Richard Pilon

Persistent HIV RNA shedding in semen despite effective antiretroviral therapy.

Effective antiretroviral therapy (ART) may reduce HIV sexual transmission by lowering genital HIV levels. A prospective study of men starting ART (n = 25) demonstrated rapid, substantial reductions in semen HIV RNA. However, despite an undetectable blood viral load, isolated semen HIV shedding was detected at more than one visit in 12 of 25 (48%) participants, with semen HIV RNA levels exceeding 5000 copies/ml in four of 25 (16%). Isolates were drug-sensitive, and this phenomenon was not associated with semen drug levels or regimen.

Human Immunodeficiency Virus (HIV) Type 1 Proviral Hypermutation Correlates with CD4 Count in HIV-Infected Women from Kenya

ABSTRACT APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.

Cross-species retroviral transmission from macaques to human beings

Cross-species transmission of simian foamy virus (SFV) to human beings from chimpanzees, baboons, and African green monkeys has been described. Although macaques are the non-human primate most often handled in research, human infection with SFV from macaques has not been reported. Two of 46 primate-facility workers tested positive for antibodies that reacted with an immunoblot that contained macaque foamy virus antigens. Phylogenetic assessment of a 96-bp fragment of amplified proviral DNA isolated from peripheral-blood mononuclear cells from one infected individual was consistent with SFV infection of macaque origin. Frequent use of macaques in biomedical research, and identification of persistent retroviral infection from macaques to human beings, could have implications for public-health policy and occupational health and safety.

Discordance in HIV-1 viral loads and antiretroviral drug concentrations comparing semen and blood plasma

For individuals not on antiretroviral therapy, the risk of heterosexual transmission of HIV appears negligible when blood plasma (BP) viral loads are <1500 HIV‐1 RNA copies/mL. It is not clear whether this observation can be extrapolated to individuals on highly active antiretroviral therapy (HAART). Because of differential tissue penetration, antiretroviral drug concentrations may be sufficient to maintain an undetectable viral load in the BP yet not achieve adequate levels to suppress HIV in the genital tract. Therefore, we wanted to correlate HIV viral loads and drug concentrations in semen plasma (SP) and BP.

Longitudinal phylogenetic surveillance identifies distinct patterns of cluster dynamics.

Objective:Through the application of simple, accessible, molecular epidemiology tools, we aimed to resolve the phylogenetic relationships that best predicted patterns of cluster growth using longitudinal population level drug resistance genotype data. Methods:Analysis was performed on 971 specimens from drug naïve, first time HIV positive subjects collected in British Columbia between 2002 and 2005. A 1240bp fragment of the pol gene was amplified and sequenced with relationships among subtype B sequences inferred using Neighbour-Joining analysis. Apparent clusters of infections having both a mean within group distance <0.031 and bootstrap value >80% were systematically identified. The entire 2002-2005 dataset was then re-analyze to evaluate the relationship of subsequent infections to those identified in 2002. BED testing was used to identify recent infections (<156 days). Results:Among the 2002 infections, 136 of 300 sequences sorted into 52 clusters ranging in size from 2 to 9 members. Aboriginal ethnicity and intravenous drug use were correlated, and both were linked to cluster membership in 2002. Although cluster growth between 2002 and 2005 was correlated with the size of the original cluster, more related infections were found in clusters seeded from nonclustered infections. Finally, all large growth clusters were seeded from infections that were much more likely to be recent. Conclusions:This population level phylogenetic analysis suggests that a greater increase in cluster size is associated with recently infected individuals, which may represent the leading edge of the epidemic. The most impressive increase in cluster size is seen originating from initially nonclustered infections. In contrast, smaller existing clusters likely describe historical patterns of transmission and do not substantially contribute to the ongoing epidemic. Application of this method for cross-sectional analysis of existing sequences from defined geographic regions may be useful in predicting trends in HIV transmission.

Transmission Patterns of HIV and Hepatitis C Virus among Networks of People Who Inject Drugs

Background The risk-related behaviours and practices associated with injection drug use remain a driver of HIV and hepatitis C virus (HCV) transmission throughout the world. Here we evaluated HIV and HCV transmission patterns in the context of social networks of injection drug users (IDU) recruited from a higher incidence region in order to better understand factors that contribute to ongoing transmission among IDU. Methods IDU recruited through a chain-referral method provided biological specimens for analysis. HIV and HCV positive specimens were sequenced and analyzed using phylogenetic methods (Neighbour-joining and Bayesian) and transmission patterns of HIV and HCV evaluated in the context of the recruitment networks. Results Among the 407 recruited IDU, HCV and HIV prevalence were 60.6% and 10.1%, respectively; 98% of HIV positive individuals were co-infected with HCV. Thirty-six percent of HCV sequences were associated with clusters, compared to 67% of HIV sequences. Four (16.7%) of the 24 HCV clusters contained membership separated by 2 or fewer recruitment cycles, compared to 10 (41.6%) derived from more than one recruitment component. Two (28.6%) of the 7 HIV clusters contained membership separated by 2 or fewer recruitment cycles while 6 (85.7%) were composed of inter component membership. Conclusions Few HIV and HCV transmissions coincided with the recruitment networks, suggesting that they occurred in a different social context or a context not captured by the recruitment network. However, among the complete cohort, a higher degree of HIV clustering indicates many are recent infections originating from within current social networks, whereas a larger proportion of HCV infections may have occurred earlier in injecting history and in the context of a different social environment.

Virus Levels in Untreated African Infants Infected with Human Immunodeficiency Virus Type 1

In developed areas, human immunodeficiency virus (HIV)-infected infants have high virus levels and rapidly progress to death. HIV levels were assessed in 1994-1997 in untreated infants in Malawi by analysis of dried blood spots tested by nucleic acid silica-bound amplification. Of 24 umbilical cord blood (CB)-positive samples, 83% had >10,000 copies/mL. The median virus level was 78,000 copies/mL. First positive sample median levels were 355,000 copies/mL among 52 perinatally infected infants and 130,000 copies/mL among 43 infants infected by breast-feeding. Virus levels were stable, and initial levels predicted levels 1 year after infection (P=.005), at which time levels did not significantly differ among in utero, perinatally, or postnatally infected infants. Thus, neither age at infection nor route of infection significantly influenced HIV levels measured 1 year after infection. Most (87%) CB-positive infants were infected before labor onset, since virus levels greatly exceeded those expected in their mothers.

Next-generation sequencing of dried blood spot specimens: a novel approach to HIV drug-resistance surveillance.

BACKGROUND HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease of collection, transportation and storage. Analysis of pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient sequencing method for determining HIV DR rates. In this study, we conducted HIV DR surveillance on DBS using NGS and identified minority variants in individual patients. METHODS A total of 48 extracts of DBS from an HIV DR surveillance study in Mexico City were re-amplified using primers tagged with multiplex identifiers, pooled and pyrosequenced. Consensus sequences were generated for each specimen with mixtures identified at positions where >20% of the reads contained a variant. Individual consensus sequences were then analysed for DR mutations and compared with those derived from Sanger sequencing. RESULTS DBS analysed with tagged pooled pyrosequencing (TPP) were highly concordant with Sanger sequencing genotypes from matching plasma and DBS (99.21% and 99.51%, respectively). An exception was an M184I mutation only detected with TPP of DBS at a frequency of 20.4%. Multiple specimens had minority variant reads below the 20% mixture threshold. CONCLUSIONS TPP using DBS is an effective method for HIV DR surveillance. TPP for genotyping results in cost savings of 40% over conventional in-house methods. The effect of low-abundance DR mutations, undetectable by conventional methods, remains to be determined. This technology might be applied to any HIV specimen (plasma/serum) and can also be used for other diagnostic assays where DNA sequencing is required.

Mucosal correlates of isolated HIV semen shedding during effective antiretroviral therapy.

Effective antiretroviral therapy (ART) suppresses the blood HIV RNA viral load (VL) below the level of detection. However, some individuals intermittently shed HIV RNA in semen despite suppression of viremia, a phenomenon termed “isolated HIV semen shedding (IHS)”. In a previously reported clinical study, we collected blood and semen samples from HIV-infected men for 6 months after ART initiation, and documented IHS at ≥1 visit in almost half of the participants, independent of ART regimen or semen drug levels. We now report the mucosal immune associations of IHS in these men. Blood and semen plasma cytokine levels were assayed by multiplex enzyme-linked immunosorbent assay, T-cell populations were evaluated by flow cytometry in freshly isolated blood and semen mononuclear cells, and semen cytomegalovirus (CMV) DNA levels were measured by PCR. Although IHS was not associated with altered blood or semen cytokine levels, the phenomenon was associated with a transient, dramatic increase in CD4+ and CD8+ T-cell activation that was restricted to the semen compartment. All participants were CMV infected, and although semen CMV reactivation was common despite ART, this was not associated with T-cell activation or IHS. Further elucidation of the causes of compartmentalized mucosal T-cell activation and IHS may have important public health implications.

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus.

ABSTRACT Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.