Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep

Small ruminants lentiviruses (SRLV) nucleotide sequences spanning the V1V2 variable regions of the env gene were amplified by nested-PCR from 38 blood samples collected from 16 naturally infected sheep flocks in France. For the rapid SRLV group determination of field isolates, the PCR-amplified fragments were subjected to a SRLV-adapted heteroduplex mobility assay (HMA). All viral sequences were clearly assignable to the SRLV group B by HMA analysis. Twenty-seven SRLV isolates were selected for DNA sequence analysis. In each case, nucleotide comparison and phylogenetic analyses confirmed the genetic relationships inferred by HMA. Six SRLV isolates belonged to subtype B1, and 21 pertained to subtype B2, one flock being infected with both subtypes. Subtypes B1 and B2 were found with different frequencies and geographic spread, but exhibited similar genetic diversities. These results give a more complete picture of the distribution and heterogeneity of SRLV env subtypes in sheep and confirmed that multiple interspecies transmission occurred in the past. Furthermore, HMA appeared to be a rapid and reliable method to differentiate caprine arthritis encephalitis virus from maedi-visna virus.     

Genetic analysis of small ruminant lentiviruses following lactogenic transmission

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.     

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.     

Seroprevalence,clinical incidence,and molecular and epidemiological characterisation of small ruminant lentivirus in the indigenous Passirian goat in northern Italy

Eight dairy flocks, comprising a total of 323 indigenous Passirian goats from northern Italy, were examined to determine the seroprevalence and clinical incidence of small ruminant lentivirus (SRLV) infections and to identify the SRLV subtypes. The seroprevalence was 81.5% (55-95%). The clinical incidence was 2.5% (0-8.3%) and was apparently low due to the practice of culling clinically affected animals. Phylogenetic analysis of eight PCR fragments (one sample from each flock) revealed that all proviruses belonged to the SRLV subtype B1, which suggests a common source of infection. Subtype B1 being the only circulating SRLV, coupled with the fact that mixed herd systems are very rare in South Tyrol, gives hope that an eradication programme in goats can be successful even without including sheep as long as sheep are kept strictly and permanently isolated.     

Genomic heterogeneity of small ruminant lentiviruses: existenceof heterogeneous populations in sheep and of the samelentiviral genotypes in sheep and goats

Summary.  We have recently shown that French small ruminant lentiviruses (SRLV) from sheep are more similar to Caprine Arthritis Encephalitis Virus (CAEV) than to visna maedi virus (VMV) in a conserved region of the pol gene. To extend these results, we have examined sequences from a variable region of the env gene in French SRLV. We found that they were nearly equally distant from both CAEV and VMV strains, suggesting a considerable divergence since the initial introduction of the virus. Analysis of separate clones from individual animals showed that some carry a population of variant viruses. The study of further pol gene sequences from both goats and sheep suggests that viral variants show little or no host species specificity. A phylogenetic tree of pol gene sequences confirmed the presence of a novel genotype of SRLV in France. Accepted January 10, 1997 Received August 12, 1996     

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).     

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.     

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.     

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

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.     

Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (±5.8%) and 95.9% (±2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.The diagnosis of small ruminant lentivirus (SRLV) infections in sheep and goats is most commonly determined by the detection of anti-SRLV antibodies in serum by an enzyme-linked immunosorbent assay (ELISA) that is typically created by the use of maedi-visna virus (MVV) or caprine arthritis-encephalitis virus (CAEV) isolates from sheep or goats of a given region or country (1). ELISA formats are typically validated against reference standard tests, including the agar gel immunodiffusion (AGID) assay, the radioimmunoprecipitation (IP) assay, or Western blot analysis. Although most seropositive sheep and goats do not show clinical signs of SRLV disease, they are persistent and potential reservoirs for transmission. Therefore, highly specific and sensitive serological diagnostic assays are essential for the early detection of SRLV.Three hundred ten of 332 serum samples from U.S. sheep from a previous CAEV competitive ELISA (cELISA) validation study (4) were tested in duplicate by using a Chekit CAEV/MVV antibody test kit (IDEXX Laboratories, The Netherlands), according to the manufacturer''s instructions. The CAEV/MVV indirect ELISA (iELISA) results were compared with those of the ovine progressive pneumonia virus (OPPV) WLC1 radio-IP assay, which has been described previously (4). The CAEV/MVV iELISA utilizes whole virus from Swiss MVV strain OLV as the antigen (15, 16). With a value of ≥60% being defined as a CAEV/MVV iELISA-positive serum sample, the sensitivity and the specificity of the CAEV/MVV iELISA were 74.0% (±7.6%) (95% confidence interval) and 98.3% (±2.0%), respectively, compared to the results of the radio-IP assay. Since the sensitivity was less than adequate, we reassessed the cutoff by calculating the mean value (in percent) ± 2 standard deviations for the radio-IP assay-negative serum samples. The results of that analysis placed the cutoff mean value at 33.1%. By using the new cutoff value, the sensitivity of the iELISA improved to 86.0% (±5.8%) and the specificity decreased slightly to 95.9% (±2.9%) compared to the results of the radio-IP assay. However, compared to the CAEV cELISA, which has a sensitivity of 98.6% and a specificity of 96.9% when the results of the radio-IP assay are used as the reference standard, the iELISA had a reduced sensitivity.Since the sera were taken from a number of different U.S. sheep kept under different husbandry and management conditions, we also wanted to test the performance of the CAEV/MVV iELISA with sera from one flock in which the sheep are exposed to the same husbandry and management conditions. Sera from an Idaho sheep flock (n = 405) consisting of sheep of the Rambouillet, Polypay, and Columbia breeds ages 3, 4, 5, and 6 years were tested by the iELISA. The results were compared to those of the CAEV cELISA by using the new iELISA cutoff value of 33.1%, and the discrepant samples were analyzed by Western blotting with OPPV WLC1 and by previously published methods (2). The positive and negative concordances of the CAEV cELISA and the CAEV/MVV iELISA were 92.5% (±3.1%) and 99.3% (±1.4%), respectively. Eighteen of 20 CAEV/MVV iELISA-negative and CAEV cELISA-positive serum samples tested positive by Western blot analysis, and the 2 remaining discrepant serum samples tested negative by Western blot analysis. One CAEV/MVV iELISA-positive and CAEV cELISA-negative sample tested negative by Western blot analysis. The 95% confidence interval for the positive and the negative concordances of the results of the CAEV/MVV iELISA relative to those of the CAEV cELISA for Idaho sheep and U.S. sheep overlapped (data not shown).A difference in the limit of detection between the CAEV/MVV iELISA and the CAEV cELISA may be a major reason for the reduced sensitivity of the CAEV/MVV iELISA (86%) compared to that of the CAEV cELISA (98.6%) with sera from U.S. sheep. Sera require dilution 1:10 for testing by the CAEV/MVV iELISA, whereas undiluted sera are used for the CAEV cELISA. To test whether the limit of detection is greater for the CAEV cELISA than the CAEV/MVV iELISA, 15 Western blot analysis-positive, CAEV cELISA-positive, and CAEV/MVV iELISA-negative serum samples from the Idaho flock were diluted 1:10 and 1:50 with 1× phosphate-buffered saline, pH 7.5, and retested by the CAEV cELISA. Twelve of these 15 serum samples tested positive by the CAEV cELISA at a 1:10 dilution, and 7 of 15 tested positive by the CAEV cELISA at a 1:50 dilution. This indicates that the CAEV cELISA has a higher dilution limit for the detection of anti-SRLV antibodies than the CAEV/MVV iELISA with sera from U.S. sheep. This high dilution limit of detection is likely due to the format of the CAEV cELISA, in which this assay is reliant upon anti-OPPV serum antibodies to inhibit the binding of a peroxidase-labeled monoclonal antibody (monoclonal antibody 74A) to a single, specific epitope on the CAEV-63 surface envelope glycoprotein.A previous report of 95.5% sensitivity and 97.2% specificity has been reported for the iELISA with sera from Swiss sheep (15). Large SRLV strain differences between Swiss MVV and U.S. OPPV strains may account for the lower sensitivity of the iELISA with sera from U.S. sheep. Therefore, gag, which encodes the capsid protein, was evaluated because it is a B-cell-immunodominant viral antigen in sheep naturally infected with MVV and OPPV and is more conserved than other viral genes (2, 7, 17). Unfortunately, no gag sequence is available for the Swiss MVV OLV (the strain used in the iELISA); however, several representative gag sequences from SRLV strains from Swiss goats and sheep that were previously reported for SRLV clades A1, A3, A4, A5, B1, and B2 were utilized (13). Genomic DNA was isolated from peripheral blood leukocytes from nine cELISA- and iELISA-positive Idaho sheep by previously described methods (6). The gag gene encoding the capsid was amplified by PCR with the following primers: primer GAGPSf (5′-TGG-CGA-CGC-AAG-GCT-CAA-A-3′) and primer GAGPSr (5′-GCG-GAC-GGC-ACC-ACA-CG-3′) (Integrated DNA Technology, Coralville, IA). The PCR mixture consisted of 100 ng of genomic DNA from sheep peripheral blood leukocytes or goat synovial membrane cells infected with WLC1, 2.5 mM_f (final concentration) MgCl₂, 0.2 mM_f deoxynucleoside triphosphates, and 1 U of Taq polymerase (Fisher Scientific, Pittsburg, PA). Primers GAGPSf and GAGPSr bind to nucleotides 734 to 752 and 2038 to 2054, respectively, of the sequence with GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY101611","term_id":"21539308","term_text":"AY101611"}}AY101611. The amplification conditions for the gag PCR were as follows: 95°C for 4 min, followed by 25 cycles of 95°C for 30 s, 58.1°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min and 4°C indefinitely. The gag PCR products were analyzed, cloned, and sequenced by previously described methods (5). The gag sequences were further refined by hand by using the Se-Al (version 2.0) program (http://tree.bio.edu.ac.uk/software/seal/), and a consensus gag sequence was generated from gag sequences from four sheep and OPPV WLC1.For phylogenetic analysis, the appropriate model of nucleotide substitution was selected by using hierarchical likelihood ratio testing with the MrModelTest2 command block (11) executed in the PAUP program (14). A general time-reversible model (also called the GTR or REV model) (12) with invariant sites and a measure of the rate of heterogeneity determined by use of the gamma distribution was chosen for full Bayesian analysis by using the program MrBayes (8). A 50%-majority-rule consensus tree based on the gag sequences was constructed by using the Mesquite software package (10). The resulting Bayesian phylogenetic tree showed that the sequences from the Idaho sheep formed one clade with OPPV WLC1, OPPV 85/34, and a single Swiss goat SRLV strain (strain 5692 A3) with high posterior probabilities (0.99 to 1.0) (Fig. ​(Fig.1).1). The finding that three gag sequences from Swiss sheep are found in other clades aside from the U.S. sheep OPPV clade (6247 A1, 5720 B2, and 5776 B1) suggests that Swiss sheep SRLV strains are quite different from U.S. OPPV strains, and these differences contribute to the reduced sensitivity of the iELISA with sera from U.S. sheep compared to that achieved with sera from Swiss sheep.Open in a separate windowFIG. 1.A 50%-majority-rule consensus tree of SRLV gag nucleotide sequences obtained by Bayesian phylogenetic analyses with the general time-reversible model with invariant sites and a measure of the rate of heterogeneity. Consensus gag nucleotide sequences from Idaho sheep (sheep 28Oct76, 29Oct10, 28Oct101, 28Oct27, 28Oct83, 28Oct18, 28Oct89, 28Oct68, and 28Oct99) and a consensus gag nucleotide sequence for WLC1 were utilized and compared to the gag nucleotide sequences from other U.S. OPPV strains (85-34), MVV strains, and CAEV strains. The GenBank accession numbers are given in parentheses next to the name or identification number given in previous studies. The numbers on the branches represent the Bayesian posterior probabilities.This combination of information suggests that U.S. sheep maintain strains of small ruminant lentiviruses phylogenetically different (determined on the basis of the gag sequences) from those from most other parts of the world. This suggests that the creation and validation of one serological assay that is based on one viral strain and that is accepted worldwide might not be successful due to the diversity of SRLV strains worldwide (3, 9).     

Genetic characterization of influenza viruses from influenza-related hospital admissions in the St. Petersburg and Valencia sites of the Global Influenza Hospital Surveillance Network during the 2013/14 influenza season

BackgroundContinuous surveillance for genetic changes in circulating influenza viruses is needed to guide influenza prevention and control.ObjectivesTo compare intra-seasonal influenza genetic diversity of hemagglutinin in influenza A strains isolated from influenza hospital admissions collected at two distinct sites during the same season.Study designComparative phylogenetic analysis of full-length hemagglutinin genes from 77 isolated influenza A viruses from the St. Petersburg, Russian Federation and Valencia, Spain sites of the Global Influenza Hospital Surveillance Network (GIHSN) during the 2013/14 season.ResultsWe found significant variability in A(H3N2) and A(H1N1)pdm09 viruses between the two sites, with nucleotide variation at antigenic positions much lower for A(H1N1)pdm09 than for A(H3N2) viruses. For A(H1N1)pdm09, antigenic sites differed by three to four amino acids from the vaccine strain, two of them common to all tested isolates. For A(H3N2) viruses, antigenic sites differed by six to nine amino acids from the vaccine strain, four of them common to all tested isolates. A fifth amino acid substitution in the antigenic sites of A(H3N2) defined a new clade, 3C.2. For both influenza A subtypes, pairwise amino acid distances between circulating viruses and vaccine strains were significantly higher at antigenic than at non-antigenic sites. Whereas A(H1N1)pdm09 viruses clustered with clade 6B and 94% of A(H3N2) with clade 3C.3, at both study sites A(H3N2) clade 3C.2 viruses emerged towards the end of the season, showing greater pairwise amino acid distances from the vaccine strain compared to the predominant clade 3C.3.ConclusionsInfluenza A antigenic variants differed between St. Petersburg and Valencia, and A(H3N2) clade 3C.2 viruses were characterized by more amino acid differences from the vaccine strain, especially at the antigenic sites.     

Genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001)

The genetic and antigenic variability of human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires from 1995 to 2001 was evaluated by partial nucleotide sequencing of the G gene and enzyme-linked immunosorbent assay analysis with anti-G monoclonal antibodies. Phylogenetic analyses showed that 37 group A strains clustered into five genotypes, whereas 20 group B strains clustered into three genotypes. Group A showed more genetic variability than group B. A close correlation between genotypes and antigenic patterns was observed. Changes detected in the G protein of viruses from both groups included (i) amino acid substitutions and(ii) differences in protein length due to either changes in stop codon usage or sequence duplications. Three B strains from 1999 exhibited a duplication of 20 amino acids, while one B strain from 2001 had 2 amino acids duplicated. The comparison among Argentinean HRSV strains and viruses isolated in other geographical areas during different epidemics is discussed.     

Genetic and epidemiological analysis of influenza virus epidemics in Taiwan during 2003 to 2006

The genetic characterization of Taiwanese influenza A and B viruses on the basis of analyses of pairwise amino acid variations, genetic clustering, and phylogenetics was performed. A total of 548, 2,123, and 1,336 sequences of the HA1 genes of influenza A virus subtypes H1 and H3 and influenza B virus, respectively, collected during 2003 to 2006 from an island-wide surveillance network were determined. Influenza A virus H3 showed activity during all periods, although it was dominant only in the winters of 2002-2003 and 2003-2004. Instead, influenza B virus and influenza A virus H1 were dominant in the winters of 2004-2005 and 2005-2006, respectively. Additionally, two influenza A virus H3 peaks were found in the summers of 2004 and 2005. From clustering analysis, similar characteristics of high sequence diversity and short life spans for the influenza A virus H1 and H3 clusters were observed, despite their distinct seasonal patterns. In contrast, clusters with longer life spans and fewer but larger clusters were found among the influenza B viruses. We also noticed that more amino acid changes at antigenic sites, especially at sites B and D in the H3 viruses, were found in 2003 and 2004 than in the following 2 years. The only epidemic of the H1 viruses, which occurred in the winter of 2005-2006, was caused by two genetically distinct lineages, and neither of them showed apparent antigenic changes compared with the antigens of the vaccine strain. For the influenza B viruses, the multiple dominant lineages of Yamagata-like strains with large genetic variations observed reflected the evolutionary pressure caused by the Yamagata-like vaccine strain. On the other hand, only one dominant lineage of Victoria-like strains circulated from 2004 to 2006.     

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.     

Antigenic and genetic variability of human respiratory syncytial viruses (group A) isolated in Uruguay and Argentina: 1993-2001

The antigenic and genetic diversity of G glycoprotein from 25 human respiratory viruses (group A) isolated during nine consecutive epidemics (1993-2001) in Montevideo, Uruguay, and 7 strains isolated in Buenos Aires, Argentina, in the same period were analyzed. Genetic variability was evaluated by partial sequence of the G protein gene. Phylogenetic analysis indicated that most Uruguayan and Argentinean group A isolates clustered into three genotypes: GA5, GA2, and GA1. Some strains clustered into the GA3 genotype characterized previously. The antigenic analysis was carried out with a panel of anti-G monoclonal antibodies that recognized conserved and strain-specific epitopes. A close correlation between the antigenic and genetic relatedness of the strains analyzed was observed.     

Amino Acid Variation within the Fusion Protein of Respiratory Syncytial Virus Subtype A and B Strains during Annual Epidemics in South Africa

Recent evidence of positive selection within the cytotoxic T-cell (CTL) epitopes of the highly conserved nucleoprotein of influenza virus raised the question of whether the CTL epitopes of Respiratory syncytial virus (RSV) are also affected by immune driven change over annual epidemics. The fusion protein (F-protein) of RSV is highly conserved within the two subtypes (A and B) and the most important target for the protective response. The position of various neutralizing epitopes has been mapped and characterized between RSV subtypes. CTL epitopes have also recently been mapped for the F-protein of subtype A, however variation within these epitopes between and within the subtypes has not been determined. To address this question, the F-proteins of 18 strains representative of all subgroup A and B genotypes identified in South Africa over a period of 5 years were sequenced. F-protein sequences were highly conserved within and between South African genotypes, with most variability occurring at the nucleotide level. Most of the amino acid differences identified within neutralizing and CTL epitopes were conserved within the subtypes, and therefore does not indicate immune selection. However, out of three CTL epitopes previously identified in subtype A, two (restricted to HLA B*57 and HLA A *01) were conserved only within subtype A, while the third (restricted to Cw*12) contained both subtype- and genotype-specific changes. These results suggest that most of the identified CTL epitopes are subtype A-specific and may not be recognized in subtype B viruses, while the HLA Cw*12 restricted epitope may also not be recognized efficiently in GA5 strains.Genbank Accession numbers: AY526550-6567.     

Amplified expression of murine leukemia virus (MuLV)-coded antigens on thymocytes and leukemia cells of AKR mice after infection by dualtropic (MCF) MuLV

Recently, we have shown that several isolates of recombinant, dualtropic (MCF) murine leukemia virus (MuLV) can induce amplification of MuLV gag and env gene-coded antigens on thymocytes of young AKR mice and, subsequently, can accelerate leukemia in these same animals. With respect to env gene-coded antigens, it is unclear whether antigen amplification represents expression of env gene products of the input virus on the surface of infected thymocytes or whether infection by MCF viruses induces the expression of other endogenous env gene sequences. Infection by antigenically marked viruses has allowed us to decide between these alternatives. We have found that AKR MCF isolates 69L1,13, and 247 can be distinguished serologically by specific patterns of reactivity with mouse and rat monoclonal antibodies that recognize 10 distinct epitopes (single antigenic determinants) of AKR ecotropic MuLV gp70 and p15(E) proteins. Accordingly, 1-monthold AKR mice were injected intrathymically with either MCF 69L1, MCF 13, or MCF 247 viruses and thymocytes of virus-injected mice were then assayed for amplification of specific epitopes at 32—36 days postinjection. Quantitative expression of MuLV antigens was determined by flow microfluorometry using a fluorescence-activated cell sorter. The patterns of reactivity with monoclonal antibodies which were originally determined for each virus in vitro were found to breed true on thymocytes infected in vivo. Moreover, the phenotype of virus recovered in vitro from virus accelerated leukemias was identical to that of the injected virus. Thus, the preleukemic change of MuLV antigen amplification appears to represent expression on thymocytes of env gene products encoded by the infecting MCF viral genome which continues to be expressed in the resulting leukemias without apparent changes in the recombinant env gene.     

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.