Displaying publications 1 - 20 of 79 in total

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  1. Fulltext The Saffold Virus-Penang 2B and 3C Proteins, but not the L Protein, Induce Apoptosis in HEp-2 and Vero Cells
    Xu Y, Victorio CBL, Meng T, Jia Q, Tan YJ, Chua KB
    Virol Sin, 2019 Jun;34(3):262-269.
    PMID: 31016480 DOI: 10.1007/s12250-019-00116-1
    Our previous work has shown that Saffold virus (SAFV) induced several rodent and primate cell lines to undergo apoptosis (Xu et al. in Emerg Microb Infect 3:1-8, 2014), but the essential viral proteins of SAFV involved in apoptotic activity lack study. In this study, we individually transfected the viral proteins of SAFV into HEp-2 and Vero cells to assess their ability to induce apoptosis, and found that the 2B and 3C proteins are proapoptotic. Further investigation indicated the transmembrane domain of the 2B protein is essential for the apoptotic activity and tetramer formation of the 2B protein. Our research provides clues for the possible mechanisms of apoptosis induced by SAFV in different cell lines. It also opens up new directions to study viral proteins (the 2B, 3C protein), and sets the stage for future exploration of any possible link between SAFV, inclusive of its related uncultivable genotypes, and multiple sclerosis.
    Matched MeSH terms: Viral Proteins/genetics*
  2. Fulltext Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
    Hassan MI, McSorley FR, Hotta K, Boddy CN
    J Vis Exp, 2017 06 27.
    PMID: 28715370 DOI: 10.3791/55187
    Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.
    Matched MeSH terms: Viral Proteins/genetics*
  3. Selection and characterization of ssDNA aptamer targeting Macrobrachium rosenbergii nodavirus capsid protein: A potential capture agent in gold-nanoparticle-based aptasensor for viral protein detection
    Ghadin N, Yusof NAM, Syarul Nataqain B, Raston NHA, Low CF
    J Fish Dis, 2024 Feb;47(2):e13892.
    PMID: 38014615 DOI: 10.1111/jfd.13892
    The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.
    Matched MeSH terms: Viral Proteins/genetics
  4. Fulltext Impact of RNA Virus Evolution on Quasispecies Formation and Virulence
    Mandary MB, Masomian M, Poh CL
    Int J Mol Sci, 2019 Sep 19;20(18).
    PMID: 31546962 DOI: 10.3390/ijms20184657
    RNA viruses are known to replicate by low fidelity polymerases and have high mutation rates whereby the resulting virus population tends to exist as a distribution of mutants. In this review, we aim to explore how genetic events such as spontaneous mutations could alter the genomic organization of RNA viruses in such a way that they impact virus replications and plaque morphology. The phenomenon of quasispecies within a viral population is also discussed to reflect virulence and its implications for RNA viruses. An understanding of how such events occur will provide further evidence about whether there are molecular determinants for plaque morphology of RNA viruses or whether different plaque phenotypes arise due to the presence of quasispecies within a population. Ultimately this review gives an insight into whether the intrinsically high error rates due to the low fidelity of RNA polymerases is responsible for the variation in plaque morphology and diversity in virulence. This can be a useful tool in characterizing mechanisms that facilitate virus adaptation and evolution.
    Matched MeSH terms: Viral Proteins/genetics*
  5. Fulltext High diversity of Cryptosporidium subgenotypes identified in Malaysian HIV/AIDS individuals targeting gp60 gene
    Iqbal A, Lim YA, Surin J, Sim BL
    PLoS One, 2012;7(2):e31139.
    PMID: 22347442 DOI: 10.1371/journal.pone.0031139
    Currently, there is a lack of vital information in the genetic makeup of Cryptosporidium especially in developing countries. The present study aimed at determining the genotypes and subgenotypes of Cryptosporidium in hospitalized Malaysian human immunodeficiency virus (HIV) positive patients.
    Matched MeSH terms: Viral Proteins/genetics*
  6. Sequence and phylogenetic analysis of S1, S2, M, and N genes of infectious bronchitis virus isolates from Malaysia
    Zulperi ZM, Omar AR, Arshad SS
    Virus Genes, 2009 Jun;38(3):383-91.
    PMID: 19242786 DOI: 10.1007/s11262-009-0337-2
    Two Malaysian infectious bronchitis virus isolates, MH5365/95 and V9/04 were characterized based on sequence and phylogenetic analyses of S1, S2, M, and N genes. Nucleotide sequence alignments revealed many point mutations, short deletions, and insertions in S1 region of both IBV isolates. Phylogenetic analysis of S1 gene and sequences analysis of M gene indicated that MH5365/95 and V9/04 belong to non-Massachusetts strain. However, both isolates share only 77% identity. Analysis based on S1 gene showed that MH5365/95 shared more than 87% identity to several Chinese strains. Meanwhile, V9/04 showed only 67-77% identity to all the previously studied IBV strains included in this study suggesting it is a variant of IBV isolate that is unique to Malaysia. Phylogenetic analysis suggests, although both isolates were isolated 10 years apart from different states in Malaysia, they shared a common origin. Analysis based on S2 and N genes indicated that both strains are highly related to each other, and there are fewer mutations which occurred in the respective genes.
    Matched MeSH terms: Viral Proteins/genetics*
  7. Existence of two forms of L protein of Newcastle disease virus isolates due to a compensatory mutation in Domain V
    Kusumaningtyas E, Tan WS, Zamrod Z, Eshaghi M, Yusoff K
    Arch Virol, 2004 Sep;149(9):1859-65.
    PMID: 15593426
    Nucleotide sequence comparison of the L gene of the Malaysian neurotropic-viscerotropic velogenic NDV strain AF2240 with other NDV strains revealed a single nucleotide insertion at position 3870. This mutation is compensated by a nucleotide deletion downstream at position 3958 which results in two forms of the L proteins containing a 30-amino acid substitution in Domain V. This compensatory mutation does not correlate with the pathogenicity of the viral strains but it may affect the viral replication as Domain V is believed to play an important role in the replication of paramyxoviruses.
    Matched MeSH terms: Viral Proteins/genetics*
  8. Fulltext Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction
    Addis SN, Lee E, Bettadapura J, Lobigs M
    Virol J, 2015;12:144.
    PMID: 26377679 DOI: 10.1186/s12985-015-0375-4
    Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed.
    Matched MeSH terms: Viral Proteins/genetics
  9. Fulltext Diversity and Evolutionary Histories of Human Coronaviruses NL63 and 229E Associated with Acute Upper Respiratory Tract Symptoms in Kuala Lumpur, Malaysia
    Al-Khannaq MN, Ng KT, Oong XY, Pang YK, Takebe Y, Chook JB, et al.
    Am J Trop Med Hyg, 2016 05 04;94(5):1058-64.
    PMID: 26928836 DOI: 10.4269/ajtmh.15-0810
    The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region.
    Matched MeSH terms: Viral Proteins/genetics
  10. Evolutionary relationships of endemic/epidemic and sylvatic dengue viruses
    Wang E, Ni H, Xu R, Barrett AD, Watowich SJ, Gubler DJ, et al.
    J Virol, 2000 Apr;74(7):3227-34.
    PMID: 10708439
    Endemic/epidemic dengue viruses (DEN) that are transmitted among humans by the mosquito vectors Aedes aegypti and Aedes albopictus are hypothesized to have evolved from sylvatic DEN strains that are transmitted among nonhuman primates in West Africa and Malaysia by other Aedes mosquitoes. We tested this hypothesis with phylogenetic studies using envelope protein gene sequences of both endemic/epidemic and sylvatic strains. The basal position of sylvatic lineages of DEN-1, -2, and -4 suggested that the endemic/epidemic lineages of these three DEN serotypes evolved independently from sylvatic progenitors. Time estimates for evolution of the endemic/epidemic forms ranged from 100 to 1,500 years ago, and the evolution of endemic/epidemic forms represents relatively recent events in the history of DEN evolution. Analysis of envelope protein amino acid changes predicted to have accompanied endemic/epidemic emergence suggested a role for domain III in adaptation to new mosquito and/or human hosts.
    Matched MeSH terms: Viral Proteins/genetics
  11. A tymovirus from Calopogonium mucunoides in Malaysia is not clitoria yellow vein tymovirus
    Gibbs AJ, Mackenzie AM, Abdul-Samad N
    Arch Virol, 1997;142(8):1697-702.
    PMID: 9672629
    A tymoyirus isolated from Malaysian crops of Calopogonium mucunoides has been shown to have virions that are serologically indistinguishable from those of clitoria yellow vein tymovirus. We have sequenced the virion protein (VP) gene of the virus and have found that although it is a member of the cluster that includes CYVV, it is the most distinct member of that cluster (< 62% sequence identity with all the others), and is clearly a separate species, which we propose should be named calopogonium yellow vein virus. Most of the serological specificity of the virions of tymoviruses seems to reside in the C-terminal hexapeptide of the virion protein.
    Matched MeSH terms: Viral Proteins/genetics*
  12. Molecular evolution and epidemiology of dengue-3 viruses
    Lanciotti RS, Lewis JG, Gubler DJ, Trent DW
    J Gen Virol, 1994 Jan;75 ( Pt 1):65-75.
    PMID: 8113741
    The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
    Matched MeSH terms: Viral Proteins/genetics*
  13. Regions on nucleocapsid protein of Newcastle disease virus that interact with its phosphoprotein
    Kho CL, Tan WS, Tey BT, Yusoff K
    Arch Virol, 2004 May;149(5):997-1005.
    PMID: 15098113 DOI: 10.1007/s00705-003-0273-8
    The nucleocapsid (NP) and phospho-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilised on a solid phase and then interacted with radio-labelled [(35)S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using this approach, a highly interactive region was identified to be resided at the first 25 amino acids of NP N-terminus. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end of NP. However, it is possible that the 49 amino acids at the C-terminal end might have another contact region for P protein, which is not as critical as the N-terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P.
    Matched MeSH terms: Viral Proteins/genetics
  14. Fulltext Application of Reverse Genetics in Functional Genomics of Potyvirus
    Kannan M, Zainal Z, Ismail I, Baharum SN, Bunawan H
    Viruses, 2020 07 26;12(8).
    PMID: 32722532 DOI: 10.3390/v12080803
    Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus-host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.
    Matched MeSH terms: Viral Proteins/genetics
  15. Fulltext Newcastle disease virus strain AF2240 as an oncolytic virus: A review
    Kalyanasundram J, Hamid A, Yusoff K, Chia SL
    Acta Trop, 2018 Jul;183:126-133.
    PMID: 29626432 DOI: 10.1016/j.actatropica.2018.04.007
    The discovery of tumour selective virus-mediated apoptosis marked the birth of an alternative cancer treatment in the form of oncolytic viruses. Even though, its oncolytic efficiency was demonstrated more than 50 years ago, safety concerns which resulted from mild to lethal side effects hampered the progress of oncolytic virus research. Since the classical oncolytic virus studies rely heavily on its natural oncolytic ability, virus manipulation was limited, thereby, restricted efforts to improve its safety. In order to circumvent such restriction, experiments involving non-human viruses such as the avian Newcastle disease virus (NDV) was conducted using cultured cells, animal models and human subjects. The corresponding reports on its significant tumour cytotoxicity along with impressive safety profile initiated immense research interest in the field of oncolytic NDV. The varying degree of oncolytic efficiency and virulency among NDV strains encouraged researchers from all around the world to experiment with their respective local NDV isolates in order to develop an oncolytic virus with desirable characteristics. Such desirable features include high tumour-killing ability, selectivity and low systemic cytotoxicity. The Malaysian field outbreak isolate, NDV strain AF2240, also currently, receives significant research attention. Apart from its high cytotoxicity against tumour cells, this strain also provided fundamental insight into NDV-mediated apoptosis mechanism which involves Bax protein recruitment as well as death receptor engagement. Studies on its ability to selectively induce apoptosis in tumour cells also resulted in a proposed p38 MAPK/NF-κB/IκBα pathway. The immunogenicity of AF2240 was also investigated through PBMC stimulation and macrophage infection. In addition, the enhanced oncolytic ability of this strain under hypoxic condition signifies its dynamic tumour tropism. This review is aimed to introduce and discuss the aforementioned details of the oncolytic AF2240 strain along with its current challenges which outlines the future research direction of this virus.
    Matched MeSH terms: Viral Proteins/genetics
  16. Identification and comparison of RCMV ALL 03 open reading frame (ORF) among several different strains of cytomegalovirus worldwide
    Balakrishnan KN, Abdullah AA, Bala J, Abba Y, Sarah SA, Jesse FFA, et al.
    Infect Genet Evol, 2017 10;54:81-90.
    PMID: 28642159 DOI: 10.1016/j.meegid.2017.06.020
    BACKGROUND: Rat cytomegalovirus ALL-03 (Malaysian strain) which was isolated from a placenta and uterus of a house rat, Rattus rattus diardii has the ability to cross the placenta and infecting the fetus. To further elucidate the pathogenesis of the Malaysian strain of Rat Cytomegalovirus ALL-03 (RCMV ALL-03), detailed analysis on the viral genome sequence is crucial.

    METHODS: Genome sequencing of RCMV ALL-03 was carried out in order to identify the open reading frame (ORF), homology comparison of ORF with other strains of CMV, phylogenetic analysis, classifying ORF with its corresponding conserved genes, and determination of functional proteins and grouping of gene families in order to obtain fundamental knowledge of the genome.

    RESULTS: The present study revealed a total of 123 Coding DNA sequences (CDS) from RCMV ALL-03 with 37 conserved ORF domains as with all herpesvirus genomes. All the CDS possess similar function with RCMV-England followed by RCMV-Berlin, RCMV-Maastricht, and Human CMV. The phylogenetic analysis of RCMV ALL-03 based on conserving genes of herpes virus showed that the Malaysian RCMV isolate is closest to RCMV-English and RCMV-Berlin strains, with 99% and 97% homology, respectively. Similarly, it also demonstrated an evolutionary relationship between RCMV ALL-03 and other strains of herpesviruses from all the three subfamilies. Interestingly, betaherpesvirus subfamily, which has been shown to be more closely related with gammaherpesviruses as compared to alphaherpesviruses, shares some of the functional ORFs. In addition, the arrangement of gene blocks for RCMV ALL-03, which was conserved among herpesvirus family members was also observed in the RCMV ALL-03 genome.

    CONCLUSION: Genomic analysis of RCMV ALL-03 provided an overall picture of the whole genome organization and it served as a good platform for further understanding on the divergence in the family of Herpesviridae.

    Matched MeSH terms: Viral Proteins/genetics
  17. FMO-guided design of darunavir analogs as HIV-1 protease inhibitors
    Chuntakaruk H, Hengphasatporn K, Shigeta Y, Aonbangkhen C, Lee VS, Khotavivattana T, et al.
    Sci Rep, 2024 Feb 13;14(1):3639.
    PMID: 38351065 DOI: 10.1038/s41598-024-53940-1
    The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.
    Matched MeSH terms: Viral Proteins/genetics
  18. Fulltext Development of a novel real-time polymerase chain reaction assay for the quantitative detection of Nipah virus replicative viral RNA
    Jensen KS, Adams R, Bennett RS, Bernbaum J, Jahrling PB, Holbrook MR
    PLoS One, 2018;13(6):e0199534.
    PMID: 29920552 DOI: 10.1371/journal.pone.0199534
    Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that can result in severe pulmonary disease and fatal encephalitis in humans and is responsible for outbreaks in Bangladesh, Malaysia, Singapore, India and possibly the Philippines. NiV has a negative-sense RNA genome that contains six genes and serves as a template for production of viral mRNA transcripts. NiV mRNA transcripts are subsequently translated into viral proteins. Traditionally, NiV quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays have relied on using primer sets that amplify a target (N that encodes the nucleocapsid) within the coding region of the viral gene that also amplifies viral mRNA. Here we describe a novel one-step qRT-PCR assay targeting the intergenic region separating the viral F and G proteins, thereby eliminating amplification of the viral mRNA. This assay is more accurate than the traditional qRT-PCR in quantifying concentrations of viral genomic RNA.
    Matched MeSH terms: Viral Proteins/genetics
  19. Fulltext Detection of east/central/south African genotype of chikungunya virus in Myanmar, 2010
    Tun MM, Thant KZ, Inoue S, Nabeshima T, Aoki K, Kyaw AK, et al.
    Emerg Infect Dis, 2014 Aug;20(8):1378-81.
    PMID: 25062511 DOI: 10.3201/eid2008.131431
    In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene.
    Matched MeSH terms: Viral Proteins/genetics
  20. Fulltext Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library
    Tham HW, Balasubramaniam VR, Chew MF, Ahmad H, Hassan SS
    J Infect Dev Ctries, 2015 Dec 30;9(12):1338-49.
    PMID: 26719940 DOI: 10.3855/jidc.6422
    INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
    METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
    RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
    CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
    Matched MeSH terms: Viral Proteins/genetics
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