Junko Hattori

Outbreak of Infections by Hepatitis B Virus Genotype A and Transmission of Genetic Drug Resistance in Patients Coinfected with HIV-1 in Japan

ABSTRACT The major routes of hepatitis B virus (HBV) infection in Japan has been mother-to-child transmission (MTCT) and blood transfusion. However, HBV cases transmitted through sexual contact are increasing, especially among HIV-1-seropositive patients. To understand the molecular epidemiology of HBV in HBV/HIV-1 coinfection, we analyzed HBV genotypes and HIV-1 subtypes in HBV/HIV-1-coinfected patients at Nagoya Medical Center from 2003 to 2007. Among 394 HIV-1-infected Japanese men having sex with men (MSM) who were newly diagnosed during the study period, 31 (7.9%) tested positive for the hepatitis B virus surface antigen. HBV sequence analyses were successful in 26 cases, with 21 (80.7%) and 5 (19.3%) cases determined as genotypes A and C, respectively. Our finding that HBV genotype A was dominant in HIV-1-seropositive patients alerts clinicians to an alternative outbreak of HBV genotype A in the HIV-1-infected MSM population and a shift in HBV genotype from C to A in Japan. The narrow genetic diversity in genotype A cases suggests that genotype A has been recently introduced into the MSM population and that sexual contacts among MSM were more active than speculated from HIV-1 tree analyses. In addition, we found a lamivudine resistance mutation in one naïve case, suggesting a risk of drug-resistant HBV transmission. As genotype A infection has a higher risk than infection with other genotypes for individuals to become HBV carriers, prevention programs are urgently needed for the target population.

A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of a peptide nucleic acid probe and catalysed signal amplification.

A novel in situ hybridization (ISH) method for detecting human immunodeficiency virus‐1 (HIV‐1) was developed by applying a peptide nucleic acid (PNA) probe and a catalysed signal amplification (CSA) method. The PNA probe used in the present study possessed 15 base sequences of the HIV‐1 protease gene, and the 5′ end of the probe was labelled with the fluorescein isothiocyanate (FITC) molecule. The hybridized probe was detected by sequential reactions of the following antibodies and reagents: horseradish peroxidase (HRP)‐conjugated anti‐FITC antibody, biotinylated tyramide (first amplification), HRP‐labelled streptavidin, biotinylated tyramide (second amplification), and streptavidin‐conjugated Alexa 488. The signal of Alexa 488 was finally detected by fluorescence microscopy. HIV‐1‐related dotted signals were clearly obtained in HIV‐1 persistently infected cell lines, MOLT4‐IIIB and ACH‐2, and CD4‐positive T lymphocytes from AIDS patients. For light microscopy, HRP‐labelled streptavidin was reacted instead of streptavidin‐conjugated Alexa 488 at the final treatment, followed by diaminobenzidine as chromogen. This method can detect HIV‐1 in either blood smear samples or paraffin‐embedded autopsy tissue and is useful as a sensitive non‐radioactive method for in situ hybridization. Copyright

Prevalence of Transmitted HIV Drug Resistance in Iran between 2010 and 2011

Objective Drug-resistant (DR) HIV emerges during combined antiretroviral treatment (cART), creating concern about widespread transmission of DR-HIV as cART is expanded in resource-limited countries. The aim of this study was to determine the predominant HIV-1 subtypes and prevalence of transmitted DR mutations among antiretroviral-naïve patients in Iran. Design To monitor transmission of DR HIV, a threshold surveillance based on the world health organization (WHO) guidelines was implemented in Iran. Methods For this HIVDR threshold surveillance study, blood samples were collected from 50 antiretroviral-naïve HIV-1-infected patients. Antiretroviral-resistant mutations were determined by sequencing HIV-1 protease, reverse transcriptase and integrase regions. The HIV-1 subtype was determined by sequencing the p17 and C2-V5 regions of the gag and env genes, respectively. Results Phylogenetic analyses of the sequenced regions revealed that 45 (95.7%) of 47 samples that were successfully obtained were CRF35_AD. The remaining two cases were subtype B (2.1%) and CRF01_AE (2.1%). Consistent results were obtained also from Env and Gag sequences. Regarding prevalence of transmitted DR viruses, two cases were found to harbor reverse transcriptase-inhibitor-resistant mutations (4.3%). In addition, although not in the WHO list for surveillance of transmitted mutations, 13 minor protease-inhibitor-resistant mutations listed in the International AIDS Society-USA panel of drug resistance mutations were found. No DR mutations were detected in the integrase region. Conclusions Our study clarified that CRF35_AD is the major subtype among HIV-1-infected patients in Iran. According to the WHO categorization method of HIVDR threshold survey, the prevalence of transmitted drug resistant HIV in Iran was estimated as moderate (5–15%).

Prevalence of infection and genotypes of GBV-C/HGV among homosexual men

Since the discovery of GB virus‐C (GBV‐C) and hepatitis G virus (HGV), many studies have been performed. These viruses are now known to be parenterally, as well as sexually transmitted. A phylogenetic analysis also revealed that GBV‐C has five major genotypes: type 1 predominates in West Africa, type 2 in Europe and the United States, type 3 in parts of Asia, type 4 in Southeast Asia, and type 5 in South Africa. Despite the number of reports so far, there have been few large‐scale surveys of homosexual men to determine the prevalence of the GBV‐C/HGV infections. We examined the levels of GBV‐C/HGV viremia in 297 homosexual men who attended the Nagoya Lesbian and Gay Revolution held in Nagoya, Japan. Reverse transcription‐polymerase chain reaction (RT‐PCR)/nested PCR of the GBV‐C/HGV 5′‐non‐coding region (NCR), and base sequence analyses showed that the infection rate was 12.5%, and genotypes in this population were classified into type 2 (32%) and type 3 (68%). None were classified as types 1, 4, or 5 in this study. Our results indicate that the GBV‐C/HGV type 2 seen mainly in Europe and the US is spreading widely in Japan, especially in the Nagoya district.

Phylodynamic Analysis Reveals CRF01_AE Dissemination between Japan and Neighboring Asian Countries and the Role of Intravenous Drug Use in Transmission

Background One major circulating HIV-1 subtype in Southeast Asian countries is CRF01_AE, but little is known about its epidemiology in Japan. We conducted a molecular phylodynamic study of patients newly diagnosed with CRF01_AE from 2003 to 2010. Methods Plasma samples from patients registered in Japanese Drug Resistance HIV-1 Surveillance Network were analyzed for protease-reverse transcriptase sequences; all sequences undergo subtyping and phylogenetic analysis using distance-matrix-based, maximum likelihood and Bayesian coalescent Markov Chain Monte Carlo (MCMC) phylogenetic inferences. Transmission clusters were identified using interior branch test and depth-first searches for sub-tree partitions. Times of most recent common ancestor (tMRCAs) of significant clusters were estimated using Bayesian MCMC analysis. Results Among 3618 patient registered in our network, 243 were infected with CRF01_AE. The majority of individuals with CRF01_AE were Japanese, predominantly male, and reported heterosexual contact as their risk factor. We found 5 large clusters with ≥5 members and 25 small clusters consisting of pairs of individuals with highly related CRF01_AE strains. The earliest cluster showed a tMRCA of 1996, and consisted of individuals with their known risk as heterosexual contacts. The other four large clusters showed later tMRCAs between 2000 and 2002 with members including intravenous drug users (IVDU) and non-Japanese, but not men who have sex with men (MSM). In contrast, small clusters included a high frequency of individuals reporting MSM risk factors. Phylogenetic analysis also showed that some individuals infected with HIV strains spread in East and South-eastern Asian countries. Conclusions Introduction of CRF01_AE viruses into Japan is estimated to have occurred in the 1990s. CFR01_AE spread via heterosexual behavior, then among persons connected with non-Japanese, IVDU, and MSM. Phylogenetic analysis demonstrated that some viral variants are largely restricted to Japan, while others have a broad geographic distribution.

Beneficial Effect of GB Virus C Co-Infection in Human Immunodeficiency Virus Type 1-Infected Individuals

Several reports have documented a better prognosis for HIV‐1‐infected patients co‐infected with GBV‐C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV‐C co‐infection on HIV‐1‐infected patients. GBV‐C RNA was detected in 37 samples in 182 HIV‐1‐infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV‐C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV‐C viral load quantified by real‐time PCR ranged from 7.8 × 103 to 3.3 × 106 copies/ml. Weakly negative correlation was observed between GBV‐C viral load and HIV‐1 viral load in 19 HAART‐naïve patients, indicating that a higher GBV‐C viral load is associated with a greater suppression of HIV‐1 replication. A previously published in vitro study suggested that GBV‐C infection would induce up‐regulation of RANTES, leading to suppression of HIV‐1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV‐C co‐infected group than in the uninfected group (190–9,959 vs. 264–31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV‐1 replication by GBV‐C co‐infection is not mediated by up‐regulated RANTES, and thus call for another as yet unknown factor.

Analysis of Near Full-Length Genomic Sequences of Drug-Resistant HIV-1 Spreading among Therapy-Naïve Individuals in Nagoya, Japan: Amino Acid Mutations Associated with Viral Replication Activity

We analyzed a total of 12 near full-length genomes of drug-resistant HIV-1 spreading among therapy-naïve individuals in Nagoya, Japan. Genomes comprised seven protease inhibitor (PI)-resistant viruses possessing an M46I (n = 6) or L90M mutation (n = 1) and five non-nucleoside reverse transcriptase inhibitor-resistant viruses possessing a K103N mutation. All 12 viruses conserved both an H87Q mutation in the cyclophilin A-binding site of Gag p24 (capsid) and a T23N mutation in the cysteine-rich domain of Tat protein. PI-resistant viruses commonly possessed two cleavage site mutations in the p6(Pol)/protease of Pol polyprotein (F48L in p6(Pol)) and the anchor/core domains of Nef protein (L57V). These amino acid mutations represent candidates for enhancing replication activity of drug-resistant viruses and supporting expansion of such viruses in therapy-naïve individuals.

Characteristics of Transmitted Drug-Resistant HIV-1 in Recently Infected Treatment-Naive Patients in Japan.

Objectives:Progress in antiretroviral treatment has led to fewer virological failure cases, but 10%–20% of treatment-naive HIV/AIDS cases are reported to harbor drug-resistant strains, suggesting transmission of drug-resistant HIV. We aimed to determine the trend in prevalence of transmitted drug-resistant (TDR) HIV in Japan, particularly in recently infected patients. Methods:Drug-resistance test was performed on 3904 HIV-1–infected cases newly diagnosed between 2007 and 2012. The number of cases infected within 6 months [recent seroconverters (RS)] was estimated by BED assay of 2700 plasma samples. Characteristics of RS cases were further analyzed. Results:The overall prevalence of TDR was 9.1%, ranging from 7.3% in 2008% to 12.5% in 2010. Among 1403 subtype B/E/D cases with >50 CD4+ T cell counts and >1000 HIV copies per milliliter, 468 (33.4%) were estimated to be RS. The prevalence of RS was significantly higher among cases who were male, Japanese, and men who have sex with men. The prevalence of TDR did not differ significantly between recent and long-term seroconverters (8.5% vs. 9.2%, respectively, P = 0.68). Common mutations in both groups were M46I/L and T215 revertants. Furthermore, sequences with these mutations, K103N and D30N/N88D formed clusters on phylogenetic trees. Conclusion:Our study clarified an increase in prevalence of TDR in Japan from 2007 to 2012. The phylogenetic clustering of cases with M46I/L or T215 revertants suggests that HIV with these mutations have become circulating strains. Furthermore, detailed analyses showed that Japanese men who have sex with men are more aware of their risk of HIV infection.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

Longitudinal Detection and Persistence of Minority Drug-Resistant Populations and Their Effect on Salvage Therapy

Background Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals. Materials and Methods Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3–2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis. Results The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment. Discussion and Conclusion Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses detectable by AS-PCR highlight the clinical importance of low-frequency drug-resistant viruses. Thus, our results highlight the usefulness of AS-PCR and support its expanded evaluation in ART clinical management.