Phylogenetic Diversity in Core Region of Hepatitis C Virus Genotype 1a as a Factor Associated with Fibrosis Severity in HIV-1-Coinfected Patients.

High hepatitis C virus (HCV) genetic diversity impacts infectivity/pathogenicity, influencing chronic liver disease progression associated with fibrosis degrees and hepatocellular carcinoma. HCV core protein is crucial in cell-growth regulation and host-gene expression. Liver fibrosis is accelerated by unknown mechanisms in human immunodeficiency virus-1- (HIV-1-) coinfected individuals. We aimed to study whether well-defined HCV-1a core polymorphisms and genetic heterogeneity are related to fibrosis in a highly homogeneous group of interferon-treated HIV-HCV-coinfected patients. Genetic heterogeneity was weighed by Faith's phylogenetic diversity (PD), which has been little studied in HCV. Eighteen HCV/HIV-coinfected patients presenting different liver fibrosis stages before anti-HCV treatment-initiation were recruited. Sampling at baseline and during and after treatment was performed up to 72 weeks. At inter/intrahost level, HCV-1a populations were studied using molecular cloning and Sanger sequencing. Over 400 complete HCV-1a core sequences encompassing 573 positions of C were obtained. Amino acid substitutions found previously at positions 70 and 91 of HCV-1b core region were not observed. However, HCV genetic heterogeneity was higher in mild than in severe fibrosis cases. These results suggest a potential utility of PD as a virus-related factor associated with chronic hepatitis C progression. These observations should be reassessed in larger cohorts to corroborate our findings and assess other potential covariates.

Virus evolution during chronic hepatitis B virus infection as revealed by ultradeep sequencing data.

Despite chronic hepatitis B virus (HBV) infection (CHB) being a leading cause of liver cirrhosis and cancer, HBV evolution during CHB is not fully understood. Recent studies have indicated that virus diversity progressively increases along the course of CHB and that some virus mutations correlate with severe liver conditions such as chronic hepatitis, cirrhosis and hepatocellular carcinoma. Using ultradeep sequencing (UDS) data from an intrafamilial case, we detected such mutations at low frequencies among three immunotolerant patients and at high frequencies in an inactive carrier. Furthermore, our analyses indicated that the HBV population from the seroconverter patient underwent many genetic changes in response to virus clearance. Together, these data indicate a potential use of UDS for developing non-invasive biomarkers for monitoring disease changes over time or in response to specific therapies. In addition, our analyses revealed that virus clearance seemed not to require the virus effective population size to decline. A detailed genetic analysis of the viral lineages arising during and after the clearance suggested that mutations at or close to critical elements of the core promoter (enhancer II, epsilon encapsidation signal, TA2, TA3 and direct repeat 1-hormone response element) might be responsible for a sustained replication. This hypothesis requires the decline in virus load to be explained by constant clearance of virus-producing hepatocytes, consistent with the sustained progress towards serious liver conditions experienced by many CHB patients.

Characterization and phylogeny of Isaria spp. strains (Ascomycota: Hypocreales) using ITS1-5.8S-ITS2 and elongation factor 1-alpha sequences.

The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

Concomitant infection of Neospora caninum and Bovine Herpesvirus type 5 in spontaneous bovine abortions / Infecção simultânea de Neospora caninum e Herpesvirus bovino tipo 5 em casos espontâneos de aborto bovino

Bovine Herpesvirus type 5 (BoHV-5) has not been conclusively demonstrated to cause bovine abortion. Brain lesions produced by Neospora caninum and Bovine Herpesvirus type 1 (BoHV-1) exhibit common features. Therefore, careful microscopic evaluation and additional diagnostic procedures are required to achieve an accurate final etiological diagnosis. The aim of the present work was to investigate the occurrence of infections due to BoHV-1, BoHV-5 and N. caninum in 68 cases of spontaneous bovine abortions which showed microscopic lesions in the fetal central nervous system. This study allowed the identification of 4 (5.9%) fetuses with dual infection by BoHV-5 and N. caninum and 33 (48.5%) cases in which N. caninum was the sole pathogen identified. All cases were negative to BoHV-1. The results of this study provide evidence that dual infection by BoHV-5 and N. caninum occur during pregnancy in cattle; however, the role of BoHV-5 as a primary cause of bovine abortion needs further research. Molecular diagnosis of BoHV-5 and N. caninum confirmed the importance of applying complementary assays to improve the sensitivity of diagnosing bovine abortion.

Não está demonstrado até ao momento, que o Herpesvírus bovino tipo 5 (BoHV-5) seja um agente causal de aborto bovino. Uma vez que as lesões cerebrais tanto de Neospora caninum como de Herpesvírus bovino tipo 1(BoHV-1) têm características similares, é necessária uma avaliação microscópica cuidadosa, bem como exames laboratoriais adicionais, para obter um diagnóstico final preciso. O objetivo do presente trabalho foi investigar a presença de infeções por BoHV-1, BoHV-5 e N. caninum em 68 casos de aborto espontâneo, nos quais se verificaram lesões microscópicas no sistema nervoso central. Foram encontrados 4 (5,9%) fetos com infeção simultânea de BoHV-5 e N. caninum e 33 (48,5%) casos com infeção exclusiva de N. caninum. Todos os casos foram negativos a BoHV-1. Os resultados deste estudo demonstram que a infeção dual por BoHV-5 y N. caninum está presente durante a gestçao dos bovinos. Apesar disso, o papel de BoHV-5 como agente primário causal de aborto, carece de mais investigaçao. O diagnóstico molecular de BoHV-5 e N. caninum confirmou a importância de se aplicar ensaios complementares para melhorar a sensibilidade do diagnóstico de aborto bovino.

Effects of UV radiation on the taxonomic composition of natural bacterioplankton communities from Bahía Engaño (Patagonia, Argentina).

In order to gain insights into the effects of solar ultraviolet radiation (UVR, 280-400 nm) on the composition of marine bacterioplankton communities from South Atlantic waters - Bahía Engaño (Patagonia, Argentina), we performed microcosms experiments during the Austral summer of 2010. Water samples were exposed to three solar radiation treatments in 25 L microcosms during 8 days: PAR+UV-A+UV-B (280-700 nm; PAB treatment), PAR+UV-A (320-700 nm; PA treatment), and PAR only (400-700 nm; P treatment). The taxonomic composition of the bacterial communities, at the beginning and at the end of the experiment, were studied by the analyses of 16S rDNA gene libraries. Multivariate and phylogenetic analyses demonstrated substantial differences in the community composition so that the samples exposed to PAR and PAR+UV-A presented more similar taxa assemblages among them than compared to the PAR+UV-A+UV-B exposed one. Our results indicate that overall, exposure to different radiation treatments can shape the taxonomic composition of marine bacterial populations, grown in microcosms, from this Patagonian area.

A clustering phenomenon among HCV-1a strains among patients coinfected with HIV from Buenos Aires, Argentina.

The human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same transmission routes which lead to high coinfection rates. Among HIV-infected individuals such rates reached 21% in Argentina, being HCV-1a the most predominant subtype. In this work, 25 HCV subtype 1a (HCV-1a) strains from Argentinean patients coinfected with HIV were studied based on E2 and NS5A sequences. Phylogenetic analyses indicated that 12 strains were highly related to each other, constituting a highly supported (posterior probability = 0.95) monophyletic group that we called "M." The remaining HCV strains (group dispersed or "D") were interspersed along the phylogenetic trees. When comparing both groups of HCV-1a, 10 amino acid differences were located in functional domains of E2 and NS5A proteins that appeared to affect eventually the peptides binding to MHC-I molecules thus favoring immune escape and contributing to the divergence of HCV genotypes. Bayesian coalescent analyses for HCV-1a cluster M isolates indicated that the time to the most recent common ancestor (tMRCA) overlaps with the age estimated recently for the HIV-BF epidemic in Argentina. Furthermore, the genomic characterization based on pol gene analysis from HIV viremic patients showed that most HIV isolates from patients coinfected with HCV-1a cluster M were BF recombinants with identical recombination patterns. In conclusion, these results suggest the presence of an HCV-1a monophyletic cluster with a potential HIV co-transmission by phylogenetic analyses.

Drug resistance mutations in HIV pol sequences from Argentinean patients under antiretroviral treatment: subtype, gender, and age issues.

We studied drug resistance mutations (DRMs) in 2623 pol sequences. Out of 94,828 amino acid substitutions that were detected, 8749 corresponded to nucleoside reverse transcriptase inhibitor (NRTI), 3765 to nonnucleoside reverse transcriptase inhibitor (NNRTI), and 7141 to protease inhibitor (PI) resistance-associated mutations. The most common DRMs were L10I, I54V, L90M, V82A, A71V, L10V, M46I, M184V, M41L, T215Y, D67N, L210W, K70R, N348I, V118I, K103N, Y181C, G190A, K101E, V108I, L100I, V90I, K101Q, and A98G. As expected, DRMs frequencies depended on viral genotype. The amounts of NRTI and PI resistance mutations among B and BF sequences from children were higher than among sequences from adults. The frequencies of PI and NRTI resistance mutations among B and BF sequences from adult men were higher than among sequences from women. Some of these observations can be explained in light of the available epidemiological information, but some cannot, indicating that further studies are needed to understand the antiretroviral resistance epidemics in Argentina.

Stable human T-cell lymphotropic virus type 1 (HTLV-1) subtype a/subgroup a endemicity in Amerindians from Northwest Argentina: a health problem to be resolved.

Jujuy province, in Northwest Argentina, is known to be endemic for HTLV-1 infection. Moreover, foci of HTLV-1 associated pathologies have also been described in this region. To gain an insight into the current situation of HTLV-1/2 in this endemic area, a seroprevalence and phylogenetic study was performed among a Kolla community from Abra Pampa city and surroundings. Out of 112 individuals, 11 (9.8%) were confirmed as HTLV-1 positive and no HTLV-2 infection was detected. The phylogenetic analysis of the LTR region showed that all the HTLV-1 sequences belonged to the Cosmopolitan subtype a/transcontinental subgroup A, and were closely related to reference sequences from Peru, Argentina, and the South of Brazil (P = 0.82). Considering the cultural and historical features of this community and in spite of the mandatory detection of anti-HTLV-1/2 antibodies in blood banks since 2005, it would be important to implement new public health measures focused on decreasing HTLV-1 transmission in this endemic area.

Glycoprotein gene sequence variation in rhesus monkey rhadinovirus.

Gene sequences for seven glycoproteins from 20 independent isolates of rhesus monkey rhadinovirus (RRV) and of the corresponding seven glycoprotein genes from nine strains of the Kaposi's sarcoma-associated herpesvirus (KSHV) were obtained and analyzed. Phylogenetic analysis revealed two discrete groupings of RRV gH sequences, two discrete groupings of RRV gL sequences and two discrete groupings of RRV gB sequences. We called these phylogenetic groupings gH(a), gH(b), gL(a), gL(b), gB(a) and gB(b). gH(a) was always paired with gL(a) and gH(b) was always paired with gL(b) for any individual RRV isolate. Since gH and gL are known to be interacting partners, these results suggest the need of matching sequence types for function of these cooperating proteins. gB phylogenetic grouping was not associated with gH/gL phylogenetic grouping. Our results demonstrate two distinct, distantly-related phylogenetic groupings of gH and gL of RRV despite a remarkable degree of sequence conservation within each individual phylogenetic group.

Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades.

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave unique names to the identified groups and no study has yet integrated all the available sequences. Thus, we hypothesized that many of the different groups previously identified actually correspond to a limited group of genotypes that are unevenly distributed worldwide. To examine this hypothesis, we sequenced the env gene from 28 BLV field strains and compared these sequences to 46 env sequences that represent all the genetic groupings already identified. By using phylogenetic analyses, we recovered six clades, or genotypes, that we have called genotypes 1, 2, 3, 4, 5 and 6. Genotypes 1-5 have counterparts among the sequence groupings identified previously. One env sequence did not cluster with any of the others and was highly divergent when compared with the six genotypes identified here. Thus, an extra genotype, which we named 7, may exist. Similarity comparisons were highly congruent with phylogenetic analyses. Furthermore, our analyses confirmed the existence of geographical clusters.

Divergent strains of human T-lymphotropic virus type 1 (HTLV-1) within the Cosmopolitan subtype in Argentina.

HTLV-1 Cosmopolitan subtype Transcontinental subgroup A has been described among aboriginal communities from the northwest endemic area of Argentina. Moreover, Transcontinental subgroup A and the Japanese subgroup B were reported among blood donors from the nonendemic central region of the country. We carried out the first HTLV-1 phylogenetic study in individuals residing in Buenos Aires capital city. Phylogenetic analysis performed on the LTR region showed that all 44 new strains clustered within the Cosmopolitan subtype, with 42 (95.4%) belonging to Transcontinental subgroup A. Of them, 20 (45.5%) strains grouped in the large Latin American cluster and 4 (9.1%) in the small Latin American cluster. The majority of them belonged to individuals of nonblack origin, grouped with Amerindian strains. Three (6.8%) were closely related to South African references and two monophyletic clusters including only HIV/HTLV-1 coinfected individuals were observed. Interestingly, two (4.5%) new sequences (divergent strains) branched off from all five known Cosmopolitan subgroups in a well-supported clade. In summary, these findings show that HTLV-1 Cosmopolitan subtype Transcontinental subgroup A is infecting residents of Buenos Aires, a nonendemic area of Argentina, and confirm the introduction of divergent strains in the country.

Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation.

The genetic diversity of 2 pairs (AH65 and AH187) of wild type bovine coronaviruses (BCoV) sequenced directly from nasal (respiratory) and rectal (enteric) swabs of two feedlot calves with respiratory and enteric symptoms [Hasoksuz, M., Sreevatsan, S., Cho, K.O., Hoet, A.E., Saif, L.J., 2002b. Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res. 84 (1-2), 101-109.]. was analyzed. Sequence analysis of the complete genomes revealed differences at 123 and 149 nucleotides (nt) throughout the entire genome between the respiratory and enteric strains for samples AH65 and AH187, respectively, indicating the presence of intra-host BCoV quasispecies. In addition, significant numbers of sequence ambiguities were found in the genomes of some BCoV-R and BCoV-E strains, suggesting intra-isolate quasispecies. The tissue culture (TC) passaged counterparts of AH65 respiratory BCoV (AH65-R-TC) and enteric BCoV (AH65-E-TC) were also sequenced after 14 and 15 passages and 1 plaque purification in human rectal tumor cells (HRT-18), respectively. Compared to the parental wild type strains, tissue culture passage generated 104 nt changes in the AH65-E-TC isolate but only 8 nt changes in the AH65-R-TC isolate. Particularly noteworthy, the majority of nucleotide changes in the AH65-E-TC isolate occurred at the identical positions as the mutations occurring in the AH65-R strain from the same animal. These data suggest that BCoV evolves through quasispecies development, and that enteric BCoV isolates are more prone to genetic changes and may mutate to resemble respiratory BCoV strains after tissue culture passage.