Prevalence and Genotypic Diversity of Human Papillomavirus Infection Among Male and Female Sex Workers in Thailand.

BACKGROUND: Human papillomavirus (HPV) is associated with cancer. Female sex workers (FSWs) are known to be at risk for HPV, but little is known about male sex workers (MSWs). METHODS: We examined HPV prevalence and associated risk factors in both populations. During 2022, HPV testing using vaginal or penile samples, HIV testing, and interviews were performed among 100 MSWs and 100 FSWs in Chiang Mai, Thailand. RESULTS: The prevalence of all HPV types was 63.5% (66% in MSW, 61% in FSW), HPV-16 prevalence was 14%, HPV-52 was 13%, and HPV-18 was 4%. There was no difference between MSW and FSW for these subtypes. The prevalence of HPV-16 or HPV-18 was 17%, and for HPV-16, HPV-18, or HPV-52, it was 26%. HIV-positive participants had a higher prevalence of all HPV types (94% vs. 60%, P = 0.004), HPV-16 or HPV-18 (39% vs. 15%, P = 0.018), and HPV-16, HPV-18, or HPV-52 (50% vs. 23%, P = 0.017). CONCLUSIONS: We demonstrated an equally high HPV prevalence across the sexes. Further studies are needed to determine if this indicates an equal risk for cancer. Increased HPV awareness, screening, and vaccination should be considered, regardless of gender.

Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Role of genomic DNA methylation in detection of cytologic and histologic abnormalities in high risk HPV-infected women.

Cervical cancer is the fourth most common malignancy affecting women worldwide. The development of disease is related to high-risk human papillomavirus (hrHPV) infection. Cytology has been the most recommended triage for primary cervical (pre)cancer screening despite relatively low sensitivity. Recently, genomic DNA methylation has been proposed as an additional marker to increase sensitivity for detecting cervical precancerous lesion. This study aimed to evaluate the performance of methylation status of three tumor suppressor genes (CADM1, FAM19A4, and MAL) and HPV genotyping in detection of cytologic and histologic abnormalities in cervical cancer screening. Two hundred and sixty samples with available frozen cell pellets including 70 randomly selected cases of negative for intraepithelial lesion or malignancy (NILM)&HPV-negative, 70 randomly selected cases of NILM&HPV-positive, and 120 cytologic abnormalities & HPV-positive from a population-based cervical cancer screening program (n = 7,604) were investigated for the DNA methylation pattern of CADM1, FAM19A4, and MAL. Of 120 cytologic abnormalities & HPV-positive cases, there were 115 available histologic results. HPV52 and HPV58 were most commonly found in histologic HSIL+. The methylation levels of CADM1, FAM19A4, and MAL were elevated with the severity of cytologic abnormality which significantly increased by 3.37, 6.65 and 2 folds, respectively, in cytologic HSIL comparing with NILM. A significant increase in methylation levels of these three genes was also observed in histologic HSIL+ compared with negative histology but only CADM1 showed a significant higher methylation level than histologic LSIL. Using the ROC curve analysis, DNA methylation levels of FAM19A4 performed best in differentiating high-grade cytology (ASC-H+ from NILM/ASC-US/LSIL), followed by CADM1 and MAL. Whilst the CADM1 methylation performed best in distinguishing histologic HSIL+ from negative/LSIL with an area under the ROC curve of 0.684, followed by MAL (0.663) and FAM19A4 (0.642). Interestingly, after combining high DNA methylation levels to HPV16/18 genotypes, rates of histologic HSIL+ detection were substantially increased from 25% to 79.55% for CADM1, 77.27% for FAM19A4, and 72.73% for MAL, respectively. The rate further increased up to 95.45% when at least one of three genes had a high methylation level. This suggests a possible role of genomic DNA methylation, especially CADM1, in detecting histologic HSIL+ lesions in combination with hrHPV testing.

Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients.

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.

Association of cytologic grade of anal "Pap" smears with viral loads of human papillomavirus types 16, 18, and 52 detected in the same specimens from men who have sex with men.

BACKGROUND: Human papilloma virus (HPV) load has been linked to cellular abnormalities of the uterine cervix, and proposed as predictors of HPV persistence and progression of dysplasia to cervical cancer. However, the association of HPV viral load and anal dysplasia and cancer has not been as thoroughly investigated. OBJECTIVES: To examine the association of the viral loads of high-risk HPV types 16, 18, and 52, with the cytologic severity grading in anal-swab specimens of MSM with and without HIV-1 co-infection. STUDY DESIGN: A cross-sectional study recruited 200 MSM in northern Thailand from July 2012 to January 2013. Real-time qPCR amplified portion of the HPV E6E7 gene, as well as the human ß-globin gene to validate adequacy of the anal specimens and to normalize interpatient viral-load comparisons. Genotyping by linear-array assay identified and distinguished types 16, 18, and 52. RESULTS: HPV-16, and -18 viral loads increased with respect to the abnormality of the cytologic diagnoses (p<0.05 for HPV-16, p<0.01 for HPV-18). HIV-1 positivity was associated with higher HPV-18 viral load (p=0.006). HPV-16 viral loads ≥102.24 copies per 5000 anal cells, and HPV-18 loads ≥103.15, were independently associated with abnormal cytology on logistic regression (p=0.022, p=0.041, respectively). Positive predictive values were 85.2% (23/27) and 80.0% (44/55) for the high viral load of a particular HPV-16 and the combined HPV-16, -18 and -52 types, respectively. CONCLUSIONS: High viral loads of HPV types 16 and 18 appear to be associated with anal cytologic abnormalities. The clinical utility of HPV viral loads to predict risk for anal cancer remains to be determined by a larger prospective cohort with sufficient frequency of high-grade dysplasia.

Cytological Anal Squamous Intraepithelial Lesions Associated with Anal High-Risk Human Papillomavirus Infections among Men Who Have Sex with Men in Northern Thailand.

BACKGROUND: Anal cancer, one of human papillomavirus (HPV) related malignancies, has increased in recent decades, particularly among men who have sex with men (MSM) and HIV-infected (HIV+) persons. We aimed to explore the prevalence of anal squamous intraepithelial lesions (ASIL) using Papanicolau (Pap) screening among MSM in northern Thailand and its associated factors. METHODS: Two hundreds MSM aged ≥18 years reporting receptive anal intercourse in the prior 6 months were recruited from July 2012 through January 2013. Medical history and behavioral data were collected by staff interview and computer-assisted self interview. Anal Pap smear, HPV genotyping, and HIV testing were performed. Two pathologists blinded to HPV and HIV status reported cytologic results by Bethesda classification. RESULTS: Mean age was 27.2 years (range 18-54). Overall, 86 (43.0%) had ASIL: 28 (14.2%) with atypical cells of undetermined significance (ASCUS), 1 (0.5%) with atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H), 56 (28.4%) with low-grade squamous intraepithelial lesion (LSIL), and 1 (0.5%) with high-grade squamous intraepithelial lesion (HSIL). ASIL was associated by univariate analysis (p ≤0.05) with older age, gender identity other than bisexual (i.e., gay men and transgender women), rectal douching, anal symptoms, genital warts, HIV positivity, and high-risk-HPV infection. However, on multiple logistic regression ASIL was associated only with high-risk HPV type (p = 0.002) and HIV infection (p = 0.01). CONCLUSIONS: ASIL is quite common in high-risk MSM in northern Thailand and is associated with high-risk HPV types and HIV infection. Routine anal Pap screening should be considered, given the high frequency of ASIL, particularly in the HIV+. High resolution anoscopy (HRA), not done here, should be to confirm PAP smears whose sensitivity and specificity are quite variable. Timely HPV vaccination should be considered for this population.

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.

High Prevalence and Genotype Diversity of Anal HPV Infection among MSM in Northern Thailand.

BACKGROUND: HPV infection is common and may cause cancer among men who have sex with men (MSM). Anal HPV infection (HPV+) was found in 85% of HIV-positive (HIV+) and 59% of HIV-negative (HIV-) MSM in Bangkok, central Thailand. As little is known about HPV in this group in northern Thailand, we studied MSM subgroups comprised of gay men (GM), bisexual men (BM), and transgender women (TGW). METHODS: From July 2012 through January 2013, 85 (42.5% of 200) GM, 30 (15%) BM, and 85 (42.5%) TGW who practiced receptive anal intercourse were recruited after informed consent, followed by self-assisted computer interview, HIV testing, and anal swabs for HPV genotyping. RESULTS: Of 197 adequate specimens, the overall prevalence of any HPV was 157 (80%). Prevalence was 89% (76/85) in GM, 48% (14/29) in BM, and 81% (67/83) in TGW. The most common high-risk types were HPV16 (27% of 197), HPV58 (23%), and HPV51 (18%). Prevalence of high-risk types was 74% in 85 GM, 35% in 29 BM, and 71% in 83 TGW. Prevalence of any HPV type, or high-risk type, was 100% and 94%, respectively, among 48 HIV+ MSM, 70% and 54% among 120 HIV- MSM. Of the 197 specimens, 36% (70) had HPV types 16 and/or 18 in the bivalent vaccine, compared to 48% (95) with ≥1 of types 16/18/06/11 in the quadrivalent, 56% (111) for 16/18/31/33/45/52/58 in the 7-valent, and 64% (126) for 16/18/31/33/45/52/58/06/11 in the 9-valent. HIV+, GM, and TGW were independently associated with HPV infection. CONCLUSIONS: We found higher rates of both any HPV and high-risk types than previous studies. Among the heretofore unstudied TGW, their equivalent HPV rates were comparable to GM. Current and investigational HPV vaccines could substantially protect GM, BM, and TGW from the serious consequences of HPV infection especially among HIV + MSM.

HPV detection and genotyping in vulvar squamous cell carcinoma in northern Thailand.

BACKGROUND: The study was aimed to evaluate the prevalence and genotype distribution of HPV infection in vulvar squamous cell carcinoma (SCC) in northern Thailand and the clinicopathological difference with regard to HPV infection status. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissue samples of vulvar SCC diagnosed between January 2006 and December 2012 were collected. HPV infection was detected by nested polymerase chain reaction (PCR) with primers MY09/11 and GP5+/6+. HPV genotyping was performed using the Linear Array Genotyping Test, followed by type-specific PCR targeting the E6/E7 region of HPV16/18/52 if the Linear Array test was negative. The histologic slides of vulvar lesions and the medical records were reviewed. RESULTS: There were 47 cases of vulvar SCC included in the study (mean patient age 57.9 ± 13.2 years). HPV infection was detected in 29 cases (62%), all of which had single HPV infections. HPV16 accounted for 23 (49%). The patients with HPV-positive SCC had a significantly younger mean age than those with HPV-negative tumors (52.7 years vs 66.2 years, p<0.001). There was no significant difference in tumor stage distribution with regard to the status of HPV infection. The presence of vulvar intraepithelial neoplasia (VIN) of usual type (basaloid or warty) was significantly more frequent in HPV-positive cases compared with HPV-negative cases (62% vs 6%, p<0.001), whereas differentiated-type VIN was more common in HPV-negative cases (24% vs 0%, p=0.019). CONCLUSIONS: HPV infection was detected in 62% of vulvar SCC in northern Thailand. HPV16 was the predominant genotype similar to the data reported from other regions. HPV-positive SCC occurred in younger patients compared with HPV-negative SCC, and was associated with usual-type VIN. Vaccination against HPV16/18 may potentially prevent almost one half of vulvar SCC in northern Thailand.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies.

HPV genotyping in adenocarcinoma of the uterine cervix in Thailand.

OBJECTIVE: To determine the distribution of human papillomavirus (HPV) genotypes in cervical adenocarcinoma in Thailand and to evaluate the clinicopathologic characteristics associated with common HPV genotypes. METHODS: Formalin-fixed, paraffin-embedded tissues from 150 patients with adenocarcinoma were collected from 4 areas of Thailand. Infection with HPV was detected by nested polymerase chain reaction (PCR) with primers MY09/11 and GP5+/6+. Genotyping was performed using a linear array assay, followed by type-specific PCR targeting the E6/E7 regions of HPV-16, HPV-18, and HPV-52 if the linear array test was negative. RESULTS: Human papillomavirus DNA was detected in 145 (97%) adenocarcinomas (132 single infections; 11 multiple infections; 2 tumors with undetermined HPV type). Genotype 18 was most common (66%), followed by HPV-16 (30%) and HPV-45 (3%). Infection with only HPV-16 and/or HPV-18 accounted for 88% of the HPV-positive tumors. Patients with HPV-18 infection had a younger age (P=0.009) and higher tumor grade (P<0.001) than patients with HPV-16 infection. CONCLUSION: The HPV detection rate in cervical adenocarcinomas in Thailand is high. The predominant genotype is HPV-18, being twice as common as HPV-16. Genotype variations are associated with patient age and tumor grade. Vaccination against HPV-16/HPV-18 might prevent almost 90% of adenocarcinomas.

Improvement of the oligonucleotide ligation assay for detection of the M184V drug-resistant mutation in patients infected with human immunodeficiency virus type 1 subtype CRF01_AE.

Methods based on genetic sequencing to monitor drug-resistance mutations in human immunodeficiency virus type 1 (HIV-1) require expensive instruments and are only capable of detecting mutant strains comprising >20% of virus populations. The National Institutes of Health's AIDS Research and Reference Reagent Program (NIH ARRRP) makes available a probe-based method, an oligonucleotide ligation assay (OLA-ARRRP), which is less expensive and more sensitive than sequencing to detect such mutations for HIV-1 subtype B. In this study, an OLA was designed to detect the Methionine to Valine mutation at codon 184 (M184V) of the reverse transcriptase (RT) gene in the circulating recombinant form AE strain of HIV-1 (HIV-1 CRF01_AE) common in Thailand, and was evaluated in Thai patients experiencing treatment failure. The subtype-specific OLA-CRF01_AE proved superior to OLA-ARRRP in detecting M184V, although this mutation existed in the genome of the multiple-drug-resistant virus at lower minimal detection levels of 3% prevalence of mutated virus, compared to 50% for OLA-ARRRP. On evaluation using clinical specimens, OLA-CRF01_AE showed excellent agreement with nucleotide sequencing (95.1% overall concordance, kappa>0.79), and the sensitivity was 100% for wild-type and 93.9% for mutant detection at codon 184. The OLA-CRF01_AE also detected M184V mutations in 2.4% (1/42) of specimens that were not detected by sequencing. The indeterminate detection by OLA-CRF01_AE was decreased, from 16.7% to 4.8%, in the samples containing mutant genotype when the strategy using unmodified- as a substitute of the modified-mutant detector probe was applied. Because of their low cost, sensitivity, and ease of use, the OLA-CRF01_AE is an attractive alternative to standard sequencing in resource-limited countries affected by this subtype of HIV-1.

Prognostic value of HPV18 DNA viral load in patients with early-stage neuroendocrine carcinoma of the uterine cervix.

OBJECTIVES: To evaluate the clinicopathologic correlation and prognostic value of HPV18 DNA viral load in patients with early-stage cervical neuroendocrine carcinoma (NECA). METHODS: Formalin-fixed, paraffin- embedded tissue of cervical NECA patients with known HPV18 infection and clinicopathologic data including follow-up results were collected. The HPV18 DNA load was assessed with quantitative PCR targeting the HPV18 E6E7 region. RESULTS: Twenty-one patients with early-stage (IB-IIA) cervical NECA were identified. HPV18 DNA viral load ranged from 0.83 to 55,174 copies/cell (median 5.90). Disease progression, observed in 10 cases (48%), was not significantly associated with any clinicopathologic variables. However, the group of patients with progressive disease tended to have a higher rate of pelvic lymph node metastasis (50% versus 9%, p=0.063) and a lower median value of HPV18 DNA viral load (4.37 versus 8.17 copies/cell, p=0.198) compared to the non-recurrent group. When stratified by a cut-off viral load value of 5.00 copies/cell, the group of patients with viral load ≤5.00 copies/cell had a significantly shorter disease-free survival than the group with viral load >5.00 copies/cell (p=0.028). The group with a lower viral load also tended to have a higher rate of disease progression (75% versus 31%, p=0.080). No significant difference in the other clinicopathologic variables between the lower and higher viral load groups was identified. CONCLUSION: THPV18 DNA viral load may have a prognostic value in patients with early-stage NECA of the cervix. A low viral load may be predictive of shortened disease-free survival in these patients.

HPV genotyping in neuroendocrine carcinoma of the uterine cervix in northern Thailand.

OBJECTIVE: To determine the distribution of HPV genotypes in cervical neuroendocrine carcinoma (NECA) in northern Thailand, and evaluate the correlation between HPV genotype and clinicopathologic features. METHODS: Samples from 111 women treated for cervical NECA at Chiang Mai University Hospital between 1992 and 2009 were tested for HPV genotype. Samples were formaldehyde-fixed, paraffin-embedded, and tested via nested PCR and dot blot hybridization. RESULTS: Ninety-seven of the 111 samples were adequate for DNA analysis. HPV DNA was detected in 93 samples, of which 76 (81.7%) were single, 14 (15.1%) were multiple, and 3 (3.2%) were untyped infections. HPV18 was the most common subtype (70 cases, 75.3%), followed by HPV16 (28 cases, 30.1%). Other genotypes included HPV58 (3.2%), HPV52 (2.1%), and HPV33 (1.1%). Collectively, HPV16 and/or HPV18 were found in 83 cases (89.3%). Women with HPV18 infection were significantly younger (42.0years) than those with non-HPV18 infections (54.1years) (P=0.003). Associated adenocarcinoma in situ was more frequently seen among women with HPV18 infection (P=0.034). CONCLUSIONS: HPV18 infection was predominant in cervical NECA. Variations in HPV genotype may be related to the clinicopathologic features and pathogenetic pathways of NECA. Vaccination against HPV16 and HPV18 might provide protection against cervical NECA in almost 90% of cases.

Influence of pr-M cleavage on the heterogeneity of extracellular dengue virus particles.

During dengue virus replication, an incomplete cleavage of the envelope glycoprotein prM, generates a mixture of mature (prM-less) and prM-containing, immature extracellular particles. In this study, sequential immunoprecipitation and cryoelectron microscopy revealed a third type of extracellular particles, the partially mature particles, as the major prM-containing particles in a dengue serotype 2 virus. Changes in the proportion of viral particles in the pr-M junction mutants exhibiting altered levels of prM cleavage suggest that the partially mature particles may represent an intermediate subpopulation in the virus maturation pathway. These findings are consistent with a model suggesting the progressive mode of prM cleavage.

Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction.

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.

Investigation of human brain tumors for the presence of polyomavirus genome sequences by two independent laboratories.

JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) may be associated with human brain tumors. These polyomaviruses have been shown to induce brain tumors in experimentally infected animals. Several studies have found polyomavirus genomic sequences in human brain tumor tissues by using polymerase chain reaction (PCR), while others have not. Inconsistencies in previous findings may be due in part to small sample sizes and differences in underlying patient populations, laboratory techniques and quality control measures. To assess the role of polyomaviruses in human brain tumors and address inconsistencies of previous reports, we investigated the prevalence of viral sequences in a series of 225 brain tumor tissue specimens in 2 independent laboratories. PCR followed by Southern hybridization was performed at the National Institute of Neurological Disorders and Stroke (NINDS). Real-time quantitative PCR was performed on the same tissues at Johns Hopkins University (JHU). Only those tumors with amplifiable DNA were tested further for polyomavirus sequences. Positive and negative control tissues were included, and all specimens were masked. Amplifiable DNA was detected in 225/225 (100%) tumors at NINDS, 9 (4%) of which contained polyomavirus sequences (3 JCV-positive, 3 BKV-positive and 3 SV40-positive). The JHU laboratory amplified DNA from 165/225 (73%) tumors, of which 1 tumor tested positive (for SV40). No tumors tested positive in both laboratories. Results for masked quality control tissues were concordant between laboratories. Nucleotide sequences for JCV, BKV and SV40 are rarely present in a large series of adult and pediatric brain tumors.

Coreceptor utilization of HIV type 1 subtype E viral isolates from Thai men with HIV type 1-infected and uninfected wives.

HIV-1 coreceptors CCR5 and CXCR4 play an important role in viral entry and pathogenesis. To better understand the role of viral tropism in HIV-1 transmission, we examined the coreceptor utilization of viral isolates obtained from men enrolled in a study of heterosexual transmission in northern Thailand. Viral isolates were obtained from HIV-1-positive males who had either HIV-1-infected spouses (RM; n = 5) or HIV-1-uninfected spouses (HM; n = 10). Viral isolates from 1 of the 5 RM males and 2 of the 10 HM males were CCR5 tropic, whereas isolates from 3 RM males and 6 of the HM male isolates were CXCR4 tropic. Of the nine X4-tropic isolates, seven also used at least one of the following coreceptors: CCR8, CCR1, CCR2b, or CX3CR1, and none employed CCR5 as an additional coreceptor. More importantly, three isolates, RM-15, HM-13, and HM-16 (one from a transmitter and two from nontransmitter), did not infect GHOST4.cl.34 cells expressing any of the known coreceptors. Further analysis using MAGI-plaque assays, which allow visualization of infected cells, revealed that RM-15 had low numbers of infected cells in MAGI-R5 and MAGI-X4 cultures, whereas HM-13 and HM-16 had high levels of plaques in MAGI-X4 cultures. Replication kinetics using activated lymphocytes revealed that these three isolates replicated in CCR5(+/+) as well as CCR5(-/-) peripheral blood mononuclear cells, suggesting that these isolates did not have an absolute requirement of CCR5 for viral entry. All three isolates were sensitive to the X4-antagonistic compounds T-22 and AMD3100. Analysis of the C2V3 region did not reveal any significant structural differences between any of the Thai subtype E isolates. Thus, there was no association between the pattern of coreceptor usage and transmissibility among these subtype E HIV-1 isolates.