Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States

We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.


The Study
We collected adult mosquitoes during June 1-August 21, 2015, by using gravid traps in a forested area (area 196,115 m 2 ) ( Figure 1). We collected 1,197 Ae. triseriatus and 690 Ae. j. japonicus adult female mosquitoes; identified them to species on the basis of morphology; and pooled them by species, trap number, and date (Table 1). Pools (626 of Ae. triseriatus and 442 of Ae. j. japonicus) consisting of 1-50 mosquitoes were stored at -80°C. We screened samples on Vero cells for cytopathic effect and confirmed the presence of virus in positive samples by using a plaque assay (8).
We amplified virus isolates on Vero cells to a titer of 10 5 PFU/mL and extracted virus RNA from infected cell supernatants by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). We used reverse transcription PCR and Bunyaviridae-specific universal primers BCS82C and BCS332V to produce a 251-bp amplicon of the small RNA segment, which was then sequenced (9). Sequencing was performed by Eton Bioscience, Inc. (San Diego, CA, USA). A BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) query indicated that the isolates were CVV.
We detected CVV in 2 Ae. j. japonicus female mosquitoes collected from different traps on August 7 (Table 1). For the week of August 4-11, we calculated the CVV minimum infection rate to be 11.5/1,000 mosquitoes (173 mosquitoes tested individually). This late-season occurrence of CVV is consistent with results of a study in Connecticut, USA (4). Although Ae. triseriatus mosquitoes were more abundant than Ae. j. japonicus mosquitoes and have a similar biology as the invasive mosquito, we did not detect CVV in Ae. triseriatus mosquitoes. We used the medium RNA segment to infer phylogeny with CVV isolates reported by Armstrong et al. (10). We translated RNA sequences to amino acid sequences and aligned them visually, which was trivial because of invariant length and lack of insertion−deletion events. The alignment was composed of 1,803 bp from 100 isolates. We partitioned sequences by codon position and evaluated alternative models of nucleotide site substitution. We inferred phylogenetic trees by using a Markov chain Monte Carlo method in MrBayes 3.2.5 with a simultaneous estimation of topology, branch lengths, and other parameters (11). Stabilization of 4 concurrent chains occurred at 1 million generations, and the first 250,000 trees were discarded as a burn-in. We averaged branch lengths and other parameters and constructed a consensus tree ( Figure 2) from the posterior distribution that contained support values for each clade in posterior probabilities.

Cache Valley Virus in
CVV isolates were grouped into 2 clades or lineages ( Figure 2) (10). Lineage 1 viruses were from the United States and Canada during 1952-2011 and lineage 2 were more recent strains from the northeastern United States. Virus isolates from Virginia were genetically similar to each other (3-bp differences) and grouped in the newly emergent lineage 2 of CVV. This monophyletic lineage shares a most recent common ancestor with a virus from Mexico isolated in 1961 (GenBank accession no. AF231118). This sister-group relationship suggests a derivation of this group from Mexico (10). However, additional historical samples from the region in Veracruz, Mexico, and elsewhere during the history of its introduction into the northeastern United States would further provide understanding of the phylogeography of the virus.
To determine vector competence of local mosquitoes for CVV, we established a laboratory strain from uninfected Ae. j. japonicus mosquitoes. Week-old female mosquitoes from the F 2 generation were offered an infectious blood meal in a membrane feeder. This blood meal contained 1 mL of the CVV-4B isolate and 9 mL of sheep blood (Colorado Serum Company, Denver, CO, USA). We transferred postfeeding, engorged mosquitoes to 0.7-L cages and held them for 14 days at 25°C, a relative humidity of 75%, and a 16:8 (L:D) photoperiod and provided 10% sucrose. We measured rates of nondisseminated and disseminated infection (virus present in legs and wings) and oral transmission (virus present in saliva). We conducted this experiment 3 times.
Infectious blood meal titers ranged from 1.6 × 10 5 to 4.6 × 10 6 PFU/mL ( Table 2). Ae. j. japonicus female mosquitoes were susceptible to oral infection with CVV and capable of transmitting the virus. After a 14-day incubation, CVV was present in 41% of abdomens, 38% of legs and wings, and 28% of saliva samples (

Conclusions
Ae. j. japonicus mosquitoes are an invasive species that has spread throughout most of the eastern United States and are a competent vector of several endemic viruses (12). Although CVV was previously isolated from Ae. j. japonicus mosquitoes in the northeastern United States (4,13), we report isolation of CVV from this species in Appalachia and show that it is a competent vector of the virus. In the laboratory, vector competence of Ae. j. japonicus mosquitoes was equivalent to that for other species believed to be part of the CVV transmission cycle. For example, transmission rates for An. quadrimaculatus mosquitoes ranged from 20% to 33% after imbibing infectious blood meals with virus titers similar to those used in our study (1). Ae. j. japonicus mosquitoes readily feed on humans and large animals, such as white-tailed deer (12). Consequently, this species probably contributes to local transmission of CVV. The study site is in close proximity to humans and pastured sheep and is frequented by deer ( Figure 1). Therefore, all components for establishment of a focus of CVV are present. If Ae. j. japonicus mosquitoes are capable of transovarial transmission, as is the case with La Crosse virus, another bunyavirus (14), these mosquitoes could then contribute to concentrating the virus within this limited geographic area. Emergence of La Crosse virus in the Appalachian region of the United States has been associated with invasions by Ae. j. japonicus and Ae. albopictus mosquitoes (15). Thus, additional studies are needed to determine the role of Ae. j. japonicus mosquitoes in the transmission, maintenance, and presence of CVV.