West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

The 3 cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3 untranslated region (3 UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3 UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5-GGAAGAGC-3, which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.Coronaviruses are large, positive-strand RNA viruses with a reproductive cycle that involves both replication of genomic RNA (gRNA) and transcription of a 3Ј nested set of subgenomic mRNAs (sgRNAs) (34). RNA sequences and structures that participate in these functions are embedded at various loci in the genome and are thought to interact with viral and host cellular components (2). The cis-acting elements that are required for coronavirus replication have been studied principally through the use of defective interfering (DI) RNAs, which are extensively deleted genomic remnants that parasitize the RNA synthetic machinery of a helper virus. Most DI RNA studies have been performed with mouse hepatitis virus (MHV) and bovine coronavirus (BCoV), closely related members of the second of the three phylogenetic groups into which coronaviruses are classified.The 3Ј end of the genome is the site of initiation of synthesis of negative-strand gRNA and sgRNA. Independent deletion analyses of MHV DI RNAs concluded that the minimal amount of the genomic 3Ј terminus that can support RNA synthesis ranges between 378 and 462 nucleotides (nt) (23,28,32). This suggested that the 3Ј genomic cis-acting element of MHV must encompass the entire 301-nt 3Ј untranslated region (3Ј UTR) as well as a portion of the adjacent nucleocapsid (N) gene. However, genetic manipulations of the intact viral genome have demonstrated that the 3Ј UTR and the 3Ј cis-acting

West Nile virus (WNV) is a mosquito-borne flavivirus that infects the central nervous system of humans and other animals. In this study, we found that C3H/HeN (C3H) mice exhibited a higher morbidity and mortality than C57BL/6 (B6) mice. We compared tissue tropism, viral replication and kinetics for C3H and B6 mice during acute viral infection. WNV was detected in multiple tissues, including novel sites such as the skin, duodenum and pancreas, but the tropism was identical for the two strains. Additionally, viral load and kinetics of spread did not differ substantially between strains. Neuroinvasion occurred in both strains by day 3 post-inoculation with early detection in the olfactory bulbs and spinal cord, suggesting that WNV neuroinvades at specific sites. Furthermore, neuroinvasion and viral load in the CNS did not predict disease outcome. Our data suggest that the disparities in morbidity and mortality between C3H and B6 mice are not due to differences in tropism, viral load or kinetics during acute WNV infection.