Jorge V. Pavan

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.

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.

Rotavirus seasonality in urban sewage from Argentina: effect of meteorological variables on the viral load and the genetic diversity.

In Argentina, the rotavirus disease exhibits seasonal variations, being most prevalent in the fall and winter months. To deepen the understanding of rotavirus seasonality in our community, the influence of meteorological factors on the rotavirus load and the genetic diversity in urban raw sewage from Córdoba city, Argentina were evaluated. Wastewater samples were collected monthly during a three-year study period and viral particles were concentrated by polyethylene glycol precipitation. RT-nested PCR was applied for rotavirus detection, and VP7/VP4 characterization and real-time PCR for rotavirus quantification. Both molecular techniques showed relatively similar sensitivity rates and revealed rotavirus presence in urban wastewater in cold and warm seasons, indicating its circulation in the local community all year round. However, a slight trend for rotavirus circulation was noted by real-time PCR in the fall and winter seasons, showing a significantly higher peak of rotavirus concentration at mean temperatures lower than 18°C and also higher, although not statistically different during drier weather. VP7 and VP4 gene characterization showed that G1 and P[8] genotypes were dominant, and temporal variations in genotype distribution were not observed. Rotavirus spread is complex and our results point out that weather factors alone cannot explain the seasonal quantitative pattern of the rotavirus disease. Therefore, alternative transmission routes, changes in human behavior and susceptibility, and the stability and survivability of the virus might all together contribute to the seasonality of rotavirus. The results obtained here provide evidence regarding the dynamics of rotavirus circulation and maintenance in Argentina.

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. J. Med. Virol. 85:363–369, 2013.

A novel human adenovirus hexon protein of species D found in an AIDS patient

To date, human adenoviruses are classified into 53 types (types 1–51 and types 53 and 54), which have been grouped into six species named A through F, and the recently identified type 52 has been proposed as member of a new species, G. Type classification is based on type-specific epitopes within loop 1 (L1) and loop 2 (L2) of the hexon protein, which contain seven hypervariable regions that are responsible for type specificity. In this paper, we present the characterization of an adenovirus strain isolated from a male AIDS patient in Cordoba, Argentina. This strain was found to be a member of species D by genomic Sma I restriction analysis. Sequencing of the L1 and L2 regions of the hexon gene and immunological characterization by virus neutralization revealed this hexon to be unique and distinct from the previously identified hexons of types within species D. A seroepidemiologic study in the human population of Cordoba showed that this strain was not endemic in the local human population.

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.

Culture amplification in human colon adenocarcinoma cell line (CaCo-2) combined with an ELISA as a supplementary assay for accurate diagnosis of rotavirus

Culture amplification in colon adenocarcinoma cell line (CaCo-2) combined with enzyme immunoassay (Pathfinder ELISA) was developed as a supplementary tool for rotavirus diagnosis. One hundred and thirty stools in which results by polyacrylamide gel electrophoresis (PAGE) were in agreement with those obtained by ELISA were amplified in the CaCo-2 cell line. After the first passage 100% specimens were revealed as positive by ELISA. This result was confirmed by PAGE and direct electron microscopy (EM) which increased the rates of rotavirus detection up to 100% after the third and fifth cell passages, respectively. All of the amplified negative stools were confirmed as negative. Among discordant results, three of the eight specimens positive by ELISA but negative by PAGE were confirmed as true positive after the third cell passage. False positive ELISA results could be discarded when the samples were culture amplified and retested by the same ELISA. Using the CaCo-2 amplification-ELISA as supplementary assay, sensitivity and specificity were 1.000 and 0.953 for ELISA and 0.917 and 1.000 for PAGE, respectively. The combined CaCo-2 cell line amplification-immunoassay method proved to be suitable both to evaluate increase in sensitivity of newly developed rotavirus assays and for rotaviral amplification before antigen assays.

Quantification of human infection risk caused by rotavirus in surface waters from Córdoba, Argentina.

Fecal contamination of water is a worrying problem because it is associated with the transmission of enteric pathogenic microorganisms that can cause many infectious diseases. In this study, an environmental survey was conducted to assess the level of viral contamination by viable enterovirus and rotavirus genome in two recreational rivers (Suquía and Xanaes) of Córdoba, Argentina. Quantitative microbial risk assessment (QMRA) was calculated to estimate the risk of rotavirus infection. Water sampling was carried out during a one-year period, the presence of total and fecal coliforms was determined and water samples were then concentrated for viral determination. Cell culture and indirect immunofluorescence were applied for enterovirus detection and RT-qPCR for rotavirus quantification. Coliform bacteria levels found in Suquía River often far exceeded the guideline limits for recreational waters. The Xanaes exhibited a lower level of bacterial contamination, frequently within the guideline limits. Enterovirus and rotavirus were frequently detected in the monitoring rivers (percentage of positive samples in Suquía: 78.6% enterovirus, 100% rotavirus; in Xanaes: 87.5% enterovirus, 18.7% rotavirus). Rotavirus was detected at a media concentration of 5.7×10(5) genome copies/L (gc/L) in the Suquía and 8.5×10(0)gc/L in the Xanaes. QMRA revealed high risk of rotavirus infection in the Suquía, at sampling points with acceptable and non-acceptable bacteria numbers. The Xanaes showed significantly lower health risk of rotavirus infection but it proved to be a public health hazard. The viral occurrence was not readily explained by the levels of bacteria indicators, thus viral monitoring should be included to determine microbiological water quality. These findings provide the first data of QMRA for recreational waters in Argentina and reveal the need for public awareness of the health implications of the use of the river waters.

IgG subclasses and DNA detection of HHV‐6 and HHV‐7 in healthy individuals

Human herpesvirus 6 (HHV‐6) and 7 (HHV‐7) are common opportunistic agents in immunocompromised hosts, although infection with HHV‐6 and HHV‐7 can also be observed in immunocompetent hosts. Despite similar biology and epidemiology, this study evaluated differences in the IgG subclass distribution associated with HHV‐6 and HHV‐7 in seropositive, healthy persons. The identified subclasses were also compared with the detection of HHV‐6 and HHV‐7 DNA. For these assays, sera, plasma, and saliva samples were obtained from 40 healthy blood donors in Argentina who were seropositive for both HHV‐6 and HHV‐7. HHV‐6 and HHV‐7 DNA were detected in saliva and plasma samples using nested PCR, and specific IgG subclasses were determined using immunofluorescent assays of sera samples. HHV‐7 DNA was detected in 90% of all plasma samples and in 100% of saliva samples. In contrast, HHV‐6 DNA was not detected in any of the plasma samples, and it was detected in only 6 of 40 saliva samples. Determination of IgG subclass distributions showed that HHV‐6 was restricted to IgG1, whereas HHV‐7 IgG subclasses included two groups, one restricted only to IgG1 and the other to IgG1 and IgG3. These results demonstrate the differences between HHV‐6 and HHV‐7 DNA range detection in saliva and plasma samples, as well as the IgG subclass patterns for each virus type, in healthy persons in Argentina. J. Med. Virol. 82:1679–1683, 2010. 2010 Wiley‐Liss, Inc.

Tracking novel adenovirus in environmental and human clinical samples: no evidence of endemic human adenovirus type 58 circulation in Córdoba city, Argentina

In recent years, several types of human adenovirus (HAdV) have arisen from the recombination between two or more previously known HAdV types, but their epidemiology is poorly understood. In this study, we investigated the circulation of HAdV-58, a recently described HAdV isolated from an HIV-positive patient in Córdoba city, Argentina. For this purpose, a 30-month survey was conducted to study the presence of this type of adenovirus in sewage samples collected at the inlet from a wastewater treatment plant in Córdoba city, Argentina. Complementarily, the virus was sought in stools of HIV-positive patients. Although HAdVs were detected in human stool samples and in a high percentage of sewage samples, no evidence of HAdV-58 circulation was detected. We suggest that there is no endemic circulation of HAdV-58 in the geographical local area. The trend is that the number of identified HAdVs increases over time. In this context, understanding the current circulating HAdVs may be biologically relevant.