Serological study of dengue virus infection in Terai region, Nepal.

A cross-sectional study was conducted to determine dengue virus IgM-positive rate in Terai region, Nepal from August to December 2007. Serum samples were collected from 183 symptomatic cases. The samples were examined for dengue virus specific IgM using particle agglutination test. Of 183 serum samples, 55 (30.0%) had positive for dengue IgM antibody. The positive rate was highest (50.0%) in Biratnagar, and lowest (19.6%) in Chitwan male to female ratio was 2:1 in IgM-positive populations. IgM-positive rate was 29.0% at ages 21-30, 25.4% at ages 11-20 and 23.6% at ages 0-10, but 10.9% at ages 31-40, and ages over 40. There was not significant association between occupation of the patients and positive rate among farmer, labour, service, business and student.

Molecular characterization of the E gene of dengue virus type 1 isolated in Guangdong province, China, in 2006.

We determined the genetic relationships and origin of the dengue virus (DENV) responsible for an outbreak of dengue fever (DF) in Guangdong province, China, in 2006. Five DENV type 1 (DENV-1) isolates were obtained from human serum samples collected from DF patients during the outbreak. The nucleotide sequences of the E (envelope) gene were compared with those of 48 previous DENV-1 isolates: 18 from Guangdong province, one from Fujian province, one from Zhejiang province, and 28 from other countries in the South Asian region. The results suggested that four DENV-1 isolates identified in Guangdong province in 2006 might be in general circulation there, although these DENV-1 viruses may have been originally introduced into the province from other countries. In contrast, one isolate from Guangzhou city in 2006, may have been introduced by a recently imported case from Cambodia.

An outbreak of psittacosis in a bird park in Japan.

An outbreak of psittacosis related to a bird park occurred in Matsue City, Shimane Prefecture, Japan, during winter 2001. Seventeen cases of psittacosis (12 visitors, three staff, and two student interns) were confirmed. A cohort study was conducted among the park staff and students to determine the risk factors for the development of acute serologically confirmed psittacosis (SCP) infection. Being 'bird staff' had an increased risk of SCP infection (RR 3.96, 95% CI 1.48-10.58). Entering the staff building, where ill birds were maintained without proper isolation, was also associated with an increased risk of SCP infection (RR 3.61, 95% CI 1.03-12.6). Isolation of ill birds and quarantine measures were found to be insufficient. Dehumidifiers and a high-pressure water spray under a closed ventilation environment may have raised the concentration of Chlamydophila psittaci in the hothouses. Bird park staff and visitors should be educated about psittacosis.

Characterization and susceptibility to antiviral agents of herpes simplex virus type 1 containing a unique thymidine kinase gene with an amber codon between the first and the second initiation codons.

A herpes simplex virus type 1 (HSV-1) containing a thymidine (TK) gene with an amber mutation at the 8th position counted from the first AUG codon was isolated from a child with acute gingivostomatitis. The virus was predicted to express a mutant viral translated from the 2nd AUG codon at the 46th amino acid position and consisting of 331 amino acids. The virus was as sensitive to acyclovir (ACV), 5-bromovinyl-2'-deoxyuridine (BVdU), 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), and 1-beta-D-arabinofuranosylthymine (araT) as a wild-type HSV-1. The mutant TK showed the same level of TK activity as the wild-type TK at reaction temperatures of 34 degrees C, 37 degrees C and 39 degrees C. ACV, BVdU, BVaraU, and araT inhibited the replication of the TK-deficient and drug-resistant HSV-1 and HSV-2 in 293T cells in which the mutant TK was expressed to the same extent as in cells in which intact HSV-1-TK was expressed, whereas BVdU and BVaraU inhibited the replication of these viruses less strongly in cells in which HSV-2-TK was expressed. It can be concluded that the mutant HSV-1 exists in nature as a variant and possesses the necessary phosphorylation activities to form ACV-monophosphate from ACV, to form BVdU-diphosphate through BVdU-monophosphate from BVdU, and to form BVaraU-diphosphate through BVaraU-monophosphate from BVaraU. These results indicate that the mutant HSV-1-TK with a deletion of the first 45 amino acid residues is phenotypically the same as that of wild-type HSV-1-TK in terms of the phosphorylation activity of TK-associated anti-herpes virus drugs.

Bacterial superantigens and T cell receptor beta-chain-bearing T cells in the immunopathogenesis of ulcerative colitis.

Ulcerative colitis (UC) is a chronic relapsing-remitting inflammatory bowel disease (IBD) that affects the colon and the rectum producing debilitating symptoms, which impair ability to function and quality of life. The aetiology of IBD is incompletely understood, but within the lymphocyte population, specific T cell subsets are known to be major factors in the development of intestinal immune pathology while different subsets are essential regulators, controlling IBD. Hence, IBD is thought to reflect dysregulated T cell behaviour. This study was to investigate if the normal molecular configuration of the T cell receptor (TCR) repertoire is compromised in patients with UC. The percentage of T cell-bearing beta-chain 4 (TCRBV4) was high in patients with UC, and T cells showed polyclonal expansion in the presence of bacterial superantigens (SA) such as streptococcal mitogenic exotoxin Z-2 (SMEZ-2), indicating that bacterial SA promote specific TCRBV family expansion. Further, in patients with UC, the duration of UC was significantly longer in patients with skewed TCRBV4 compared with patients without TCRBV4 skewing, suggesting that long-term exposure to bacterial SA such as SMEZ-2 might promote systemic immune disorders like the remission-relapsing cycles seen in patients with UC. In conclusion, our observations in this study support the perception that the systemic activation of T cells by enteric bacterial SA might lead to a dysregulated, but exuberant immune activity causing the remission and flare-up cycle of mucosal inflammation in patients with UC. Future studies should strengthen our findings and increase understanding on the aetiology of IBD.

An improved procedure for rapid determination of viral RNA sequences of avian RNA viruses.

A system for rapid determination of viral RNA sequences, RDV, was improved for detection of avian RNA virus in allantoic fluids. We detected avian paramyxovirus nucleotide sequences using RDV method ver 2.0.

Confirmation of dengue virus infection by detection of dengue virus type 1 genome in urine and saliva but not in plasma.

We successfully detected dengue virus (DENV) genome in urine and saliva but not in plasma samples from a Japanese dengue fever patient. The results of the present study suggest that detection of DENV genome in urine and saliva can be an effective diagnostic method, particularly for children with viral hemorrhage.

Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

Detection of antibodies to Japanese encephalitis virus in the wild boars in Hiroshima prefecture, Japan.

Serum specimens were collected from 25 wild boars in Hiroshima prefecture located in the western region of Japan from November 2004 to February 2005. The sera were tested for antibodies to Japanese encephalitis virus (JEV) by IgM capture and IgG enzyme-linked immunosorbent assays (ELISA), and plaque reduction neutralization test. Seventeen samples (68%) were positive for neutralizing antibody to JEV. All the neutralizing antibody-positive samples were positive for IgG-ELISA. One was also positive for IgM. The results indicate that approximately 70% of the wild boars were positive for anti-JEV antibody, and raises the possibility that wild boars may play a role in the infectious cycle of JEV in this region.

[Molecular characterization of full-length genome of Japanese encephalitis virus (02-76) newly isolated in China.].

BACKGROUND: To sequence and analyze the complete nucleotide sequence of the Japanese encephalitis virus (JEV) strain 02-76, newly isolated in 2002 in China and to provide information for the genomic structure of JEV and the characteristics of virulence. METHODS: Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments, sequencing was performed and all the nucleotides were connected to acquire the full-length genome. Computer software was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees including Clustal X(1.8), DNASTAR, GENEDOC(3.2). RESULTS: The result of sequence analysis showed that the genome of 02-76 strain was 10,977 nucleotides long. An open reading frame from 95 to 10,391 including 10,296 bases was found capable of coding for a 3432 amino acid polyprotein. Compared with the Beijing 1 strains isolated in 1949 in China, there was a 248 nucleotide divergence and 16 amino acid divergence. Comparison of the complete genome sequences of different JEV isolates showed a 0.6%-15.1% nucleotide sequence divergence among them, which resulted in 0.2%-4.6% amino acid sequence divergence. Phylogenetic analysis through PrM/C,E,3'NTR and full-length genome showed that the 02-76 strain belonged to genotype 3. CONCLUSION: Analysis based on the complete genome sequences of different JEV isolates showed that the 02-76 isolate in 2002 belonged to genotype 3 and was close to the old Chinese isolates SA-14.

Specific IgM and IgG responses in primary and secondary dengue virus infections determined by enzyme-linked immunosorbent assay.

IgM- and IgG-capture ELISAs are widely used as diagnostic tests for confirmation of dengue virus infection. The positive rate of anti-dengue IgM and IgG detection was examined in primary and secondary dengue virus infections in the setting of a provincial hospital using IgM- and IgG-capture ELISAs. Disease day 1 was defined as the day of onset of symptoms. In total, 232 plasma samples were collected from 106 confirmed dengue cases consisting of 12 primary and 94 secondary infections. In primary infection, anti-dengue IgM was detected in 4 out of 5 samples collected on disease day 5 and in all the 21 samples collected on disease day 6 or later. Specific IgG was detected in 2 out of 5 samples collected on day 12, and in 5 out of 6 samples collected on disease days 13-15, but was not detected in samples collected on disease day 10 or earlier. In secondary infection, IgM was not detected in the samples on disease days 2 and 3, but detected in 20 out of 79 samples collected on days 4-6, in 44 out of 65 on disease days 7-11 and in 40 out of 51 samples on disease days 12-14. In contrast, specific IgG was detected in 21 out of 60 samples on disease days 4 and 5, in 13 out of 19 on disease day 6, in 62 out of 65 on disease days 7-11 and in all the samples collected on disease day 12 or later. The result indicate that seroconversion rates of IgM and IgG are different between primary and secondary infections, and suggest that detection of specific IgM and IgG is necessary for determining dengue virus infection and for differentiating primary and secondary dengue infections.

New criteria for immunofluorescence assay for Q fever diagnosis in Japan.

A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of > or =1:128 and/or IgG titer of > or =1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of < or =1:16 and an IgG titer of < or =1:32 by the IFA test were negative by both ELISA and WBA. In the proposed "equivocal area," with an IgM titer of > or =1:32 and < or =1:64 and/or an IgG titer of > or =1:64 and < or =1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an "equivocal area" should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.

Immunoglobulin A antibody responses in dengue patients: a useful marker for serodiagnosis of dengue virus infection.

We determined the usefulness of an immunoglobulin A (IgA) antibody-capture enzyme-linked immunosorbent assay for serodiagnosis of dengue virus infections. The results indicate that the presence of IgA and IgM in serum samples assures recent primary dengue virus infection even with a single serum sample.

Serological and virological features of dengue fever and dengue haemorrhagic fever in Thailand from 1999 to 2002.

Serological and virological features of dengue fever (DF) and dengue haemorrhagic fever (DHF) in Thailand were analysed in 2715 patients from 1999 to 2002. The illness was caused by DEN-1 in 45%, DEN-2 in 32%, DEN-3 in 18% and DEN-4 in 5% of patients. Almost all of the DHF cases caused by DEN-2 and DEN-4 were in secondary infection, while approximately 20% of the DHF cases caused by DEN-1 and DEN-3 were in primary infection. Male:female ratio and age distribution were not different among four serotypes in primary and secondary infections. These results indicate that DEN-1 and DEN-3 induce DHF in both primary and secondary infections, and suggest that DEN-2 and DEN-4 in Thailand are less likely to cause DHF in primary infections.

Review on flavivirus vaccine development. Proceedings of a meeting jointly organised by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand.

In light of the continuous spread of human pathogenic flaviviruses, in particular the mosquito-transmitted species, vaccine development remains a high priority on the public health agenda. On 26-27 April 2004, a conference was held in Bangkok, Thailand, to review current status of flavivirus vaccine development and related issues, focussing on dengue (DEN) and Japanese encephalitis (JE). This event, co-sponsored by the World Health Organization (WHO) and the Thai Ministry of Public Health, reviewed the progress made with vaccine development, sero-epidemiological studies and other accompanying activities critical for vaccine development and vaccination. The considerable interest in and awareness of the flavivirus diseases and their prevention by public health decision makers, as well as the establishment of two dedicated programmes for dengue and Japanese encephalitis vaccine development raise hopes that new or improved vaccines will become available in the coming years.

Modification of endothelial cell functions by Hantaan virus infection: prolonged hyper-permeability induced by TNF-alpha of hantaan virus-infected endothelial cell monolayers.

Serious vascular leakage is central to the pathogenesis of hantavirus infections. However, there is no evidence suggesting the hantavirus infection of endothelial cells directly causes obvious cell damage or morphological alteration either in vivo or in vitro. In this study, we examined whether Hantaan virus (HTNV) infection modifies the barrier function of endothelial cell monolayers upon the exposure to pro-inflammatory cytokines. Low levels (1 ng/ml) of tumor necrosis factor-alpha initially increased the permeability in both HTNV-infected and uninfected monolayers similarly. Thereafter, however, these monolayers showed significant difference. The HTNV-infected monolayers remained irreversibly hyper-permeable during the experimental period up to 4 days, while the uninfected monolayers completely recovered the barrier function. The prolonged hyper-permeability of HTNV-infected monolayers was not associated with cell death or gap formation in the monolayers, and was independent from their nitric oxide or prostaglandin production. These results are the first evidence that hantavirus infection modifies barrier function of endothelial cell monolayers and suggest that HTNV-infection of endothelial cells may contribute to the increased vascular leakage through the prolonged response to cytokines.