Insight into the origin of chikungunya virus in Malaysian non-human primates via sequence analysis.

Chikungunya virus (CHIKV) is maintained in the sylvatic cycle in West Africa and is transmitted by Aedes mosquito species to monkeys. In 2006, four verified CHIKV isolates were obtained during a survey of arboviruses in monkeys (Macaca fascicularis) in Pahang state, Peninsular Malaysia. RNA was extracted from the CHIKV isolates and used in reverse transcription polymerase chain reactions (RT-PCR) to amplify PCR fragments for sequencing. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the whole viral sequence. A total of 11,238 base pairs (bp) corresponding to open reading frames (ORFs) from our isolates and 47 other registered isolates in the National Center for Biotechnology Information (NCBI) were used to elucidate sequences, amino acids, and phylogenetic relationships and to estimate divergence times by using MEGA 7.0 and the Bayesian Markov chain Monte Carlo method. Phylogenetic analysis revealed that all CHIKV isolates could be classified into the Asian genotype and clustered with Bagan Panchor clades, which are associated with the chikungunya outbreak reported in 2006, with sequence and amino acid similarities of 99.9% and 99.7%, respectively. Minor amino acid differences were found between human and non-human primate isolates. Amino acid analysis showed a unique amino acid at position 221 in the nsP1region, at which a glycine (G) was found only in monkey isolates, whereas arginine (R) was found at the same position only in human isolates. The time to the most recent common ancestor (MRCA) estimation indicated that CHIKV probably started to diverge from human to non-human primates in approximately 2004 in Malaysia. The results suggested that CHIKV in non-human primates probably resulted from the spillover of the virus from humans. The study will be helpful in understanding the movement and evolution of CHIKV in Malaysia and globally.

Epidemiological and clinical features of enteroviral acute lower respiratory tract infections in hospitalized paediatric patients in a Malaysian tertiary center.

BACKGROUND: Human enteroviruses (HEVs) have been recognized to cause a significant number of respiratory tract infections in many regions. Previous studies conducted to analyse enteroviral respiratory tract infections focused on outbreaks. Data in the Southeast Asian region is still rather limited to date. OBJECTIVES: We conducted a prospective analysis to understand the epidemiological characteristics of enteroviral lower respiratory tract infections (LRTIs) among paediatric patients admitted to Hospital Ampang, a tertiary hospital in Malaysia. METHODOLOGY: Nasopharyngeal aspirates for common respiratory viruses and throat swabs for enteroviruses were obtained for rtPCR analyses. All positive enteroviral results were then cultured for species identification. RESULTS AND CONCLUSION: Of the total 211 recruited patients, enteroviral LRTIs made up 8%. The clinical features of enteroviral infections are mostly clinically indistinguishable from that of respiratory syncytial virus (RSV) infections. However, RSV appears to be more lymphocytosis causing than enteroviruses (EV). We found a higher asthma incidence within the enteroviral group compared to RSV group. Enteroviral infections continue to play an important role in LRTIs in children beyond infancy and up to school age. Among the enteroviral strains, EV71 contributes a major role in enteroviral LRTIs in our center. Routine testing for enterovirus would certainly help identify a significant proportion of unexplained viral LRTIs among paediatric patients. However, further cost analyses studies would be helpful to determine if incorporating testing for enteroviruses into routine respiratory viruses panel tests is economically feasible.

Entomological study of chikungunya infections in the State of Kelantan, Malaysia.

BACKGROUND & OBJECTIVES: Chikungunya infection has become a public health threat in Malaysia since the 2008 nationwide outbreaks. Aedes albopictus Skuse has been identified as the chikungunya vector in Johor State during the outbreaks. In 2009, several outbreaks had been reported in the State of Kelantan. Entomological studies were conducted in Kelantan in four districts, namely Jeli, Tumpat, Pasir Mas and Tanah Merah to identify the vector responsible for the virus transmission. METHODS: CHIKV cases records were obtained from State Health Department, Kelantan and localities involved were identified. Larva survey was conducted to collect the immature mosquito stages. Modified aspirators were used to collect the adult mosquitoes. All samples on dry ice were transferred to laboratory and the presence of the virus was detected using reverse transcriptase PCR. RESULTS: A total of 1,245 mosquito larvae were collected during larval survey and 2,019 adult mosquitoes were collected using aspirator. From these collections, 640 mosquito pools were tested for the presence of CHIKV by RT-PCR but none found positive. Ae. albopictus was the most abundant mosquito collected, followed by Culex sp., Armigeres sp. and Anopheles sp. A total of 2, 814 artificial containers were inspected during the study. INTERPRETATION & CONCLUSIONS: Since none of the mosquito samples was found to be positive for chikungunya virus, the vector(s) of chikungunya virus in these localities could not be identified.

Use of dengue NS1 antigen for early diagnosis of dengue virus infection.

Accurate and timely diagnosis of dengue virus is important for early detection of dengue virus infection. In this study, the usefulness of the dengue NS1 antigen test was evaluated as a routine, rapid diagnostic test for dengue virus infection. A total of 208 sera from patients suspected of having dengue virus infection were collected and tested for dengue antibody, dengue genome and dengue NS1 antigen. Dengue antibody test, dengue PCR test and dengue antigen test were able to detect dengue virus infection from Days 1 to 8 in 72.8, 52.8 and 44.0% of samples, respectively. Of the 208 sera tested, 69.2% (144/208) of the acute sera were positive for dengue virus infection based on IgM antibody, IgG antibody, NS1 antigen and PCR tests. Thirty-two point two percent of the samples (67/208) were found positive for dengue NS1 antigen, 38.5% (80/208) were PCR positive, 40.9% (85/208) were IgM positive and 36.1% (75/208) were IgG positive for dengue virus. The results reveal the detection rate of dengue virus infection was similar for PCR and dengue antibody (65.9%) and for NS1 antigen and dengue antibody (62.0%) combinations. Therefore, the dengue NS1 antigen test can be used to complement the current antibody test used in peripheral laboratories. Thus, the combination of the NS1 antigen and antibody tests could increase the diagnostic efficiency for early diagnosis of dengue infection.

Identification of Chikungunya virus strains circulating in Kelantan, Malaysia in 2009.

Malaysia experienced its first outbreak of chikungunya virus (CHIKV) infection in late 1998 in Klang District in Selangor; six years later the virus re-emerged in the state of Perak. All the CHIKV isolates in 1988 and 2006 shared high sequence similarities and belonged to the Asian genotype. In 2007 and 2008 CHIKV infection again reemerged but the genotype was the Central/East African genotype. This strain was found to be similar to the strains causing outbreaks in the India Ocean. In 2009, the strains circulating in Malaysia, including the state of Kelantan, based on the partial E1 gene, also belong to the Central/East African genotype.