present, GII.4 being the main one, with winter seasonality. In contrast, in the much more heavily populated area of Buenos Aires city the prevalent GII.4 was accompanied by several additional genotypes (GII.4, GII.b, GII.2, GII.7, GII.17, GII.e and GII.g) and one ungenotyped GII NoV, with no clear seasonality. GII.4 2006b was the main variant detected (60.9%). Phylogeographic and phylodynamic analyses performed in region D of the VP1 gene showed a most recent common ancestor in 2002 and a substitution rate of 3.7×10(-3) substitutions per site per year (HPD95%=2.3×10(-3)-5.2×10(-3)) for this variant still involving a significant population size with a slight decrease since 2008. The spatio-temporal diffusion analysis proposed Europe as an intermediate path between the American Continent and the rest of the World for NoV dissemination. Given the importance of NoV as a cause of epidemic gastroenteritis and the likelihood of its environmental transmission, the results of this work should increase public and institutional awareness of the health risk involved in sewage discharges into the environment. Environmental surveillance of enteric viruses could be a very useful tool not only to prevent waterborne outbreaks, but also to describe the epidemiology of the viruses. The detailed analysis of the viral genomes disposed into the environment contributed to the characterization of the dissemination, diversity and seasonality of NoV in its natural host population. In future studies, environmental surveillance and molecular analysis should be complemented with a quantitative viral risk assessment for estimating the disease burden from viruses in the environment.

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.

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.

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.

Environmental poliovirus surveillance during oral poliovirus vaccine and inactivated poliovirus vaccine use in Córdoba Province, Argentina.

This study compares the presence of environmental poliovirus in two Argentinean populations using oral poliovirus vaccine (OPV) or inactivated poliovirus vaccine (IPV). From January 2003 to December 2005, Córdoba City used IPV in routine infant immunizations, with the exception of intermittent OPV use in August 2005. Between May 2005 and April 2006, we collected weekly wastewater samples in Córdoba City and the province's three major towns, which continued OPV use at all times. Wastewater samples were processed and analyzed for the presence of poliovirus according to WHO guidelines. During the months of IPV use in Córdoba City, the overall proportion of poliovirus-positive samples was 19%. During an intermittent switch from IPV to OPV, this proportion increased to 100% within 2 months. During the 3 months when IPV was reintroduced to replace OPV, a substantial proportion of samples (25%) remained positive for poliovirus. In the OPV-using sites, on average, 54% of samples were poliovirus positive. Seventy-seven percent of poliovirus isolates showed at least one mutation in the VP1-encoding sequence; the maximum genetic divergence from the Sabin strain was 0.7%. Several isolates showed mutations on attenuation markers in the VP1-encoding sequence. The frequency or type of virus mutation did not differ between periods of IPV and OPV use or by virus serotypes. This study indicates that the sustained transmission of OPV viruses was limited during IPV use in a middle-income country with a temperate climate. The continued importation of poliovirus and genetic instability of vaccine strains even in the absence of sustained circulation suggest that high poliovirus vaccine coverage has to be maintained for all countries until the risk of reintroduction of either wild or vaccine-derived poliovirus is close to zero worldwide.

Molecular characterization of porcine picobirnaviruses and development of a specific reverse transcription-PCR assay.

The molecular characterization of partial- length genomic segment 2 of porcine picobirnavirus (PBV) strains and the development of a specific reverse transcription-PCR (RT-PCR) assay for detection of virus in feces are reported. Phylogenetic analysis indicated that the studied porcine isolates were more closely related (>85% identity) to human PBV belonging to genogroup I than to the other porcine PBV described so far. Analysis by RT-PCR and polyacrylamide gel electrophoresis of fecal samples collected in Venezuela and Argentina showed that PBV circulate at high frequencies in piglets.

Two instances of large genome profile picobirnavirus occurrence in Argentinian infants with diarrhea over a 26-year period (1977-2002).

BACKGROUND: Picobirnavirus' (PBV) association with diarrhea in children is not reliably established and the potential role of pathogenic PBV needs further investigations. OBJECTIVE: The aim of this study is to clarify the role of PBV in diarrhea illness in children. METHODS: Between January 1977 and December 2002, 2224 stool specimens were collected from children <3 years old with diarrhea illness. All samples were analyzed by the polyacrylamide gel electrophoresis technique (PAGE) for the presence of bisegmented dsRNA virus genomic pattern. Gels were dried and archived. This study procedure allowed us to keep a laboratory electrophoretic record of each sample assayed. In the present study, all the electrophoretic records were reviewed in order to identify PBV positive samples. RESULTS: Two out of 2224 (0.09%) stools were positive for large genome profile of PBV. These two positive samples were collected from hospitalized children <1 year old; one of them presenting rotavirus co-infection. CONCLUSIONS: The findings obtained in the present report support strong evidence that large genome profile PBV can be considered more an occasional viral agent rather than an etiological agent associated with diarrheal illness.

Long-lasting poliovirus-neutralizing antibodies among Argentinean population immunized with four or five oral polio vaccine doses 1 month to 19 years previously.

The persistence of poliovirus-neutralizing antibodies was investigated in 297 individuals residing in Argentina who had completed the vaccination cycle with four or five oral polio vaccine (OPV) doses 1 mo to 19 yr before this study. Seropositivity for the three polio types in individuals who had received four OPV doses remained high and stable, showing rates not less than 94.6, 98.2, and 91.1% for types 1, 2, and 3, respectively, for a period of at least 6 yr. Almost identical rates were found in children who completed a vaccination schedule of five OPV doses 1 to 2 yr earlier. However, humoral immunity to poliovirus types 3 and 1 declined significantly 9 and 17 yr, respectively, after the booster dose had been administered; in contrast, type 2 immunity remained fairly stable during the 19-yr study period. Overall, geometric mean titer values for poliovirus types 1 and 2 were higher than those for poliovirus type 3. This is likely a result of low initial poliovirus type 3 antibody titers that eventually fell below the limits of detection at later time points. The results indicate that although antibody titers primed by OPV decline over time, they are remarkably long-lived, immunity to poliovirus types 1 and 2 being more prevalent than that against type 3 at late intervals postvaccination.

Measles virus-specific IgG4 antibody titer as a serologic marker of post-vaccinal immune response.

In previous research, we concluded that measles virus specific IgG4 antibody titer could be used to differentiate between natural [IgG4 GMT 80 (95% CI, 33 to 191)] and vaccinal source of measles infection [IgG4 GMT 13 (95% CI, 7 to 26)]. The aim of this paper is to show that this new serologic marker (IgG4 measles antibody titer) can be applied to help interpret rare but well documented cases of measles Ig M-positive results in vaccinated individuals who, 1-2 months after vaccination, developed rash and fever and therefore do not meet the criteria for post-vaccinal measles infection. Six measles IgM-positive serum samples obtained from measles vaccinated individuals who developed rash/fever 1 to 2 months post-vaccination were studied by Immunofluorescence assay for the quantification of IgG4 measles specific antibody. IgG4 antibody titers from all these samples were between 1:10-1:20, consequently, the IgM positive results from the study cases could be ascribed to post-vaccinal immune response. Thus, measles virus specific IgG4 antibody titer could be used as a serologic marker of post-vaccinal immune response.

Discrepancies in viral gastroenteritis diagnosis: an unusual dual reovirus-adenovirus infection case.

BACKGROUND: A variable rate of false-positive results may be observed with commercial assays for the detection of rotavirus and adenovirus antigen in stool specimens, depending on the quality of the reagents and the presence of potentially interfering substances in stool samples. OBJECTIVE: The present report analyse the discrepant results that could be obtained by the commercially available diagnostic tests and that can mask the reliable viral diagnosis. STUDY DESIGN: One fecal sample was collected from a hospitalized child aged 6 months with acute watery diarrhea and dehydration. The fecal specimen was processed the same day for the rotavirus and adenovirus antigen detection. RESULTS: The sample was positive for rotavirus antigen by one-step membrane test based on immunochromatographic assays (ICA) and enzyme immunoassays (EIA) monoclonal test but it was negative by an EIA polyclonal test, polyacrylamide gel electrophoresis (PAGE) and RT-PCR assays. In the other hand, the sample was positive for adenovirus antigen by ICA and EIA adenovirus type 40/41. Finally, the sample showed by PAGE an electrophoretic profile resembled that of reovirus. CONCLUSION: The use of a wide repertory of diagnosis tests allowed to reach an unusual reovirus-adenovirus type 40/41 dual infection. This case also point out the potential participation of reovirus in the ethiology of the diarrhea illness.

Childhood astrovirus-associated diarrhea in the ambulatory setting in a Public Hospital in Cordoba city, Argentina.

Human astroviruses have been increasingly identified as important agents of diarrheal disease in children. However, the disease burden of astrovirus infection is still incompletely assessed. This paper reports results on the epidemiological and clinical characteristics of astrovirus-associated diarrhea, as well as the impact of astrovirus infection on the ambulatory setting at a Public Hospital in Córdoba city, Argentina. From February 2001 through January 2002, 97 randomly selected outpatient visits for diarrhea among children < 36 months old were enrolled. A single specimen of stool from each child was collected and tested for astrovirus antigen by enzyme immunoassay. Astroviruses were detected in 12.37% of the diarrheal episodes. All the positive cases occurred in children 4 to 18 months, but the highest rate was in children aged 4 to 6 months (23.80%). The clinical symptoms of astrovirus associated-diarrhea were fever 41.66%, vomiting 25.00% and dehydration 8.33%; overall 16.66% required hospitalization. Astrovirus was identified through the year and no seasonally pattern was detected (cool semester 15.21% versus warm semester 9.80% p > 0.05). According to our estimation about one out of seventy-four children in this cohort would be assisted annually for an astroviral-diarrheal episode in the Public Hospital and one out of eight diarrheal cases could be attributed to astrovirus infection. Astrovirus is a common symptomatic infection in pediatric outpatient visits in the public hospital in the study area, contributing 12.37% of the overall morbidity from diarrhea.

Childhood astrovirus-associated diarrhea in the ambulatory setting in a Public Hospital in Cordoba city, Argentina

Os astrovírus humanos têm sido identificados como importantes agentes de diarréias em crianças embora o impacto da sua infecção não tenha sido esclarecido. Este estudo não só mostra os resultados das características epidemiológicas e clínicas, mas também o impacto da infecção por astrovírus em pacientes ambulatoriais de um Hospital Público da cidade de Córdoba na Argentina. Escolheram-se randomicamente 97 pacientes ambulatoriais com menos de 36 meses, entre fevereiro de 2001 e janeiro de 2002, que consultaram por diarréia. Pesquisou-se antígeno de astrovírus por ensaio imuno-enzimático em uma única amostra de fezes por paciente estudado. Determinou-se a presença de astrovírus em 12,37 por cento dos casos de diarréia. Todos os casos positivos foram em crianças de 4 a 18 meses, mas o índice mais elevado se apresentou em crianças de 4 a 6 meses (23,80 por cento). Os sintomas de diarréia associada a astrovírus foram febre 41,66 por cento; vômitos 25,00 por cento e desidratação 8,33 por cento; ou seja, 16,66 por cento dos pacientes precisaram hospitalização. A presença de astrovírus foi anual sem se observar comportamento sazonal, semestre frio 15,21 por cento versus semestre quente 9,80 por cento p > 0,05. Em nossa pesquisa, uma de cada 74 crianças seria atendida anualmente por apresentar um episódio de diarréia associada a astrovírus no hospital público e um de cada 8 casos de diarréia poderia atribuir-se à infecção por astrovírus. Astrovírus é uma infecção sintomática em pacientes pediátricos ambulatoriais, representando 12,37 por cento da morbidade por diarréia.

Molecular diversity of partial-length genomic segment 2 of human picobirnavirus.

OBJECTIVES: This study was carried out in order to evaluate the efficacy of the recently developed picobirnavirus (PBV) sets of primers and to establish the phylogenetic relationships of Argentine strains with PBV strains isolated in China and the USA. METHODS: Thirteen fecal specimens tested as positive for PBV by polyacrylamide gel electrophoresis were analyzed by reverse transcription-polymerase chain reaction assays using primers target to the genomic segments 2 of PBV strains isolated in China and the USA. The amplicons were sequenced and analyzed. RESULTS: Primers derived from the China strain produced amplicons in only 4 of the 13 specimens (30.76%). No sample was revealed as positive with the primers derived from the US strain. DNA sequencing of polymerase chain reaction products differed in nucleic acid and amino acid sequences by 13.9-42.28% and 18.1-51.1%, respectively. Despite this strain diversity, three domains of conserved nucleotide sequences as well as the amino acid motif D-S-D typical of RNA-dependent RNA polymerase gene of double-strand RNA viruses were identified. Comparatively, these conserved regions were also identified in homologous PBV strains from the USA and China. Phylogenetic analysis showed no time or geographic clustering. CONCLUSIONS: These findings provide evidence that PBV may represent an emerging heterogeneous group of viruses.

Twenty year study of the occurrence of reovirus infection in hospitalized children with acute gastroenteritis in Argentina.

We studied the occurrence of reovirus infection in children hospitalized with acute gastroenteritis in Argentina during a 20-year interval (1981 through 2001). Three of 2854 (0.10%) stools were positive for reovirus but negative for adenovirus, astrovirus and rotavirus. Children infected with reovirus were <1 year old; one had meningoencephalitis in addition to gastroenteritis. This study indicates that reovirus is an uncommon cause of childhood gastroenteritis requires medical assistance.

The epidemiology of acute viral gastroenteritis in hospitalized children in Cordoba city, Argentina: an insight of disease burden

Information concerning the disease burden of viral gastroenteritis has important implications for the use and monitoring the impact of public health policies. The present study, carried out in Córdoba city, Argentina, documents the epidemiology of severe viral diarrhea as well as the burden of viral gastrointestinal disease in the hospital children admission. A total of 133 stools were collected from hospitalized children (Town Childhood Hospital) suffering from acute diarrhea and studied for the presence of Group A rotavirus, astrovirus and adenovirus 40/41 by enzyme-immuno assay, between November 1997 and October 1998. Enteric viruses accounted for 42.1 percent of the total diarrheal cases analyzed. Group A rotaviruses, astroviruses, adenoviruses 40/41 and mixed infections were found in 35.3, 4.5, 1.5, and 0.8 percent studied specimens respectively. We estimated that 1 in 27 children in the 0-35 month-old cohort/range would be annually hospitalized for a viral gastroenteritis illness. The major impact on viral diarrhea lies on rotaviral infection, accouting for 84.0 percent of the viral diarrheal cases analyzed and for approximately one third of severe diarrheas requiring hospital admission in Córdoba City, Argentina

Agentes virales asociados al sindrome diarreico en pacientes trasplantados renales / Viral agents related to diarrheic syndrome in kidney transplanted patiens

El objetivo del presente estudio fue documentar la frequencia de agentes virales clásicos y emergentes y su asociación etiológica con el síndrome diarreico en pacientes transplantados renales en Córdoba, Argentina. Se estudiaron 42 muestras fecales de individuos transplantados renales, internados y ambulatórios, con o sin diarrea, todas obtenidas después del transplante. Los pacientes se encontraban bajo triple terapia inmunosupresora con esteroides, azatioprina y ciclosporina ó tacrolimus. Los resultados obtenidos revelaron la presencia de ratovirus grupo A y picobirnavirus en tres de nueve pacientes con síndrome diarreico severo (33.33 porciento), en ausencia de otros patógenos bacterianos entéricos. La presencia de estos agentes virales se correlacionó con niveles elevados de ciclosporina en sangue (> 290 ng/ml) o bien con un tratamiento inmunosupresor prolongado. En contraste, no se detectó ningún virus en la etiología de cuadros diarreicos severos en pacientes transplantados renales. (AU)

Agentes virales asociados al sindrome diarreico en pacientes trasplantados renales / Viral agents related to diarrheic syndrome in kidney transplanted patiens

El objetivo del presente estudio fue documentar la frequencia de agentes virales clásicos y emergentes y su asociación etiológica con el síndrome diarreico en pacientes transplantados renales en Córdoba, Argentina. Se estudiaron 42 muestras fecales de individuos transplantados renales, internados y ambulatórios, con o sin diarrea, todas obtenidas después del transplante. Los pacientes se encontraban bajo triple terapia inmunosupresora con esteroides, azatioprina y ciclosporina ó tacrolimus. Los resultados obtenidos revelaron la presencia de ratovirus grupo A y picobirnavirus en tres de nueve pacientes con síndrome diarreico severo (33.33 porciento), en ausencia de otros patógenos bacterianos entéricos. La presencia de estos agentes virales se correlacionó con niveles elevados de ciclosporina en sangue (> 290 ng/ml) o bien con un tratamiento inmunosupresor prolongado. En contraste, no se detectó ningún virus en la etiología de cuadros diarreicos severos en pacientes transplantados renales.