ABSTRACT
A reverse transcription-PCR (RT-PCR) technique was used to detect La Crosse (LAC) virus RNA in the central nervous system (CNS) tissues of two patients who died of LAC encephalitis in 1960 and 1978. Viral RNA was readily detected by RT-PCR although the tissues had been stored frozen for up to 37 years. LAC virus was detected in the cerebral cortex but not in other CNS tissues. RT-PCR allowed detection of replicative forms of the virus, indicating that the virus was actively replicating in the specific CNS tissues. The small (S) RNA segments of the viruses from the CNS samples were demonstrated to be genetically similar by single-strand conformation polymorphism analyses. These S RNA segments were then sequenced; only two base changes were demonstrated between the 1960 and the 1978 samples, suggesting that LAC virus is genetically stable in areas of endemicity. The RT-PCR analyses of analyte directly from CNS tissues allows study of the virus without passage in cell culture.
Subject(s)
Encephalitis, California/virology , La Crosse virus/genetics , La Crosse virus/isolation & purification , RNA, Viral/genetics , RNA, Viral/isolation & purification , Animals , Base Sequence , Cell Line , Central Nervous System/virology , Cerebral Cortex/virology , Cricetinae , DNA Primers/genetics , Encephalitis, California/diagnosis , Genome, Viral , Humans , Mice , Molecular Sequence Data , Polymorphism, Single-Stranded Conformational , Reverse Transcriptase Polymerase Chain Reaction , Virology/methods , Virus CultivationABSTRACT
Nucleotide sequences were determined for the 5' termini of La Crosse virus (LAC) S segment mRNA from persistently infected mosquito cell cultures (C6/36 from Aedes albopictus) and embryos (Aedes triseriatus). LAC primes transcription of its mRNA with "scavenged" 5' caps and adjacent oligonucleotides from host mRNAs, and these non-virus-encoded 5'-terminal extensions are heterogeneous in infected mammalian cells. The nature of mosquito host-derived primers has not been previously investigated. During early C6/36 cell infection, LAC mRNA 5'-terminal sequences were heterogeneous, but variability decreased as infection persisted. One predominant sequence, 5' CCACTCGCCACT (sequence 1), was observed throughout C6/36 cell infection but was more prevalent after 15 days postinfection. This LAC mRNA 5'-terminal sequence comprised 81% of the scavenged host oligonucleotides from vertically infected A. triseriatus eggs during embryogenesis. As these embryos progressed in the dormant overwintering stage (diapause), the predominant scavenged sequence became 5' AGGAAAAGATGGT (sequence 2), and sequence 1 became less prevalent. As the eggs emerged from diapause, the LAC mRNA 5' termini were more variable; 33% had sequence 1, and the remainder were heterogeneous. In post-diapausing eggs, 100% of viral mRNAs had sequence 1 at their 5' termini. Molecular analyses thus revealed continuous but selective LAC cap scavenging during persistent C6/36 cell infection and during embryogenesis and diapause in A. triseriatus eggs. The variety of host-derived sequences was limited in both biosynthetically active (embryonating) and dormant (diapausing) eggs.
