BHK-21 (BHK; baby hamster kidney), C6/36 (Aedes albopictus), DF-1 (chick), and Vero E6 cells were obtained from American Type Culture Collection (Manassas, VA) or from the CDC cell culture reference section. The Neuro-2a (N2a) murine neuroblastoma cell line was a gift from Dr. Mark Zabel (Colorado State University) and was originally obtained from ATCC (catalog #CCL-131). All cells were propagated in minimal essential medium with 10% heat-inactivated fetal calf serum at 37°C (28 °C for C6/36) in a 7% CO₂ incubator.

WEEV McM, originally isolated in 1941 from the brain of an infected human, isolate was obtained from the Arbovirus Reference Collection at the Centers for Disease Control and Prevention, Fort Collins, Colorado, USA. The WEEV McM infectious clone was constructed by Dr. Thomas Welte, CSU. WEEV IMP was isolated from a Cx. tarsalis in 2005 in Imperial County, CA. The WEEV IMP infectious clone was constructed by Dr. Michael Anishchenko and obtained from Dr. Aaron Brault (CDC, Ft. Collins, CO). Passage history prior to cloning was as follows: WEEV McM-MP2, SMB1, V2; WEEV IMP-V2 (MP, mouse; SMB, suckling mouse brain; V, Vero cells) (Logue 2009). Seed stocks for these experiments were made by electroporation of viral RNA from infectious clones into BHK cells grown in minimal essential medium with 10% fetal calf serum. Cell culture supernatant was collected when 90% of cells showed cytopathic effect (48–72 hrs) and stored in aliquots supplemented with 20% FBS at −80°C. Virus titer was determined by plaque assay on Vero cells as previously described [10].

Where possible, chimeric viruses were constructed using the existing conserved, unique restrictions sites, KpnI and AvrII, present at WEEV McM nucleotides 7576 and 9682, respectively. Construction of the chimeras receiving material not bordered by these sites and the point chimeras was based on a strategy using type IIs restriction enzymes adapted from Blakqori and Weber [12] and is outlined in detail by Saxton-Shaw et al for the generation chikungunya/o′nyong nyong chimeric viruses [13]. This method allowed the generation of chimeras and point mutants without the introduction of nonnative nucleotides to the virus.

In general, cloning was completed in a three step process. First, an amplicon was generated by PCR from the parental backbone virus clone extending from the AvrII or KpnI site toward the other (inward). The PCR primers would add a site for convenient cloning into the pUC19 polycloning site. To the other end of the amplicon, a SapI recognition site oriented inward was added so that the restriction site was the exact sequence at which the change was to occur, the chimera junction. A second restriction site was added outside the SapI site for cloning into pUC19. This amplicon was cloned into a modified pUC19 plasmid that previously had the BsmBI sites and SapI site removed. A second PCR amplicon was generated from the clone donating the fragment from the AvrII or KpnI site to the chimera junction. Outside the AvrII or KpnI site, this amplicon contained the same convenient recognition site as was added outside the SapI site in the previous amplicon. On the opposite terminus was added a SapI site oriented inward such that restriction would occur at the same site as on the first amplicon, leaving complementary overhangs. Digestion of both the previous plasmid and the second amplicon with SapI and the terminal convenient restriction site and subsequent ligation generated a plasmid cassette containing a chimeric AvrII-KpnI region. Digestion of this plasmid and the parental virus clone with AvrII and KpnI and subsequent ligation resulted in a full-length chimeric WEEV clone free of artifactual base changes introduced by the cloning process.

Generation of pair 5, which exchanges a region not bordered by AvrII or KpnI, required an additional step. Generation of the first cloning plasmid was performed with an amplicon that contained a linker enzyme site (MfeI) between the added SapI site and selected pUC19 cloning site. The second amplicon was generated from the same parent and also contained a pUC19 cloning enzyme site outside AvrII or KpnI and an MfeI site outside the SapI site. This amplicon was cloned into the first plasmid using a second pUC19 cloning enzyme site and MfeI, generating a -SapI-MfeI-SapI- motif at the point of foreign DNA insertion. The DNA to be inserted was amplified from the other parental clone, adding SapI sites to both termini, oriented inward such that the restriction sites were situated over the desired cloning junction. Digestion of this amplicon and plasmid with SapI and ligation created a cloning cassette containing the chimeric region from the AvrII to KpnI sites that were then inserted back into the parental clone as above.

Finally, the point mutants were generated by a three-step process. The region of interest, including the site to be mutated and flanking sequence to include convenient, ideally unique, cloning sites, was broken into two PCR fragments. Each PCR fragment was comprised of the sequence from the mutation site through the cloning site. The PCR primers contained SapI recognition sites oriented inward toward the mutation site and cut sites modified to leave complementary overhangs containing the desired base change. Digestion of the PCR amplicons with SapI and the relevant enzymes to restrict the opposite ends followed by a three-part ligation with a modified pUC19 plasmid yielded a plasmid construct containing the region of interest with the desired base change. Once the change was confirmed by sequence analysis, the region was digested and removed from the provisionary plasmid construct and inserted into the full-length WEEV plasmid. Full-length plasmid constructs were confirmed to have incorporated the desired change by sequence analysis.

T-25 flasks or 24-well plates (Corning, Corning, NY) were seeded with BHK, N2a, C6/36, or DF-1 cells. When approximately 90% confluent, cell monolayers were infected with virus at low multiplicity (MOI = 0.01–0.05). At specified times, supernatant was removed from three wells or two flasks for each virus on each cell type and placed in a screw-cap cryovial at −70°C until titration by plaque assay. Titration results for each virus were compared at all time points by the two tailed t-test.

The use of chickens was reviewed and approved by the Animal Care and Use Committee at Colorado State University (protocol #12-3418A). Care and handling of chicks was in accordance with the PHS Policy and Guide for the Care and Use of Laboratory Animals. Three day old chicks (n = 6/virus) were infected with 10⁴ plaque-forming units (pfu) of McM and IMP viruses by subcutaneous injection and monitored daily by a veterinarian for 7 days. Chicks were bled at days 1, 2, 3, and 4 dpi to determine viremia. The humane endpoint for these experiments was at the termination of the experiment (7 dpi).

The use of all mice was reviewed and approved by the Animal Care and Use Committee at Colorado State University (Protocol #11-2605A). Five week-old female CD-1 mice (The Jackson Laboratory, Bar Harbor, ME or Charles River Labs, Wilmington, MA) were allowed to acclimate for 7–10 days. Viral stocks were diluted in serum-free MEM to a concentration of 2×10⁴ pfu per mL. Mice were infected in groups of 5–10, at least two replications per virus (final n = 10–15), by subcutaneous (s.c.) injection inside the left thigh with 10³ pfu in 50 µL. Inocula were titered by plaque assay on Vero cells to confirm dosage.

All infected and uninfected control mice were visually monitored by laboratory personnel at a minimum of two times daily. Additionally, mice were monitored once daily by Laboratory Animal Resources and results of these observations reported to the University Veterinarian. Our protocols allow continuation until clinical signs are evident. Mice exhibiting lethargy, unresponsiveness, or neurological signs (postural instability, seizures, piloerection, and/or paralysis) were euthanized by CO₂ inhalation. The day a mouse was euthanized was considered the day of death for calculation of mean time to death (MTD). Survivorship was followed for a period of 14 days and compared among virus constructs by Fisher's exact test.

Three to four day-old adult Cx. tarsalis mosquitoes were fed on a blood meal containing WEEV. The blood meal contained equal parts of virus, FBS with 10% sucrose, and goose blood (Colorado Serum CO., Boulder, CO) washed with phosphate-buffered saline and packed by centrifugation. A Hemotek feeding system (Discovery Workshops, Accrington, Lancashire, UK) was used to deliver the blood meal to the mosquitoes at 37°C. The mosquitoes were allowed to feed for 1 hour on the apparatus. The fully engorged females were separated and placed into a humidified environmental chamber (Thermo Scientific 3960, Houston, TX) and held at 28°C for 8 days until processing. Blood meal titers were determined by plaque assay and ranged from 2.0×10⁵–2.0×10⁶ pfu/mL.

After the 8 day holding period, mosquitoes were cold anesthetized and decapitated, placing the bodies into 1.7 ml tubes (Eppendorf, Hauppauge, NY). A 400 µl aliquot of Dulbecco's minimal essential medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and streptomycin, 1 U/ml of fungizone and gentamycin was placed into each tube and the sample was homogenized using a pestle (Kontes, Vineland, NJ). The supernatant was clarified by filtration through a 0.2 µM syringe filter (Pall, Ann Arbor, MI) into a clean tube and placed at −70°C until use.

Virus presence was determined by pipetting 100 µL of clarified supernatant onto Vero cells in a 96-well plate. The plate was incubated at 37°C under 5% CO₂ and monitored daily for cytopathic effect (CPE). Samples were considered virus positive if CPE was observed.

A panel of McM/IMP chimeras was constructed to identify the determinants of WEEV McM virulence in CD-1 mice (figure 1). Rescued virus was administered by subcutaneous inoculation of 6 week-old CD-1 mice. Chimera pair 1 containing exchanges in the E1 gene region retained phenotypes similar to the parental viruses with 86% mortality and 0% mortality for the McM-based and IMP-based chimeras, respectively. Exchange of the region from the unique AvrII restriction site at McM nt 7576 to the unique KpnI site at McM nt 9552 (pair 2; figure 1) did transfer the virulent phenotype with the McM-based chimera losing virulence and the IMP-based chimera becoming virulent (80%) but with a relatively delayed mean time to death of 7.9 days. These results suggested that virulence is determined by capsid, E3, and/or E2 genes.

Chimera pair 3 and 4 exchanged the capsid gene from the AvrII site to the 3′ end and the E2 gene from the 5′ terminus to the KpnI site (figure 1). Exchanging the capsid gene did not confer or remove the virulent phenotype. The IMP and McM-based chimeras exhibited 0% and 80% mortality in CD-1 mice, respectively. However, exchanging the E2 gene did have an effect on virulence. The McM-based chimera containing the IMP E2 gene completely lost its virulence while the IMP-based chimera with the McM E2 gene exhibited 40% mortality (MTD = 7.7 days). The E2 gene was further manipulated by exchanging E2 nts 1–518 or E2 nts 519–1226. Pair 5, exchanging E2 nts 1–518 retained the phenotype of the parental viruses, though mortality was further decreased for the McM-based chimera (30%). Placing the IMP-derived E2 nts 519–1226 in the McM backbone reduced mortality to 0% (pair 6, figure 1). The reciprocal chimera also exhibited reduced mortality (10%). Therefore the minimal necessary determinant essential for mouse virulence was narrowed to a region of McM E2 from nt 519 to the KpnI site at 1226.

Alignment of the IMP and McM E2 sequences (accession numbers GQ287641 and GQ287640, respectively) revealed 19 nucleotide differences between the strains in this region. Of these, 11 were silent and eight led to seven amino acid differences (two nucleotide changes are involved in a single amino acid change; figure 2). Fourteen point mutants were constructed representing the reciprocal chimeras for each of the seven loci. Exchanges at six of the seven loci (aa 181, 217, 224, 231, 277, and 390) retained the parental neurovirulence phenotype in the mouse. Mortality rates associated with the McM-based point mutants ranged from 70–100% (table 1). Despite retaining a generally virulent phenotype, placement of the IMP-like amino acid at positions 181 and 224 yielded significantly less mortality than the McM parent (p<0.05). Mortality among IMP-based mutants ranged from 0–10%, none significantly different than the IMP parent. Only the exchange of amino acid 214 (Q to R) affected the pathogenic phenotype. Mortality following infection with the IMP-based virus containing the McM E2 Q214 (IMP E2 R214Q) residue remained at the previously observed 0%. The reciprocal chimera [McM-based mutant containing IMP-like E2 R214 (McM E2 Q214R)] showed significant loss of virulence relative to the McM parent virus as only a single animal succumbed to disease (n = 10; p<0.001) indicating that while not sufficient to confer virulence in an otherwise attenuated backbone, the McM E2 amino acid 214 is necessary for virulence in our mouse model of WEEV infection. The single animal that succumbed following infection with the McM E2 Q214R appeared to follow an atypical disease course (hind limb paralysis only) that will be examined in future studies. That animal and the single mouse that succumbed to infection with IMP E2 S390A are the only examples in these studies of infection with a virus containing arginine at E2 aa 214 resulting in a lethal outcome.

As avians are the natural reservoirs for WEEV, we further sought to examine the characteristics of these viruses in a readily available avian model. 10⁴ pfu of McM, IMP, McM E2 Q214R and IMP E2 R214Q viruses were subcutaneously injected into 3 day old chicks to observe any differences among the viruses in their resulting viremia within an avian model. Viremia in chicks was detected for McM at 1 dpi but no viremia was detected at days 2–3 dpi (figure 3). Viremia was detected at 1–3 dpi in chicks infected with IMP or IMP E2 R214Q virus. Though none of the viremia differences reached statistical significance, the highest measurable viremias occurred on day two (figure 3) and were associated with IMP or IMP E2 R214Q viruses. In the present study, none of the infected chicks showed signs of disease during the 7 dpi monitoring period. Hardy et al showed 1 day old chicks were highly susceptible to WEEV-induced disease [14]. It is not clear if the different results are due to an age-dependent susceptibility to WEEV disease or some other detail of our model. However, that viremia was more often associated with IMP infection suggests that the observed McM virulence in mice is not due to an extraordinary general virulence in vertebrates. Additional studies in avian hosts, including passerine birds, are proposed for the future.

In addition to the mouse virulence phenotype, we used McM/IMP chimeras to investigate the determinants of the observed difference in mosquito infectivity. Cx. tarsalis mosquitoes were allowed to feed on blood meals containing each of the 12 structural chimeras and 14 E2 point mutation exchanges (figure 1, table 1). The infectivity of the parent strains was 10.0% and 78.9% for McM and IMP, respectively. Clone pairs 1 and 3 yielded no significant change from the original parent infection phenotype; however pair 2 restored the infection phenotype and suggested a determinant in the capsid, E3, or E2 regions. 87.9% of Cx. tarsalis given McM backbone virus were infected and 30% of mosquitoes given IMP backbone virus were infected. Clone pairs 4, 5, and 6 further identified the determinant of infection as residing in the same region of E2 (nts 519–1226) that determined mouse virulence. Pair 6 had a demonstrated infectivity of 23.8% for both rescued viruses, representing a deviation from the parental infection phenotype.

Infectivity for the point mutations at E2 amino acids 181 and 214 were 5.1% and 11.7%, respectively, for the IMP strain mutants and 13.3% and 0% for the McM mutants, representing a significant reduction of the IMP parental mosquito infection phenotype (p<0.001 for both IMP-based viruses), but not restoration (Table 1). The remaining pairs resulted in a slight reduction and an approximately 3-fold increase in infectivity for the IMP and McM-based chimeras, respectively (the pairs exchanging amino acid 224 and 231 will all significantly different than the parent viruses, p<0.05). These results suggest that while there may be multiple determinants, the IMP arginine at E2 position 214 is critical but not sufficient for mosquito infectivity.

To determine whether the observed phenotypes were due to general replicative differences, multi-step growth curves of McM, IMP, or the McM or IMP-based E2 aa 214 point chimeras were performed in both mammalian and insect cells. In BHK cells, all four viruses grew similarly well, rapidly increasing in titer from 10–100 pfu/mL to 10⁶–10⁷ pfu/mL (figure 4A). No consistent statistical differences were observed among the four viruses, though the titer of McM E2 Q214R was significantly greater than McM, IMP, and IMP E2 R214Q 72 hpi (p<0.05).

N2a murine neuroblastoma cells were used to determine whether growth differences among viruses might account for the observed differences in mouse mortality due to neuron infection. Again, no overt growth difference was observed among, McM, IMP, McM E2 Q214R, and IMP E2 R214Q viruses through 72 hpi (figure 4B). Though at 72 hpi, IMP trended toward having lower virus titers than McM, at both 48 and 72 hpi, no statistically significant difference in virus titer was observed between any of the four viruses.

Cultured DF-1 chicken embryo fibroblast cells were infected with McM, IMP, McM E2 Q214R, and IMP E2 R214Q viruses to observe any growth differences that would be regulated by the virus-vertebrate reservoir dynamics. In this cell line, significant growth differences were observed, especially through 24 hpi (figure 4C). All 4 viruses were different at 18 hpi, with the McM and McM E2 Q214R McM viruses 1–2.5 logs higher than IMP and IMP E2 R214Q viruses at 24 hpi. Peak viral titers were significantly different between McM (24 hpi) and IMP (36 hpi), but no other viruses. The titers for the point mutants were significantly higher than the parent viruses at 48 and 72 hpi.

In C6/36 mosquito cells, all four viruses showed similar replication kinetics with no statistically significant differences observed (figure 4D). Mean titer measurements at 72 hpi ranged from 6.8 Log10 pfu/mL for IMP (E2-R214Q) to 7.5 Log10 pfu/mL for McM.