Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

ABSTRACT Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

The role of human adenoviruses type 41 in acute diarrheal disease in Minas Gerais after rotavirus vaccination

Abstract Human adenovirus species F (HAdV-F) type 40 and 41 are commonly associated with acute diarrheal disease (ADD) across the world. Despite being the largest state in southeastern Brazil and having the second largest number of inhabitants, there is no information in the State of Minas Gerais regarding the role of HAdV-F in the etiology of ADD. This study was performed to determine the prevalence, to verify the epidemiological aspects of infection, and to characterize the strains of human adenoviruses (HAdV) detected. A total of 377 diarrheal fecal samples were obtained between January 2007 and August 2011 from inpatient and outpatient children of age ranging from 0 to 12 years. All samples were previously tested for rotavirus, norovirus, and astrovirus, and 314 of 377 were negative. The viral DNA was extracted, amplified using the polymerase chain reaction and the HAdV-positive samples were sequenced and phylogenetically analyzed. Statistical analyses were performed using the Chi-square test (p < 0.05), considering two conditions: the total of samples tested (377) and the total of negative samples for the remaining viruses tested (314). The overall prevalence of HAdV was 12.47% (47/377); and in 76.60% (36/47) of the positive samples, this virus was the only infectious agent detected. The phylogenetic analysis of partial sequences of 32 positive samples revealed that they all clustered with the HAdV-F type 41. The statistical analysis showed that there was no correlation between the onset of the HAdV infection and the origin of the samples (inpatients or outpatients) in the two conditions tested: the total of samples tested (p = 0.598) and the total of negative samples for the remaining viruses tested (p = 0.614). There was a significant association in the occurrence of infection in children aged 0–12 months for the condition 1 (p = 0.030) as well as condition 2 (p = 0.019). The occurrence of infections due to HAdV did not coincide with a pattern of seasonal distribution. These data indicate the significant involvement of HAdV-F type 41 in the etiology of ADD in Minas Gerais, which demonstrates the importance of other viral agents in the development of the disease after the introduction of rotavirus vaccine immunization.

Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Studies published in indexed journals on lawsuits for medicines in Brazil: a systematic review / Estudios publicados en revistas indexadas acerca de decisiones judiciales para el acceso a los medicamentos en Brasil: una revisión sistemática / Estudos publicados em periódicos indexados sobre decisões judiciais para acesso a medicamentos no Brasil: uma revisão sistemática

The aim of this systematic review was to identify and characterize articles in indexed scientific journals with quantitative data surveys on administrative or legal proceedings for access to medicines. The SciELO, LILACS, MEDLINE via PubMed, Embase, and Scopus databases were used. We identified 45 articles, of which 17 were selected. The larger studies, each covering between 2,000 and 2,927 lawsuits, were done in the states of São Paulo, Rio de Janeiro, and Santa Catarina, Brazil. Eleven studies specified the type of legal representation, of which six examined cases with public attorneys and five with private attorneys. Only two studies reported whether the lawsuit was individual or class action, and in both the claims were individual. Since the majority of the medicines requested in the lawsuits were medium to high-cost, the review indicates that lawsuits contributed to the incorporation of these drugs into current pharmaceutical care in Brazil.

El objetivo de esta revisión sistemática fue identificar y caracterizar los artículos disponibles en revistas científicas indexadas en bases de datos electrónicas, que llevaron a cabo un estudio cuantitativo de datos, procedimientos administrativos o judiciales sobre la cuestión del acceso a los medicamentos a través de demandas judiciales. Los estudios fueron localizados en las bases de datos SciELO, LILACS, MEDLINE vía PubMed, Embase, Scopus. Se identificaron 45 artículos, de los cuales se seleccionaron 17. Los estudios que se llevaron a cabo engloban de 2.000 a 2.927 procesos judiciales en São Paulo, Río de Janeiro y Santa Catarina, Brasil. En once estudios se realizaron encuestas a los representantes legales de la acción judicial. En seis estudios predominó la representación pública legal y en cinco abogados privados. Sólo dos estudios examinaron si la acción era individual o colectiva y en los dos hubo prevalencia de acciones individuales. Como la mayoría de los medicamentos estaba involucrada en acciones legales de medio y alto coste, se cree que las demandas han contribuido a la incorporación de fármacos en la política pública actual.

O objetivo desta revisão sistemática foi identificar e caracterizar artigos disponíveis em periódicos científicos indexados em bases eletrônicas, que realizaram levantamento de dados quantitativo, em processos administrativos ou judiciais, sobre a questão do acesso a medicamentos por meio de ações judiciais. Foram usadas as bases de dados SciELO, LILACS, MEDLINE via PubMed, Embase e Scopus. Identificamos 45 artigos, dos quais foram selecionados 17 artigos. Os estudos com faixa de 2.000 a 2.927 processos foram conduzidos em São Paulo, Rio de Janeiro e Santa Catarina, Brasil. Em 11 estudos foram pesquisadas qual a representação jurídica da ação. Em seis estudos predominaram a representação de advogados públicos e em cinco particulares. Somente dois estudos observaram se a ação era coletiva ou individual, sendo que nas duas pesquisas a prevalência era de ações individuais. Como a maioria dos medicamentos envolvidos nas ações é de médio e alto custo, acredita-se que as demandas judiciais tenham contribuído para incorporação de medicamentos nas ações de assistência farmacêutica atuais.

Teat papillomatosis associated with bovine papillomavirus types 6, 7, 9, and 10 in dairy cattle from Brazil

This study describes the clinical, histopathological, and virological characterization of teat papillomatosis from Brazilian dairy cattle herds. Four types of bovine papillomavirus were identified (BPV6, 7, 9, and 10); one of these (BPV7) is being detected for the first time in Brazilian cattle.

Liver histology and HBV DNA levels in chronically HBV infected patients with persistently normal alanine aminotransferase

Data on histological activity and HBV DNA levels in patients with chronic HBV infection and persistently normal alanine aminotransferase levels are sparse. We aimed to investigate the histological activity and HBV DNA levels in these patients. There were 132 patients with HBeAg negative chronic HBV infection and persistently normal alanine aminotransferase levels that were included prospectively. Data were dichotomized according to the median levels. Associations of histology with HBV DNA and other variables were assessed. A total of 80 patients were male. The median age was 36 years. The median baseline HBV DNA was 2.9Log10 IU/mL. There were 50 cases [38%] with a total score >/= 5, 53 cases [40.2%] had grade >/= 4 and 40 cases [30.3%] had stage >/= 2. A baseline HBV DNA <2000 IU/mL was seen in 24 cases [48%] of those with total score >/= 5, 28 cases [53%] of those with grade >/= 4 and 9 cases [22.5%] with stage >/= 2. Multivariate analysis of baseline HBV DNA above the median level significantly predicted the total score, grade and stage with an adjusted odds ratio of 5.43, 3.47, and 4.23, respectively when compared to below median values. A second liver biopsy was performed in 61 patients. The median time interval between the two biopsies was 40 months. Total scores of 23 cases [38%] progressed by >/= 2 scores and the HBV DNA of 18 cases [22.5%] increased by >/= 1 Log[10] IU when compared to baseline values. HBeAg negative chronic HBV infection with persistently normal alanine aminotransferase is not a silent disease. Active liver disease may be seen in such patients with viral loads less than 2000 IU/mL

Characterization of canine oral papillomavirus by histopathological and genetic analysis in Korea

In August 2008, forty dogs out of 400 developed oral warts in a breeding farm in Korea. Canine oral papilloma infection is a common disease in dogs. However, there has been no report of an outbreak of canine oral papillomavirus (COPV) in a group of dogs or in dog breeding farms in Korea, and the genetic analysis of COPV in Korea has yet to be performed. This study diagnosed canine oral papilloma from the oral samples of these dogs based on histopathological examination and immunohistochemistry. Polymerase chain reaction was applied to amplify the corresponding products using pre-existing primer sets for COPV and a universal human papillomavirus targeting L1 gene. Further genetic analysis of the major viral capsid gene L1 confirms the sequences of Korean COPV, which shows a close relationship to previously reported COPV. This study describes the histopathological and immunohistochemical characteristics of canine oral papilloma in a group of breeding dogs in Korea and discloses the complete L1 gene sequences of Korean COPV.

Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.

High genetic diversity in human immunodeficiency virus: type 1 in Jamaica / Elevada diversidad genética del virus de la inmunodeficiencia humana: tipo 1 en Jamaica

The subtypes of the human immunodeficiency virus - type 1 (HIV-1) strains from 54 HIV-1 - infected persons including 44 strains which were typed previously by heteroduplex mobility assay (HMA) were determined by DNA sequencing and phylogenetic analysis. Of 54 HIV- infected persons, 92.5% were infected with HIV-1 subtype B and 7.5% with other HIV-1 subtypes including subtypes D (3.7%), A (1.9%) and J (1.9%). In the phylogenetic analysis, the subtype A virus found in the sample clustered with subtype A reference strains and a circulating recombinant form (CRF) reference strain which originates in Central Africa and is circulating in Cuba indicating a close relationship between these viruses. There was 86% concordance between HMA and DNA sequencing in assigning subtype B viruses. For the non-B subtype viruses, there was less concordance between the two methods (67%). The results confirm the predominance of HIV-1 subtype B strains and the high genetic diversity of HIV-1 strains in circulation in Jamaica. The efficacies and some limitations of the HMA as a method of HIV-1 subtyping also were noted. It is important that the HIV/AIDS epidemic in Jamaica be monitored meticulously for possible expansions in non-B subtypes and the emergence of inter-subtype recombinant forms. We recommend that the more expensive DNA sequencing and phylogenetic analysis, including HIV-1 genotyping for antiretroviral drug resistance testing, be used as an adjunct to the more cost-effective HMA to track the HIV/AIDS epidemic in Jamaica.

Los subtipos de cepas de virus de la inmunodeficiencia humana-tipo-1 de 54 personas infectadas con el VIH-1, que incluyeron 44 cepas previamente clasificadas según su tipo mediante ensayo de movilidad de heterodúplex (HMA), fueron determinados mediante secuenciación de ADN y análisis filogenético. De 54 personas infectados con VIH, 92.5% estaban infectadas con VIH-1 subtipo B y 7.5% con otros subtipos de VIH-1 incluidos los subtipos D (3.7%), A (1.9%), J (1.9%). En el análisis filogenético, el virus de subtipo A hallado en la muestra, se agrupa con las cepas de referencias del subtipo A y una cepa de referencia de forma recombinante circulante (CRF), que tienesu origen en África Central y está circulando en Cuba, lo que indica una estrecha relación entre estos virus. Hubo un 86% de concordancia entre el HMA y la secuenciación del DNA en la asignación de virus de subtipo B. Para los virus de subtipo no B, hubo menos concordancia entre los dos métodos (67%). Los resultados confirman el predominio de las cepas del subtipo B del VIH-1, y la alta diversidad genética de las cepas del VIH-1 en circulación en Jamaica. También se señalaron las eficacias y algunas limitaciones del HMA como método de clasificación del VIH-1 en subtipos. Es importante monitorear meticulosamente la epidemia de VIH/SIDA en Jamaica, a fin de detectar posibles expansiones de subtipos no B y la aparición de formas recombinantes inter-subtipos. Recomendamos que por ser ambos métodos más costosos, tanto la secuenciación de ADN como el análisis filogenético - incluyendo el genotipado del VIH-1 para probar la resistencia antiretroviral del medicamento - sean usados como complementos del HMA, el cual es más costo-efectivo, para seguir de cerca el rastro de la epidemia VIH/SIDA en Jamaica.

Rapid and real-time detection technologies for emerging viruses of biomedical importance.

The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/or detection of antibodies,which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. We have developed two real-time assays ie., SYBR green I based real time reverse transcription polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (LAMP) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, Japanese encephalitis, chikungunya, west Nile, severe acute respiratory syndrome virus (SARS) etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specificity. One of the most important advantages of LAMP is its field applicability, without requirement of any sophisticated equipments. Both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of India. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

Optimization of in situ hybridization assay using non-radioactive DNA probes for the detection of canine herpesvirus (CHV) in paraffin-embedded sections

Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

Epstein-Barr virus in nasopharyngeal carcinoma in Indian patients.

Formalin fixed paraffin embedded tissue blocks from 40 cases of nasopharyngeal carcinoma were investigated for the presence of Epstein-Barr virus (EBV). Heminested polymerase chain reaction was employed to amplify U 2 region of EBV DNA coding for EBNA 2 gene. This is the first study of its kind carried out in India. EBV was detected in 28 out of 40 (70%) nasopharyngeal carcinomas. Type A EBV was identified in 20 cases (71.4%), type B in 7 cases (25%) and coinfection in one case (3.5%). Either type A, or type B EBV, was identified in 22 out of 23 cases (95.7%) of undifferentiated nasopharyngeal carcinoma and 6 out of 15 cases (40%) of non keratinized squamous carcinoma. The higher incidence of EBV in non keratinized squamous carcinoma of nasopharynx assumes importance in view of the contrary reports in other studies. 26 cases were from Western India, of which 15 (57.6%) showed presence of type A EBV and 2 (7.6%) type B EBV. From Eastern Indian particularly Assam, 4 our of 5 cases showed positivity for type B EBV and 1 a coinfection. Whereas, from Bihar 3 out of 4 cases showed presence of type A EBV. From Middle East (Arabs) type A EBV was detected in 3 out of 5 cases of nasopharyngeal carcinomas. Geographically, type A EBV was far more prevalent in Western India, while in Eastern India particularly Assam, all five vases were positive for type B EBV. Thus, a significant variation in the type of EBV infection was observed in nasopharyngeal carcinoma in different ethnic populations in India.

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 3 virus, Bangkok genotype.

The nucleotide sequence of the nonstructural protein gene (1,610 bases) of dengue 3 virus (Bangkok genotype; CH53489 isolated in 1973) has been determined in both forward and reverse directions. The PCR based cycle sequencing technic by the enzymatic method of Sanger et al using a sequencing primer 5'-end labeled with gamma-32P-ATP was the method of our choice for sequence analysis. Two cDNA templates were prepared by RT-PCR technique starting from the nucleotides 6,306-6,969 and 6,925-7,915 of the dengue 3 genome with the lengths of 663 and 990 base pairs respectively. In our cycle sequencing experiments, it has been observed that the substitution of 7-deaza-dG for dG in DNA eliminated most of the secondary structures that produce gel artifacts. The final sequence results of these two cDNA templates were established from their sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequences as well as their deduced amino acid sequences of the Bangkok dengue 3 (CH53489) virus and the published sequence data of the dengue 3 prototype (H87) was manipulated by the PC-DOS-GIBIO DNASIS TM 06-00 software. The homology of the nucleotide sequences between the two dengue 3 viruses was 96.65%. The deduced amino acid sequence from nucleotides 6,306-7,915 of the two viruses showed conserved amino acids of the nonstructural protein NS4a and 6 amino acid changes in NS4b and NS5.