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Immunological characterization of rice tungro spherical virus coat proteins and differentiation of isolates from the Philippines and India

Druka A, Burns T, Zhang S, Hull R

J Gen Virol, 1996 Aug;77 ( Pt 8):1975-83.
PMID: 8760450

Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.

Matched MeSH terms: DNA, Viral
Emergence of dengue virus type 4 genotype IIA in Malaysia

AbuBakar S, Wong PF, Chan YF

J Gen Virol, 2002 Oct;83(Pt 10):2437-2442.
PMID: 12237425 DOI: 10.1099/0022-1317-83-10-2437

Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.

Matched MeSH terms: DNA, Viral
Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia

AbuBakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK

Virus Res, 1999 May;61(1):1-9.
PMID: 10426204

Thirteen enterovirus 71 (EV71) isolates were obtained from both fatal and non-fatal infections of patients seen in Peninsula Malaysia and in Sarawak during an outbreak of hand, foot and mouth disease (HFMD) in Malaysia in 1997, with incidences of fatal brainstem encephalomyelitis. The isolates were identified using immunofluorescence staining, neutralization assays, and partial sequencing of the 5' untranslated regions (UTR). Assessment of the potential genetic relationships of the isolates using the partial 5'UTR sequences suggested clustering of the isolates into at least two main clusters. Isolates from Peninsula Malaysia were found in both clusters whereas Sarawak-derived isolates clustered only in cluster II. Isolates derived from fatal infections, however, occurred in both clusters and no distinctive nucleotide sequences could be attributed to the fatal isolates. Examination of the nucleotide sequences revealed at least 13 nucleotide positions in all the isolates which differ completely from the previously reported EV71 5'UTR sequences. In addition, at least 11 nucleotide position differences within the 5'UTR were noted which differentiated cluster I from cluster II. Predicted secondary RNA structures drawn using the nucleotide sequences also suggested differences between isolates from the two clusters. These findings suggest the presence of at least two potentially virulent EV71 co-circulating in Malaysia during the 1997 HFMD outbreak.

Matched MeSH terms: DNA, Viral
Recombinant Newcastle Disease virus capsids displaying enterovirus 71 VP1 fragment induce a strong immune response in rabbits

Sivasamugham LA, Cardosa MJ, Tan WS, Yusoff K

J Med Virol, 2006 Aug;78(8):1096-104.
PMID: 16789020

The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.

Matched MeSH terms: DNA, Viral
Genotyping of hepatitis B virus in Malaysia based on the nucleotide sequence of preS and S genes

Ong HT, Duraisamy G, Kee Peng N, Wen Siang T, Seow HF

Microbes Infect., 2005 Mar;7(3):494-500.
PMID: 15792534

Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.

Matched MeSH terms: DNA, Viral
Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

Jazayeri SD, Ideris A, Zakaria Z, Yeap SK, Omar AR

Comp Immunol Microbiol Infect Dis, 2012 Sep;35(5):417-27.
PMID: 22512819 DOI: 10.1016/j.cimid.2012.03.007

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.

Matched MeSH terms: DNA, Viral/administration & dosage; DNA, Viral/immunology
Fulltext Genetic diversity of hepatitis B co-infection with hepatitis C, D and E viruses among Malaysian chronic hepatitis B patients

Hudu SA, Niazlin MT, Nordin SA, Tan SS, Omar H, Shahar H, et al.

Afr Health Sci, 2018 Dec;18(4):1117-1133.
PMID: 30766578 DOI: 10.4314/ahs.v18i4.33

Background: Hepatitis B virus co-infection with other strains of viral hepatitis is associated with increased risk of liver cirrhosis and hepatic decompensation.
Objectives: This is a prevalence study that assessed the genetic diversity of chronic hepatitis B patients and coinfection.
Methods: Chronic hepatitis B patients enrolled in this study were tested for antibodies of other hepatitis viruses using ELISA kits. Patient clinical profiles were collected and partial genes of HBV, HCV, and HEV were amplified, sequenced, and analyzed using phylogenetic analysis. The associations between variables were determined using the chi-squared test.
Results: Of the 82 patients recruited for this study, 53.7% were non-cirrhotic, 22.0% cirrhotic, 20.7% acute flare and 3.7% hepatocellular carcinoma. Majority (58%) of patients had a high level of ALT (≥34 U/L). Sequence analysis showed HBV (63.9%) belonged to genotype B, HEV belonged to genotype 4 while HCV belonged to genotype 3a and the genotypes were found to be significantly associated with the clinical stage of the patients (χ2=56.632; p<0.01). Similarly, Hepatitis B e antigen was also found to be significantly associated with the clinical stage of infection (χ2=51.952; p<0.01).
Conclusion: This study revealed that genetic diversity was found to have a significant impact on the severity of infection.

Matched MeSH terms: DNA, Viral
Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998

Brown BA, Oberste MS, Alexander JP, Kennett ML, Pallansch MA

J Virol, 1999 Dec;73(12):9969-75.
PMID: 10559310

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.

Matched MeSH terms: DNA, Viral
Fulltext Epstein-Barr virus latent membrane protein-1 (LMP-1) 30-bp deletion and Xho I-loss is associated with type III nasopharyngeal carcinoma in Malaysia

See HS, Yap YY, Yip WK, Seow HF

World J Surg Oncol, 2008;6:18.
PMID: 18275617 DOI: 10.1186/1477-7819-6-18

Nasopharyngeal carcinoma (NPC) is a human epithelial tumour with high prevalence amongst Chinese in Southern China and South East Asia and is associated with the Epstein-Barr virus (EBV). The viral genome harbours an oncogene, namely, the latent membrane protein 1 (LMP1) gene and known variants such as the 30-bp deletion and loss of XhoI restriction site have been found. Less is known about the relationship between these variants and the population characteristics and histological type.

Matched MeSH terms: DNA, Viral
Hepatitis B: review of development from the discovery of the "Australia Antigen" to end of the twentieth Century

Yap SF

Malays J Pathol, 2004 Jun;26(1):1-12.
PMID: 16190102

"Parenteral" or "serum" hepatitis is known to have afflicted man for centuries. However, it was not until the mid-1960s that the causative agent of this infection, the hepatitis B virus, was discovered. Since then, the biology and the replication strategy of the virus, and the clinical features and the epidemiology of the hepatitis B infection have been determined. Knowledge about the virus and the infection it causes led to the development of firstly, a plasma-derived vaccine and later a recombinant vaccine for the prevention of the infection. Integration of the hepatitis B vaccine into newborn vaccination programmes on a worldwide basis represents a major step in the effort to eliminate this infectious disease and its complications. Laboratory tests are available for the diagnosis and monitoring of the disease. Therapies have been developed to halt the progress of the chronic infection in affected individuals. While these developments have resulted in a decrease of the frequency of infection in many countries, particularly those that have implemented universal immunization of newborns, the chronic infection remains a significant global problem. Worldwide, over 300 million individuals are infected and each year, an estimated 1 million persons die from chronic complications of the disease including hepatocellular carcinoma and hepatic failure. The therapies currently available result in elimination of the virus in only a relatively small proportion of subjects and carry with it serious side effects. Geopolitical, economic and other factors hinder the vision of elimination of the infection through immunization programmes. Nevertheless, work continues to clarify further the underlying pathological mechanism of the infection, the host and viral factors that promote elimination or persistence of the virus in the human host. It is hoped that such investigations will reveal viral targets for the design of newer and potentially more effective drugs to treat the infection.

Matched MeSH terms: DNA, Viral
Fulltext Naturally occurring hepatitis B virus surface antigen mutant variants in Malaysian blood donors and vaccinees

Hudu SA, Harmal NS, Saeed MI, Alshrari AS, Malik YA, Niazlin MT, et al.

Eur J Clin Microbiol Infect Dis, 2015 Jul;34(7):1349-59.
PMID: 25792010 DOI: 10.1007/s10096-015-2358-1

Hepatitis B virus surface mutants are of enormous importance because they are capable of escaping detection by serology and can infect both vaccinated and unvaccinated populations, thus putting the whole population at risk. This study aimed to detect and characterise hepatitis B-escaped mutants among blood donors and vaccinees. One thousand serum samples were collected for this study from blood donors and vaccinees. Hepatitis B surface antigen, antibodies and core antibodies were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. DNA detection was performed via nested polymerase chain reaction (PCR), and the S gene was sequenced and analysed using bioinformatics. Of the 1,000 samples that were screened, 5.5% (55/1,000) were found to be HBsAg-negative and anti-HBc- and HBV DNA-positive. All 55 isolates were found to belong to genotype B. Several mutations were found across all the sequences from synonymous and non-synonymous mutations, with the most nucleotide mutations occurring at position 342, where adenine was replaced by guanine, and cytosine at position 46 was replaced by adenine in 96.4% and 98% of the isolates, respectively. Mutation at position 16 of the amino acid sequence was found to be common to all the Malaysian isolates, with 85.7% of the mutations occurring outside the major hydrophilic region. This study revealed a prevalence of 5.5% for hepatitis B-escaped mutations among blood donors and vaccinated undergraduates, with the most common mutation being found at position 16, where glutamine was substituted with lysine.

Matched MeSH terms: DNA, Viral
Fulltext [Study of pathogenicity of Nipah virus and its vaccine development]

Yoneda M

Uirusu, 2014;64(1):105-12.
PMID: 25765986 DOI: 10.2222/jsv.64.105

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans, and incurred a high fatality rate in humans. We established a system that enabled the rescue of replicating NiVs from a cloned DNA. Using the system, we analyzed the functions of accessory proteins in infected cells and the implications in in vivo pathogenicity. Further, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins, which appeared to be an appropriate to NiV vaccine candidate for use in humans.

Matched MeSH terms: DNA, Viral
Infectious cDNA clone of attenuated Langat tick-borne flavivirus (strain E5) and a 3' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice

Pletnev AG

Virology, 2001 Apr 10;282(2):288-300.
PMID: 11289811

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.

Matched MeSH terms: DNA, Viral/genetics*
Antibody and T cell responses induced in chickens immunized with avian influenza virus N1 and NP DNA vaccine with chicken IL-15 and IL-18

Lim KL, Jazayeri SD, Yeap SK, Mohamed Alitheen NB, Bejo MH, Ideris A, et al.

Res Vet Sci, 2013 Dec;95(3):1224-34.
PMID: 23948357 DOI: 10.1016/j.rvsc.2013.07.013

We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.

Matched MeSH terms: DNA, Viral/immunology
The relationships between West Nile and Kunjin viruses

Scherret JH, Poidinger M, Mackenzie JS, Broom AK, Deubel V, Lipkin WI, et al.

Emerg Infect Dis, 2001 Jul-Aug;7(4):697-705.
PMID: 11585535

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.

Matched MeSH terms: DNA, Viral

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