Molecular prevalence of Bartonella spp. in bat flies in east coast Malaysia.

Bats are a significant reservoir for numerous pathogens, including Bartonella spp. It is one of the emerging zoonotic bacterial diseases that can be transmitted to humans and may cause various unspecific clinical manifestations. Thus, bartonellosis is rarely diagnosed and is regarded as a neglected vector-borne disease (VBD). Bat flies have been hypothesised to be a vector in the transmission of pathogens among bats. They are host-specific, which reduces the likelihood of pathogen transmission across bat species; however, they are likely to maintain high pathogen loads within their host species. To explore the presence of Bartonella spp. in bat flies from Peninsular Malaysia; bat fly samples collected from various sites at the east coast states were subjected to molecular detection for Bartonella spp. It was discovered that 38.7 % of bats from Terengganu and Kelantan were infested with bat flies; however, no bat fly was found in bats collected from Pahang. The collected bat flies belonged to the families Nycteribiidae (79.6 %) and Streblidae (20.4 %). The collected bat flies were pooled according to the locations and species into 39 pools. Out of these 39 pools, 66.7 % (n = 26) were positive for Bartonella spp. by PCR. Sequence analyses of five randomly selected PCR-positive pools revealed that pools from Kelantan (n = 3) have the closest sequence identities (99 %) to Bartonella spp. strain Lisso-Nig-922 from Nigeria. However, the other pools from Terengganu (n = 2) were closely related to Bartonella spp. strain KP277 from Thailand and Bartonella spp. strain Rhin-3 from the Republic of Georgia with 99 % and 100 % sequence identity, respectively. This suggests that the Bartonella spp. found in Malaysian bat flies are genetically diverse and can potentially serve as reservoirs for pathogenic Bartonella spp.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment.

BACKGROUND: Colistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments. RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 µg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia. CONCLUSION: Our findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Promoting cardioprotection with fenugreek: Insights from CoCl2-induced hypoxia in neonatal rat cardiomyocytes.

Objectives: This study aimed to investigate the protective effects of fenugreek on CoCl2-induced hypoxia in neonatal rat cardiomyocytes. Materials and Methods: Primary cardiomyocytes were isolated from Sprague Dawley rats aged 0-2 days and incubated with various concentrations of fenugreek (10-320 µg/ml) and CoCl2-induced hypoxia for different durations (24, 48, and 72 hr). Cell viability, calcium signaling, beating rate, and gene expression were evaluated. Results: Fenugreek treatments did not cause any toxicity in cardiomyocytes. At a concentration of 160 µg/ml for 24 hr, fenugreek protected the heart against CoCl2-induced hypoxia, as evidenced by reduced expression of caspases (-3, -6, -8, and -9) and other functional genes markers, such as HIF-1α, Bcl-2, IP3R, ERK5, and GLP-1r. Calcium signaling and beating rate were also improved in fenugreek-treated cardiomyocytes. In contrast, CoCl2 treatment resulted in up-regulation of the hypoxia gene HIF-1α and apoptotic caspases gene (-3, -9, -8, -12), and down-regulation of Bcl-2 activity. Conclusion: Fenugreek treatment at a concentration of 160 µg/ml was not toxic to neonatal rat cardiomyocytes and protected against CoCl2-induced hypoxia. Furthermore, fenugreek improved calcium signaling and beating rate and altered gene expression. Fenugreek may be a potential therapeutic agent for promoting cardioprotection against hypoxia-induced injuries.

First Report of Trypanosoma theileri in Equine Host and Tabanus sp. in Malaysia.

Trypanosoma (Megatrypanum) theileri is a non-pathogenic or weakly pathogenic parasite of domestic cattle that is cyclically transmitted by blood-sucking insects, mainly tabanid flies. It has been reported in several countries like Brazil, Venezuela, Japan, Taiwan, Thailand, Vietnam, and the Philippines. Although the ruminant industry is actively expanded in Malaysia, T. theileri and T. theileri-like trypanosomes have never been reported from Malaysia. The low pathogenicity of this species might be the main reason for overlooking T. theileri in this country. This paper describes an unforeseen finding of T. theileri from the outbreak of T. evansi in the state of Kelantan, Malaysia. This is the first time T. theileri reported in Malaysia, and also the first time T. theileri is reported in equid. Clinical signs compatible with infection by blood protozoa were observed; however, it was uncertain whether they were due to T. theileri infection. The detection of T. theileri from the blood sample and Tabanus sp. were confirmed through molecular analysis with PCR and DNA sequencing. In the present study, T. theileri from one horse and one Tabanus sp. were clustered with sequences of the previously described phylogenetic lineages from Japan, Chad and Brazil cattle. Even though this species is claimed to be host-specific with ruminant host restriction, the finding from this study suggested that T. theileri can infect equine whilst other isolates are known to infect ruminant species only. It is suspected there were two genotypes of T. theileri circulating in at least two districts of Kelantan. Thus, further study on multiple DNA regions should be conducted to determine the strains of detected T. theileri in Malaysia. Its impact on the horse and cattle industry should also be revised.

Prevalence, antibiotic susceptibility, and presence of drug resistance genes in Aeromonas spp. isolated from freshwater fish in Kelantan and Terengganu states, Malaysia.

BACKGROUND AND AIM: The emergence of antibiotic-resistant bacterial pathogens has been increasingly reported, which has resulted in a decreasing ability to treat bacterial infections. Therefore, this study investigated the presence of Aeromonas spp., including its antibiotic resistance in various fish samples, Oreochromis spp., Clarias gariepinus, and Pangasius hypophthalmus, obtained from Kelantan and Terengganu, Malaysia. MATERIALS AND METHODS: In this study, 221 fish samples, of which 108 (Oreochromis spp., n=38; C. gariepinus, n=35; and P. hypophthalmus, n=35) were from Kelantan and 113 (Oreochromis spp., n=38; C. gariepinus, n=35; and P. hypophthalmus, n=40) were from Terengganu, were caught using cast nets. Then, samples from their kidneys were cultured on a Rimler Shott agar to isolate Aeromonas spp. Polymerase chain reaction (PCR) was used to confirm this isolation using specific gene primers for species identification. Subsequently, the isolates were tested for their sensitivity to 14 antibiotics using the Kirby-Bauer method, after which the PCR was conducted again to detect resistance genes: sul1, strA-strB, aadA, bla TEM, bla SHV, tetA-tetE, and tetM. RESULTS: From the results, 61 isolates were identified as being from the genus Aeromonas using PCR, of which 28 were Aeromonas jandaei, 19 were Aeromonas veronii, seven were Aeromonas hydrophila, and seven were Aeromonas sobria. Moreover, 8, 12, and 8 of A. jandaei; 4, 3, and 12 of A. veronii; 6, 0, and 1 of A. hydrophila; and 3, 3, and 1 of A. sobria were obtained from Oreochromis spp., C. gariepinus, and P. hypophthalmus, respectively. In addition, the isolates showed the highest level of resistance to ampicillin (100%), followed by streptomycin (59.0%), each kanamycin and nalidixic acid (41.0%), neomycin (36.1%), tetracycline (19.7%), sulfamethoxazole (14.8%), and oxytetracycline (13.1%). Resistance to gentamicin and ciprofloxacin both had the same percentage (9.8%), whereas isolates showed the lowest resistance to norfloxacin (8.2%) and doxycycline (1.6%). Notably, all Aeromonas isolates were susceptible to chloramphenicol and nitrofurantoin. Results also revealed that the multiple antibiotic resistances index of the isolates ranged from 0.07 to 0.64, suggesting that the farmed fish in these areas were introduced to the logged antibiotics indiscriminately and constantly during their cultivation stages. Results also revealed that the sul1 gene was detected in 19.7% of the Aeromonas isolates, whereas the tetracycline resistance genes, tetA and tetE, were detected in 27.9% and 4.9% of the isolates, respectively. However, ß-lactam resistance genes, bla TEM and bla SHV, were found in 44.3% and 13.1% of Aeromonas isolates, respectively, whereas strA-strB and aadA genes were found in 3.3% and 13.1% of the isolates, respectively. CONCLUSION: This study, therefore, calls for continuous surveillance of antibiotic-resistant Aeromonas spp. in cultured freshwater fish to aid disease management and better understand their implications to public health.

Infectious bronchitis associated with Escherichia coli infection in commercial broiler chickens: a case report.

OBJECTIVE: In this case report, we have investigated the infectious bronchitis (IB) virus (IBV) outbreak with the co-infection of Escherichia coli in 28-33-day-old broiler chickens in Malaysia. MATERIALS AND METHODS: A farmer complained that Cobb 500 chickens, raised in the open house, were having bloody diarrhea, open mouth breathing, non-uniform growth, and ruffled feathers. The mortality was about 100 birds (from about 7000 birds) per day. The sick birds were isolated and subjected to physical examination, postmortem, and histopathological analyses. Gross lesions were observed and recorded. The lung samples have proceeded with histopathological evaluations. The lungs, kidneys, trachea, air sac, and heart samples were collected to isolate bacteria and fungi through a series of conventional cultural methods, followed by molecular confirmation of the IBV. RESULTS: Postmortem examination revealed air sacculitis, hemorrhagic tracheitis, pulmonary congestion, fibrin deposition in the liver and air sac, hemorrhagic enteritis, and renomegaly. The bacterial culture and biochemical tests revealed E. coli in the lungs, trachea, liver, intestine, and kidney samples. However, no fungus could be isolated from those samples. Histological evaluation of lung samples demonstrated infiltration of inflammatory cells in the pulmonary tissues. Apart from this, reverse transcription-polymerase chain reaction confirmed the presence of avian coronavirus responsible for infectious bronchitis (IB). CONCLUSION: The chickens were diagnosed with IB concurrent with E.coli. The chickens exhibited typical nephropathogenic strain of IBV infection, causing high mortality.

Complex chronic respiratory disease concurrent with coccidiosis in broiler chickens in Malaysia: A case report.

OBJECTIVE: The case study describes the cause of an increase in mortality rates among 35-day-old broilers that developed respiratory distress and bloody diarrhea on a farm in Malaysia. MATERIALS AND METHODS: The organ samples were subjected to laboratory testing and postmortem inspection. Escherichia (E.) coli and Mycoplasma (M.) gallisepticum were detected using bacterial isolation and molecular diagnostics using polymerase chain reaction. RESULTS: Chickens with the infection had widespread fibrin buildup in several organs and hemorrhages on the duodenal mucosa. Additional histology and laboratory analysis of organ samples revealed infection with M. gallisepticum, E. coli, and enteric Eimeria spp., all of which are consistent with complex chronic respiratory disease (CCRD) associated with coccidiosis. Tylosin tartrate 20% (w/w) (2.5 gm/l) was prescribed for 1 week along with a combination of the broad-spectrum bacteriostatic drug streptomycin (25 mg/kg) and coccidiostat (2 gm/5 l). CONCLUSION: CCRD and coccidiosis are both infectious diseases that can infect chicken flocks, resulting in production losses and carcass quality degradation. Early disease detection and proper treatment should be provided promptly, and tight farm biosecurity should be implemented to prevent chicken mortality on the farm, as was achieved successfully.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample.

BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model. RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites. CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Contemporary Strategies and Current Trends in Designing Antiviral Drugs against Dengue Fever via Targeting Host-Based Approaches.

Dengue virus (DENV) is an arboviral human pathogen transmitted through mosquito bite that infects an estimated ~400 million humans (~5% of the global population) annually. To date, no specific therapeutics have been developed that can prevent or treat infections resulting from this pathogen. DENV utilizes numerous host molecules and factors for transcribing the single-stranded ~11 kb positive-sense RNA genome. For example, the glycosylation machinery of the host is required for viral particles to assemble in the endoplasmic reticulum. Since a variety of host factors seem to be utilized by the pathogens, targeting these factors may result in DENV inhibitors, and will play an important role in attenuating the rapid emergence of other flaviviruses. Many experimental studies have yielded findings indicating that host factors facilitate infection, indicating that the focus should be given to targeting the processes contributing to pathogenesis along with many other immune responses. Here, we provide an extensive literature review in order to elucidate the progress made in the development of host-based approaches for DENV viral infections, focusing on host cellular mechanisms and factors responsible for viral replication, aiming to aid the potential development of host-dependent antiviral therapeutics.

Antibacterial and cytotoxic activities of the Syzygium polyanthum leaf extract from Malaysia.

BACKGROUND AND AIM: The increasing prevalence of drug resistance eventually leads scientist to discover new drugs that could solve the problem. Since ancient immemorial times, medicinal plants generally known as herbs were widely used in every culture throughout the world. In fact, currently up to 70,000 plant species have been screened for biological activities and about 70% ends up for commercialization. Therefore, this study was aimed to evaluate the potential cytotoxic and antibacterial effect of Syzygium polyanthum leaves which are local Malaysia plants, against 4T1 and MCF-7 mammary carcinoma cells, respectively, and also against bacteria causing mastitis in cows. MATERIALS AND METHODS: The cytotoxic effect of hydromethanolic extract of S. polyanthum against 4T1 and MCF-7 mammary carcinoma cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells were treated with the concentration of extracts ranging from 15.63 µg/mL to 1000 µg/ml for 72 h, and the percentage of cell survivability was determined based on minimum concentration that was able to allow at least 50% growth of cancer cells (IC50) after 72 h. The antibacterial activity was tested against common bacteria causing mastitis in cow. The bacteria were isolated from milk samples. The antibacterial activity of the extract was determined by disk diffusion method and susceptibility test based on minimum inhibitory concentration (MIC). RESULTS: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius were isolated from the milk samples that positive for mastitis. The MIC values range from 7.12 mm to 13.5 mm. The extract exhibits the widest zone of inhibition (13.5±0.20 mm) at 1000 mg/ml of concentrations. The extract relatively has low cytotoxicity effect against 4T1 and MCF-7 cells with IC50 values ranging from 672.57±59.42 and 126.05±50.89 µg/ml, respectively. CONCLUSION: S. polyanthum exerts weak antibacterial activity and cytotoxic effect to mammary carcinoma cells. The extract does not toxic to cells. However, further study is recommended, especially, this plant should be tested for in vivo.

An ambient temperature stable and ready-to-use loop-mediated isothermal amplification assay for detection of toxigenic Vibrio cholerae in outbreak settings.

Cholera, caused by Vibrio cholerae is a foodborne disease that frequently reported in food and water related outbreak. Rapid diagnosis of cholera infection is important to avoid potential spread of disease. Among available diagnostic platforms, loop-mediated isothermal amplification (LAMP) is regarded as a potential diagnostic tool due to its rapidity, high sensitivity and specificity and independent of sophisticated thermalcycler. However, the current LAMP often requires multiple pipetting steps, hence is susceptible to cross contamination. Besides, the strict requirement of cold-chain during transportation and storage make its application in low resource settings to be inconvenient. To overcome these problems, the present study is aimed to develop an ambient-temperature-stable and ready-to-use LAMP assay for the detection of toxigenic Vibrio cholerae in low resource settings. A set of specific LAMP primers were designed and tested against 155 V. cholerae and non-V. cholerae strains. Analytical specifity showed that the developed LAMP assay detected 100% of pathogenic V. cholerae and did not amplified other tested bacterial strains. Upon testing against stool samples spiked with toxigenic V. cholerae outbreak isolates, the LAMP assay detected all of the spiked samples (n = 76/76, 100%), in contrast to the conventional PCR which amplified 77.6% (n = 59/76) of the tested specimens. In term of sensitivity, the LAMP assay was 100-fold more sensitive as compared to the conventional PCR method, with LOD of 10 fg per µL and 10 CFU per mL. Following lyophilisation with addition of lyoprotectants, the dry-reagent LAMP mix has an estimated shelf-life of 90.75 days at room temperature.

Development of a thermostabilised triplex LAMP assay with dry-reagent four target lateral flow dipstick for detection of Entamoeba histolytica and non-pathogenic Entamoeba spp.

This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.

Cellular transcripts regulated during infections with Highly Pathogenic H5N1 Avian Influenza virus in 3 host systems.

BACKGROUND: Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and the virus replicates in high levels in the respiratory tract inducing severe lung lesions. The pathogenesis of the disease is actually the outcome of the infection as determined by complex host-virus interactions involving the functional kinetics of large numbers of participating genes. Understanding the genes and proteins involved in host cellular responses are therefore, critical for the elucidation of the mechanisms of infection. METHODS: Differentially expressed transcripts regulated in a H5N1 infections of whole lung organ of chicken, in-vitro chick embryo lung primary cell culture (CeLu) and a continuous Madin Darby Canine Kidney cell line was undertaken. An improved mRNA differential display technique (Gene Fishing™) using annealing control primers that generates reproducible, authentic and long PCR products that are detectable on agarose gels was used for the identification of differentially expressed genes (DEGs). Seven of the genes have been selected for validation using a TaqMan® based real time quantitative PCR assay. RESULTS: Thirty seven known and unique differentially expressed genes from lungs of chickens, CeLu and MDCK cells were isolated. Among the genes isolated and identified include heat shock proteins, Cyclin D2, Prenyl (decaprenyl) diphosphate synthase, IL-8 and many other unknown genes. The quantitative real time RT-PCR assay data showed that the transcription kinetics of the selected genes were clearly altered during infection by the Highly Pathogenic Avian Influenza virus. CONCLUSION: The Gene Fishing™ technique has allowed for the first time, the isolation and identification of sequences of host cellular genes regulated during H5N1 virus infection. In this limited study, the differentially expressed genes in the three host systems were not identical, thus suggesting that their responses to the H5N1 infection may not share similar mechanisms and pathways.

Characterization of Nipah virus from naturally infected Pteropus vampyrus bats, Malaysia.

We isolated and characterized Nipah virus (NiV) from Pteropus vampyrus bats, the putative reservoir for the 1998 outbreak in Malaysia, and provide evidence of viral recrudescence. This isolate is monophyletic with previous NiVs in combined analysis, and the nucleocapsid gene phylogeny species.

Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein.

Viruses in the genus Orthobunyavirus, family Bunyaviridae, have a genome comprising three segments (called L, M, and S) of negative-sense RNA. Serological studies have classified the >170 named virus isolates into 18 serogroups, with a few additional as yet ungrouped viruses. Until now, molecular studies and full-length S-segment nucleotide sequences were available for representatives of eight serogroups; in all cases, the S segment encodes two proteins, N (nucleocapsid) and NSs (nonstructural), in overlapping open reading frames (ORFs) that are translated from the same mRNA. The NSs proteins of Bunyamwera virus (BUNV) and California serogroup viruses have been shown to play a role in inhibiting host cell mRNA and protein synthesis, thereby preventing induction of interferon (IFN). We have determined full-length sequences of the S segments of representative viruses in the Anopheles A, Anopheles B, and Tete serogroups, and we report here that these viruses do not show evidence of having an NSs ORF. In addition, these viruses have rather longer N proteins than those in the other serogroups. Most of the naturally occurring viruses that lack the NSs protein behaved like a recombinant BUNV with the NSs gene deleted in that they failed to prevent induction of IFN-beta mRNA. However, Tacaiuma virus (TCMV) in the Anopheles A serogroup inhibited IFN induction in a manner similar to that of wild-type BUNV, suggesting that TCMV has evolved an alternative mechanism, not involving a typical NSs protein, to antagonize the host innate immune response.