title: Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses.
abstract: BACKGROUND
  La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.
  
  
  RESULTS
  To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.
  
  
  CONCLUSIONS
  SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.
authors.affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-1692, USA.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Reese
authors.forename: Eric C
authors.initials: EC
authors.lastname: Mossel
authors.forename: Meaghan K
authors.initials: MK
authors.lastname: Beaty
authors.forename: Eric T
authors.initials: ET
authors.lastname: Beck
authors.forename: Dave
authors.initials: D
authors.lastname: Geske
authors.forename: Carol D
authors.initials: CD
authors.lastname: Blair
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Beaty
authors.forename: William C
authors.initials: WC
authors.lastname: Black
mesh_terms: D000330:Aedes / Q000821:virology*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000956:Antigens, Viral / Q000302:isolation & purification; D005260:Female; D018062:La Crosse virus / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D000956:Antigens, Viral; D012367:RNA, Viral