The detection of SI+ in addition to I+ mosquitoes (Figure 2) was surprising. I+ mosquitoes were only detected because we assayed individual mosquitoes by IFA and PCR. They did not yield infectious virus and would not be detected by cell culture bioassay . I+ mosquitoes are >10-fold more common in nature than SI+ mosquitoes, and thus could pose a significant risk to humans (Table 1). When first detected, we thought I+ mosquitoes might be a laboratory artifact resulting from less than optimal collecting and processing conditions, which could have reduced virus stability, infectivity, and titer, resulting in the I+ phenotype. However, the finding was replicated in multiple years. I+ mosquitoes may have low titer infections in certain organs and tissues that could be rescued by environmental and physiological stimuli. For example, isolation of St. Louis encephalitis and West Nile viruses from overwintering mosquitoes [37, 38] is enhanced when field collected Culex pipiens mosquitoes are held at ambient insectary temperatures and provided sugar meals before virus assay. However, I+ mosquitoes were hatched and reared to adults in the insectary before virus assay, which would have provided ample time and metabolic and cellular activity for LACV to replicate to detectable titer. Perhaps additional physiological stimuli (e.g., mating, oogenesis, gonadotrophic cycles) would rescue infectious virus from I+ mosquitoes. The sensitivity of virus isolation by cell culture bioassay may also be a confounding factor. In early studies, virus was isolated by inoculation of samples into suckling mice [5, 7], which is more sensitive than virus isolation in cell culture . However, the equally sensitive method of intrathoracic inoculation of Ae. triseriatus mosquitoes also failed to yield infectious virus (data not shown). These observations suggest that I+ mosquitoes represent non-productive or abortive infections of the mosquito. The I+ mosquitoes must be controlling or clearing the infection. If so, they could yield critical and fundamental information concerning the molecular, immunologic, and physiological bases of vector competence.
The SI+ mosquitoes are also of great interest. If they are stably-infected, a relatively small number of these females could maintain LACV in nature with a low general field infection rate . SI+ mosquitoes were detected in the population at a low level (prevalence rate = 0.0009) (Table 1) but were widely distributed in the collection area (Figure 1). The prevalence of SI+ mosquitoes ranged from 0.0084 (NAT/Crawford County, WI/2007) to 0.12 (CAL-GA/Houston, MN/2007) (Table 2). The identification of SI+ mosquitoes in the same NAT site in two different years suggests that LACV is stabilized in mosquitoes in this collection site. Stabilized infection of D. melanogaster with SIGMAV occurs when the germarium of females become infected [16, 17], resulting in virus TOT to nearly 100% of progeny. A relatively low number of stably-infected females can maintain SIGMAV at a low prevalence indefinitely in nature [39, 40]. Studies are needed to determine if LACV infects the germarium of SI+ mosquitoes and is passed to all or most progeny in ensuing generations.
This also raises the question of whether individual mosquito families or populations are responsible for the SI+ phenotype or whether some LACV quasispecies arise by accident or at random in particular populations, and cause SI+ infections in mosquitoes in geographic islands. There is significant gene flow in Ae. triseriatus populations  including in our study area in the LaCrosse region . Nonetheless, it is possible that in geographic islands an innate immune arms race could emerge between the virus and the vector resulting in stabilized infection.
Our molecular evolutionary analyses strongly argue that SI+ quasispecies evolve at random in particular breeding sites or geographic islands. SI+ isolates are phylogenetically similar but are polyphyletic. Synonymous substitution rates greatly exceeded replacement rates in all genes and isolates. Overall, no general differences in RSCU were detected between previously published and SI+ isolates. However, the overall comparison of the LACV NSm gene from I+ and SI+ mosquitoes did identify a single QTN (U ↔ A transversion) in a Leu (CUN) codon in the NSm gene (Figure 8b). The significance of this QTN remains to be determined.
A consistent and significant trend was detected by Fu and Li's F* analysis of singleton versus shared mutations (Figure 8a) and in direct comparison of I+ and SI+ mosquitoes (Figure 8b). S and L segments evolved in a manner consistent with purifying selection and subsequent neutralism. But the SI+ M segment had a large and significant excess of intermediate-frequency alleles while the M segment had a large, significant excess of singletons in previously published LACV sequences and in sequences from I+ mosquitoes. It is difficult to make any definite conclusions from the previously published sequences, which were collected over a period of 45 years and did not arise from an intensive spatial or temporal field sample. Thus, the chances of finding intermediate frequency ηi alleles is small. Nevertheless, Figures 7a and 8a illustrate that most of the evolution of the past 45 years has occurred in the M segment.
The patterns in direct comparison of NSm sequences from I+ and SI+ mosquitoes in Figures 7b and 8b are consistent with an hypothesis that the error prone RNA-dependent RNA polymerase generates a constellation of genotypes and the majority of these generate I+ phenotypes in their mosquito host. The large accumulation of singletons in I+ isolates in third codon positions reflect the activity of error prone RNA-dependent RNA polymerase, filtered by purifying selection. In contrast, occasionally a SI+ genotype arises and, because they survive and are maintained at a higher rate in stably-infected mosquito lineages than I+ genotype, SI+ maintain an excess of intermediate-frequency ηi alleles. These intermediate-frequency alleles may confer reduced detection and/or destruction via the RNAi or apoptotic/autophagic pathways. Disruptive selection would then increase to moderate frequencies those genotypes that avoided destruction in the mosquito host and become manifested as SI+ mosquitoes. A much larger proportion of novel gene sequences in the M segment would yield viable virus but which would only partially avoid detection and/or destruction. Selection would not increase the frequencies of these genotypes and instead they would be manifested as a large frequency of singleton ηe alleles. This trend was especially pronounced in the M segment in general and in the NSm gene in SI+ isolates in particular. There was >9 fold nucleotide diversity in the NSm gene among SI+ isolates as compared with I+ isolates (Table 4). Despite having 37 segregating sites, none of these were singletons in SI+ while four of only five sites were singletons in I+ individuals. This suggests that disruptive selection was increasing the frequency of shared (ηi) genotypes in SI+ mosquitoes while no such selection favored any of the I+ genotypes resulting in singletons (ηe) in those mosquitoes. Overall, LACV NSm sequences in SI+ mosquitoes differed between sites, suggesting that different polymorphisms in NSm may condition the SI+ phenotype. These polymorphisms would be expected in a quasispecies model of LACV infection in mosquitoes, and different polymorphisms in NSm and Ae. triseriatus innate immune genes could condition the same SI+ phenotype in different sites or geographic islands in the endemic area.
In one site (NAT), we were able to characterize NSs and NSm gene sequences from SI+ and I+ mosquitoes (Figure 9). There were no differences in NT sequence in the NSs genes (Additional File - Figure 1), but there were multiple NT and four amino acid differences between LACV NSm gene from SI+ and I+ mosquitoes in the site (Figure 9). The role that these changes may have in conditioning the establishment of the respective phenotypes remains to be determined. Sequence analysis of the Ae. triseriatus inhibitor of apoptosis-1 (AtIAP1) gene in mosquitoes from our study area revealed extensive polymorphisms (approximately 3 fold greater diversity than in a typical mosquito gene). One would assume that this gene would be highly conserved to prevent apoptosis .
In the D. melanogaster-SIGMAV system, the ref(2)P gene, which determines stabilized virus infection of the host, is also highly variable, presumably as a result of the host counteracting genetic changes in the virus . The fly ref(2)P locus has two principal alleles, ref(2)P
P and ref(2)P
O, respectively, which determine whether or not the fly will restrict Sigma virus infection or be permissive to stabilized infection . There is a well characterized innate immune arms race between the ref(2)p gene and SIGMAV N protein epitopes , which determines productive infection of the host, TOT of the virus, and stabilized infection. The gene product of the ref(2)P locus is a protein kinase in the Toll innate immune pathway . Perhaps a similar major innate immune gene-for-gene interaction conditions the LACV SI+ and I+ phenotypes in Ae. triseriatus. The accumulating evidence suggests that interactions between the LACV NSm and the AtIAP1 gene may somehow condition the SI+ phenotype. In this regard, apoptosis and autophagy have been show to share common caspase regulatory pathway components, autophagy and apoptosis both occur during degeneration of ovarian follicles, and autophagy has just been recognized as a key antiviral response in D. melanogaster [23–26]. All of this is especially provocative in the context of the LACV-Ae. triseriatus system in which virus amplification and maintenance in nature is predicated upon productive infection of ovarian follicles. Infection of Ae. triseriatus ovarian follicles with West Nile virus, but not LACV, induces an autophagosomic response in the follicles (BJB, unpublished data). Reverse genetics capability is now available for LACV, and we can exploit this robust approach to investigate and identify potential viral determinants of the SI+ and I+ phenotypes .
Mating efficiency studies with the newly discovered SI+ and I+ mosquitoes are also needed. Previous studies demonstrated a fitness advantage with increased mating efficiency for LACV-infected mosquitoes from the field, but the mosquitoes were not phenotyped in terms of SI+ or I+ infection . The mating advantage may be more pronounced in SI+ mosquitoes. Mathematical models of LACV and KEYV [13–15] need to consider such factors as stabilized infection or mating advantages resulting from such infection. These two factors may help maintain stable LACV prevalence from year to year in the vector population.