Phylogeography and evolutionary history of hepatitis E virus genotype 3 in Argentina.

BACKGROUND: Few studies about the evolutionary history of the hepatitis E virus (HEV) have been conducted. The aim of our work was to investigate and make inferences about the origin and routes of dispersion of HEV-3 in Argentina. METHODS: Phylogenetic, coalescent and phylogeographic analyses were performed using a 322-bp ORF2 genomic fragment of all HEV-3 sequences with known date and place of isolation published at GenBank until May 2018 (n=926), including 16 Argentinian sequences (isolated from pigs, water and humans). RESULTS: Phylogenetic analysis revealed two clades within HEV-3: abchij and efg. All Argentinian samples were grouped intermingled within clade 3abchij. The coalescent analysis showed that the most recent common ancestor for the clade 3abchij would have existed around the year 1967 (95% highest posterior density (HPD): 1963-1970). The estimated substitution rate was 1.01×10-2 (95%HPD: 9.3×10-3-1.09×10-2) substitutions/site/y, comparable with the rate previously described. The phylogeographic approach revealed a correspondence between phylogeny and place of origin for Argentinian samples, suggesting many HEV introductions in the country, probably from Europe and Japan. CONCLUSIONS: This is the first evolutionary inference of HEV-3 that includes Argentinian strains, showing the circulation of many HEV-3 subtypes, obtained from different sources and places, with recent diversification processes. ACCESSION NUMBERS: [KX812460], [KX812461], [KX812462], [KX812465], [KX812466], [KX812467], [KX812468], [KX812469].

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.

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.

First detection of hepatitis E virus in Central Argentina: environmental and serological survey.

BACKGROUND: The hepatitis E virus (HEV) is an emergent causative agent of acute hepatitis worldwide, transmitted by fecal-oral route. In Argentina it is considered rare, so differential laboratory testing is not routinely performed. Besides, in Argentina's central area epidemiological and molecular characteristics of HEV are still unknown. OBJECTIVES: Provide evidence of local circulation of HEV by molecular detection on environmental samples and by serological survey in healthy adult population of Córdoba city, Argentina. STUDY DESIGN: Environmental surveillance was conducted in river and sewage samples collected between 2007 and 2009-2011. Viral detection was performed by RT-Nested PCR of ORF-1 and ORF-2 partial regions. Anti-HEV IgG was determined by EIA in 433 serum samples collected between 2009 and 2010. RESULTS: HEV was detected in 6.3% of raw sewage samples and in 3.2% of riverine samples. Nucleotide sequencing analyses revealed that all isolates belonged to genotype 3, subtypes a, b and c. The prevalence of IgG anti-HEV was 4.4%. Seroprevalence increased with the age of the individuals (OR: 3.50; 95% CI 1.39-8.87; p=0.0065) and, although the prevalence was higher in low income population, no statistical relation was found between anti-HEV and socioeconomic level. CONCLUSIONS: The environmental findings added to serological results, demonstrate that HEV circulates in central Argentina. Contamination of water with HEV could represent a route of transmission for local populations, which have a high number of susceptible individuals. This fact alerts local health care systems in order to include detection of HEV in the diagnostic algorithm of viral hepatitis.

Evidence of hepatitis A virus circulation in central Argentina: seroprevalence and environmental surveillance.

BACKGROUND: Hepatitis A virus (HAV) has shown intermediate endemicity in Argentina, but notification of clinical cases has decreased since the introduction of the vaccine in 2005. OBJECTIVES: In order to get insight into the local circulation of this virus after four years of the official introduction of the vaccine, the aims of this study were to provide information on HAV immune status of the adult population of Córdoba city and to conduct environmental surveillance of HAV in sewage and river samples in the same region. STUDY DESIGN: The prevalence of anti-HAV was determined by EIA in 416 samples of people (without prior vaccination) from Córdoba city (2009-2010). Spline regression models were estimated under generalized additive models. Environmental surveillance was conducted in river and sewage samples collected in the same period. Viral detection was performed by RT-Nested PCR of the 5'UTR. RESULTS: In Córdoba, the global prevalence of anti-HAV was 73.5%. It increased with age (p<0.0001) and it was associated with the low-income population (OR: 1.14; 95% CI 1.05-1.25). This prevalence decreased in younger age groups, especially in the high-income population. Environmental monitoring revealed the presence of HAV (IA) in 20.8% and 16.1% of wastewater and river samples, respectively. CONCLUSIONS: As a consequence of a decrease in HAV circulation due to improvements in immunization, socio-economic and hygienic conditions, young adults are becoming increasingly susceptible to HAV infections. Environmental monitoring demonstrated that HAV circulates in the local population; therefore, health care systems should consider the implementation of preventive measures for susceptible adults in order to reduce the risk of HAV infection.

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 analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene.

In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.

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.

Randomized trial of inactivated and live polio vaccine schedules in Guatemalan infants.

BACKGROUND: The immunogenicity of inactivated poliovirus vaccine (IPV) in developing countries is not well documented. This study compared the immune response to IPV with that to oral poliovirus vaccine (OPV) in Guatemalan infants. METHODS: This was an open-label, randomized comparison of IPV only, OPV only, or IPV followed by OPV in Guatemalan public health clinics. Serum samples were tested for neutralizing antibodies, and stool samples were tested for Sabin strain polioviruses. RESULTS: Seropositivity rates 2 months after 2 doses of IPV were 98%-100% for polio types 1, 2, and 3 and were 97.1%, 99.3%, and 92.1% for OPV-only recipients (P<.001 for the response to type 3). One month after the third dose, 100% of IPV-only recipients had protective antibodies against all 3 types, compared with 99%, 100%, and 97% against polio types 1, 2, and 3 respectively, among recipients of OPV only. Infants who received IPV only had higher geometric mean titers than infants who received OPV only. Maternal antibodies lowered the final antibody responses to IPV but did not prevent the development of protective levels of antibody. Of 191 stool samples from infants who received IPV only, 5 (2.6%) were positive for poliovirus vaccine strains. CONCLUSIONS: IPV alone and IPV followed by OPV are safe and effective for Guatemalan infants.

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.