Evaluation of immunochromatography tests for detection of novel GII.17 norovirus in stool samples.

A novel GII.17 norovirus has emerged as a major cause of epidemic and endemic acute gastroenteritis in several countries in Asia. We used a small panel of stool samples in which GII.17 virus had been quantified by real-time RT-PCR to evaluate four commercially available norovirus immunochromatography (IC) kits. At least 10(8) copies/mL of GII.17 virus were required by each IC kit for a positive result, which is 1,000-fold more than that reported for these assays for GII.4 viruses.

Correlation between mutations in the core and NS5A genes of hepatitis C virus genotypes 1a, 1b, 3a, 3b, 6f and the response to pegylated interferon and ribavirin combination therapy.

Several studies have reported correlation between mutations in core and NS5A proteins of hepatitis C virus (HCV) and response to interferon (IFN) therapy. In particular, mutations in NS5A protein have been shown to correlate with responsiveness to IFN treatment of HCV-1b in Japanese patients. This study investigated whether amino acid (aa) mutations in the core and NS5A proteins of HCV-1a, 1b, 3a, 3b and 6f correlated with the response to pegylated interferon (Peg-IFN) plus ribavirin (RBV) therapy in Thai patients. The entire sequences of core and NS5A of HCV from 76 HCV-infected patients were analysed in comparison with corresponding reference sequences. The data revealed that the number of aa mutations in full-length NS5A, its C-terminus, IFN sensitivity-determining region, variable region 3 (V3) and V3 plus flanking region of HCV-1b NS5A protein were significantly higher in responders than in the treatment failure group (P = 0.010, 0.031, 0.046, 0.020 and 0.006, respectively). Similar results were found in a putative protein kinase R binding domain region in HCV-6f NS5A protein (P = 0.022). Moreover, specific aa substitutions in NS5A that appeared to be associated with responders or the treatment failure group were observed at positions 78 and 305 for HCV-1b (P = 0.028), 64 and 52 for HCV-1a (P = 0.033) and 6f (P = 0.045). Nevertheless, analysis of aa sequences of core protein revealed highly conserved sequences among HCV genotypes and no significant differences between the viruses from responders and the treatment failure group. Our findings indicate that mutations in aa residues of NS5A of HCV-1a, 1b and 6f correlated well with responsiveness to Peg-IFN and RBV combination therapy.

Genetic characterization of group C rotavirus isolated from a child hospitalized with acute gastroenteritis in Chiang Mai, Thailand.

During an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand, from 2002 to 2004, in which 263 stool specimens tested, one isolate of group C rotavirus was detected from a two-year-old child admitted to hospital with acute gastroenteritis. The human group C rotavirus, named CMH004/03, was characterized further by molecular analyses of its VP4, VP6, and VP7 gene segments as well as determination of RNA pattern by polyacrylamide gel electrophoresis (PAGE). Molecular characterization of VP4, VP6, and VP7 genes by sequence analyses showed high levels of sequence identities with those of human group C rotavirus reference strains isolated worldwide at 95.2% to 99.4% on nucleotide and 97.5% to 100% on amino acid levels. In contrast, the CMH004/03 strain exhibited far lesser nucleotide and amino acid sequence identities at 67.7% to 84.1% and 68.7% to 91.3%, respectively, when compared with those of porcine and bovine group C rotaviruses. Phylogenetic analyses of VP4, VP6, and VP7 genes clearly confirmed that the CMH004/03 strain clustered in a monophyletic branch with other human group C rotavirus reference strains and distantly related to the clusters of animal group C rotavirus strains. In addition, the RNA electrophoretic migration pattern of CMH004/03 showed a typical pattern (4-3-2-2) of group C rotavirus. To our knowledge, this study is the second report of group C rotavirus infection in pediatric patients in Thailand after it was reported for the first time about two decades ago.

Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997.

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus-positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa-group with long electropherotype (e-type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN-group with a short e-type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4-96.3%, amino acid identity) and 116E from India (92-92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996-1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1-14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes.

Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid.

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25 degrees C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts.

Epidemiology of rotavirus infection in Thailand.

Rotavirus is a major cause of acute severe diarrhea in children worldwide and an important cause of death among young children in developing countries. Group A rotaviruses are antigenically complex and multiple serotypes infect humans. Reassortant rotavirus vaccines are now available which offer protection against severe illness caused by rotavirus serotypes G1-4. Before vaccines are introduced into target populations, it is necessary to establish the baseline data of the epidemiology of rotavirus infection in those countries. The purpose of the present study is to provide information related to the epidemiology of rotavirus infection in Thailand. All rotavirus studies performed in Thailand were found through Medline and Thai Index Medicus searches. A total of 26 of the most relevant studies published in international and national journals are reviewed. Most studies reported that the prevalence of rotavirus infection in Thailand was 27-34%, although a few studies have reported a prevalence above this range. The peak seasonal distribution of rotavirus infection among children hospitalized with diarrhea in Thailand was seen in the dry cool seasons: October to February. The prevalence of rotavirus infection was most frequently found in children aged 6-11 months up to 2 years. G1 was the most prevalent serotype in Thailand, followed by G2, G4 and G3, respectively. At least three G serotypes, mostly G1, G2 and G4, are seen to coexist in Thailand each epidemic year and in some studies all four G-serotypes were reported in the same epidemics. In a 1996-1997 study, G9 was the third prevailing serotype after G1 and G2, respectively. These results indicate that rotavirus epidemics occur in Thailand every year and children are the most affected population. In Thailand, although G1-G4 have been reported, G1 is the most prevalent serotype in each epidemic and G9 is becoming increasingly common.

Thalidomide stimulates T cell responses and interleukin 12 production in HIV-infected patients.

We performed a placebo-controlled study to evaluate the effects of immunomodulatory treatment with thalidomide on HIV levels, TNF-alpha levels, and immune status of 31 HIV-infected individuals, after temporary suppression of viral replication with antiretroviral drugs. Treatment with a combination of zidovudine and lamivudine (ZDV/LMV) for 14 days resulted in a median decline in plasma viremia of 1.94 log10 RNA equivalents/ml. After discontinuation of ZDV/LMV, thalidomide therapy (200 mg/day for 4 weeks) did not retard the prompt return of HIV titers to the pretreatment levels, and had no effect on plasma levels of TNF-alpha. In contrast, thalidomide treatment resulted in significant immune stimulation. We observed increased levels of plasma soluble IL-2 receptor, soluble CD8 antigen, and IL-12 (p < 0.01 for all parameters), as well as increased cutaneous delayed-type hypersensitivity reactions to recall antigens (p < 0.01) in thalidomide-treated patients. These changes were associated with a median increase in HIV titer of 0.2 log10 RNA equivalents/ml in the thalidomide-treated group (p < 0.05), which resolved after stopping the drug. Further studies were performed in vitro to elucidate the mechanism of thalidomide-induced immune stimulation. When purified T cells from HIV-infected individuals were stimulated by immobilized anti-CD3 in the presence of thalidomide, a costimulatory effect of the drug was observed, resulting in increased production of IL-2 and IFN-gamma, and increased T cell-proliferative responses. Further experiments showed that thalidomide increased IL-12 production by antigen-presenting cells in a T cell-dependent manner. Our findings suggest a potential application for thalidomide as a novel immune adjuvant in HIV disease.

Possible occurrence of a genetic bottleneck in dengue serotype 2 viruses between the 1980 and 1987 epidemic seasons in Bangkok, Thailand.

Cocirculation of two genetic subtypes of dengue serotype 2 viruses was first observed in the 1980 epidemic season in Thailand. To further delineate the evolutionary history and the contribution of these subtypes to subsequent epidemics, we determined the envelope glycoprotein gene sequence of 20 dengue serotype 2 viruses isolated from infected patients during 1987 and compared them with those derived from earlier years. Subtype IIIa strains represented the majority (18 of 19) of dengue type 2 viruses derived from Bangkok metropolitan area, whereas all three strains from a province in the northeastern region belonged to subtype IIIb, indicating uneven local distribution of dengue subtypes within the same year. Three types of sequence variation were identified in both subtypes: substitutions that were unique to individual strains; substitutions that were shared among all subtype IIIa or IIIb viruses of both the 1980 and 1987 epidemics; and those that were shared only among all subtypes IIIa or IIlb viruses of the 1987 epidemic, but were absent from the corresponding subtypes of 1980. While the first and second types of substitution were indicative of the most recent random mutations and previous mutations that had been fixed in virus populations, respectively, the third type suggested possible occurrence of a genetic bottleneck and subsequent expansion of one or a limited number of subtype IIIa strains in Bangkok between 1980 and 1987. Immunoblot analysis of intracellular NS1 antigen with anti-NS1 monoclonal antibodies also revealed antigenic heterogeneity of the NS1 protein that correlated with the subdivision based on envelope protein variation.

Antigenic analysis of influenza viruses isolated in Thailand between 1991 and 1994.

We studied the epidemiology of influenza viruses in Thailand by isolating them and comparing their antigenic features with those of Japanese isolates. Between 1991 and 1994, 32 strains were isolated from 186 throat swab specimens. Twenty-one of the 32 isolates were of type A, subtype H3N2, and 11 strains were type B. It was suggested that the isolates of type A, subtype H3N2, drifted antigenically from A/Beijing/352/89-like to A/Kitakyusyu/159/93-like variants used as reference strains for comparison. The type B isolates in 1991 were suspected to be antigenically different from those of B/Bangkok/163/90, Thailand, in HI tests. These 1991 isolates were similar to B/Mie/1/93, which was isolated in the latter half of the epidemic in Japan in winter 1992/1993.

Primary sequence of the envelope glycoprotein of a dengue type 2 virus isolated from patient with dengue hemorrhagic fever and encephalopathy.

Dengue viruses exist in nature as a collection of highly similar but not identical members (quasispecies). In order to correlate the presence of viral quasispecies with rare occurrence of unusual clinical manifestations in dengue-infected individuals, a dengue type 2 virus was isolated from the peripheral blood of a 12-year-old boy who presented with fever, headache, drowsiness and tonic seizure of the left arm, and subsequently manifested symptoms and signs of dengue hemorrhagic fever. Analysis of the envelope glycoprotein sequence of the encephalopathy-associated virus and two other dengue type 2 viruses from the same epidemic season in Chiang Mai, Thailand revealed that all three viruses belonged to the subtype IIIa of the five-subtype phylogenetic nomenclature system for dengue type 2 virus. The encephalopathy-associated dengue virus was more divergent from the others and was characterized by an Ala-->Val substitution at the position 173 of the envelope glycoprotein. This substitution mapped to the central domain 1 which was not known to be involved directly in envelope-receptor interaction.

Production of mouse immunoglobulin G by a hybrid plant derived from tobacco-mouse cell fusions.

Mouse-tobacco hybrid calli, and complete plants producing mouse gamma-3 heavy and lambda light chains, have been generated by somatic cell fusions of mouse spleen cells and tobacco mesophyll protoplasts. Both gamma 3 and lambda chains were detected in hybrid calli and complete plants by enzyme-linked immunosorbent assay, immunofluorescent staining, and Western blotting. When cellular DNA from hybrid tobacco mesophyll protoplasts was amplified by the polymerase chain reaction (PCR) using two pairs of gamma 3 chain DNA primers and one pair of lambda chain DNA primers, the PCR products contained gamma 3 and lambda chain DNAs, which could be detected by southern blotting and DNA hybridization, using specific synthetic oligonucleotide probes for gamma 3 and lambda respectively. In situ hybridization of hybrid tobacco mesophyll protoplasts with specific recombinant DNA probes of gamma 3 and lambda chains showed the presence of gamma 3 and lambda chain DNAs in the hybrid protoplasts.

Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes.

The effect of interferon-gamma (IFN-gamma) on dengue virus multiplication in human peripheral blood monocytes was investigated. Enriched monocytes were treated with IFN-gamma and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-gamma caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-gamma also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-gamma-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-gamma in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937.

Studies on serological cross-reaction in sequential flavivirus infections.

Acute- and convalescent-phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre-existing flavivirus antibodies were tested for cross-reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent-phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross-reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross-reactive. Of three JE cases with pre-existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross-reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.

Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera.

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration of interleukin-1 producing monocytes from human peripheral blood mononuclear leukocytes by agar plating technique.

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10(5) monocytes, with mean +/- SEM = 1344 +/- 182 cells/10(5) monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10(5) monocytes/ml, with mean +/- SEM = 42.8 +/- 11.2 U/10(5) monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean +/- SEM = 33.5 +/- 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated (r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-alpha.

Determination of antibody from typhoid patients against lipopolysaccharide and protein antigens of Salmonella typhi.

Although the Widal test is simple, inexpensive and the most widely used for serodiagnosis of typhoid fever, the sensitivity and specificity of the test is sometimes doubtful. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum IgG and IgM antibodies to protein and lipopolysaccharide (LPS) antigens of Salmonella typhi which was compared with the Widal test in various groups of subjects. In typhoid patients with hemocultures positive for S. typhi (TP group), ELISA positivity was found on 100% for IgG antiprotein, 94.44% for IgG anti-LPS and 88.89% for IgM to both the protein and LPS antigens. In contrast, the Widal test was positive in only 61.11% for anti-O and 83.33% for anti-H antibodies. In healthy control subjects (HC group), only 5% of serum samples were positive for IgG anti-protein and none was positive for IgG anti-LPS or IgM to either the protein or LPS. In contrast, the Widal test was positive in 7.5% of HC group for anti-O and 17.5% for anti-H antibodies. In blood bank donors (BB group), both ELISA and Widal tests were positive in 23-40% of sera. Since the hospital records of BB group were incomplete. It might be possible that some of these subjects had recently been infected with S. typhi. Our data indicate that the standard Widal test was associated with false negative reactions in 16-39% of blood culture positive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Procedure for titration of interleukin-2 based on direct observation of factor-dependent growth of cells.

A semi-quantitative procedure for titration of interleukin-2 (IL-2) without the use of radioactive labeling of IL-2 target cells was developed. The procedure is based on the observation that those cells cultivated in round-bottom wells formed visible cultures and that the dimension of these cultures decreased at higher dilutions of IL-2 in the medium. The end-point of the titration can be easily established by observing the cultures with a concave mirror placed under the titration plate. The procedure has been used for the determination of IL-2 during its purification and characterization.