Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented.
Introduction: Dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) are Arboviruses that are transmitted by the same vector, Aedes aegypti. Dengue has become a global problem since the Second World War and is common in more than 110 countries. In Malaysia, dengue is a major disease burden as total economic costs to the country as a result of dengue is close to RM1.05 billion in 2010 and estimated to rise to 1.3 billion by 2020. Apart from Dengue, Zika and Chikungunya are the other important mosquito borne diseases in Malaysia. The aim of this study was to develop a multiplex real-time assay for simultaneous detection of DENV, ZIKV and CHIKV in clinical specimens. Methods: The published singleplex protocols were used with key modifications to implement a triplex assay. A one-step multiplex real-time RT-PCR assay was developed that can simultaneously detect RNA of DENV, ZIKV and CHIKV with good performance for a routine diagnostic use. The assay was evaluated for inter- and intra-reproducibility by mean CT value. The diagnostic sensitivity was tested with 135 archived samples which had been defined positive or negative by routine singleplex assays. Whole blood, plasma and urines were used in this study. Results: Intra- and inter-reproducibility and sensitivity varied from 0.10% to 4.73% and from 0.45% to 5.98% for each virus respectively. The specificity of detection was 100%. The multiplex real-time RT-PCR assay showed concordance with test results performed by routine singleplex assays. No cross reaction was observed for any of the clinical samples. Conclusion: The development of a rapid, sensitive and specific molecular assay for DENV, ZIKV and CHIKV infections will produce a greater diagnostic capacity in our laboratory. This multiplex approach is cost effective and robust with the concurrent detection of 3 viruses of public health concern.
The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus.
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.
The rapid spread of highly aggressive arboviruses, parasites, and bacteria along with the development of resistance in the pathogens and parasites, as well as in their arthropod vectors, represents a huge challenge in modern parasitology and tropical medicine. Eco-friendly vector control programs are crucial to fight, besides malaria, the spread of dengue, West Nile, chikungunya, and Zika virus, as well as other arboviruses such as St. Louis encephalitis and Japanese encephalitis. However, research efforts on the control of mosquito vectors are experiencing a serious lack of eco-friendly and highly effective pesticides, as well as the limited success of most biocontrol tools currently applied. Most importantly, a cooperative interface between the two disciplines is still lacking. To face this challenge, we have reviewed a wide number of promising results in the field of green-fabricated pesticides tested against mosquito vectors, outlining several examples of synergy with classic biological control tools. The non-target effects of green-fabricated nanopesticides, including acute toxicity, genotoxicity, and impact on behavioral traits of mosquito predators, have been critically discussed. In the final section, we have identified several key challenges at the interface between "green" nanotechnology and classic biological control, which deserve further research attention.