Genomic characterization of hepatitis C virus isolates from Argentina

Thirty-three Argentinian patients infected with hepatitis C virus (HCV) were studied for viral genotyping. The patients included 10 hemophiliac and 4 polytransfused children and 19 adults: 3 polytransfused, 7 dialyzed and 9 sporadic cases. Core-based genotyping permitted the classification of 31 samples. Genotypes II, I and V were the most frequent: 21 (63.6%), 16 (48.4%) and 10 (30.3%) of the 33 patients, respectively. Only one polytransfused patient carried genotype IV. Genotype II was detected in 7 out of 9 sporadic cases. Thirteen patients (39.3%) were coinfected with two genotypes, and 2 others were coinfected with three genotypes. The remaining 2 samples which could not be typed were characterized following the restriction fragment length polymorphism (RFLP) method, and were classified as type 1. One of these had two consecutive transitional mutations in the 5′ untranslated region (5′ UTR). © 1995 Wiley-Liss, Inc.     

Molecular characterization of hepatitis A virus isolates from Argentina

Hepatitis A, a vaccine preventable disease, is now of transitional or intermediate endemicity in Argentina, as the epidemiologic pattern of the disease has shifted with improvements in living conditions in some parts of the country. Increase in the susceptibility of older children and adults has led to increasing disease incidence. Molecular epidemiology has played an important role in the understanding of HAV infection by identifying modes of spreading and by permitting the monitoring of changes in circulating virus brought about by prevention programs. South American isolates characterized are limited. Eighty-two sporadic and outbreak isolates from Argentina were sequenced in the VP1/2A region of HAV genome over a 9-year period. All the isolates belonged to subgenotype IA. All our sequences grouped into two big clusters. Apparently, at least two lineages have been co-circulating in the same place at the same time. Despite great genetic variability, few point amino acid changes could be deduced. Four sequences showed an Arg --> Lys substitution at 1-297 which characterized the genotype IB at the amino acid level. Many isolates carried a conservative amino acid substitution Leu --> Ile at position 42 of the 2A domain, previously described as a possible fingerprint of HAV sequences in Brazil. The other rare changes have been found before, except for a 1-277 Asn --> Ser substitution displayed in two isolates that has not been previously reported. Argentina recently implemented universal vaccination in 1-year-old children. Molecular tools would be useful in an active surveillance program.     

Increased prevalence of genotype F hepatitis B virus isolates in Buenos Aires, Argentina.

The genomic coding region of the hepatitis B surface antigen (HBsAg) was partially sequenced from 12 HBsAg-positive sera of carriers residing in Buenos Aires, Argentina. A phylogenetic analysis groups the 12 isolates into genotypes A (n = 4), B (n = 1), D (n = 2), and F (n = 5). The occurrence of genotypes A and D is not unexpected, considering the mainly European origin of the studied population. The high prevalence of genotype F and its genetic composition support the suggestion that F strains originated in native populations of the New World.     

Genetic analysis of dengue virus type 3 isolated in Buenos Aires, Argentina

Dengue virus as a member of mosquito-borne flaviviruses is responsible for an increasing number of human infections worldwide, mainly in tropical and subtropical urban areas. The agent, a single-stranded positive sense RNA virus, is comprised of four serotypes and genetic variation within each serotype can be further divided into different genotypes. Dengue outbreaks were reported in bordering countries during the last years; the latest reported in Paraguay in 2006-2007. In Buenos Aires, 32 dengue cases were confirmed in travelers coming from this country by anti-dengue IgM antibodies detection, RT-PCR and/or isolation in C6/36 cell line. Structural proteins C, prM/M, E and non-structural proteins 1 and 2 from eight viruses were genetically characterized. Phylogenetic inference was performed for the E-protein and all viruses clustered with dengue virus 3 Genotype III. This is the first report of genetic characterization of dengue virus 3 in Argentina.     

Diversity of hepatitis C virus genotype 1b in Buenos Aires, Argentina: description of a new cluster associated with response to treatment

Hepatitis C virus (HCV) genotype 1b is the most prevalent in Argentina. As observed in most HCV-genotype 1b described previously worldwide, the population analyzed in this work is growing at exponential rate. Ten out of 22 samples tested comprise a well-defined cluster. This new cluster appears to be highly susceptible to therapy with non-pegylated interferon plus ribavirin (80% rate of sustained response). The comparative analysis of amino acid sequences yielded a characteristic pattern for responder samples defined by amino acids S75, V147, V158 (Core region) and AC2217-8 (NS5A region). In conclusion, this study describes the epidemic spread of genotype 1b in Buenos Aires, Argentina, and shows the emergence of a new cluster that would result from a founder effect of an unusual sequence or a quasispecies variant that evolved through the time. This cluster, which appears to be highly susceptible to therapy, suggests that the virus itself might be more important than individual patient characteristics when responding to treatment.     

Hepatitis B virus in Mar del Plata,Argentina: Genomic characterization and evolutionary analysis of subgenotype F1b

The aim is to describe the molecular epidemiology and perform a genomic characterization of hepatitis B virus (HBV) circulating in Mar del Plata and to identify the origin and diversification patterns of the most prevalent genotype. The S gene and the region encompassing the X gene, basal core promoter (BCP), and precore (preC) was analyzed in 56 samples. They were genotyped as: 80% F1b, 9% A2, 7% D3, and 2% D1. A recombinant F4/D2 genome was detected. The double substitution G1764A/A1762T at the BCP (reduced HBeAg expression) was found in 20% F1b, 2% A2, 2% D1, and 2% D3 samples. A unique D3 presented the G1896A substitution at the preC (HBeAg negative phenotype). A 13% of the samples showed mutations at the HBsAg “a” immunodeterminant (escape from neutralizing antibodies). Mutations at the polymerase (antiviral resistance) were found in 52% of the samples. Coalescent analysis of subgenotype F1b, the most prevalent in the city, showed that viral diversification in Mar del Plata started by year 2000. F1b was the most prevalent genotype detected, being a characteristic of actual HBV infections in Mar del Plata. Local HBV exhibit clinically relevant mutations, but a minority of them was shown to be associated to potential vaccination escape or antiviral resistance. Nevertheless, further studies are needed to determine whether any of these mutants could pose a threat to prevention, diagnosis, or treatment.     

Phylogenetic characterization of genotype 4 hepatitis C virus isolates from Argentina

Among 114 patients infected with hepatitis C virus, three genotype 4 isolates, unusual in Argentina, were detected by phylogenetic analysis over different genomic regions. The patients were not related. One sample was associated with Egyptian sequences, and the others were associated with a Zairean isolate, a fact which reinforces the idea that they are from independent sources.     

Genotyping of acute hepatitis a virus isolates from China, 2003-2008

Hepatitis A virus (HAV) is usually transmitted by an oral–fecal route and is prevalent not only in developing countries but also in developed countries. In the present study, the phylogenetic characterization of the VP1/2A junction region (321 nucleotides) of China HAV isolates was examined. Anti‐HAV IgM‐positive serum samples were collected from 8 provinces, including 20 cities or counties in China from 2003 to 2008; 337 isolates from 406 HAV patients' serum samples were amplified by RT‐PCR, sequenced at the VP1/2A junction region and aligned with the published sequences from GenBank to establish phylogenetic analysis. All China HAV isolates in this study belonged to genotype I, with 98.8% (333/337) of samples clustering in sub‐genotype IA and 1.2% (4/337) in sub‐genotype IB. In addition, sub‐genotype IA isolates clustered into four groups (92.7–100% nucleotide identity), and the samples collected from all China HAV isolates in this investigation showed 87.5–100% nucleotide identity, but the amino acids in this region were more conserved (95.2–100% identity). Few unique amino acid changes could be deduced (VP1‐253: Glu → Gly; 2A‐34: Pro → Ala; 2A‐33: Leu → Phe). Genetically identical or similar HAV strains existed in some investigated areas in China during different years, suggesting that an indigenous strain has been circulating in those regions. This report provides new data on the genetic relatedness and molecular epidemiology of HAV isolates from China as well as the distribution of sub‐genotype IA and IB in this part of the world. J. Med. Virol. 83:1134–1141, 2011. © 2011 Wiley‐Liss, Inc.     

Changes in risk behavior and dynamics of hepatitis C virus infections among young drug users in Amsterdam, the Netherlands

To elucidate the character and magnitude of the hepatitis C virus (HCV) epidemic among drug users in Amsterdam, 197 young drug users from the period 2000 to 2004 were compared with 215 counterparts from 1985 to 1989. Although injection risk behavior and HCV seroprevalence decreased sharply over time, HCV seroprevalence remains high (44%) among young drug users who have ever injected. Phylogenetic analysis shows that current HCV infections originate from diversification of strains already circulating in the past, but also from the recent introduction of new subtypes. HCV subtypes 1a and 3a remain the most prevalent among drug users in Amsterdam, but other subtypes such as 4d and 2b have entered the population. In conclusion, both the unpopularity of injecting drug use and the success of prevention campaigns are likely to be responsible for the decline in the seroprevalence of HCV and increased median time to seroconversion. Treatment of those infected chronically, in combination with the continuation of prevention programs, might decrease future HCV transmission.     

Full genome characterization of hepatitis B virus strains from blood donors in Iran

Iran is a low to medium endemic country for hepatitis B virus (HBV), depending on the region, where genotype D is dominant. Samples from 170 asymptomatic HBsAg‐positive blood donors were quantified and the median viral load was 6.7 × 10² IU/ml with 10.6% samples unquantifiable. Fifty complete genome sequences of these strains were characterized. Phylogenetic analysis identified 98% strains as subgenotype D1 and 2% as D2. Deduced serotypes were ayw2 (94%), ayw1 (4%), and adw (2%). The nucleotide diversity of the complete genome subgenotype D1 Iranian strains was limited (2.8%) and comparison with D1 strains from Egypt and Tunisia revealed little variation between strains from these three countries (range 1.9–2.8%). The molecular analysis of the individual genes revealed that the G1896A mutation was present in 86.2% of the strains and in 26 strains (29.9%) this mutation was accompanied by the G1899A mutation. The double mutations A1762T/G1764A and G1764T/C1766G were found in 20.7% and 24.1% of the strains, respectively. The pre‐C initiation codon was mutated in five strains (5.8%). One strain had a 2‐amino acid (aa) insertion at position s111 and another sP120Q substitution suggesting a vaccine escape mutant. J. Med. Virol. 83:948–952, 2011. © 2011 Wiley‐Liss, Inc.     

Complete genome sequencing of dengue virus type 1 isolated in Buenos Aires, Argentina

Dengue (DEN) constitutes a major viral arthropod-borne human illness. South America was last considered free of dengue two decades ago when a dramatic increase in the number of dengue fever and hemorrhagic dengue cases had been reported. Five viruses were isolated in Buenos Aires City from the 1999-2000 Paraguay outbreak. RT-PCRs obtained directly from plasma were cloned into pGemT vectors and sequences of the structural genes and NS1 were analyzed. Three viruses were full-length sequenced from RT-PCR obtained from cell-culture isolates. Excess of synonymous over non-synonymous mutations suggested that the structural proteins were under strong functional constraints while a weak purifying selection was operating in the whole polyprotein. Sequence diversity and selective pressures varied among patients but results were significantly above the procedure threshold. One sample showed small-plaque phenotype and impaired growth coupled to 3'untranslated region mutations. Phylogenetic analysis of full-length sequences split Buenos Aires isolates into two clusters within American DEN-1 genotype V: Clade I was phylogenetically linked to Brazilian samples and Clade II with samples from Paraguay and Northeastern Argentina. In Buenos Aires City, only dengue virus serotype 1 imported from Paraguay has been detected, though without evidence of local transmission.     

Molecular epidemiology of human respiratory syncytial virus subgroup A over a six-year period (1999-2004) in Argentina

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections in children. Little information about the molecular epidemiology of HRSV in developing countries, such as Argentina, is available. By RT-PCR, we subgrouped 353 HRSV isolates over six consecutive epidemic seasons (1999-2004) and few isolates from 1997. Between them, 232 (65.7%) belonged to subgroup A and 121 (34.3%) to subgroup B. Therefore, the nucleotide, amino-acid variability and phylogenetic relations of 78 HRSV subgroup A isolates, were analyzed using RFLP and sequence analysis of the G-protein gene. The results showed that there were two main restriction patterns (PA1 and PA2) and two previously described genotypes (GA2 and GA5) cocirculating in Buenos Aires, without evidence of alternation between them during the studied period. The Argentine sequences were compared with previously reported molecular data from other countries. It showed that viruses genetically related circulated the same years within neighboring countries and the sequences from long-distant places were closely related to Argentine sequences, but they belonged to different sampling years. The data reported here support the growing database on the molecular diversity of HRSVA circulating in Latin America in children under 2 years of age and contributes to describe the pattern of global spread of this virus.     

Exceptional genetic variability of hepatitis B virus indicates that Rwanda is east of an emerging African genotype E/A1 divide

In Western Africa, hepatitis B virus (HBV) genotype E predominates throughout a vast crescent spanning from Senegal to Namibia and at least to the Central African Republic to the East. Although from most of the eastern parts of sub‐Saharan Africa only limited sets of strains have been characterized, these belong predominantly to genotype A. To study how far the genotype E crescent extends to the East, a larger number of HBV strains from Rwanda were analyzed. Phylogenetic analysis of 45 S fragment sequences revealed strains of genotypes A (n = 30), D (n = 10), C (n = 4), and B (n = 1). Twelve genotype A sequences formed a new cluster clearly separated from the reference strains of the known sub‐genotypes. Thus, with four genotypes and at least six sub‐genotypes and a new cluster of genotype A strains, HBV shows an exceptional genetic variability in this small country, unprecedented in sub‐Saharan Africa. Despite this exceptional genetic variability, not a single genotype E virus was found indicating that this country does not belong to the genotype E crescent, but is east of an emerging African genotype E/A1 divide. J. Med. Virol. 81:435–440, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular characterization of hepatitis B virus isolates from Zimbabwean blood donors

Hepatitis B virus (HBV) is endemic in Africa, being hyperendemic in sub-Saharan Africa. Genotypes A, D, and E circulate in Africa, showing a distinct geographical distribution. The aim of the present study was to determine the HBV genotype distribution in blood donors from different geographical locations in Zimbabwe. Using a restriction fragment polymorphism assay, sequencing of the basic core promoter/precore region and of the complete S open reading frame showed that 29 HBV isolates from geographically distinct regions belong to subgenotype A1. The complete genome of two of these Zimbabwean HBV isolates was sequenced. Forty-four percent of the Zimbabwean HBV isolates (11/23) were characterized by a G1862C missense mutation, which causes a Val to Leu amino acid substitution at position 17 of the precore region. The majority of Zimbabwean HBV isolates clustered with a number of South African HBV isolates, with which they shared characteristic amino acids in the preS1, preS2, and polymerase spacer regions. The wide distribution of subgenotype in Africa, as well as the high intragroup divergence and the geographical clustering of the African and Asian subgenotype A1 HBV isolates indicate that this subgenotype has a long period of endemicity in these regions.     

Hepatitis in Albanian children: molecular analysis of hepatitis A virus isolates

Hepatitis A is a common disease in developing countries and Albania has a high prevalence of this disease associated to young age. In spite of the occurrence of a unique serotype there are different genotypes classified from I to VII. Genotype characterisation of HAV isolates circulating in Albania has been undertaken, as well as the study of the occurrence of antigenic variants in the proteins VP3 and VP1. To evaluate the genetic variability of the Albanian hepatitis A virus (HAV) isolates, samples were collected from 12 different cities, and the VP1/2A junction amplified and sequenced. These sequences were aligned and a phylogenetic analysis performed. Additionally, the amino half sequence of the protein VP3 and the complete sequence of the VP1 was determined. Anti-HAV IgM were present in 66.2% of all the sera. Fifty HAV isolates were amplified and the analysis revealed that all the isolates were sub-genotype IA with only limited mutations. When the deduced amino acid sequences were obtained, the alignment showed only two amino acids substitutions at positions 22 and 34 of the 2A protein. A higher genomic stability of the VP1/2A region, in contrast with what occurs in other parts of the world could be observed, indicating high endemicity of HAV in Albania. In addition, two potential antigenic variants were detected. The first at position 46 of VP3 in seven isolates and the second at position 23 of VP1 in six isolates.     

Genetic analysis of hepatitis A virus isolates from Brazil

A limited number of hepatitis A virus (HAV) isolates from South America have been characterised at the genomic level. IgM anti-HAV positive serum samples collected from patients with hepatitis A living in the five geographical regions of Brazil (North, Northeast, Central, South, and Southeast) were used to obtain HAV isolates and determine their genetic relatedness. Of the 232 case isolates, sequence data were obtained from the VP1/2A junction region of the HAV genome. All isolates were classified in genotype I; 231 belonged to subgenotype IA, and one to subgenotype IB. HAV isolates from four States formed distinct clusters of highly related sequences. However, isolates from other states did not cluster and the sequences from those states were intermingled with sequences found in the other states. The amino acid sequences of all but two isolates showed a Leu --> Ile substitution at position 42 in the 2A protein. This substitution appeared to be a characteristic geographic fingerprint of HAV sequences within Brazil.     

Genetic typing of equine arteritis virus isolates from Argentina

We report the nucleotide sequence and genetic diversity of four Equine Arteritis Virus (EAV) ORF 5 and 6 from Argentina isolates, obtained from asymptomatic virus-shedding stallions. Nucleic acid recovered from the isolates were amplified by RT-PCR and sequenced. Nucleotide and deduced amino acid sequences from the Argentine isolates were compared with 17 sequences available from the GenBank. Phylogenetic analysis revealed that the Argentine isolates grouped together in a definite cluster near European strains. Despite the greater genetic variability among ORF 5 from different isolates and strains of EAV, phylogenetic trees based on ORF 5 and 6 are similar. Both trees showed that virus sequences from America and Europe segregate into distinct clades based on sequence analysis of either ORF 5 or 6. This study constitutes the first characterization of Argentine EAV isolates. M. G. Echeverría, S. Díaz, E. Nosetto Members of CONICET (Scientific Research Council)