Approximately 57% of clinical and 33% of poultry isolates examined produced a cytotoxin. Cytotoxic activity was detected in 25 (50%) isolates of Campylobacter of which 12 were isolated from bloody diarrhea and 9 from watery stools. The cytotoxin titers were low, ranging from 2 to 16. The crude filtrates from 50 Campylobacter isolates showed no cytotoxic effect in Vero cells, no fluid accumulation in suckling mice and no hemolytic activity.
Previous studies have found Kunjin (KUN) virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.
Eighteen strains of Aeromonas hydrophila from patients with bacteraemia were investigated for possible virulence factors. Cytotoxin and haemolysin were produced by all strains, whereas cholera toxin-like factor was produced by 33% of strains only. Enterotoxin production was not detected. Haemagglutination of guinea-pig, fowl and rabbit erythrocytes was demonstrated by 83%, 67% and 61% of strains, respectively. Fucose- and mannose-sensitive haemagglutinins were predominant. None of the strains agglutinated sheep erythrocytes. Extrachromosomal DNA was detected in 17 strains, 16 of which had a plasmid (3.6-5.1 MDa), the majority being between 4.6 and 5.1 MDa.
Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.
By means of the gentamicin HEp-2 cell invasion assay, it was demonstrated that 82% of the Campylobacters tested were cell-invasive, including 83% of isolates from bloody diarrhoea and 80% of isolates from watery diarrhoea. The large number of invasive strains from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. Whether this stage can progress further to more severe symptoms such as bloody diarrhoea remains to be elucidated. Whether this progression to bloody diarrhoea occurs as a result of toxin production is still debatable. In Vero cells, invasion was less efficient and intracellular multiplication was not observed.
Metaldehyde is used widely in Malaysia for the control of molluscs. This communication reports the cytotoxic effects of this chemical on cultured cells as assessed by cell morphology and the DNA synthesising capability as well as its transport into cells. After 15 days of exposure with 20.0 ppm of the compound, the DNA synthesising capability was shown to be unaffected. The IC50 for Vero cells was 276.0 ppm. Transport of thymidine across cells was found to be not significantly affected even at high metaldehyde concentrations (up to 320.0 ppm) suggesting integrity of cells were not significantly affected. The present cellular studies have therefore shown that the cytotoxic effects of this chemical is rather low.
Metaldehida digunakan dengan meluas di Malaysia untuk mengawal perosak moluska. Kesan sitotoksik bahan kimia ini di peringkat sel dari segi ciri-ciri perubahan moifologi dan keupayaan mensintesis DNA serta kajian awal kesannya terhadap proses kemasukan ke dalam sel dilaporkan di sini. Keupayaan mensintesis DNA didapati tidak terjejas secara signifikan selepas diberikan 20.0 ppm metaldehida secara berterusan selama 15 hari. Nilai IC50 bagi sel Vero adalah 276.0 ppm. Kemasukan timidina ke dalam sel tidak terjejas secara signifikan apabila sel diperlakukan dengan metaldehida, walaupun pada kepekatan yang agak tinggi iaitu sehingga 320.0 ppm. Kajian telah menunjukkan bahawa kesan sitotoksik oleh metaldehida adalah rendah.
In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.
Infectious agent(s) causing the fatal Sarawak acute childhood viral infection (SACVI) has not been identified. In the present study, results indicating that inocula prepared from the fatal cases of SACVI induced apoptosis in Vero cell cultures are presented. These findings suggest the possible involvement of apoptotic cellular responses in SACVI.
Identification of the aetiologic agent(s) associated with an outbreak of fatal childhood viral infection in Sarawak, Malaysia, in mid 1997 remains elusive. It is reported here that African green monkey kidney (Vero) and human monocytic (U937) cells treated with inocula derived from clinical specimens of some of these fatal cases showed the presence of cellular genomic DNA degradation when the extracted DNA was separated by pulsed field gel electrophoresis (PFGE), oligonucleosomal DNA ladders characteristic of apoptotic cells when the infected cells' DNA was separated by agarose gel electrophoresis, and apoptotic cellular DNA fragmentation when cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). These results suggest that inocula derived from the patients' clinical specimens contain factors which stimulate apoptotic cellular responses in vitro.
Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.
Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.
In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.
Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.
Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.
In late 1998, Nipah virus emerged in peninsular Malaysia and caused fatal disease in domestic pigs and humans and substantial economic loss to the local pig industry. Surveillance of wildlife species during the outbreak showed neutralizing antibodies to Nipah virus mainly in Island flying-foxes (Pteropus hypomelanus) and Malayan flying-foxes (Pteropus vampyrus) but no virus reactive with anti-Nipah virus antibodies was isolated. We adopted a novel approach of collecting urine from these Island flying-foxes and swabs of their partially eaten fruits. Three viral isolates (two from urine and one from a partially eaten fruit swab) that caused Nipah virus-like syncytial cytopathic effect in Vero cells and stained strongly with Nipah- and Hendra-specific antibodies were isolated. Molecular sequencing and analysis of the 11,200-nucleotide fragment representing the beginning of the nucleocapsid gene to the end of the glycoprotein gene of one isolate confirmed the isolate to be Nipah virus with a sequence deviation of five to six nucleotides from Nipah virus isolated from humans. The isolation of Nipah virus from the Island flying-fox corroborates the serological evidence that it is one of the natural hosts of the virus.
Emerging infectious diseases involving zoonosis have become important global health problems. The 1998 outbreak of severe febrile encephalitis among pig farmers in Malaysia caused by a newly emergent paramyxovirus, Nipah virus, is a good example. This disease has the potential to spread to other countries through infected animals and can cause considerable economic loss. The clinical presentation includes segmental myoclonus, areflexia, hypertension, and tachycardia, and histologic evidence includes endothelial damage and vasculitis of the brain and other major organs. Magnetic resonance imaging has demonstrated the presence of discrete high-signal-intensity lesions disseminated throughout the brain. Nipah virus causes syncytial formation in Vero cells and is antigenically related to Hendra virus. The Island flying fox (Pteropus hypomelanus; the fruit bat) is a likely reservoir of this virus. The outbreak in Malaysia was controlled through the culling of >1 million pigs.
Dengue virus type 2 (DENV-2) infection induced apoptotic cellular DNA fragmentation in Vero cells within 8 days of infection. The addition of high concentrations of extracellular Zn(2+) but not Ca(2+), Mg(2+) or Mn(2+) to the cell culture medium hastened the detection of apoptosis to within 4 h after infection. No apoptotic cellular DNA fragmentation was detected in the cell culture treated with Zn(2+) alone or infected with heat- or ultraviolet light-inactivated DENV-2 in the presence of Zn(2+). These results suggest that (i) apoptosis is induced in African green monkey kidney cells infected with live DENV-2 and (ii) the addition of high extracellular Zn(2+) accelerates detection of apoptosis in the DENV-2-infected cells.
A virus, named Oya virus, was isolated in Vero cell cultures from the lungs of a pig suspected of Nipah virus infection. The virus was revealed as a spherical enveloped RNA virus with a diameter of 79 nm. For identification of Oya virus, RT-PCR was performed. A common primer set for S-RNA of the Simbu serogroup of the genus Bunyavirus was able to amplify a cDNA from Oya virus RNA. The sequence data of the product revealed that the partial gene of Oya virus S-RNA segment had 65-70% homology with published cDNA sequences of Simbu serogroup viruses. The phylogenetic analysis of the data showed that the Oya virus is grouped in Simbu serogroup, but is genetically distinct from the serogroup viruses that have been analyzed molecularly. Serological surveys revealed that the virus distributed widely and densely in Malaysia.
The coding region of the nucleocapsid (N) gene was amplified from the viral RNA and inserted into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. Hence, the N(fus) protein produced in E. coli could serve as an alternative antigen for the detection of anti-NiV in swine.
Both wild-type virulent and mutant strains of pseudorabies virus (PrV) were used in this study. Mutants used were derived from the plaque purified strain PrVmAIP. A total of six drug resistant mutants, three bromodeoxyuridine (BUdR) resistant and three iododeoxyuridine (IUdR) resistant, respectively, were isolated and passaged in chicken embryo fibroblast (CEF) cells. The DNA of these PrVs were compared with the wild-type isolates by means of the restriction fragment pattern (RFP) findings produced with Bam HI, Kpn I, Hind III and Bgl II restriction enzymes (RE). Compared to the wild-type PrVs (PrV-VBA1-parental strain of PrVmAIP; PrV-VBA2; PrV-VBA3), the RFP of PrVmAIP showed the presence of mutations within the RE sites studied. Both PrV-VBA1 and PrV-VBA2 appeared to be closely related but their RFPs differed from PrV-VBA3. Significant differences either in the number, size or migrations of the DNA fragments could also be detected in the BUdR resistant strains. Even though different features of cytopathic effect (GPE) were observed in the IUdR resistant PrVs, the RFP findings remained identical. The PrVs studied showed considerable differences from the reference PrV (PrV-CD).