Virological surveillance,molecular phylogeny,and evolutionary dynamics of hepatitis C virus subtypes 1a and 4a isolates in patients from Saudi Arabia

Hepatitis C virus (HCV) subtypes are pre-requisite to predict endemicity, epidemiology, clinical pathogenesis, diagnosis, and treatment of chronic hepatitis C infection. HCV genotypes 4 and 1 are the most prevalent in Saudi Arabia, however; less consensus data exist on circulating HCV subtypes in infected individuals. This study was aimed to demonstrate the virological surveillance, phylogenetic analysis, and evolutionary relationship of HCV genotypes 4 and 1 subtypes in the Saudi population with the rest of the world. Fifty-five clinical specimens from different parts of the country were analyzed based on 5′ untranslated region (5′ UTR) amplification, direct sequencing, and for molecular evolutionary genetic analysis. Pair-wise comparison and multiple sequence alignment were performed to determine the nucleotide conservation, nucleotide variation, and positional mutations within the sequenced isolates. The evolutionary relationship of sequenced HCV isolates with referenced HCV strains from the rest of the world was established by computing pairwise genetic distances and generating phylogenetic trees. Twelve new sequences were submitted to GenBank, NCBI database. The results revealed that HCV subtype 4a is more prevalent preceded by 1a in the Saudi population. Molecular phylogeny predicts the descendants’ relationship of subtype 4a isolates very close to Egyptian prototype HCV strains, while 1a isolates were homogeneous and clustering to the European and North American genetic lineages. The implications of this study highlight the importance of HCV subtyping as an indispensable tool to monitor the distribution of viral strains, to determine the risk factors of infection prevalence, and to investigate clinical differences of treatment outcomes among intergenotypic and intragenotypic isolates in the treated population.     

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.     

Whole genome sequence analysis of Cryptococcus gattii from the Pacific Northwest reveals unexpected diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.     

ClassyFlu: Classification of Influenza A Viruses with Discriminatively Trained Profile-HMMs

Accurate and rapid characterization of influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA) sequences with respect to subtype and clade is at the basis of extended diagnostic services and implicit to molecular epidemiologic studies. ClassyFlu is a new tool and web service for the classification of IAV sequences of the HA and NA gene into subtypes and phylogenetic clades using discriminatively trained profile hidden Markov models (HMMs), one for each subtype or clade. ClassyFlu merely requires as input unaligned, full-length or partial HA or NA DNA sequences. It enables rapid and highly accurate assignment of HA sequences to subtypes H1–H17 but particularly focusses on the finer grained assignment of sequences of highly pathogenic avian influenza viruses of subtype H5N1 according to the cladistics proposed by the H5N1 Evolution Working Group. NA sequences are classified into subtypes N1–N10. ClassyFlu was compared to semiautomatic classification approaches using BLAST and phylogenetics and additionally for H5 sequences to the new “Highly Pathogenic H5N1 Clade Classification Tool” (IRD-CT) proposed by the Influenza Research Database. Our results show that both web tools (ClassyFlu and IRD-CT), although based on different methods, are nearly equivalent in performance and both are more accurate and faster than semiautomatic classification. A retraining of ClassyFlu to altered cladistics as well as an extension of ClassyFlu to other IAV genome segments or fragments thereof is undemanding. This is exemplified by unambiguous assignment to a distinct cluster within subtype H7 of sequences of H7N9 viruses which emerged in China early in 2013 and caused more than 130 human infections. http://bioinf.uni-greifswald.de/ClassyFlu is a free web service. For local execution, the ClassyFlu source code in PERL is freely available.     

HCV Genome-Wide Genetic Analyses in Context of Disease Progression and Hepatocellular Carcinoma

Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV’s sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.     

Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I.

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.     

Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia,North Africa

HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20^th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869–1902) before the introduction of HCV-2k in 1901 (1867–1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization.     

A novel genotype of GB virus C: its identification and predominance among injecting drug users in Yunnan, China

GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5'UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5'UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a "non-pathogenic virus".     

Phylogeny,morphology and pathogenicity of Elsinoë ampelina,the causal agent of grapevine anthracnose in Brazil and Australia

Anthracnose caused by Elsinoë ampelina is one of the most important table grape diseases in humid regions in Brazil and Australia. The objective of this study was to characterize E. ampelina isolates from Brazil and Australia by means of phylogenetic analyses, morphological features and pathogenicity tests. Phylogenetic relationships among 35 isolates were determined based on a data set of internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences. In phylogenetic tree analyses, using a combined ITS and TEF sequence alignment, all E. ampelina isolates were clustered together in a single well‐supported clade. In contrast to the absence of genetic variability within ITS and TEF sequences, HIS3 sequences showed 54 polymorphic sites. The haplotype network generated from HIS3 data set showed four distinct haplotypes. EA1 was the predominant haplotype including 29 isolates from both countries. High genetic variability was observed in two Brazilian isolates, haplotype EA4, which may have lost the intron region during species evolution. Colony colours differed between Brazilian and Australian isolates, but showed similar wrinkled colony texture, absence of spores, sparse‐to‐absent white aerial mycelium and slow growth (0.049–0.060 mm/day). Brazilian isolates produced conidia of 5.65 × 2.65 μm, larger than conidia from Australian isolates, which measured 5.14 × 2.30 μm. In pathogenicity tests, all nine Australian isolates inoculated were pathogenic on detached canes and potted vines of table grape.     

Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.Hepatitis C virus (HCV) infection is a major global health issue leading to persistent viral infection in the majority of those infected and is associated with progressive liver disease, cirrhosis, and hepatocellular carcinoma. Six major genotypes of HCV have been described that have evolved in geographically distinct regions and that share approximately. 80% nucleotide homology with one another. HCV viral genotypes have been further classified into subtypes (25). HCV subtype 3a infection is now the most common subtype in the United Kingdom (11), although it is globally distributed and frequently associated with intravenous drug use.The classification of HCV viral strains by genotype and subtype has proven informative not only in terms of the epidemic and evolutionary history of the virus but also in terms of clinical outcomes. In particular, the response rates to current gold standard therapy (9) and the prevalence of hepatic steatosis (20) are significantly higher for subtype 3a than for genotype 1 infections. The reasons for this are not understood but must relate to viral genetic and phenotypic differences between strains, or to differences in the ability of hosts to exert an effective immune response against particular viral sequences, or to a combination of both factors.To date, detailed assessment of the HCV genome has largely focused on HCV genotype 1. Indeed, only a few full-length HCV subtype 3a viral sequences are currently published and available within the major HCV databases (Los Alamos; http://hcv.lanl.gov/components/hcv-db/combined_search/searchi.html and euHCVdb; http://euhcvdb.ibcp.fr/euHCVdb/) (16).To characterize HCV subtype 3a in detail, we performed whole-genome analysis of a cohort of patients with persistent HCV subtype 3a infection. We subsequently focus on the highly variable regions observed in the envelope protein E2 in both acute and chronic infection, since it was apparent that these regions were not restricted to the well-documented hypervariable region 1 (HVR1) that is found at the 5′ end of E2 in all HCV genotypes.Viral genomic variability can be assessed at a number of different levels; first, intergenotypic variability may arise in genomic regions that are conserved within the same subtype but are distinct between subtypes. Second, there is intragenotypic variability, which may be defined as regions of viral variability within the same genotype or subtype. Finally, intrahost variability is where viral genomic variability occurs within the same viral subtype and also the same host when individual clonal sequences are assessed. Although intergenotypic variability may simply be a feature of the existence of geographically distinct HCV subtypes, intragenotypic and intrahost variability may reflect viral regions subject to specific selection pressures, with important functional implications.We observed two distinct regions of intrahost and intragenotypic hypervariability within genotype 3a envelope 2 (E2)—in addition to the previously described HVR1—that we have named HVR495 and HVR575. We show that these regions are subject to positive selection pressure, sometimes very early in acute infection. Although HVR575 has been previously recognized as a site of intergenotypic variation (18), the identification of this region as a hypervariable site within genotype 3a and as a site under early selection pressure leading to variability within the same host has not been previously described.     

Timing and evolution of the most recent common ancestor of the Korean clade HIV subtype B based on <Emphasis Type="Italic">Nef</Emphasis> and <Emphasis Type="Italic">Vif</Emphasis> sequences

Molecular phylogenetic studies of the HIV-1 isolated from Koreans have suggested the presence of the so-called “Korean clade”, which can be defined as a cluster free of foreign isolates. The Korean clade accounts for more than 60% of Korean isolates and exerts characteristic amino acid sequences. Thus, it is merited to estimate when this Korean clade first emerged in order to understand the evolutionary pattern of the Korean clade. We analyzed and reconstructed the most recent common ancestor (MRCA) sequences from nef (n=229) and vif (n=179) Korean clade sequences. Linear regression analyses of sequence divergence estimates were plotted against sampling years to infer the year in which there was zero divergence from the MRCA sequences. MRCA sequences suggested the Korean clade was first emerged around 1984, before the first detection of HIV-1 in Korea in 1985. Further studies on synonymous and nonsynonymous substitution rates suggested positive selection event for the Korean clade, while other subtype B had undergone negative to neutral evolution.     

中国产禾本科植物内生真菌的遗传多样性及rDNA-ITS系统发育分析

采用RAPD技术对分离自我国的13株Epichlo?spp.和9株Neotyphodiumspp.的禾本科植物内生真菌进行了遗传多样性分析,同时对其中的4株进行了rDNA-ITS序列分析及系统发育研究。RAPD分析结果显示:原产自我国的菌株与原产自欧洲的菌株N.uncinatum之间亲缘关系较远;我国的21个菌株之间也存在一定的遗传多样性。rDNA-ITS序列分析表明:原产自我国的菌株聚为一枝,表明我国的Neotyphodium属真菌很有可能直接由我国的Epichlo?属真菌演化而来的新的类群;我国的Epichlo?属真菌有可能和国外推测的一个未确定的Neotyphodium属真菌的杂交进化起源(LAC)有关。     

Evolution of the human immunodeficiency virus type 1 subtype-specific V3 domain is confined to a sequence space with a fixed distance to the subtype consensus.

Human immunodeficiency virus type 1 (HIV-1) strains can be separated into genetic subtypes based on phylogenetic analysis of the envelope gene. Once it had been shown that population-wide intrasubtype genetic variation of HIV-1 strains increases in the course of the AIDS epidemic, it remained uncertain whether HIV-1 subtypes are phenotypic entities spreading as distinct virus populations. To examine this, we applied Eigen's concepts of sequence geometry and fitness topography to the analysis of intrasubtype evolution of the gp120 V3 domain of HIV-1 subtypes A, B, C, and D in the course of the global AIDS epidemic. We observed that despite the high evolution rate of HIV-1, the nonsynonymous distances to the subtype consensus of sequences obtained early in the epidemic are similar to those obtained more than 10 years later, in contrast to the synonymous distances, which increased steadily over time. For HIV-1 subtype B, we observed that the evolution rate of the individual sequences is independent of their distance from the subtype B consensus, but for the individual sequences most distant from the consensus evolution away from the consensus is constrained. As a result, individual HIV-1 genomes fluctuate within a sequence space with fixed distance to the subtype consensus. Our findings suggest that the evolution of the V3 domain of HIV-1 subtypes A, B, C, and D is confined to an area in sequence space within a fixed distance to the consensus of a respective subtype. This in turn indicates that each HIV-1 subtype is a distinct viral quasispecies that is well adapted to the present environment, able to maintain its identity in the V3 region over time, and unlikely to merge during progression of the AIDS epidemic.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Characterization and signature pattern analysis of Korean clade HIV-1 using nef gene sequences

Phylogenetic studies of the HIV-1 gene sequences isolated from Korean patients have suggested that most of Korean isolates belong to the subtype B strain. This study aims to characterize the Korean clade by molecular phylogenetic analysis using all of the Korean nef gene sequences registered in the NCBI GenBank (N=422), in addition to 41 reference strains and 94 foreign isolates. Through phylogenetic analyses, we verified that most of the Korean isolates belonged to the subtype B, where 78.8% are clustered exclusively of foreign isolates. This cluster has been named the Korean clade subtype B (KCB) in order to distinguish it from other subtype B clusters. Genetic distance analysis suggested that the KCB cluster was more homogeneous and clearly distinctive from the non-Korean clade subtype B (NKCB). Comparison of consensus amino acid sequences from KCB and NKCB revealed that characteristic KCB signature amino acid patterns composed of 11 amino acid residues, whose frequencies in the KCB were significantly higher than in the NKCB. The KCB signature amino acid residues were critical in identifying KCB from NKCB, since substitution of the NKCB sequences with KCB signature amino acids relocated them to the Koran clade, and vice versa.     

Full-genome sequences of hepatitis B virus subgenotype D3 isolates from the Brazilian Amazon Region

The Brazilian Amazon Region is a highly endemic area for hepatitis B virus (HBV). However, little is known regarding the genetic variability of the strains circulating in this geographical region. Here, we describe the first full-length genomes of HBV isolated in the Brazilian Amazon Region; these genomes are also the first complete HBV subgenotype D3 genomes reported for Brazil. The genomes of the five Brazilian isolates were all 3,182 base pairs in length and the isolates were classified as belonging to subgenotype D3, subtypes ayw2 (n = 3) and ayw3 (n = 2). Phylogenetic analysis suggested that the Brazilian sequences are not likely to be closely related to European D3 sequences. Such results will contribute to further epidemiological and evolutionary studies of HBV.     

Identification of human immunodeficiency virus type 1 envelope genes recombinant between subtypes B and F in two epidemiologically linked individuals from Brazil.

Sequence analysis of a human immunodeficiency virus type 1 env gene PCR amplified from a Brazilian woman's peripheral blood mononuclear cell DNA (sample RJIO1) showed that it was likely to have been derived from a double recombination event between human immunodeficiency virus type 1 subtypes B and F. The major portion of the gp120 coding sequence belonged to the B lineage, but a segment of the C2 to V3 region (approximately 135 nucleotides) clearly associated with sequences of the F lineage. The subtype F-like segment had 15 noncontiguous signature nucleotides in common with Brazilian subtype F sequences that were not found, or were rare, in subtype B sequences. In contrast, this same segment had only 3 signature nucleotides shared with subtype B sequences and not present in the Brazilian subtype F sequences. Phylogenetic analysis, amino acid signature pattern analysis, and the pattern of synonymous mutations all supported the hypothesis of a recombinational origin of the RJIO1 sequence. Related recombinant genes were also detected in peripheral blood mononuclear cell DNA obtained from the woman's recent sexual partner, indicating that the recombination event probably occurred at some previous time in the chain of virus transmission. Divergent viral sequences in the V3 region were found in the male sexual partner, while a relatively homogeneous viral population was detected in the woman, consistent with her recent infection.