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.

Figure 1

Aedes triseriatus mosquito collection sites in Minnesota, Wisconsin, and Iowa. Circles represent the collection sites. Red circles are the sites where LACV super-infected mosquitoes were collected in 2006 and 2007. Site 1 - BEN2 Lafayette County, WI, Site 2 - NAT, Crawford County, WI. Site 3 - SVP Vernon County, WI, and Site 4 - CAL-GA Houston County, MN. La Crosse, WI is identified with the X.

Figure 2

La Crosse virus antigen in infected, field-collected Aedes triseriatus mosquitoes. Mosquitoes were collected as eggs from the sites. Eggs were induced to hatch in the laboratory and emerged adults were assayed directly for the presence of LACV antigen by IFA (see Methods and Materials).

Figure 3

Maximum Likelihood (ML) tree derived for the LACV S segment using RAxML. The top figure is a cladogram; numbers over branches indicate % bootstrap support. The bottom figure is a phylogram. Length of scale bar = 0.01. Colored branches correspond to sites in Figure 1.

Figure 4

Maximum Likelihood (ML) tree derived for the LACV M segment using RAxML. The top figure is a cladogram; numbers over branches indicate % bootstrap support. The bottom figure is a phylogram. Length of scale bar = 0.01. Colored branches correspond to sites in Figure 1.

Figure 5

Maximum Likelihood (ML) tree derived for the LACV L segment using RAxML. The top figure is a cladogram; numbers over branches indicate % bootstrap support. The bottom figure is a phylogram. Length of scale bar = 0.01. Colored branches correspond to sites in Figure 1.

Figure 6

Maximum Likelihood (ML) tree derived for the LACV NSm sequence of SI+ and I+ mosquitoes using RAxML. The top figure is a cladogram; numbers over branches indicate % bootstrap support. The bottom figure is a phylogram. Length of scale bar = 0.01. Colored branches correspond to sites in Figure 1.

Figure 7

Molecular evolution of LACV: Comparison of segregating sites, haplotypes, and singletons between the three RNA segments of SI+ isolates and previously published isolates (7a) and between NSm genes from I+ and SI+ mosquitoes (7b).

Figure 8

Nucleotide differences (D_a) between RNA segments from LACV SI+ and previously published isolates (a) and between NSM sequences from SI+ and I+ mosquitoes (b).

Figure 9

LACV NSm NT (a) and AA (b) differences between SI+ and I+ mosquitoes from one site. Sequences from the NAT site are highlighted in yellow.