Effect of Difference in Consensus Sequence between HIV-1 Subtype A/E and Subtype B Viruses on Elicitation of Gag-Specific CD8+ T Cells and Accumulation of HLA-Associated Escape Mutations.

The Gag280 mutation is associated with HLA-C*01:02 but not with HLA-B*52:01 in subtype A/E-infected individuals, whereas this mutation is associated with HLA-B*52:01 but not with HLA-C*01:02 in subtype B infections. Although it is known that the Gag280 mutant is selected by HLA-B*52:01-restricted GagRI8 (Gag275-282)-specific T cells in subtype B infections, it remains unknown why this Gag280 mutation is associated with HLA-C*01:02 rather than HLA-B*52:01 in subtype A/E infections. The subtype B and A/E viruses have different consensus sequence, with Thr and Val at Gag280, respectively. To clarify the effect of this difference in Gag280 consensus sequence, we investigated the role of HLA-C*01:02-restricted GagYI9 (Gag277-285)-specific T cells in selection of Gag280 mutations in subtype A/E-infected Vietnamese and subtype B-infected Japanese individuals. GagYI9-4V-specific T cells, which were frequently elicited in Vietnamese individuals infected with the consensus-type A/E virus, failed to recognize GagV280T mutant A/E virus-infected cells. GagYI9-4T mutant epitope-specific T cells, which were weakly elicited in individuals infected with the mutant A/E virus, had weak or no ability to recognize the mutant virus. These results account for the mechanism for selection and accumulation of GagV280T mutants in the case of subtype A/E infections. In contrast, HLA-C*01:02-restricted GagYI9-4T-specific T cells were weakly elicited in Japanese individuals infected with the subtype B virus, explaining why HLA-C*01:02-restricted Gag280 mutations are not accumulated in the case of a subtype B infection. The present study demonstrated that a difference in the Gag280 consensus sequence influenced the elicitation of the GagYI9-specific T cells involved in the accumulation of HLA-C*01:02-associated Gag280 mutations.IMPORTANCE HIV-1 mutations escaped from HIV-specific CD8+ T cells are mostly detected as HLA-associated mutations. A diversity of HLA-associated mutations is somewhat distinct to each race and region, since HLA allele distribution differs among them. A difference in the consensus sequence among HIV-1 subtypes may also influence the diversity of HLA-associated mutations. HLA-C*01:02-associated GagV280T and HLA-B*52:01-associated GagT280A/S mutations were previously identified in HIV-1 subtype A/E-infected and subtype B-infected individuals, respectively, though these subtype viruses have a different consensus sequence at Gag280. We demonstrated that the GagV280T mutant virus was selected by HLA-C*01:02-restricted GagYI9-4V-specific T cells in subtype A/E-infected Vietnamese but that HLA-C*01:02-restricted GagYI9-4T-specific T cells were weakly elicited in subtype B-infected Japanese. Together with our recent study which demonstrated the mechanism for the accumulation of HLA-B*52:01-associated mutations, we clarified the mechanism for the accumulation of different Gag280 mutations and the effect of the difference in the consensus sequence on the accumulation of escape mutations.

Unraveling HIV-1 diagnosis in special pediatric cases.

BACKGROUND: Early HIV-1 diagnosis and initiation of antiretroviral treatment is essential to prevent AIDS, and reduce mortality in children. HIV-1 molecular diagnosis in children before 18 months of age require, two independent samples to confirm a result. However, some patients have discordant virologic results in different samples, raising uncertainty for a conclusive diagnosis. We defined these patients as "special pediatric cases". OBJECTIVES: The aim of our study was to characterize the "special pediatric cases" among HIV-1 infected children diagnosed in a five-year period at our laboratory and evaluate the impact on the time to HIV-1 diagnosis. STUDY DESIGN: A total of 44 perinatally HIV-1 infected infants with molecular diagnostic performed at the Pediatric Garrahan Hospital were analyzed from 2013 to 2017. RESULTS: We identified eight "special pediatric cases". In the first samples, all of them had negative results by different DNA-PCR assays. Three infants had undetectable plasma viral load (pVL), four had low detectable pVL value, and one infant had no available pVL. All samples with detectable pVL, including those with low pVL (ie: 65copies/mL), had high pVL values at the end of the diagnosis. Considering the age of the HIV-1 infected children at the end of the diagnosis, five "special pediatric cases" (62 %) had a "late" positive diagnosis [mean (range) = 146 (89-268) days old]. CONCLUSIONS: These "special pediatric cases" are not as unusual as previously thought and are important diagnostic challenges. Also, this study add evidence to include the viral load assay in the molecular diagnostic algorithm for perinatal HIV-1 infection.

Immune escape mutations in HIV-1 controllers in the Brazilian Amazon region.

BACKGROUND: Human immunodeficiency virus (HIV-1) infection is characterized by high viral replication and a decrease in CD4+ T cells (CD4+TC), resulting in AIDS, which can lead to death. In elite controllers and viremia controllers, viral replication is naturally controlled, with maintenance of CD4+TC levels without the use of antiretroviral therapy (ART). METHODS: The aim of the present study was to describe virological and immunological risk factors among HIV-1-infected individuals according to characteristics of progression to AIDS. The sample included 30 treatment-naive patients classified into three groups based on infection duration (> 6 years), CD4+TC count and viral load: (i) 2 elite controllers (ECs), (ii) 7 viremia controllers (VCs) and (iii) 21 nonviremia controllers (NVCs). Nested PCR was employed to amplify the virus genome, which was later sequenced using the Ion PGM platform for subtyping and analysis of immune escape mutations. RESULTS: Viral samples were classified as HIV-1 subtypes B and F. Greater selection pressure on mutations was observed in the group of viremia controllers, with a higher frequency of immunological escape mutations in the genes investigated, including two new mutations in gag. The viral sequences of viremia controllers and nonviremia controllers did not differ significantly regarding the presence of immune escape mutations. CONCLUSION: The results suggest that progression to AIDS is not dependent on a single variable but rather on a set of characteristics and pressures exerted by virus biology and interactions with immunogenetic host factors.

Domesticated gag Gene of Drosophila LTR Retrotransposons Is Involved in Response to Oxidative Stress.

Drosophila melanogaster is one of the most extensively used genetic model organisms for studying LTR retrotransposons that are represented by various groups in its genome. However, the phenomenon of molecular domestication of LTR retrotransposons has been insufficiently studied in Drosophila, as well as in other invertebrates. The present work is devoted to studying the role of the domesticated gag gene, Gagr, in the Drosophila genome. The Gagr gene has been shown to be involved in the response to stress caused by exposure to ammonium persulfate, but not in the stress response to oligomycin A, zeomycin, and cadmium chloride. Ammonium persulfate tissue specifically activates the expression of Gagr in the tissues of the carcass, but not in the gut. We found that the Gagr gene promoter contains one binding motif for the transcription factor kayak, a component of the JNK signaling pathway, and two binding motifs for the transcription factor Stat92E, a component of the Jak-STAT signaling pathway. Remarkably, Gagr orthologs contain the second binding motif for Stat92E only in D. melanogaster, D. simulans and D. sechellia, whereas in D. yakuba and D. erecta, Gagr orthologs contain a single motif, and there are no binding sites for Stat92E in the promoters of Gagr orthologs in D. ananassae and in species outside the melanogaster group. The data obtained indicate the formation of the protective function of the Gagr gene during evolution.

First report on the prevalence and genetic relatedness of Feline Foamy Virus (FFV) from Turkish domestic cats.

Feline Foamy Virus (FFV) is an important retroviral agent affecting domestic cats in Turkey that has been studied less intensively than Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV). Accordingly, we aimed to investigate the presence and prevalence of FFV among domestic cats by molecular techniques. PCR was used to amplify the gag-pol gene overlap in order to detect the presence of FFV. The gene encoding bet, an important accessory gene, was also characterized. Molecular characteristics were analyzed and phylogenetic trees were constructed. We determined the positivity rate as 10% in all samples (20/200) based on the gag-pol test. The phylogenetic analysis indicated that the Turkish FFV sequences form a separate cluster among other isolates in the constructed maximum likelihood (ML) tree. bet-based products were obtained for two samples (1%; 2/200) that were also positive for gag-pol. These bet gene sequences confirm the presence of a separate cluster for the Turkish FFV isolates. The results suggest that FFV is prevalent and widespread in Turkish domestic cats. Additionally, these new FFV sequences represent the first FFV sequences from Turkey to be submitted to GenBank. This study paves the way for studies on the pathogenicity of FFV.

Two-year cross-sectional studies reveal that single, young MSMs in Shenzhen, China are at high risk for HIV infection.

BACKGROUND: Shenzhen City is a rapidly growing area with a large number of floating populations, thus making it difficult to control HIV. Serial cross-sectional studies are helpful for the prediction of epidemiological tendency. In this study, two parallel cross-sectional studies were compared to explore changes in HIV epidemiology in Shenzhen, China. METHODS: Two hundred and fifty newly reported HIV-positive cases were randomly selected in Shenzhen City in 2013 and 2015. Socio-demographical information was collected with informed consent. Full-length gag and partial pol genes were amplified using nested RT-PCR followed by sequencing and phylogenetic analysis. The genotypes of anti-HIV drug resistance were also analyzed. The characteristics of the HIV epidemics of 2013 and 2015 were compared to identify patterns. RESULTS: The proportion of single, young MSMs dramatically increased in 2015 compared to 2013. Many subtypes, including CRF07_BC (36.4%), CRF01_AE (34.1%), CRF55_01B (10.2%), B (6.4%), CRF08_BC (3.4%), CRF59_01B (0.9%), C (0.7%), D (0.2%), CRF68_01B (0.2%), CRF67_01B (0.2%), and unique recombinant forms (URFs, 7.3%), were identified. Close phylogenetic relationships between strains prevalent in Shenzhen and other areas of China was observed. No epidemic cluster confined to single, young MSMs was identified. 0.4 and 2.8% of the strains contained transmitted drug-resistant mutations in 2013 and 2015, respectively. CONCLUSION: Although the interval period is short, changes in HIV epidemiology in Shenzhen City are distinct. Frequent surveillance of HIV epidemics in Shenzhen City is thus necessary. Single, young MSMs have become a high-risk population for HIV infection and should be considered as focus population for HIV prevention and behavior intervention in Shenzhen City.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1ß in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

BACKGROUND: There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. METHODS: miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes: long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. RESULTS: The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. CONCLUSIONS: In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line.

HIV-1 CRF07_BC with a Seven Amino Acid Deletion in the gag p6 Region Dominates in HIV-1-Infected Men Who Have Sex with Men in China.

We examined sequence variation in the HIV-1 gag p6 region from 27 individuals infected with HIV-1 CRF07_BC. An additional 269 gag p6 sequences of CRF07_BC from the Los Alamos National Laboratory database were also analyzed. A unique deletion of seven amino acid (aa) (p6Δ7) (aa 30-36, PIDKELY, in the HXB2 genome) was observed to exist exclusively in CRF07_BC. Indeed, 54.1% (160/296) of the CRF07_BC sequences contained the p6Δ7 mutation. The prevalence of the p6Δ7 mutation was 37.2% (29/78) and 92.3% (48/52) in CRF07_BC-infected intravenous drug users and men who have sex with men (MSM), respectively. Our results demonstrate that the p6Δ7 mutation dominates in MSM infected by HIV-1 CRF07_BC in China and suggests that this deletion could serve as a useful marker for monitoring HIV-1 evolution and epidemic. In future studies, it will be of interest to determine whether such genotypic variation influences viral replication capacity and disease progression.

HIV-1 is spreading out of former high-risk population through heterosexual transmission in Hebei, China.

INTRODUCTION: Multiple specific populations, including MSMs, IDUs, and FPDs, are involved in HIV epidemic in China. In the recent years, HIV transmission due to heterosexual transmission also contributed greatly to HIV epidemic in China. Very few studies have been fulfilled to characterize relationships of HIV-1 strains prevalent in different populations. In this study, the phylogenetic relationships of HIV-1 spreading in different populations were investigated. MATERIALS AND METHODS: HIV-1 sero-positive patients infected through different routes were enrolled into the study. Nested RT-PCR was used to amplify HIV gag and pol genes followed by sequencing. RESULTS: Multiple subtypes, including subtype B (52.1%), CRF01_AE (34.4%), CRF07_BC (6.3%), subtype C (4.2%), CRF02_AG (1.0%), CRF08_BC (1.0%) and unique recombination forms (1.0%) were identified. Phylogenetic analysis showed that strains from MSM, IDU, and FPDs grouped into clusters separately. However, strains identified in heterosexual transmitted population intermixed with all of other high risk populations. DISCUSSION AND CONCLUSION: The genetic data supposed that HIV-1 was spreading out of MSMs, IDUs, and FPDs through heterosexual transmission in Hebei, China. Urgent prevention and behavior intervention in the population will be necessary. Furthermore, the detailed sequence data will help the design of HIV-1 vaccines in China. Sequence Data: All of sequences have been deposited into the GenBank with the accession number: KJ820007-KJ820144.

Induction of HIV-1 gag specific immune responses by cationic micelles mediated delivery of gag mRNA.

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA.

HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil.

HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil. .

Genetic diversity in the feline leukemia virus gag gene.

Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field.

The fitness landscape of HIV-1 gag: advanced modeling approaches and validation of model predictions by in vitro testing.

Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model), generalizing our previous approach (Ising model) that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these) predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = -0.74, p = 3.6×10-6) are strongly correlated, and this was further strengthened in the regularized Ising model (r = -0.83, p = 3.7×10-12). Performance of the Potts model (r = -0.73, p = 9.7×10-9) was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion, and harnessing this knowledge for immunogen design.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting.

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000-25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Isolation of maedi/visna virus from a sheep in Japan.

Maedi/visna (MV) is a lentiviral disease of sheep caused by the maedi/visna virus (MVV). Although MV is prevalent in many countries, it had not been reported in Japan. In 2011, however, three sheep in northern Japan were reported to be seropositive against the MVV antigen, indicating a persistent MVV infection. In the present study, we isolated MVV from one sheep to confirm MVV infection and conducted genomic classification of the virus. The co-culture of leukocytes from a seropositive sheep with fetal goat lung cells resulted in the formation of syncytial cells and the amplification of a long terminal repeat sequence of MVV by polymerase chain reaction. The isolate was confirmed as being MVV, rather than the caprine arthritis-encephalitis virus based on phylogenetic analysis of the gag gene sequence. Although the sheep was asymptomatic, nonpurulent meningitis and demyelination were found in the spinal cord. These were considered to be early lesions associated with pathogenic MVV infection. Therefore, the present study demonstrated that MVV is distributed in Japan.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Emerging variability in HIV-1 genetics among recently infected individuals in Yunnan, China.

BACKGROUND: Yunnan has the longest endured Human Immunodeficiency Virus-1 (HIV-1) epidemic in China, and the genetic diversity of HIV-1 constitutes an essential characteristic of molecular epidemiology in this region. To obtain a more comprehensive picture of the dynamic changes in Yunnan's HIV-1 epidemic, a cross-sectional molecular epidemiological investigation was carried out among recently infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced partial gag (HXB2∶781-1861) and env (HXB2∶7002-7541) genes from 308 plasma samples of recently infected patients. With phylogenetic analysis, 130 specimens generated interpretable genotyping data. We found that the circulating genotypes included: CRF08_BC (40.8%), unique recombinant forms (URFs, 27.7%), CRF01_AE (18.5%), CRF07_BC (9.2%), subtype B (2.3%) and C (1.5%). CRF08_BC was the most common genotype, and was predominant in both intravenous drug users (IDUs) and heterosexually transmitted populations. CRF08_BC and CRF07_BC still predominated in eastern Yunnan, but CRF08_BC showed increasing prevalence in western Yunnan. Strikingly, the URFs raised dramatically in most regions of Yunnan. Seven different types of URFs were detected from 12 prefectures, suggesting that complicated and frequent recombination is a salient feature of Yunnan's HIV-1 epidemic. Among URFs, two BC clusters with distinctive recombination patterns might be potential new CRF_BCs. CRF01_AE was no longer confined to the prefectures bordering Myanmar, and had spread to the eastern part of Yunnan, especially the capital city of Kunming, with a large number of infections in the transient population. The ratios of the main genotypes showed no statistical differences between infected IDUs and heterosexually transmitted infections. CONCLUSIONS/SIGNIFICANCE: The changing patterns of the dominant HIV-1 genotypes in Yunnan indicate the complex evolving dynamic nature of the epidemic. Understanding new trends in molecular epidemiology of HIV-1 infection is critical for adjusting current prevention strategies and vaccine development in Yunnan.

Short communication: high natural polymorphism in the gag gene cleavage sites of non-B HIV type 1 isolates from Gabon.

The main goal of the present study was to determine the frequency of substitutions in the cleavage sites (CS) of gag gene among non-B HIV-1 isolates from Gabon. Fifty plasma specimens, collected in 2010-2011, from HIV-1-infected patients failing first-line antiretroviral (ARV) regimens (constituted of two nucleoside reverse transcriptase inhibitors+one nonnucleoside reverse transcriptase inhibitor) (n=38) and from HIV-1-infected individuals untreated with ARV (n=12) were analyzed in the gag and gag-pol cleavage sites. Compared to HXB2 reference sequence, the total median number of substitutions in gag and gag-pol CS was 10 (range, 5-18). The cleavage site p2/NC was the most variable of the four gag CS with 100% (50/50) isolates carrying at least 1 substitution (range, 1-9). The two gag-pol TFP/p6pol and p6pol/PR CS sites were also highly variable (at least one substitution, 50/50, 100% in both cases). Substitutions at position G381 (p2/NC), L449 (p1/p6gag), and K444 (TFP/p6pol) were significantly more frequent in CRF02_AG strains, compared to other non-B strains (30.4% vs. 3.7%, p=0.03; 87.0% vs. 59.3%, p=0.03; and 91.3% vs. 59.3%, p=0.01, respectively). Other non-B subtypes were significantly more likely to harbor substitutions at position N487 (p6pol) (70.4%) than CRF02_AG (39.1%) (p=0.02). In Gabon, gag and gag-pol cleavage sites were highly polymorphic in protease inhibitor-naive patients harboring non-B HIV-1 strains. In sub-Saharan Africa, further studies are definitively required to better understand the impact of gag mutations among subjects receiving second-line LPV/r-containing regimens (monotherapy or triple combinations).