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Replication‐defective mutants of mouse cytomegalovirus protect against wild‐type virus challenge
Author(s) -
Gill Tracey A.,
Morley Peter J.,
Sweet Clive
Publication year - 2000
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/1096-9071(200010)62:2<127::aid-jmv2>3.0.co;2-h
Subject(s) - biology , mutant , virology , capsid , microbiology and biotechnology , viral replication , virus , transcription (linguistics) , gene , virus quantification , reverse transcriptase , polymerase chain reaction , genetics , linguistics , philosophy
Five temperature‐sensitive mutants ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 30) of murine cytomegalovirus have been shown previously not to produce infectious virus in mice. In the present study, the stage at which these mutants are blocked in their replication in vitro was examined by transcriptional analysis of 4 temporally regulated marker genes (IE‐1, E‐1, gB and gH) using a semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) coupled with an electron microscopic analysis of infected cells incubated at permissive (33°C) and non‐permissive (39 and/or 40°C) temperatures. Replication of tsm 13 appeared to be blocked at a late phase of replication after capsid formation while the block appeared to be as early as the immediate‐early phase in tsm 22‐ infected cells. In contrast, mutants tsm 9, tsm 20 and tsm 30 were blocked at a maturation step, probably of capsid formation, as gene transcription of all 4 marker genes occurred, albeit at reduced level, at 39 and 40°C but no capsids or virions were produced at 40°C. Replication and transcription of mutants tsm 13, tsm 20 and tsm 30 were also examined in infected mice. Mutant tsm 13 showed no gene expression or infectious virus while mutants tsm 20 and tsm 30 produced no infectious virus from days 3–60 post infection, except unusually for a low titre of tsm 30 (2.3 x 10 3 pfu/ml) in salivary glands 21 days post infection. Gene transcription of all 4 marker genes was observed in one or more tissues (salivary glands, spleen, kidneys, liver, thymus, heart, lungs) at one or more time points (3, 7, 10, 14, 21 days post‐infection) with both mutants. Mice became infected latently with tsm 20 but not tsm 30, and mice previously infected with tsm 20 or tsm 30 were protected against a sub‐lethal challenge with virulent parental virus; tsm 30 also protected against a lethal challenge. This suggests that these two mutants may be good model vaccines for further studies on the mechanism of protection induced and for identification of the ts genes. J. Med. Virol. 62:127–139, 2000. © 2000 Wiley‐Liss, Inc.