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ABSTRACT: Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.
PLoS ONE 01/2013; 8(4):e61530. · 4.09 Impact Factor
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ABSTRACT: RD-114 virus is a feline endogenous retrovirus and produced as infectious viruses in some feline cell lines. Recently, we reported the contamination of an infectious RD-114 virus in a proportion of live attenuated vaccines for dogs and cats. It is very difficult to completely knock out the RD-114 proviruses from cells, as endogenous retroviruses are usually integrated multiply into the host genome. However, it may be possible to reduce the risk of contamination of RD-114 virus by regulating the viral release from cells.
In this study, to understand the molecular mechanism of RD-114 virus budding, we attempted to identify the viral and cellular requirements for RD-114 virus budding. Analyses of RD-114 L-domain mutants showed that the PPPY sequence in the pp15 region of Gag plays a critical role in RD-114 virus release as viral L-domain. Furthermore, we investigated the cellular factors required for RD-114 virus budding. We demonstrated that RD-114 virus release was inhibited by overexpression of dominant negative mutants of Vps4A, Vps4B, and WWP2.
These results strongly suggest that RD-114 budding utilizes the cellular multivesicular body sorting pathway similar to many other retroviruses.
Virology Journal 12/2011; 8:540. · 2.34 Impact Factor
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ABSTRACT: In this study, a simple one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Lassa virus (LASV) was established. The two primer sets were designed to detect LASV circulating in Sierra Leone and northeastern Nigeria. The RT-LAMP assay using these primer sets was able to detect 100 copies of the in vitro transcribed artificial LASV RNA within 25 min. The assay was also evaluated using intact viral RNA extracted from cell culture-propagated viruses and confirmed to be highly specific for LASV. The RT-LAMP assay developed in this study is rapid, simple, and highly specific for the detection of LASV, although its sensitivity is slightly lower than that of real-time RT-PCR. In addition, because the RT-LAMP assay does not require the use of sophisticated equipment, it would be advantageous for clinical diagnosis of LASV infection in developing countries. It might also be employed in cases of deliberate release during bioterrorism attacks or in epidemiological surveillance for disease outbreaks.
Microbiology and Immunology 01/2011; 55(1):44-50. · 1.30 Impact Factor
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ABSTRACT: Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection.
PLoS ONE 01/2011; 6(3):e18247. · 4.09 Impact Factor
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ABSTRACT: Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.
Journal of clinical microbiology 07/2010; 48(7):2330-6. · 4.16 Impact Factor
