Phylogenetic analysis of two new complete genomes of the hepatitis E virus (HEV) genotype 3 from Thailand.

Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis globally. Evolutionary phylogeny classifies the HEV into eight genotypes that correlate with the viral transmission. Only four genotypes have been proven to be responsible for transmission in humans. However, there has been no report on the genomics and genotyping of HEV in Thailand during the past ten years. Here, we identified the genotype distributions of the Thai isolates of HEV and we sequenced two HEV genomes. We screened for 18 Thai isolates of HEV from Siriraj Hospital in Bangkok, from 2014-2016. The HEV genomes were sequenced from the serum and feces of a patient. The results showed that all Thai isolates of HEV were identified as genotype 3 (HEV-3). The ORF2 and genome phylogenies suggested two subgenotypes, called 3.1 and 3.2. The Thai isolates of HEV were frequently found in the subgenotype 3.1. The genome sequences of the two Thai isolates of HEV from the serum and fecal samples of the same patient showed 91% nucleotide similarity with the HEV genotype 3. Comparisons between the HEV genome and the ORF2 phylogenies illustrated that the ORF2 tree can be used to identify HEV genotypes, but it has less phylogenetic power for the HEV evolution. The two new genome sequences of HEV-3 from Thailand could contribute valuable information to the HEV genome study. (226 words).

A Nationwide Survey of the Seroprevalence of Hepatitis E Virus Infections Among Blood Donors in Thailand.

Hepatitis E virus (HEV) is a common cause of acute hepatitis infections. Our previous 3-year study at two large Thai hospitals established an occurrence of 4-5% of HEV infections from swine HEV genotype 3 in suspected acute hepatitis patients, with the high incidence in older adults. This study was a serosurvey to determine the prevalence of HEV infections among Thai adults. We obtained sera from 630 healthy blood donors with a median age of 38 (18-64) years who attended Thai Red Cross transfusion units throughout Thailand. The donors were domiciled in 16 provinces in the northern (n = 159), central (n = 193), northeastern (n = 158), and southern (n = 120) regions. The seroprevalence of IgG antibody to HEV (anti-HEV) was determined by the EUROIMMUN test kit, using indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant antigens derived from ORF2 of HEV genotypes 1 and 3. Demographic data, including information related to HEV infection risk (the number of pigs and the proportion of Muslims in each province), were also obtained. The overall anti-HEV prevalence among Thai adults was 29.7%. The frequencies of anti-HEV found in the northern (28.9%, 95% confidence interval [CI] = 22.4-36.4), northeastern (34.8%, 95% CI = 27.8-42.5), and central (35.8%, 95% CI = 29.3-42.7) regions were similar, whereas the frequency in the southern (14.2%, 95% CI = 9.0-21.5) region, known to have a large Muslim population, was low. An increasing frequency of the specific antibody was observed among the elderly. A low HEV infection rate was associated with an Islamic population where there are low number of pigs and low swine consumption. Furthermore, the higher anti-HEV incidences in the northeastern provinces might relate to the local cultural practice of consuming undercooked pork. Besides the need for an HEV vaccination in the future, there is a requirement for rapid early diagnosis; the undertaking of prevention-management campaigns might also reduce the number of infected patients.

Cross-reactive antibodies against H7N9 and H5N1 avian influenza viruses in Thai population.

BACKGROUD: Avian influenza H5N1 and H7N9 viruses have jumped across species from avian to humans and become a threat to public health. Not much is known about pre-existing cross-reactive antibodies against these avian viruses in human population. OBJECTIVE: To determine the prevalence of cross-reactive anti-HA and anti-NA antibodies to avian influenza H5N1 and H7N9 viruses in Thai population. METHOD: Archival serum samples from 100 blood donors and 21 patients infected with 2009 pandemic influenza A (H1N1) (pdmH1N1) virus were investigated by hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays for anti-HA and anti-NA antibodies, respectively. The test antigens comprised 2 human viruses (pdmH1N1 and H3N2 viruses), and 6 reassortant viruses carrying HA and NA genes of avian H5N1 or H7N9 virus generated by reverse genetics. RESULTS: HAI antibody titers ≥ 10 were found in 58, 89, 0 and 15% of blood donors as tested against pdmH1N1, H3N2, H5N1 and H7N9 viruses, respectively. On the other hand, NAI antibodies were detected in 98, 94, 73 and 53% of blood donors when reverse genetic-derived viruses harboring NA gene from pdmH1N1, H3N2, H5N1 or H7N9 virus were used as the test antigens. Moreover, 66.7% of pdmH1N1 patients who had > 4 fold increase in HAI antibody titers in paired sera developed > 4 fold increase in NAI antibody titers. CONCLUSIONS: Anti-NA antibody has broader reactivity than anti-HA antibody, therefore, it can be a supplement to anti-HA antibody in the prevention against novel influenza viruses.

Safety and immune responses following administration of H1N1 live attenuated influenza vaccine in Thais.

BACKGROUND: Emergence and rapid spread of influenza H1N1 virus prompted health authorities to develop a safe and effective influenza vaccine for domestic use. The Thai Government Pharmaceutical Organization (GPO) with technical support from Russia through WHO had prepared a pandemic live attenuated vaccine (PLAIV) using ca-ts attenuated candidate strain A/17/CA/2009/38 (H1N1) for Thais. METHODS: Each participant received two doses of intranasal H1N1 vaccine or placebo 21 days apart. All were followed up at 7, 21, 42 and 60 days after first immunization. Blood was drawn for hemagglutination inhibition (HAI) assay from all participants at days 1, 21, 42, and 60 after first immunization. A subset of 40 participants aged 19-49 years was randomly selected for nasal washing at days 1, 21, 42, and 60 to assess IgA using direct enzyme-linked immunosorbent assay (ELISA) along with serum HAI and microneutralization (MN) assay determination. RESULTS: A total of 363 subjects aged 12-75 years were randomized into 2 groups (271 vaccinees:92 placebos). Almost all AEs were mild to moderate. Local reactions were stuffy nose (22.3%), runny nose (25.1%), scratchy throat (27.2%) and sore throat (19.3%). Systemic reactions included headache (21.7%), myalgia (13.8%), fatigue (16.8%) and postnasal drip (19.9%). On day 60, HAI seroconversion rates for vaccine:placebo group were 30.3:6.0 for ITT and 29.4:5.1 for PP analysis. Children showed highest seroconversion rate at 44, but it decreased to 39.4 when all 3 assays (HAI, MN assay and ELISA) from subgroup analysis were considered. CONCLUSION: The vaccine candidate is safe. The use of more than one assay may be needed for evaluation of immune response because live attenuated vaccines could effectively induce different kinds of responses. Different individuals could also mount different kinds of immune response, even to the same antigen.

The distribution of hepatitis B virus genotypes in Thailand.

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Serological response to the 2009 pandemic influenza A (H1N1) virus for disease diagnosis and estimating the infection rate in Thai population.

BACKGROUND: Individuals infected with the 2009 pandemic virus A(H1N1) developed serological response which can be measured by hemagglutination-inhibition (HI) and microneutralization (microNT) assays. METHODOLOGY/PRINCIPAL FINDINGS: MicroNT and HI assays for specific antibody to the 2009 pandemic virus were conducted in serum samples collected at the end of the first epidemic wave from various groups of Thai people: laboratory confirmed cases, blood donors and health care workers (HCW) in Bangkok and neighboring province, general population in the North and the South, as well as archival sera collected at pre- and post-vaccination from vaccinees who received influenza vaccine of the 2006 season. This study demonstrated that goose erythrocytes yielded comparable HI antibody titer as compared to turkey erythrocytes. In contrast to the standard protocol, our investigation found out the necessity to eliminate nonspecific inhibitor present in the test sera by receptor destroying enzyme (RDE) prior to performing microNT assay. The investigation in pre-pandemic serum samples showed that HI antibody was more specific to the 2009 pandemic virus than NT antibody. Based on data from pre-pandemic sera together with those from the laboratory confirmed cases, HI antibody titers ≥ 40 for adults and ≥ 20 for children could be used as the cut-off level to differentiate between the individuals with or without past infection by the 2009 pandemic virus. CONCLUSIONS/SIGNIFICANCE: Based on the cut-off criteria, the infection rates of 7 and 12.8% were estimated in blood donors and HCW, respectively after the first wave of the 2009 influenza pandemic. Among general population, the infection rate of 58.6% was found in children versus 3.1% in adults.

Occult hepatitis B virus infection in Thai blood donors.

BACKGROUND: An evaluation by the National Blood Center, the Thai Red Cross Society, of two commercial multiplex nucleic acid tests (NATs; the Chiron PROCLEIX ULTRIO test and the Roche Cobas TaqScreen MPX test) for screening Thai blood donors for hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency virus Type 1 identified 175 HBV NAT-reactive/hepatitis B surface antigen (HBsAg)-negative donors. The classification of the HBV infection of these donors was confirmed by follow-up testing. STUDY DESIGN AND METHODS: Index samples were tested for HBV serologic markers and HBV viral loads were determined. Donors were followed for up to 13 months and samples were tested with both NAT assays and for all HBV serological markers. RESULTS: Of 175 HBV NAT-yield donors, 72 (41%) were followed. Based on the follow-up results, the majority of donors who were followed had an occult HBV infection (66.7%), followed by donors with a primary, acute infection (26.4%). The majority of donors in this latter group (20.8%) were in the window period. Three donors (4.2%), who were anti-HBs positive, had a reinfection or breakthrough infection. CONCLUSION: The majority of donors detected during routine screening, who were HBsAg negative and NAT reactive, had an occult HBV infection, thus validating the decision to introduce NAT for blood donations in Thailand.

Erythrocyte binding preference of 16 subtypes of low pathogenic avian influenza and 2009 pandemic influenza A (H1N1) viruses.

All 16 subtypes of avian influenza viruses of low pathogenicity (LPAIV) as well as their hemagglutinin (H) antigens, and four 2009 pandemic influenza A (H1N1) virus isolates were assayed for hemagglutinating activity against 5 erythrocyte species: goose, guinea pig, human group O, chicken and horse. Of all viruses and antigens assayed, the highest hemagglutination (HA) titers were obtained with goose and guinea pig erythrocytes. Hemagglutinating activity of replicating LPAIV and LPAIV antigens decreased, in order, with chicken and human group O; meanwhile, horse erythrocytes yielded lowest or no HA titer. Moreover, the 2009 pandemic viruses did not agglutinate both horse and chicken erythrocytes. Our study concluded that goose and guinea pig erythrocytes are the best in HA assay for all subtypes of influenza viruses.

HIV-1 subtype B Tat gene activities and disease progression in HIV-1 CRF01_AE infection.

HIV-1 tat gene function and immunogenicity of HIV-1 Tat protein from 3 low (PS01, PS40, PS58) and 3 high (PS19, PS65, LP22) viral load infected, untreated and asymptomatic individuals from Thailand were compared. Levels of Tat-dependent chloramphenicol acetyltransferase (CAT) induced in HL3T1 cells with tat1 gene from HIV-1 isolates of high viral load group was significantly higher than those from low viral load group. HIV-1 subtype determination using env (C2-V4) gene demonstrated that 2/3 (PS01 and PS40) and 1/3 (PS58) from low viral load group were CRF01_AE and subtype B, while all 3 HIV-1 isolates from high viral load group were CRF01_AE. However, all 3 HIV-1 tat nucleotide sequences from low viral load group, which contained env CRF01_AE sequence, belonged to subtype B whereas all those from high viral load group contained CRF01_AE sequence. HIV Tat recombinant proteins from these groups were tested for immunogenicity in mice. All recombinant Tat proteins (except from PS58) were immunogenic in a dose-dependent manner, but with significantly differences of the immunogenicity levels between high and low viral load groups. These results indicated that HIV-1 subtype B tat gene activities might be associated with reduced disease progression of HIV-1 CRF01_AE infected individuals.

Kinetics and longevity of antibody response to influenza A H5N1 virus infection in humans.

Anti-H5N1 antibody was determined by microneutralization, hemagglutination inhibition, and Western blotting assays in serial blood samples collected from eight Thai patients, including four fatal cases and four survivors. The antibody was detected as early as 5 days and, typically, with an increase in titer in paired blood at about 15 days after disease onset. The anti-H5 antibody response was long-lasting, for almost 5 years in cases which can be followed that far. In addition, cross-neutralizing activity to related clade 1 viruses was observed.

One-year experience of nucleic acid technology testing for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations.

BACKGROUND: Blood donations collected at the National Blood Center, the Thai Red Cross Society, Bangkok, in 2007 were tested by nucleic acid amplification technology (NAT) using the Chiron TIGRIS/Procleix Ultrio test and the Roche cobas s 201/cobas TaqScreen multiplex (MPX) test. STUDY DESIGN AND METHODS: The sensitivity, specificity, and robustness were determined by testing 486,676 seronegative blood donations. Samples from each day of collection were divided into two sets; the odd-numbered samples were tested individually on the TIGRIS and the even-numbered samples were tested in pools of 6 on the cobas s 201. The status of reactive samples was confirmed by duplicate testing of samples from the plasma bag to calculate the test specificity. Reactive samples were tested on the alternate system and followed up. RESULTS: The analytical sensitivity of both systems met the 95% limits of detection claimed by the respective package inserts. No cross contamination was seen with either system. Test specificity was 99.93 and 99.90% for the Procleix Ultrio and cobas TaqScreen tests, respectively. The NAT yield rates for human immunodeficiency virus Type 1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) were 1:97,000, 1:490,000, and 1:2800, respectively. Several occult HBV donors, the majority of whom were detected by both tests, were also identified. The HIV-1 and HCV window cases were detected with both tests. CONCLUSION: The performances of the systems and tests indicated that both were acceptable for routine NAT by the National Blood Center, the Thai Red Cross Society. However, the Procleix Ultrio test appeared to be less sensitive than the cobas TaqScreen test for HBV.

Serum levels of MIP-1beta and RANTES in HIV-1 subtype CRF01_AE infected patients with different rates of disease progression.

The beta-chemokines have been shown to inhibit HIV replication in vitro. To evaluate the role of serum beta-chemokines in disease progression and their anti-viral role in vivo, we determined serum levels of macrophage inflammatory protein-1beta (MIP-1beta) and regulated upon activation normal T-cell expressed and secreted (RANTES) of twenty HIV-1 subtype CRF01_AE infected patients: nine progressors (PRs, follow-up CD4+ cell count < 200/mm3 and progression to AIDS or death) and eleven slower progressors (SPs, asymptomatic and/or follow-up CD4+ cell counts > 350/mm3 at the end of follow-up) and determined their plasma viral loads. The subjects were followed for at least 36 months. All had initial CD4 values > 350 cells/mm3. In this longitudinal study, serum levels of MIP-1beta and RANTES in specimens obtained either early or later in the course of HIV infection did not differ significantly between progressors and slower progressors (p > 0.05). There were no significant changes in serum MIP-1beta and RANTES levels over time in either patient group (p > 0.05). No significant associations were observed between plasma viral loads and the measured beta-chemokines (r = -0.205, p = 0.21 for MIP-1beta and r = -0.12, p = 0.492 for RANTES). The results suggest these chemokines do not play a major systemic role in control of viremia or protection against the progression of HIV disease.

Novel 2-chloro-8-arylthiomethyldipyridodiazepinone derivatives with activity against HIV-1 reverse transcriptase.

Based on the molecular modeling analysis against Y181C HIV-1 RT, dipyridodiazepinone derivatives containing an unsubstituted lactam nitrogen and 2-chloro-8-arylthiomethyl were synthesized via an efficient route. Some of them were evaluated for their antiviral activity against HIV-1 RT subtype E and were found to exhibit virustatic activity comparable to some clinically used therapeutic agents.

An avian influenza H5N1 virus that binds to a human-type receptor.

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.

Erythrocyte binding preference of avian influenza H5N1 viruses.

Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays.

Trends of HIV seropositivity at Siriraj Hospital: 13 year's observation from 1992-2004.

OBJECTIVE: The aim of the present report was to observe the trend of seroprevalence rates of HIV seropositivity for routine services at Siriraj Hospital for 13 years. MATERIAL AND METHOD: The prevalence rate of HIV seropositivity was analyzed in three groups of subjects: 1) patients who attended the hospital with HIV related diseases; 2) pregnant women at first visit to the antenatal care clinic; 3) emigrating workers who have applied for employment in foreign countries. RESULTS: Of the 13 year-observation, HIV seroprevalence rates in the groups of patients, pregnant women and emigrating workers was 10.6% (95%CI 8.9-12.3%), 2.0% (95%CI 1.8-2.2%) and 0.6% (95%CI 0.4-0.8%), respectively. CONCLUSION: The low prevalence of HIV seropositivity in the group of emigrating workers may be due to self selection, whereas the prevalence in pregnant women, which was rather consistent at about 2.0%, may represent the infection rate in the general population. The seroprevalence rate measured in the group of pregnant women demonstrates that Thailand should increase efforts to confine the spread of HIV infection in the community.

HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant.

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.

Hepatitis B virus DNA in unusual serological profiles of hepatitis B surface antigen-positive sera.

On the basis of a seroepidemiological survey of hepatitis B virus (HBV) infection conducted on 6208 random serum samples from four provinces of Thailand, we found 19 of 246 (7.7%) hepatitis B surface antigen (HBsAg)-positive samples with unusual serological constellations of HBV infection. Ten samples tested positive for HBsAg, anti-HBc (anti-hepatitis B core antibody), and anti-HBs (anti-hepatitis B surface antibody) markers (group I), 3 specimens were HBsAg and anti-HBs positive without detectable anti-HBc (group II), and the remaining 6 specimens showed only HBsAg (group III). In group I, 7 of 10 HBsAg-positive sera could be confirmed by HBsAg neutralization, yielding positive results for all samples. None of the group II sera were available in sufficient amounts for confirmation. In group III, five of six sera were confirmed by HBsAg neutralization, with four showing a positive reaction. HBV DNA was detected in 7 of 10 (70%) specimens in group I, in 1 of 3 (33.3%) specimens in group II, and in 3 of 6 (50%) specimens in group III. On the basis of HBsAg neutralization, HBV DNA was found in five of seven (71.4%) HBsAg-positive samples in group I and in three of four (75%) HBsAg-positive samples in group III, whereas the one confirmed HBsAg-negative sample in group III also remained negative for HBV DNA. Amino acid sequences were compared with those specifying the "a" determinant of the wild-type virus, particularly focusing on HBV-S protein variations between positions 110 and 160. Among 11 HBV DNA-positive sera, G145A was detected in 2 samples in group I, with the remaining samples identical to the wild-type virus. These unusual serological profiles may be due to the altered immune response of the host or to HBV variants.