Molecular Characteristics and Pathogenicity of a Novel Transplacental Rat Cytomegalovirus
Cytomegalovirus (CMV) is a species-specific betaherpesvirus which causes acute, persistent and latent infections in both humans and animals. CMV is the most frequent congenital infection in humans. RCMV strain ALL-03 was the first CMV ever isolated from the placenta and uterus of the house rat (R...
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Format: | Thesis |
Language: | English |
Published: |
2005
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Online Access: | http://psasir.upm.edu.my/id/eprint/63/1/1000548934_TFPV_2005_1.pdf |
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Summary: | Cytomegalovirus (CMV) is a species-specific betaherpesvirus which causes acute,
persistent and latent infections in both humans and animals. CMV is the most frequent
congenital infection in humans. RCMV strain ALL-03 was the first CMV ever isolated
from the placenta and uterus of the house rat (Rattus rattus diardii). As such,
hypothetically, this RCMV should be a distinct strain from the existing isolates that is
capable to cross placenta and infect the fetus. The objectives of the study were (i) to
identify the novelty of the RCMV strain ALL-03, (ii) to characterize its immediateearly
(IE) genes, and (iii) to determine its pathogenicity by developing the in utero
transmission and neonatal infection models in rats. Overall, the present study signifies
the virological and molecular detection of the RCMV antigen, DNA and mRNA in
addition to the serological demonstration of the RCMV-specific immune response.
Other than the traditional diagnostic methods, the study had also used advanced
techniques, for examples, double antibody sandwich enzyme-linked immunosorbent
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assay (DAS-ELISA), quantitative real-time reverse transcription-polymerase chain
reaction (RT-PCR) and real-time PCR. The study was commenced by characterizing the
strain ALL-03. Upon infection, the virus showed delayed cytopathology, cellassociation,
low maximum titres, the presence of herpesviral inclusion bodies and
herpesvirus related particles in infected rat embryonic fibroblast (REF) cells; specific
antigen-antibody reaction with RCMV strain Maastricht; and rat-specific are all in
accord with a RCMV. The genetic difference at the genome level with that of
Maastricht, English, UPM/Sg and UPM/Kn strains had confirmed its novelty. The first
recognized genes expressed during CMV infection, the IE genes were studied by
analyzing the mRNA transcripts of infected-REF cells. The cDNA libraries were cloned
into plasmids for sequencing. Each sequence was then probed towards the databanks for
an identity search. Following the PCR and hybridization techniques, two distinct
transcripts of unknown identities within the databanks were confirmed to be of the
strain ALL-03 origin. These two IE transcripts were found considerably different to the
IE genes of RCMV strains Maastricht and English. Meanwhile, a real-time RT-PCR
assay was developed specifically to quantify the in vitro transcription levels of the two
RCMV IE mRNAs. The kinetic transcription profiles and the bioinformatics analyses
suggested them as exon 4 or IE1 and exon 5 or IE2. An in utero infection model
demonstrated the clinical signs, pathological changes and anatomical virus distribution
to the uterus, placenta, embryo, fetus, lung, kidney, spleen, liver and salivary gland of
rats. The placenta was observed to be involved in the maternofetal RCMV infection.
The maternal viremia leading to uterine infection which subsequently transmitting to
the fetus through the placenta is the most likely phenomenon of congenital CMV
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infection in the model. The study has established a useful rat model that mimics the
neonatal CMV infection in humans especially for the virus dissemination in different
organs, viremia and immune response. The kinetic quantitation of the viral antigen,
DNA and antibody was assessed by DAS-ELISA, real-time PCR and ELISA
respectively. This neonatal rat model demonstrated a characteristic splenomegaly and
acute virus dissemination in blood, spleen, liver, lung and kidney. The salivary gland
infection is suggested to augment the antibody response that may be responsible for a
reduction of viremia. The study has provided important new insights of CMV disease
particularly for a congenital infection in humans. The exploitation of the major IE
regions has permitted greatest advances as a candidate of viral-vectored
immunocontraception for rat control and generation of eukaryotic expression vectors. |
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