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Fulltext Complete Genome Sequence of Rat Cytomegalovirus Strain ALL-03 (Malaysian Strain)

Balakrishnan KN, Abdullah AA, Camalxaman SN, Quah YW, Abba Y, Hani H, et al.

Genome Announc, 2015;3(3).
PMID: 26044413 DOI: 10.1128/genomeA.00451-15

The complete genome sequence of the ALL-03 strain of rat cytomegalovirus (RCMV) has been determined. The RCMV genome has a length of 197,958 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. This strain is closely related to the English strain of RCMV in terms of genetic arrangement but differs slightly in size.

Matched MeSH terms: Muromegalovirus
Characterization of a novel rat cytomegalovirus (RCMV) infecting placenta-uterus of Rattus rattus diardii

Loh HS, Mohd-Azmi ML, Lai KY, Sheikh-Omar AR, Zamri-Saad M

Arch Virol, 2003 Dec;148(12):2353-67.
PMID: 14648291

A new rat cytomegalovirus (RCMV) isolated from the placenta/uterus of a house rat (Rattus rattus diardii) was found to productively infect rat embryo fibroblast (REF) cells. The virus produced typical herpesvirus-like cytopathic effects characterized by a lytic infection. The well-known herpesvirus morphology was confirmed by electron microscopy. Its slow growth in cell culture indicated that the virus is belonging to subfamily Betaherpesvirinae. Electron microscopy techniques and immunohistochemistry confirmed the presence of herpesviral inclusion bodies and virus related particles in the cytoplasm and nucleus of infected cells. Hyperimmune serum against the Maastricht strain of RCMV revealed the virus identity in neutralization test, immunoperoxidase and immunofluorescence techniques. Despite typical characteristics of CMV, the viral genome is significantly different from that of Maastricht, English, UPM/Sg and UPM/Kn strains. The dissimilarities, which have not been reported before, had been confirmed by mean of restriction endonuclease analysis. The new RCMV strain, a virus that infects placenta and uterus of rats, has been named as ALL-03.

Matched MeSH terms: Muromegalovirus/growth & development; Muromegalovirus/isolation & purification*; Muromegalovirus/pathogenicity
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: Muromegalovirus/classification; Muromegalovirus/genetics*
Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Camalxaman SN, Zeenathul NA, Quah YW, Loh HS, Zuridah H, Hani H, et al.

In Vitro Cell Dev Biol Anim, 2013 Mar;49(3):238-44.
PMID: 23435855 DOI: 10.1007/s11626-012-9553-5

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.

Matched MeSH terms: Muromegalovirus/isolation & purification*; Muromegalovirus/metabolism; Muromegalovirus/pathogenicity
Fulltext Cross-reactivity of Malaysian rat cytomegalovirus strains with its human counterpart

Camalxaman SN, Zeenathul NA, Quah YW, Loh HS, Zuridah H, Sheikh-Omar AR, et al.

Trop Biomed, 2011 Dec;28(3):661-7.
PMID: 22433897 MyJurnal

This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments.

Matched MeSH terms: Muromegalovirus/immunology*
Development of a quantitative real-time RT-PCR for kinetic analysis of immediate-early transcripts of rat cytomegalovirus

Loh HS, Mohd-Azmi ML

Acta Virol., 2009;53(4):261-9.
PMID: 19941390

One-step real-time RT-PCR assay was developed for quantification of the immediate-early (IE), namely IE1 and IE2 transcripts of Rat cytomegalovirus (RCMV), strain ALL-03 in rat embryonic fibroblast cells (REF). This in-house SYBR Green I based RT-PCR was shown to have higher amplification efficiency and detection limit as compared to a commercially available real-time RT-PCR kit in quantifying these two transcripts. The quantification histogram revealed the divergence of transcription activities of the two IE genes. The IE1 transcript had a concentration peak at 7 hrs post infection (p.i.), whereas IE2 transcript at 20 hrs p.i. Regulation of IE expression is critical for determination, whether the infection is going to be abortive, lytic or latent. Therefore, this in-house developed quantitative RT-PCR assay offers an alternative for diagnosis and monitoring of the acute cytomegalovirus (CMV) infection directed at IE transcript detection.

Matched MeSH terms: Muromegalovirus/genetics; Muromegalovirus/metabolism*
Pathogenesis and antibody response to a cytomegalovirus infection in newborn rats

Loh HS, Mohd-Azmi ML, Sheikh-Omar AR, Zamri-Saad M, Tam YJ

Acta Virol., 2007;51(1):27-33.
PMID: 17432941

The present study described the kinetics of Rat cytomegalovirus (RCMV) infection in newborn rats by monitoring infectious virus and viral antigens in various organs, viral DNA in the blood (DNAemia) and antibody response. These parameters were evaluated quantitatively using double-antibody sandwich ELISA (DAS-ELISA), real-time PCR, indirect ELISA and virus infectivity assay. For the first time DAS-ELISA was used for detection of RCMV antigen directly from organ samples. The relationships between the presence of viral antigens in the infected organs and antibody levels were established by the Spearman's rank test. It was found that the virus was present in the blood, spleen, liver, lungs, and kidneys earlier than in the salivary glands. Furthermore, the early immunity of the newborn rats led to a delayed seroconversion. We suggested that the prolonged presence of the virus in salivary glands could augment the antibody response that conversely might be responsible for a reduction of viremia. This study expanded our understanding of RCMV pathogenesis leading to improved therapeutic and preventive treatment regimens particularly for the neonatal Human cytomegalovirus (HCMV) infections. Additionally, the detection procedures developed in this study such as DAS-ELISA and real-time PCR could serve as alternative techniques for rapid screening of large number of samples.

Matched MeSH terms: Muromegalovirus/growth & development*; Muromegalovirus/immunology*; Muromegalovirus/isolation & purification
Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro

Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.

Infect Genet Evol, 2021 06;90:104783.
PMID: 33640483 DOI: 10.1016/j.meegid.2021.104783

OBJECTIVE: This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human.
METHODS: Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells.
FINDINGS: Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P

Matched MeSH terms: Muromegalovirus/physiology*
Pathogenesis and vertical transmission of a transplacental rat cytomegalovirus

Loh HS, Mohd-Lila MA, Abdul-Rahman SO, Kiew LJ

Virol J, 2006;3:42.
PMID: 16737550

Cytomegalovirus (CMV) congenital infection is the major viral cause of well-documented birth defects in human. Because CMV is species-specific, the main obstacle to developing animal models for congenital infection is the difference in placental architecture, which preludes virus transmission across the placenta. The rat placenta, resembling histologically to that of human, could therefore facilitate the study of CMV congenital infection in human.

Matched MeSH terms: Muromegalovirus*