Molecular analysis of La Crosse virus transcription and replication in host cells

La Crosse (LAC) virus is an arthropod-borne virus (family Bunyaviridae, genus Orthobunyavirus) transmitted by Aedes triseriatus mosquitoes and is an important cause of pediatric arboviral encephalitis in the United States. Like other arthropod-borne viruses, LAC virus can be very cytolytic in mammalian cells, but establishes a persistent, nonpathogenic infection in the mosquito following an infectious blood meal. Indeed, LAC virus is maintained in nature through transovarial transmission from an infected female mosquito to her progeny. The potential molecular basis of these long-term, persistent, nonpathogenic infections was investigated in this dissertation. LAC virus primes transcription of its mRNA by using the scavenged 5' terminal cap and adjacent nucleotides from host mRNA. Previous studies demonstrated that certain 5' nonviral sequences predominated during LAC infection of mosquito cells. To characterize this further, the temporal specificity of LAC cap scavenging for transcription of S segment mRNA was analyzed for 42 days after infection of Aedes albopictus (C6/36) and Aedes triseriatus (MAT) cells and between 4 and 48 hours after infection of baby hamster kidney (BHK-21) cells. Two predominant 5' nonviral sequences [sequence A (S'-CAGTTACGTT- 3') and sequence B (5'-TTGGTCGTCATCG-3')] were observed on LAC mRNA. Sequence A comprised 66.0% of sequences observed and sequence B comprised 5.2% of sequences observed. Also, 19.0% of sequences were unique and heterogeneous. The sequences of caps scavenged by snowshoe hare (SSH) and Tahyna (TAH) viruses and various LAC virus isolates in cell cultures and mosquito tissues were similar. Cap scavenging in cells dually infected with LAC virus and Sindbis (SIN) virus (family Togaviridae, genus Alphavirus) revealed interactions between unrelated viruses within the same cell, including altered growth kinetics of both viruses and differential targeting of host mRNAs. Scavenged 5' nonviral- and SIN mRNA sequences were shown to be present on LAC virus mRNA. Comparison of scavenged sequences with available databases indicated similarities with mitochondrial genes and ribosomal RNA genes. Previous studies utilizing a reverse transcription-PCR (RT-PCR) based assay examined co-regulation of LAC virus RNA synthesis with host metabolic activity. A more sensitive real-time quantitative PCR (Q-PCR) based assay was developed during this study to detect and quantitate the LAC virus negative-sense small (S) segment RNA genome (vRNA), the full-length positive-sense complement (vcRNA), and the positive-sense subgenomic messenger (mRNA) transcript in mosquito tissues. Results showed that over a five-week period following virus infection of the midgut there was a slight reduction in mRNA and vcRNA quantities, and stable vRNA quantities. Following a second blood meal or a sugar meal at day 30, quantities of the LAC RNAs decreased in midguts. In ovaries, quantities of the LAC RNAs increased following infection, and as the ovaries shifted from biosynthetically active to quiescent metabolism after the initial infectious blood meal, the quantities remained stable. Within 24 hours following a subsequent blood meal, quantities of the LAC RNAs increased, suggesting co-regulation of LAC virus RNA synthesis with host metabolic activity. Interestingly, the same up-regulation was observed in ovaries from mosquitoes fed a sugar meal. Quantitative analysis of virus infection in mosquito and mammalian cell cultures was also performed. This approach quantitatively confirmed coregulation of viral and host metabolic activity as a determinant of efficient transovarial transmission.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Microbiology, Immunology, and Pathology

Grantor dc:publisher

Colorado State University. Libraries

Year dc:date.issued

2003

Authors dc:creator

• Kempf, Brian Joseph, author
• Beaty, Barry J., advisor
• Blair, Carol D., advisor
• Miller, Barry, committee member
• Roberts, Elaine, committee member
• Olson, Kenneth, committee member

• microbiology

Statement dc:rights

• Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.

Language dc:language.iso

eng, English

Identifier URI

https://doi.org/10.25675/3.025817

OAI identifier oai:identifier

oai:mountainscholar.org:10217/242961