Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland

Small ruminant lentivirus (SRLV) infections are widespread in Poland, but the genetic features of sheep viruses are still lacking and limited to partial gag sequences for goat viruses. In this study, segments from the gag and env genes of Polish SRLV strains screened by heteroduplex mobility assay were subjected to genetic analyses. Subtype A1 was found in both sheep and goats, while subtypes B1 and B2 were found in goats and sheep, respectively. In addition, two novel subtypes (named A12 and A13) were found in sheep. Their close phylogenetic relatedness with SRLV strains previously isolated from Polish goats indicated that these new subtypes are predominant and circulate in both species. The antigenic relationships of subtypes A12 and A13 with other SRLV subtypes were tested in an ELISA assay based on recombinant antigens carrying the immunodominant domains of structural proteins (MA, CA and SU). Antigenic cross-reactivity in the Gag epitopes was evident among genotype A subtypes and, to a lower extent, between genotypes A and B. In contrast, a subtype-specific immunoresponse was detected in the SU epitopes. These results emphasize the broad genetic and antigenic diversity of SRLV strains circulating in Europe and confirmed the need to consider all viral genotypes to choose the antigens in serological tests in order to avoid misdiagnosis in control and eradication programs.     

Detection of intersubtype recombinants with respect to env and nef genes of HIV-1 among female sex workers in Calcutta, India

HIV-1 detected among female sex workers in Calcutta, India was characterized in respect to env and nef genes. A total of 39 HIV-1 seropositive samples were used in the study. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 22 out of 39 samples (56.4%) were infected with subtype C with respect to both env and nef genes; however, 17 samples (43.6%) showed distinct subtype discordance. Simplot analysis of these samples showed the presence of intersubtype recombination between subtypes C and B. Both env C/nef B and env B/nef C recombinants were found to be present; 16 samples were found to be env C/nef B and 1 sample was detected as env B/nef C. This result indicates the emergence of intersubtype recombinants of HIV-1 for the first time in this eastern part of India.     

Phylogenetic analysis of small-ruminant lentivirus subtype B1 in mixed flocks: evidence for natural transmission from goats to sheep

Small-ruminant lentiviruses (SRLV), consisting of the caprine arthritis-encephalitis virus (CAEV) and the maedi-visna virus (MVV), cause chronic multisystemic infections in goats and sheep. The SRLV subtype B1, characterized by the prototypic strain CAEV-CO, has a worldwide distribution and, remarkably, has been isolated exclusively from goats, suggesting potential host specificity. To test this hypothesis, SRLV pol sequences were obtained by PCR amplification from blood samples of seropositive dairy goats and sheep living in mixed flocks. Phylogenetic analysis of these sequences demonstrates that SRLV subtype B1 does cross the species barrier under field conditions through direct contact between adult animals. This implies that SRLV control programs targeting only sheep or goats can no longer be proposed (based on a putative species specificity of the SRLV subtype B1).     

中国HIV-1型B、C亚型主要流行株外膜蛋白基因V3-V4区序列的变异分析

目的　研究我国人类免疫缺陷病毒 1型 (HIV 1)B、C亚型主要流行株在感染过程中基因变异的特点及其与选择压力的关系。方法　应用巢式聚合酶链反应 (nested PCR)对 2 5 8例HIV 1感染者血样中的HIV 1外膜蛋白 (env)基因进行扩增 ,并使用ABI 377型测序仪对扩增产物测序后 ,选择其中 37份B亚型和 35份C亚型HIV 1毒株env基因包括V3～V4区的序列进行比较分析 ,并计算和分析氨基酸同义替换与非同义替换的比值 (Ks Ka)。结果　B亚型毒株V3～V4区的基因离散率高于C亚型毒株。无论B亚型 ,还是C亚型毒株 ,其V4区基因序列较V3区变异更大。在C亚型毒株中 ,V3区基因序列变异甚至比V3上游区和C3区小。B和C亚型毒株整个V3～V4基因区的Ks Ka比值均 <1,差异有非常显著性 (P <0 0 0 1) ,其中B亚型毒株以V3区的Ks Ka比值最小 ,而C亚型毒株则以V4区的Ks Ka比值最小。结论　B和C亚型毒株env基因的变异主要发生在V4区而不是V3区。C亚型毒株V3区较V3上游区和C3区还要保守 ,是本研究的特殊发现。这两种亚型在我国快速流行中发生的变异是在选择压力下发生的 ,而不是随机进化的结果 ,而且选择压力对这两种亚型毒株V3、V4区的作用程度也不一样。这将为我国艾滋病防治策略的制定和疫苗研究提供科学的依据。     

Discrimination of subtype B and non-subtype B strains of human immunodeficiency virus type 1 by serotyping: correlation with genotyping

The ability of a peptide-based serotyping assay to differentiate human immunodeficiency virus (HIV) type 1 (HIV-1) subtype B infections from non-subtype B infections was investigated with 166 anti-HIV-1- and HIV RNA-positive (by PCR) serum or plasma specimens. The specimens were divided genetically into those infected with subtype B and non-subtype B by application of a screening heteroduplex mobility assay (HMA) that used plasmids for subtypes A and B alone. Specimens that were not clearly infected with HIV-1 subtype B by HMA or for which the two methods had discordant results in distinguishing those infected with subtype B from those infected with non-subtype B were then investigated with a full HMA plasmid panel and, for selected specimens, env sequencing. For the 141 genotyped and serotypically reactive specimens, the correlation between genotyping and serotyping (all subtypes) was 69%. Of the 67 specimens that reacted monotypically as serotype B, 64 were shown to be infected with genotype B (positive predictive value, 96%). Of the 82 specimens that contained genotype B nucleic acid, 64 reacted monotypically as serotype B (sensitivity, 78%), and 4 specimens reacted with a single non-subtype B peptide; the viruses in 14 specimens could not be assigned a serotype. Initial screening results had indicated that 12 samples had results discordant between restricted HMA and serotyping. The V3 loop amino acids of the infecting HIV strains from the seven specimens with discordant serology results were analyzed. For five specimens discordance occurred when the amino acid sequence of the infecting virus closely resembled those of more than one consensus peptide antigen or when the observed V3 crown motif of the strain was atypical for the genetic subtype present. For the other two specimens no explanation for the discordance was identified. Five specimens gave unclear or discordant results in the initial HMA screen, but the results were resolved when the full plasmid panel was used. Serotyping, although of limited sensitivity, distinguishes between subtype B and non-subtype B infections with a high degree of specificity. However, it poorly differentiates the major non-subtype B subtypes, particularly subtypes A and C. When HIV-1 subtype B predominates, serological typing and/or subtype-restricted HMA screening usefully distinguishes between subtype B and non-subtype B infections.     

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.     

Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations

Serological surveys for small ruminant lentivirus (SRLV) infections have revealed seropositive sheep in several mixed herds, where sheep are kept together with seropositive goats. Here we have examined the genetic relationships in LTR, pol and env surface unit (SU) and the growth patterns in goat (GSM) and sheep (FOS) synovial membrane cell cultures of SRLV isolates obtained from both mixed and single species herds. Phylogenetic analyses of pol and env SU revealed that Norwegian SRLVs derived from both goat and sheep in mixed herds are distributed into group C, while isolates obtained from unmixed sheep flocks cluster in group A, together with maedi-visna-like representatives of the A1 subtype. In this study, the direction of group C virus transmission is proposed to be from goat to sheep. The replication efficiency in GSM and FOS cultures and the cytopathic phenotype induced by the SRLV isolates gave no indication of any species-specific characteristics. No particular nucleotide sequences of the LTR-U3 region or env SU were identified that could be related to cytopathic phenotype. This study shows that sheep in Norway harbour SRLVs belonging to phylogenetic groups A and C, and this provides further evidence for cross-species infection being a regular characteristic of SRLVs, which may represent an important source for viral persistence.     

Screening of HIV-1 isolates by reverse heteroduplex mobility assay and identification of non-B subtypes in Italy

OBJECTIVE: The increasing prevalence of HIV-1 transmission through heterosexual contacts and the growing number of immigrants from non-Western countries, where non-B subtypes and recombinant forms are prevalent, suggest the possible emergence in Italy of a new epidemic wave of HIV-1 non-B subtypes as well as recombinant forms. METHODS: The distribution of HIV-1 subtypes has been evaluated in 63 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the last 5 years. A modified heteroduplex mobility assay (HMA) strategy, reverse HMA (rHMA), has been developed in our laboratory, allowing rapid identification of divergent-from-B-subtype isolates, which have been subsequently characterized by detailed molecular and phylogenetic analyses. RESULTS: Five samples show, on rHMA, an electrophoretic pattern compatible with a non-B subtype classification. Their phylogenetic analysis, performed on both env and gag regions, confirms the rHMA subtyping prediction, given that 3 samples fall into the "A-family" subtype and 2 into the G subtype. The 5 non-B-subtype HIV-1 isolates have been identified among 23 variants (prevalence, 21.74%) isolated during the 2000 to 2001 period in heterosexuals. In parallel, B-subtype isolates show high levels of intrasubtype nucleotide divergence, compatible with a constant HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes, and the data indicate that rHMA represents a powerful tool for HIV-1 biomolecular screening in epidemics characterized by a mono-/dual-subtype predominance.     

Biological phenotypes of HIV-1 subtypes A and B strains of diverse origins

The identification of specific biologic phenotypic traits that can be correlated with different HIV-1 genetic subtypes was sought. The genetic subtypes were determined by either sequencing (Cameroonian strains, n = 18) or by the heteroduplex mobility assay (HMA) (Belgian strains, n = 21 and lvorian strains, n = 25). Seventeen (81%) of the 21 Belgian isolates belonged to subtype B and 4 (17%) were subtype A strains. Subtype A variants were predominant in the two African countries studied; 11 (61%) of 18 strains from Cameroon and 23 (92%) of 25 strains from the Ivory Coast. Of the 64 isolates, 38 (58%) and 19 (29%) belonged to subtypes A and B, respectively. No significant difference was observed for biological phenotypes (slow/low and rapid/high) of both genetic subtypes. In symptomatic individuals, however, a significantly higher number of subtype B isolates were of rapid/high phenotype, compared with subtype A (5 of 10; 50%) vs. 2 of 22; 9%), respectively; X² = 6.7, P = 0.02). The findings suggest that overall HIV-1 isolates belonging to genetic subtype B are not distinguishable from subtype A variants on the basis of their biological phenotypes. Syncytium-inducing variants were less prevalent regardless of the geographic origin of the isolates. © Wiley-Liss, Inc.     

A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle

A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5′-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104 bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID₅₀ of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep.     

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.     

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.     

Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade

We performed a phylogenetic analysis of caprine and ovine lentiviruses using long sequences in gag and pol of 104 new Swiss isolates and six available corresponding database sequences. Forty-five isolates, forming five sequence clusters, were unclassifiable by the present classification. Pairwise DNA distance analysis indicated different categories of relatedness, requiring a new classification system. We propose four principal sequence groups, A-D, which differ by 25-37%. Groups A and B are further divided into subtypes which differ by 15-27%. Group D and four of the seven group A subtypes, A3, A4, A5 and A7, are formed by new Swiss isolates. Molecular epidemiology revealed that Swiss B1 strains differed no more from French, Brazilian or US strains than from each other, suggesting virus propagation through international livestock trade. Furthermore, infection of goats by subtypes A3 or A4 was significantly associated with documented contact with sheep, which also harbor these subtypes, thus indicating regularly occurring sheep-to-goat transmission.     

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.     

In-utero transmission of quasispecies among human immunodeficiency virus type 1 genotypes

Samples from infants infected in-utero by the human immunodeficiency virus type 1 (HIV-1) subtypes A, C, D, and recombinants from Dar es Salaam, Tanzania, were examined for the presence of viral genetic quasispecies. HIV-1 envelope diversity was measured on peripheral blood mononuclear cells collected within the first 48 h of life from 53 infants. Phylogenetic analysis of C2-C5 envelope nucleotide sequences was used for HIV-1 subtype classification. Forty-two of 53 samples (79%) showed a heteroduplex mobility assay (HMA) suggestive of transmission of a single quasispecies, while 21% showed infection with multiple quasispecies. No differences among HIV-1 subtypes were found in the proportion of single to multiple quasispecies transmitted in-utero (Likelihood ratio test, P = 0.83), nor were differences found among single to multiple quasispecies transmitted and maternal viral load (Mann-Whitney test, P = 0.44). This suggests that differences in perinatal transmission between subtypes we previously observed in this cohort could not be associated with the likelihood for multiple independent infections during in-utero infections.     

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.     

Molecular subtyping of the HIV-1 V3 loop sequences detected in HIV-1-positive patients in southern Taiwan

Polymerase chain reaction and nucleotide sequence analysis were performed to amplify and determine the V3 loop sequences of human immunodeficiency virus type 1 (HIV-1) from ten seropositive patients at National Cheng Kung University Hospital, Tainan. The nucleotide sequences and the deduced amino acid (a. a.) sequences of these V3 regions were compared with those of known HIV-1 prototypes. The V3 loop a. a. sequences detected in eight individuals belong to subtype B which predominates in North America and Europe, whereas two individuals were infected with HIV-1 subtype E which is mainly found in the heterosexual populations of Thailand. Sequence analysis of these variant HIV-1 strains revealed a number of interesting features and a phylogenetic tree was also constructed according to the V3 loop nucleotide sequences of these variant strains and HIV-1 isolates from other parts of the world. Furthermore, our results suggest that the north vs south geographical separation in terms of HIV-1 epidemiology in Taiwan is insignificant.     

Phylogenetic analysis of small ruminant lentivirus (SRLV) in Italian flocks reveals the existence of novel genetic subtypes

In order to investigate the genetic heterogeneity of small ruminant lentivirus (SRLV) isolates in Italy, 55 clinical samples collected between 1998 and 2010 were analysed. The phylogenetic study was based on analysis of gag–pol sequences. Our findings revealed that the SRLVs belonged to the subtype A9 (n = 3, sheep), B1 (n = 5, goat), B2 (n = 3, sheep) and E2 (n = 5, goat). Interestingly, 39 isolates from both sheep and goat, significantly differed from all the other SRLVs previously described and formed two separate clusters within genotypes A and B tentatively named A11 (n = 27, goat and sheep) and B3 (n = 12, goat and sheep), which have never been shown before. These results revealed a marked diversity among Italian field SRLV strains which might reflect the absence of any systematic control measures.     

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.