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Background: Tumor cells express programmed death ligand-1 (PD-L1) through several biological processes, thereby having different clinical significance depending on the underlying mechanism of expression. Currently, mechanisms leading to PDL1 gene expression in colorectal cancer (CRC) are not fully understood. Methods: We investigated 98 Indonesia CRC patients to determine PD-L1 protein expressions and their correlations with PD-L1 gene copy number status, tumor infiltrating lymphocytes (TILs), tumor mutational profile, as well as clinicopathologic features. Results: Our investigation demonstrated that 18% of patients positively expressed PD-L1. Further analysis on PD-L1 copy number revealed that all PD-L1+ tumors had normal copy number, indicating that the expression of PD-L1 was not a consequence of genetic amplification of PD-L1. From TILs analysis, there was a significant increase of CD8 in all tumor cells expressing PD-L1 (P=0.0051), indicating that the inducible PD-L1 expression was the prominent mechanism occurred in CRC. Furthermore, the expression of PD-L1 in this CRC population was significantly associated with high frequency of MSI compared to the remainder PD-L1- tumors (P=0.0001), suggesting the natural immunogenicity of tumors via MSI status plays role in attracting immune response. On the other hand, p53 mutations which were frequently observed within Indonesian CRCs (76.5%), they were not associated with PD-L1 expression (p=0.1108), as well as KRAS gene (29.6%; p=0.5772) and BRAF gene mutations (5%; p=0.2171). Conclusion: Our study demonstrated that PD-L1 expressions in CRC were predominantly found as a consequence of infiltrating CD8 T lymphocytes that in part arise in the setting of microsatellite instability. Taken together, our findings further support the role of adaptive immune resistance to drive PD-L1 induction in tumor microenvironment and may provide important rationale for strategy implementation of immunotherapy for CRC cases.

82 Background: The presence of the programmed death ligand-1 (PD-L1) has been used as biomarker to select patients and analyze responses to anti-PD-1/L1 antibodies. PD-L1 positivity may be a result of genetic mechanism leading to constitutive PD-L1 expression or inducible mechanism by T-cell presence. Each mechanism may have different significance and response to such therapy. In colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). This study aimed to investigate the mechanism of PD-L1 gene activation in colorectal cancer (CRC) patients, particularly in MSI population. Methods: We have investigated 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using Single Tube TaqMan CNA Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Results: Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) patients (15%; 8/53) (P = 0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). We found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlated with PD-L1 expression. Conclusions: Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.

Background Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. Aims This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Methods Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Results Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Conclusion Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

Objective: The aim of this study was to investigate the distribution of hepatitis C virus (HCV) genotype and the possible association between genotype and HCV-associated liver disease in Indonesia. Methods: 32 anti-HCV-positive asymptomatic carriers (AC), 55 chronic hepatitis (CH), 41 liver cirrhosis (LC), and 35 hepatocellular carcinoma (HCC) patients were included in this study. HCV genotyping was performed by phylogenetic analysis of the NS5B and 5′-UTR regions. Results: The HCV subtype 1b (36.5%), based on NS5B region, was the most prevalent, followed by subtypes 3k (15.4%), 2a (14.4%), 1a (12.5%) and 1c (12.5%), and 2e (4.8%). Subtypes 2f, 3a, 3b, and 4a were also found in some of the samples. HCV subtypes 3k (40.0%) and 1a (35.0%) were the two major subtypes in AC. HCV subtype 1b was not found in AC, but it was common in CH (31.3%), LC (50.0%), and HCC (57.1%). Conclusion: HCV subtype 1b was prevalent in samples of HCV-associated liver disease patients, including CH, LC and HCC. The percentage of subtype 1b was increased with the disease severity (AC < CH < LC < HCC).

Many genetic recombinations of poliovirus (PV) are to be found in excreted viruses, including viruses from vaccineassociated paralytic poliomyelitis (VAPP) as well as healthy vaccine recipients. Most recombinations were among different serotypes of PVs. However, recombination can also occur between PV and other enteroviruses. It was predicted that the hot spot of the recombination is in the nonstructural protein-coding regions, but the exact site is may be different in each recombination. We have demonstrated that the construct recombinant virus between PV and coxsackie A virus serotype 11 (CAV-11), or with CAV-17 with recombination site in the N-term of 2C-coding region, were viable. However, the recombination of PV with CAV-18 at this site was not viable. To determine if the recombination between PV and CAV-18 can occur at other sites, eight chimeric cDNAs (between PV [isolate PJ156] and CAV-18 [PJ156/CAV-18]), all having different recombination sites (2C-8, 2C-133, 2C-235, 2C-268, 2C-287, 2C-327, 3A-67, 3C-60) were constructed using the long-PCR method. The cDNA was then transcribed in vitro and then transfected into the HEp-2 cell-line. As expected, the recombinant virus PJ156/CAV-18, with recombination sites 2C-327, 3A-67, and 3C-60 were viable, while all the others were not. The recombinant viruses displayed a slightly smaller plaque size, but emonstrated quite similar growth as compared to the parental control PJ156. Since analysis for similarity has shown that the homology between PV and CAV-18 was high around these regions, these results supported the copy-choice mechanism of enterovirus recombination.

The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.

Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p

Background The Minangkabau is one of the major ethnic groups in Indonesia. Previous studies with a limited number of samples have shown a different prevalence of HBV/C in the Minangkabau compared to the Indonesian population in general. The aim of this study was to assess the HBV genotype distribution pattern and the prevalence of pre-S, T1753V and A1762T/G1764A mutations among the Minangkabau HBV carriers. The samples were collected from Padang, West Sumatera and from western Java. Mixed primers for specific genotypes were used to determine the HBV genotype. Pre-S or S genes were amplified, sequenced and aligned with reference sequences from GenBank to derive a phylogenetic tree for subgenotyping. Pre-S genes were also analyzed for mutations. The basal core promoter (BCP) region was amplified and directly sequenced to analyze T1753V and A1762T/G1764A mutations. Results The predominant HBV genotype among the Minangkabau HBV carriers (n=117) was C (72.6%) followed by B (24.8%) and co-infection with B and C (2.6%). The prevalence of pre-S mutations, including both the pre-S deletion and pre-S2 start codon mutation, was 41.0%, and the T1753V and A1762T/G1764A mutations were found in 51.9% and 71.2% respectively. HBV/C1 was the predominant HBV subgenotype in the Minangkabau HBV carriers, and was found in 66.2%, followed by B3, B7, C8, B2, B9, C2, and C10 (18.3%, 7.0%, 2.8%, 1.4%, 1.4%, 1.4%, and 1.4% respectively). From samples that were found to be co-infected with HBV B and C, two samples were successfully cloned and subgenotyped, including one with mixed subgenotypes of B3 and C1, and another one with mixed subgenotypes of B7, C1, putative intergenotypic of B/A, and C/A. Furthermore, three samples from donors of non-Minangkabau ethnicity from Padang were found to be infected with an intragenotypic recombination form, including a putative recombinant of B8/B3 and B9/B7. Conclusion HBV/C with subgenotype C1 was the predominant HBV genotype among HBV carriers of Minangkabau ethnicity. The prevalence of pre-S, A1762T/G1764A, and T1753V mutations was higher among the Minangkabau compared to Indonesian HBV carriers in general.

AIM: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS: The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher’s exact test, χ2 test, and t-test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P < 0.05 was considered significant. RESULTS: Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P < 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg(+) group, but it was similar between CH, LC and HCC in HBeAg(-) group. CONCLUSION: Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg(+) patients.

Background CYP2C9 and VKORC1 are two major genetic factors associated with inter-individual variability in warfarin dose. Additionally, genes in the warfarin metabolism pathway have also been associated with dose variance. We analyzed Single Nucleotide Polymorphisms (SNPs) in these genes to identify genetic factors that might confer warfarin sensitivity in Indonesian patients. Methods Direct sequencing method was used to identify SNPs in CYP2C9, VKORC1, CYP4F2, EPHX1, PROC and GGCX genes in warfarin-treated patients. Multiple linear regressions were performed to model the relationship warfarin daily dose requirement with genetic and non-genetic variables measured and used to develop a novel algorithm for warfarin dosing. Results From the 40 SNPs analyzed, CYP2C9 rs17847036 and VKORC1 rs9923231 showed significant association with warfarin sensitivity. In our study population, no significant correlation could be detected between CYP2C9*3, CYP2C9C-65 (rs9332127), CYP4F2 rs2108622, GGCX rs12714145, EPHX1 rs4653436 and PROC rs1799809 with warfarin sensitivity. Conclusions VKORC1 rs9923231 AA and CYP2C9 rs17847036 GG genotypes were associated with low dosage requirements of most patients (2.05 ± 0.77 mg/day and 2.09 ± 0.70 mg/day, respectively). CYP2C9 and VKORC1 genetic variants as well as non-genetic factors such as age, body weight and body height account for 15.4% of variance in warfarin dose among our study population. Additional analysis of this combination could allow for personalized warfarin treatment in ethnic Indonesians.

AIM: To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients. METHODS: Sixty-four patients with chronic hepatitis, 65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study. HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing. Viral load was measured by real-time polymerase chain reaction. RESULTS: Of 179 patients, 108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted. The A1896 mutation was not found in HBeAg(+) patients, however, this mutation was detected in 70.7% of HBeAg(-) patients. This mutation was frequently found when HBeAg was not expressed (87.7%), compared to that found in HBeAg seroconverted patients (65.1%). The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P = 0.004). The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients, however, the prevalence of this mutation did not significantly differ among the two groups (P = 0.054). In HBeAg(+) patients, the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001). The A1899 mutation did not correlate with HBV DNA (P = 0.609). In HBeAg(-) patients, the T1762/A1764 mutation alone was not correlated with HBV DNA (P = 0.095), however, the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001). CONCLUSION: The percentage of HBeAg(-) patients is high in Indonesia, and most of the HBeAg(-) patients had been seroconverted. The A1896 mutation was most likely the major cause of HBeAg loss. The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients, but not in HBeAg(-) patients.

Previous study revealed that a recombinant virus between poliovirus (isolate PJ156) and coxsackie A virus serotype 17 (CAV-17), namely PJ156/CAV-17, was temperature sensitive. It is well known that two amino acids in 3D region (His-73 and Ile-362) are determinants for temperature sensitivity of poliovirus, in particular for Sabin 1 strain. However, it is not known whether those amino acids affect the temperature sensitivity for other enteroviruses. Sequence analysis of 3D region of PJ156/CAV-17 showed that amino acid in 3D-73 and -362 were Tyr and Ile, respectively, similar with the sequence of parental CAV-17 virus. Since amino acid in 3D-73 of PJ156/ CAV-17 was not His, it is suggested that the temperature sensitivity of the PJ156/CAV-17 was associated with the Ile-362. To confirm this suggestion, the temperature-sensitive escape mutants of PJ156/CAV-17 were generated by blind passaging at 39.5 oC. The escape mutants were then recovered and plaque purified, and the sequence of 3D region was determined. It was found that the amino acid in 3D-362 was Thr, instead of Ile. Consequently, Ile- 362 was proved to be involved in temperature sensitivity of PJ156/CAV-17. Full sequences of both viruses were also determined and compared. Furthermore, the characteristics of the temperature resistant of PJ156/CAV-17 variant were analyzed. It is confirmed that the recovered PJ156/CAV-17 virus could grow well at 39.5 oC, and there was strong correlation between temperature sensitivity and attenuation.

Background Alpha-fetoprotein (AFP) is a tumor-associated glycoprotein that functions in regulation of both ontogenic and oncogenic growth. Recent study showed that AFP can induce apoptosis or impair monocyte-derived dendritic cell (MDDC) function. However, it is still unclear which AFP domain (D-AFP) plays major role in this function. Results As expected monocytes cultured in the presence of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Interleukin-4 (IL-4) developed into MDDC. Up-regulation of HLA-DR and CD11c as well as loss of CD14 molecules could be observed. Full length AFP (FL-AFP), domain 2 AFP (D2-AFP) and D3-AFP, but not D1-AFP, significantly inhibited the expression of HLA-DRhigh/CD11chigh and CD80+/CD86high molecules. In contrast, CD83 expression was substantially down-regulated in all samples. Expression of CD40 was significantly suppressed by FL-AFP but not by any D-AFPs. Finally, both FL-AFP and D-AFP impaired the MDDC ability to secrete IL-12 (p70). Conclusions D2- and D3- but not D1-AFP extensively suppresses the MDDC function. All the recombinant AFP proteins impaired the ability of MDDC to secrete IL-12.

Japanese encephalitis virus (JEV) is a neuropathogenic virus commonly caused cen-tral nervous diseases such as meningitis and severe encephalitis. Although vaccine has been developed, no specific and effective drug is available so far. We previously carried out a screening of inhibitor of JEV RNA helicase, an enzyme that essential for virus replication, from Actinomycetes and found that Streptomyces chartreusis produce the inhibitor of JEV RNA helicase. In this study, an extracellular protein which has inhibition activity on ATPase activity of JEV RNA helicase was purified from supernatant of Streptomyces chartreusis culture by ammonium sulfate pre-cipitation and size exclusion chromatography. SDS-PAGE analysis showed a single band with aproximate molecular mass of 11,4 kDa, suggesting that the inhibitor was successfully purified into a single protein. Key words : Japanese encephaliti virus, RNA helikase Inhibitor, ATPase, Strepto-myces chartreusis.

Hepatocellular carcinoma (HCC) atau kanker hati adalah salah satu dari kanker yang paling umum dan menjadi penyebab utama kematian di negara-negara Asia. HCC biasanya berkembang pada pasien dengan penyakit hati kronis dan sirosis. Pengamatan HCC pada pasien sirosis biasanya dilakukan menggunakan ?-fetoprotein (AFP) dan ultrasonografi. Akan tetapi, sensitivitas AFP untuk deteksi HCC sangat rendah, sedangkan penggunaan ultrasonografi cukup mahal dan sangat tergantung pada keahlian operator. Hal inilah yang menjadi ide dasar tentang perlunya mencari suatu strategi baru (biomarker baru) dalam mendeteksi HCC secara dini. Biomarker yang ideal harus lebih sensitif, spesifik, noninvasif, murah, dan dapat diterima oleh pasien. Berdasarkan tinjauan literatur yang kami lakukan (2001-2009), dilaporkan beberapa penemuan biomarker baru yang cukup menjanjikan, seperti AFP-L3, des-gamma carboxyprothrombin (DCP), golgi protein 73 (GP73), glypican-3 (GPC3), squamous cell carcinoma antigen (SCCA), transforming growth factor-?1 (TGF?b1), insulin-like growth factor-II (IGF-II), insulin-like growth factor-binding protein-2 (IGFBP-2), human cervical cancer oncogene (HCCR), hepatocyte growth factor (HGF), KL-6 and ?-acid glycoprotein (AAG). Akan tetapi, penggunaannya secara klinis atau uji validasi belum pernah dilaporkan. Beberapa kendala dan keterbatasan yang dilaporkan dalam penemuan biomarker ini antara lain jumlah sampel yang digunakan kurang memadai, metoda analisis yang beragam (heterogenity), terbatas pada wilayah tertentu, predictive analysis biomarker, dan masih sedikitnya studi longitudinal untuk mengevaluasi kemampuan biomarkers dalam mendeteksi penyakit pada taraf preklinik. Uji klinik dan metoda validasi yang tepat saat ini menunggu lahirnya suatu generasi biomarker baru untuk pasien HCC.Kata kunci: Hepatocellular carcinoma (HCC), biomarker, metoda pengujian

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.

Background: Recently, there have been large outbreaks of hand, foot and mouth disease (HFMD) mainly caused by enterovirus 71 (EV71) associated with severe neurological diseases in the Western Pacific Region (WPR). To monitor the realtime trend of EV71 transmission throughout the WPR, the authors conducted a molecular epidemiological analysis of EV71 infection. Methods: Viruses were isolated from clinical samples from patients with HFMD or those with neurological complications. The EV71 isolates were identified by microneutralization assay. The VP4 and/or VP1 regions of recent EV71 isolates were sequenced and subjected to phylogenetic analysis using reference EV71 strains. Results: The phylogenetic analysis of EV71 isolates from the WPR revealed two major genogroups, B and C, based on the nucleotide sequence alignment of the VP1 or VP4 region. These two major genogroups were further divided into subgenogroups, B1, B2, B3, and B4 and C1, C2, C3 and C4, respectively. Conclusion: The molecular epidemiological analyses of recent and previous EV71 isolates in the WPR indicated that two major genogroups of EV71 are co-circulating in Australia, Malaysia, Singapore, Taiwan and Japan. Recent EV71 isolates in Mainland China constitute a new distinct genetic cluster, subgenogroup C4. Two major lineages of EV71 are the major causative agents of the present HFMD epidemics in the WPR and both are considered to be neurovirulent.

Enterovirus 71 (EV71), one of the major causative agents for hand, foot and mouth disease (HFMD), is sometimes associated with severe central nervous system diseases. In 1997, in Malaysia and Japan, and in 1998 in Taiwan, there were HFMD epidemics involving sudden deaths among young children, and EV71 was isolated from the HFMD patients, including the fatal cases. The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis. EV71 strains from previously reported epidemics belonged to genotype A-1, while those from recent epidemics could be divided into two genotypes, A-2 and B. In Malaysia, genotype A-2 was more prevalent, while in Japan and Taiwan, B genotype was more prevalent. Two isolates from fatal cases in Malaysia and one isolate from a fatal case in Japan were genotype A-2. However, all isolates from three fatal cases in Taiwan belonged to genotype B. The severity of the HFMD did not link directly to certain genotypes of EV71.

AIM: To identify the distribution of hepatitis B virus (HBV) subgenotype and basal core promoter (BCP) mutations among patients with HBV-associated liver disease in Indonesia. METHODS: Patients with chronic hepatitis (CH, n = 61), liver cirrhosis (LC, n = 62), and hepatocellular carcinoma (HCC, n = 48) were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing. RESULTS: HBV genotype B (subgenotypes B2, B3, B4, B5 and B7) the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C (subgenotypes C1, C2 and C3) was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 (84.9%) and C1 (82.2%) were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 (84.9%) and adrq+ (89.4%) were the most prevalent in HBV genotype B and C, respectively. Double mutation (A1762T/G1764A) in the BCP was significantly higher in LC (59.7%) and HCC (54.2%) than in CH (19.7%), suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC (46.8%), but lower in HCC (22.9%) and CH (18.0%), suggesting that this mutation may be an indicator of cirrhosis. CONCLUSION: HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.

The role of the conserved DExH motif of the Japanese encephalitis virus (JEV) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV NS3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly(U). Thus, the mutational effect at His in motif II was different in the HCV and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activities are separable enzymatic functions.

Background Hepatitis B virus (HBV) genotype appears to show varying geographic distribution. Molecular epidemiological study of HBV in particular areas in Indonesia is still limited. This study was aimed to identify the prevalence of HBV genotype/subgenotype and mutations in basal core promoter (BCP) region in voluntary blood donors in Makassar, one of the biggest cities in east part of Indonesia. A total of 214 hepatitis B surface antigen (HBsAg)-positive samples were enrolled in this study. HBV genotype/subgenotype was identified by genotype-specific PCR method or direct sequencing of pre-S region. Mutations in BCP were identified by direct sequencing of the corresponding region. Results HBV/B and HBV/C were detected in 61.21% and 25.23% of the samples, while mix of HBV/B and HBV/C was found in 12.62% of the samples. Based on pre-S region, among HBV/B and HBV/C, HBV/B3 (95.00%) and HBV/C1 (58.82%) were predominant. Interestingly, HBV/D was identified in two samples (22.165.07 and 22.252.07). Complete genome sequences of two HBV/D strains (22.165.07 and 22.252.07) demonstrated that both strains belong to HBV/D6, and the divergence between the two strains were 1.45%, while divergences of both 22.165.07 and 22.252.07 strains with reference strain (AM422939/France) were 2.67%. A1762T/G1764A mutation was observed in 1.96% and 5.36%, whereas T1753V mutation was found in 2.94% and 1.79% of HBV/B and HBV/C, respectively. Conclusion HBV/B and HBV/C are dominant in Makassar, similar to most areas in Indonesia. Mutations in BCP which might be associated with severity of liver disease are less common.