Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein,and second matrix and small hydrophobic proteins

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys(3)-His(1) motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but < or = 25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.     

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.     

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.     

HIV-1 diversity in Brazil: genetic, biologic, and immunologic characterization of HIV-1 strains in three potential HIV vaccine evaluation sites. Brazilian Network for HIV Isolation and Characterization

The Brazilian Network for HIV Isolation and Characterization was established for the surveillance of HIV variability in Brazil. Here, we report characterization of HIV strains and virus-specific immune responses from 35 clinical samples collected from three potential HIV vaccine sites. Three genetic subtypes of HIV-1 were identified by heteroduplex mobility assay (HMA) B (in 82.9% of the samples), F (14.3%), and C (2.9%). Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with HMA results. Four variants of subtype B with different tetrapeptides at the tip of the V3 loop were found: the GPGR motif (North American), GWGR motif (Brazilian B"), and two minor variants, GFGR and GPGS, as previously detected. No significant association was found between HIV-1 subtypes and the mode of transmission or biologic properties of HIV-1 isolates (derived from 88.6% of the specimens). Only 5 of 16 isolates studied were neutralized by the autologous sera. Consistent with previous results, no relation between viral subtype and peptide enzyme-linked immunosorbent assay (ELISA) seroreactivity or neutralization was evident. This study also demonstrated the effectiveness of the collaborative approach followed by Brazilian scientists when addressing a complex subject such as HIV variability.     

Isolation, cloning, and complete nucleotide sequence of a phenotypically distinct Brazilian isolate of human T-lymphotropic virus type II (HTLV-II)

Analysis of human T-lymphotropic virus type II (HTLV-II) isolates from North America and Europe have demonstrated the existence of two molecular subtypes of the virus, HTLV-IIa and HTLV-IIb. Recently, studies on HTLV-II infections in Brazil have revealed isolates that are related phylogenetically to the HTLV-IIa subtype but have a HTLV-IIb phenotype with respect to the transactivating protein, tax. To more clearly define this relationship, HTLV-II was isolated from peripheral blood of an IVDA from Sao Paulo, Brazil (SP-WV), and the complete provirus was cloned and sequenced. Comparison of HTLV-II(SP-WV) nucleotide sequences to other available complete HTLV-II proviral sequences revealed that HTLV-II(SP-WV) is most closely related to HTLV-II(Mo), the prototypic HTLV-IIa subtype sequence. Phylogenetic analysis of LTR, env, and tax regions unequivocally demonstrated that HTLV-II(SP-WV) and all other Brazilian sequences examined are members of the IIa subtype. The predicted amino acid sequences of the major coding regions of HTLV-II(SP-WV) are also most closely related to HTLV-II(Mo), with the important exception of tax. The tax protein encoded by HTLV-II(SP-WV) is 96-99% identical to the tax of IIb isolates and is similar in that it has an additional 25 amino acids at the carboxy-terminus compared to the HTLV-II(Mo) tax with which it shares 91% identity. Analysis of tax stop codon usage of a number of HTLV-IIa isolates from North American, Europe, and Brazil demonstrated that isolates from the last region appear to be unique in their extended tax phenotype. It could be demonstrated that the extended tax proteins in the HTLV-IIb and Brazilian isolates had equivalent ability to transactivate the viral LTR, and studies with deletion mutants indicated that the extended C-terminus is not essential for transactivation. In contrast, the HTLV-IIa tax was found to have a greatly diminished ability to transactivate the viral LTR, which appeared to be a consequence of reduced expression of the protein. The studies show that although the Brazilian strains do not represent an entirely new subtype based on nucleotide sequence analysis they are a phenotypically unique molecular variant within the HTLV-IIa subtype.     

Molecular characterization of the <Emphasis Type="Italic">env</Emphasis> gene from Brazilian field isolates of Bovine Leukemia Virus

Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions. In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found in the aminoacid sequences of the gp51 protein were localized in the G and H epitopes. Using the SIFT software, it was predicted that they should not alter the protein functions. Phylogenetic analyses showed that partial or complete env gene sequences grouped in three or four phylogenetic clusters, respectively. The sequences from the Brazilian isolates had similar mutation rates as compared to samples from other countries, and belonged to at least two phylogenetic clusters.     

HIV-1 subtyping in Salvador, Bahia, Brazil: a city with African sociodemographic characteristics

To investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDU's sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions.     

Molecular characterization of human astroviruses isolated in Brazil, including the complete sequences of astrovirus genotypes 4 and 5

Summary. Human astroviruses (HAstV) are recognized as an important cause of gastroenteritis in young children worldwide. This study describes the molecular characteristics of astroviruses isolated in Brazil, using RT-PCR and molecular sequencing of segments of all three viral ORFs. Genetic analysis of a 348-nucleotide segment from ORF 2 demonstrated that the Brazilian isolates belong to HAstV genotypes 1 to 5 and 8. ORF 1b sequences displayed a high degree of nucleotide identity even between different genotypes, which disfavours HAstV genotyping in this region. ORF 1a sequence analysis classified all Brazilian samples as genogroup A. The complete sequences of HAstV genotype 4 (putative serotype 4) and genotype 5 (putative serotype 5) were determined for the first time.     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

Sequence and phylogenetic analysis of the sigmaA-encoding gene of avian reovirus

The full-length sigmaA-encoding gene nucleotide sequences (1251 bp) of ten avian reovirus (ARV) field-isolates and three vaccine strains were determined and analyzed to study the degree of genetic divergence and evolution. Strains were isolated over a 23-year period from different hosts, pathotypes, and geographic locations. A phylogenetic tree constructed from variation in the sigmaA nucleotide sequences among ARV isolates showed that Taiwanese isolates from different dates of isolation were grouped into two distant groups, indicating that they have evolved in nature. In paired identity analysis, there was over 97.3% nucleotide sequence identity in the sigmaA-encoding genes between group I Taiwanese isolates (T6, 750505, 919, and 918) and Japanese isolate OS161 as well as three US vaccine strains, suggesting that they might have descended from a common ancestor. However, the nucleotide sequences of these sigmaA-encoding genes varied extensively from those of group II Taiwanese isolates (601SI, R2/TW, 1017-1, 916, and 601G), displaying only 86% identity. These results revealed that the genetic diversity in the sigmaA-encoding gene of ARV correlated with the date of isolation and geographic locations.     

Phylogenetic analysis of partial S1 and N gene sequences of infectious bronchitis virus isolates from Italy revealed genetic diversity and recombination

A total of ten infectious bronchitis virus (IBV) isolates collected from commercial chickens in Italy in 1999 were characterized by RT-PCR and sequencing of the S1 and N genes. Phylogenetic analysis based on partial S1 gene sequences showed that five field viruses clustered together with 793/B-type strains, having 91.3–98.5% nucleotide identity within the group, and one isolate had very close sequence relationship (94.6% identity) with 624/I strain. These two IBV types have been identified in Italy previously. The other three variant isolates formed novel genotype detected recently in many countries of Western Europe. For one of these variant viruses, Italy-02, which afterwards became the prototype strain, the entire S1 gene was sequenced to confirm its originality. In contrast, phylogenetic analysis of more conserved partial N gene sequences, comprising 1–300 nucleotides, revealed different clustering. Thus, three variant IBVs of novel Italy-02 genotype, which had 96.7–99.2% S1 gene nucleotide identity with each other, belonged to three separate subgroups based on N gene sequences. 624/I-type isolate Italy-06 together with Italy-03, which was undetectable using S1 gene primers, shared 97.7% and 99.3% identity, respectively, in N gene region with vaccine strain H120. Only one of the 793/B-type isolates, Italy-10, clustered with the 793/B strain sharing 99.3% partial N gene identity, whereas the other four isolates were genetically distant from them (only 87.7–89.7% identity) and formed separate homogenous subgroup. The results demonstrated that both mutations and recombination events could contribute to the genetic diversity of the Italian isolates. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database and have been assigned the accession numbers AJ458958, AJ457137, AJ458959, AJ491327, AJ458960–AJ458964 for the S1 gene and AM260961–AM260970 for the N gene.