Viral titers in HOFI inocula were determined by plaque assay titration and ranged between 4.1–4.4 log₁₀ PFU/mL with little deviation among samples (Table 1). Similarly, titers in blood meals and five fully engorged mosquitoes collected after blood feeding showed matching titers among groups with 6.3–6.8 log₁₀ PFU/mL in blood meals and 4.5–4.7 log₁₀ PFU/mL in mosquitoes (Table 2). These titers represent biologically relevant doses typically found during natural WNV transmission between HOFIs and Cx. tarsalis [21]. Analysis by qRT-PCR amplicons revealed slightly higher COAV997-5nt RNA copy numbers compared to the wildtype viruses (COAV997-IC, NY99-IC, CA-04) for all inocula. Despite equal viral titers, we previously noted increased COAV997-5nt RNA copies after in vitro replication [24]. Therefore results from final samples were normalized to the initial dose using the calculated RNA ratios between wildtype and COAV997-5nt at the time of inoculation as determined in Tables 1 and 2.

Day 0 temperature reads from all birds averaged 41.3°C and served as the baseline temperature for this study. Surprisingly, none of the infected birds exhibited fever during peak viremia (Table 3) between 2–4 dpi, with cloacal body temperatures not differing significantly from the baseline temperature on any day (Fig 1). In fact, elevated viremia was accompanied by hypothermia during terminal stages of disease in HOFIs 678, 636, 683, 664 and 611, but not in surviving infected birds or in mock-inoculated birds (Fig 1). These 5 terminal birds presented a “puffed-up” plumage appearance, most likely in an attempt to upregulate their body temperatures. This observation contrasts studies in corvids that reported fevers up to 45°C [15], thereby potentially impairing replication of temperature-sensitive strains of WNV [11, 15]. In addition to a host-specific response, factors secondary to acute WNV infection such as starvation and loss in body condition also may be associated with hypothermia.

Peak viremia was detected at 3 dpi for all HOFIs except for 679 (4 dpi) and 664 (2 dpi) (Table 3). Birds infected singly with COAV997-IC or COAV997-5nt developed peak viremias ranging 6.8–8.7 log₁₀ PFU/mL (Table 3, Fig 1), demonstrating that replication was not impaired in vivo in the absence of fever and was potentially sufficient to infect Culex mosquitoes. In contrast, growth of COAV997-IC was greatly reduced at 44°C in duck embryonic fibroblast (DEF) cells, suggesting attenuation in febrile avian hosts [11]. Cell culture conditions may not mimic all aspects of viral replication in avian and vector hosts and that highlights the importance of relevant in vivo models for phenotypic characterization of WNV and other arboviruses [25].

Infection with COAV997-IC and COAV997-5nt was followed by death in 0 of 6 and 1 of 6 birds, respectively, indicating comparable low mortality in HOFIs (Figs 1 and 2). Using a plaque assay in Vero cells [26], we determined serum viral titers (Fig 1) and compared them utilizing a repeated measures two-way ANOVA of variance. Serum titers of HOFIs singly infected with either COAV997-IC or COAV997-5nt were not significantly different (p>0.05), and there were also no titer differences between the competitively infected group (COAV997-IC + COAV997-5nt) and the singly infected COAV997-IC and COAV997-5nt groups (p>0.05). Next, we quantified RNA copies of COAV997-IC and COAV997-5nt by qRT-PCR in the sera of competitively infected HOFIs (Fig 3A) and found no significant differences (p = 0.25) at any time points between 1–7 dpi. When comparing COAV997-IC RNA copies from singly and dually infected birds, we found significant differences on 4 dpi, but not on any other days. This also was found for COAV997-5nt from singly and dually infected birds on 4 dpi, indicating increased variability when making comparisons among different groups. In summary, COAV997-IC and COAV997-5nt replicated to equal viral titers and RNA copy numbers during viremia in dually infected HOFIs indicating fitness neutrality in vivo.

Identical mortality rates of 67% and 50% were observed for N99-IC and CA-04, respectively, in both competitively and singly infected groups (n = 6), suggesting that the specific virulence of NY99 and CA-04 may be reproduced in the presence of the less virulent COAV997-5nt (Figs 1 and 2). The same NY99-IC strain previously caused similar mortality of 63% in HOFIs [19]. NY99-IC showed increased fitness with RNA copy numbers being significantly greater (p = 0.003) compared to COAV997-5nt (Fig 3B). Similarly, CA-04 replicated to higher RNA copies (p = 0.01) than COAV997-5nt (Fig 3C). There were no differences (p>0.05) in RNA copies of NY99-IC and CA-04 when comparing the results from competitively and singly infected birds.

Plaque reduction neutralization titers (PRNT) were determined in end point sera when the experiment was terminated on 14 dpi or on the last available sample before death (Table 3). All sera were tested at a 1:20 dilution and were considered positive when 90% of 50–100 PFU were neutralized (PRNT₉₀). From a total of 28 HOFIs euthanized on 14 dpi, 21 showed detectable neutralizing antibodies (nAb), whereas serum from seven birds did not neutralize 90% of 50–100 PFU of WNV. Four of those seven birds were infected singly with COAV997-IC, potentially indicating that the immune response might be slow or low after COAV997 infection compared to other strains including COAV997-5nt, despite the fact that these birds developed peak viremias of 6.9–7.4 log₁₀ PFU/mL (Table 3, Fig 1E); other negative birds were infected with COAV997-IC + COAV997-5nt, NY99-IC + COAV997-5nt and CA-04 and had viremias >7 log₁₀ PFU/mL.

Individuals that succumbed during acute infection with detectable WNV titers in their sera (Fig 1) also showed higher RNA copy numbers in tissues compared to birds euthanized on 14 dpi (Fig 4). Tissues from birds infected competitively with COAV997-IC + COAV997-5nt (Fig 4A) did not show significant differences in the RNA copy numbers detected from the two viruses in any of the tissues (p = 0.67), providing more evidence that the two viruses have equal fitness in HOFIs. Similar to results obtained from sera, more RNA copies of NY99-IC (p = 0.004, Fig 4B) and CA-04 (p = 0.007, Fig 4C) were detected in the tissues of dually infected birds compared to COAV997-5nt. Therefore, the fitness outcome determined in multiple tissues at necropsy was not different compared to serum, indicating that there was no tissue-specific fitness observed for these strains (Figs 3 and 4).

Females were collected 14 days after blood feeding and the bodies, legs and expectorants were analyzed using plaque assay titration (Fig 5A) and qRT-PCR (Fig 6A). Viral titers obtained from bodies, legs and expectorants from competitively and singly COAV997-IC and COAV997-5nt infected mosquitoes showed no difference in magnitude (p>0.05). Rates of infection (63%, 70%, 59%), dissemination (63%, 70%, 59%) and transmission (53%, 63%, 55%) also were similar for COAV997-IC + COAV997-5nt, COAV997-IC and COAV997-5nt groups, respectively (Fig 5A, 5D and 5E). We then analyzed the bodies of competitively infected females by qRT-PCR and found that six bodies contained COAV997-IC RNA only, five contained COAV997-5nt RNA only, and seven bodies were positive for RNA from both viruses (Fig 6A). The quantity of RNA copies was not different (p = 0.23) between COAV997-IC and COAV997-5nt in mosquito bodies. When determining RNA copies in Vero cell passaged mosquito expectorant, we found 30% transmission for COAV997-IC and 27% transmission for COAV997-5nt (Fig 7A). Overall, this indicated that the genetically marked COAV997-5nt infected, replicated and was transmitted equally to the COAV997-IC virus in Cx. tarsalis (Fig 7A).

Viral titers determined by plaque assay titration did not differ among bodies, legs and expectorants of mosquitoes from all groups collected at 14 dpi (p>0.05; Fig 5). However, NY99-IC showed 43% infection, 43% dissemination and 40% transmission, whereas CA-04 demonstrated 80% infection, 77% dissemination and 63% transmission as determined by plaque assay titrations of singly infected females (Fig 5F and 5G), indicating lower fitness of NY99-IC than CA-04 in Cx. tarsalis. Competitive infection of NY99-IC and CA-04 with the genetically marked COAV997-5nt virus resulted in rates that were similar to those averaged between singly infected groups (Fig 5B and 5C).

As described above, bodies, legs and expectorants yielded titers that were not statistically different (p>0.05; Fig 5), but the number of NY99-IC infected mosquitoes was lower than for the other viruses. We assessed the RNA copy number of NY99-IC and COAV997-5nt in bodies of dually infected females, and found that these were not quite significantly different (p = 0.056; Fig 6B). However, only one mosquito became singly infected with NY99-IC, whereas nine mosquitoes became infected singly with COAV997-5nt, and seven mosquitoes tested positive for both NY99-IC and COAV997-5nt when both viruses were presented in the blood meal (Fig 6B) leading to a calculated infection rate of 27% and 53%, respectively, in mixed infections. Overall, our data showed that on day 14 there were fewer Cx. tarsalis infected with NY99-IC compared to COAV997-5nt, but both viruses replicated to similar titers and RNA copy numbers.

In the current study, the CA-04 virus, originally isolated from a dead Yellow-billed Magpie in 2004, exhibited high fitness and virulence in HOFIs (Figs 1C, 1H, 2, 3C, 3G and 4C, 4G). This isolate also demonstrated high fitness in Cx. tarsalis mosquitoes with infection rates of 80% determined by plaque assay titration of singly infected bodies (Fig 5G). When analyzing bodies after exposure to dual infection with COAV997-5nt, we found three CA-04 singly infected, five COAV997-5nt singly infected, and 14 dually infected mosquito bodies (Fig 6C), resulting in 57% infection rate for CA-04 and 63% infection for COAV997-5nt, with no significantly different RNA copy numbers between the two viruses (p = 0.22). We therefore did not observe a fitness trade-off effect for CA-04 as described previously for arboviruses during host alteration [16, 27].

All mosquito expectorants were analyzed by qRT-PCR before and after one passage in Vero cell culture (Fig 7) to assess fitness transmission differences. In NY99-IC singly infected mosquitoes, we detected virus-specific RNA in 40% of Vero cell passaged expectorants (Fig 7F). In contrast, 27% and 37% of Vero cell passaged expectorants from dually infected mosquitoes contained NY99-IC and COAV997-5nt RNA, respectively (Fig 7B). Vero cell propagated expectorant of CA-04 + COAV997-5nt yielded 27% transmission of CA-04 and 53% of COAV997-5nt (Fig 7C). In summary, the transmission rates calculated from Vero cell passaged expectorant using qRT-PCR agreed well with infectious virus detected by plaque assay titration for singly infected groups (Figs 5D–5G and 7D–7G). In dually infected groups, the combined transmission rates matched well with infectious virus detected by plaque assay, but the virus-specific transmission rates did not correspond well with virus-specific infection rates detected for NY99-IC and CA-04 (Figs 5B–5C and 7B–7C). This lack of concordance may be attributed to multiple aspects of vector competence including midgut infection, dissemination and transmission barriers. Also, decreased ability of NY99-IC to infect the midgut cells of Cx. tarsalis (infection rate) may be compensated by increased infection of the salivary gland cells leading to more efficient transmission (transmission rate) whereas the opposite may be true for CA-04.

By utilizing competitive replication of genetically related WNV isolates in HOFIs and Cx. tarsalis, we were able to reproduce the phenotypic hallmarks of NY99-IC and two WN02 strains, COAV997-IC and CA-04. COAV997-IC and COAV997-5nt exhibited neutral fitness in birds and mosquitoes, indicating our genetic labelling did not alter fitness and validating our use of this founding strain in future competition studies. Although characterization of the fitness of NY99-IC and CA-04 was straight-forward in birds, interpretation of results from competitively infected mosquitoes proved to be more complex and will require the definition of fitness on more than one level. However, the ability to discern between singly and dually infected mosquitoes might provide valuable insights into the complexity of infection dynamics within vector mosquitoes. Limitations also included fitness being determined in only two moderately susceptible host species; however, this model may be expanded to include high and low competent hosts, because natural transmission dynamics involve rural and urban transmission cycles with a wide range of different avian and Culex species [6]. In the current study, we used day 14 to quantify mosquito infection and transmission, but collection of mosquitoes could be extended to multiple time points to interrogate infection and transmission dynamics during early and late competition.

In the current study, competition in dually infected individuals facilitated the rapid screening for relative fitness differences between two viruses, while eliminating inter-host variability and reducing the number of animals. Small genetic changes in WNV can result in significant phenotypic changes and selected nucleotide substitutions may give rise to new and better adapted viruses. Our in vivo fitness competition model will help track the spatio-temporal phenotypic evolution of WNV in California compared to the founding COAV997-2003 isolate. It is unclear what facilitates WNV epidemics and whether outbreak isolates are associated with increased fitness in either host. The 2012 outbreak of WNV in Texas was associated with increased incidence in humans, but sequencing of isolates failed to demonstrate an association between genetic factors and outbreak magnitude [28]. Our model may help investigate whether outbreak isolates exhibit elevated fitness in birds and/or mosquitoes and may contribute to outbreak predictions based on the characterization of the viral fitness of emerging WNV strains.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the American Veterinary Medical Association (AVMA). The University of California is approved for animal experimentation under the National Institutes of Health (NIH) Animal Welfare assurance number A3433. Collection, care, maintenance and infection of House Finches was approved under the University of California Davis Institutional Animal Care and Use Committee (IACUC) approved protocol 15895, US Federal Fish and Wildlife permit MB082812-1, and the State Fish and Game collecting permit SC-002281. House Finches were trapped using modified Australian Crow traps operated by contractors at multiple vineyards near Bakersfield, CA under depredation permits which afforded us permission to remove birds for experimental purposes. No endangered species were caught when trapping wild House Finches. We made significant efforts to avoid death as an endpoint through daily observations of animals for the duration of 1–3 hours a day throughout the entire ABSL-3 phase of the experiment. During these observations, the health condition of each animal was assessed individually and within the cage group by a veterinarian (G. Worwa). Moribund animals (lethargic or unresponsive birds) and animals suffering from visible pain or distress were euthanized via inhalation of carbon dioxide as specified in IACUC protocol 15895. Death by carbon dioxide inhalation is listed as an acceptable euthanasia method for birds by the American Veterinary Medical Association (AVMA) in the AVMA Guidelines for Euthanasia of Animals 2013 Edition. However, death in House Finches due to acute West Nile virus infection often occurs very suddenly and therefore prevented a timely intervention for most birds in this study. Blood used to infect mosquitoes was collected from chickens maintained in accordance with IACUC protocol 15892. Chickens were purchased by the Kern Mosquito and Vector Control District in Bakersfield who gave permission for us to collect blood in accordance with procedures described and approved in IACUC protocol 15892. WNV strain COA977-2003 was isolated from Cx. tarsalis mosquitoes collected by dry-ice baited traps operated at the Wister State Wildlife Area in Imperial County, CA [33.277, 115.577] as part of the California Mosquito-Borne Encephalitis Virus Surveillance Program; specific collection permits were not required to operate mosquito traps. The biosafety level 3 facility was approved under Biological Use Authorization 0872 by the University of California, Davis, Environmental Health and Safety Institutional Biosafety Committee and USDA Permit 47901.

The COAV997-2003 isolate (NCBI GenBank accession no. JF703162) was made from a Cx. tarsalis mosquito pool collected in Imperial Valley, California, in July 2003 and represents historically the WN02-representative founding isolate from California [4]. Infectious-clone derived viruses of COAV997-2003 (COAV997-IC) and NY99 strain 382–99 (NY99-IC) were generated previously [11, 15], and the NY99-IC was a representative of the East Coast genotype. SAC-04-7168 (CA-04; NCBI GenBank accession no. DQ080059) was isolated from a Yellow-billed Magpie (Pica nuttalli) found dead in 2004 in Sacramento, California, one year after invasion of WNV into California [14]. The CA-04 isolate was passaged three times in Vero cells before use in this study. We previously described the construction of COAV997-5nt through substitution of five synonymous nucleotides (CTCTCC → TTGAGT) in the envelope gene of COAV997-IC at nucleotide positions 2449 and 2451–2454 [24]. Infectious clone derived viruses and COAV997-5nt were rescued initially from baby hamster kidney cells (BHK-21, clone 13, ATCC no. CCL-10) and subsequently grown in African green monkey kidney cells (Vero cells; obtained from the ATCC no. CCL-81) for stock production. The genotypes and phenotypes of the above viruses previously have been well characterized [11, 13, 24, 29]. We demonstrated similar replication dynamics and RNA copy yields for COAV997-IC and COAV997-5nt in Vero and C6/36 cells indicating that the two viruses are equally fit in vitro [24]. Although titers were almost identical, COAV997-5nt yielded slightly higher RNA copy numbers compared to COAV997-IC in Vero and C3/36 cells. Although these differences were not statistically significant [24], we accounted for this variation by normalizing all results to the actual RNA copy input at time of inoculation (Tables 1 and 2).

A total of eight experimental groups of HOFIs and Cx. tarsalis mosquitoes were used in this study: three competitively (dually) infected groups, four singly infected and one mock-infected group (Tables 1, 2 and 3). For the competitively infected groups the genetically marked COAV997-5nt was mixed 1:1 with COAV997-IC, NY99-IC or CA-04. The 1:1 mixture was achieved using equal numbers of plaque forming units (PFU) of each virus determined by plaque assay titration [26]. The COAV997-IC + COAV997-5nt group represented the group to be tested for neutral fitness in vivo, whereas NY99-IC + COAV997-5nt and CA-04 + COAV997-5nt groups were analyzed for differential fitness outcome in the in vivo model. Each of the four viruses was inoculated singly into birds and fed within an artificial blood meal to mosquitoes to compare results from single and dual infections. Mock-infection was performed using Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Gibco, Carslbad, CA) without any additives and using the same sterile inoculation techniques as described below for each species.

Groups of six HOFIs were inoculated with a total of 1000 PFUs and five birds were mock-inoculated with DMEM by needle injection subcutaneously above the right pectoral muscle. This dose has been shown to be biologically relevant as it corresponded to the virus concentration expectorated by Culex mosquitoes [19, 30]. Inocula were prepared on ice by adjusting the titer of all viruses to 8 log₁₀ PFU/mL and subsequent dilution to 4.3 log₁₀ PFU/mL in DMEM. Birds infected with a single virus were inoculated with 50μL of the 4.3 log₁₀ PFU/mL virus dilution. For animals infected dually with COAV997-5nt, a 1:1 mixture of the two viruses was made at 4.3 log₁₀ PFU/mL of which 50μL were administered leading to a total calculated concentration of 500 PFU for each virus. Aliquots of all inocula were saved from the tubes in which the inoculum was prepared before inoculation and from the syringes used for injection after administration, and all samples were stored at -80°C until analysis. All inocula aliquots were analyzed by plaque assay titration [26] and qRT-PCR [24] to determine the titer and RNA copy number that was effectively used in the inocula (Table 1).

Mosquitoes were orally exposed to viruses using a virus-spiked infectious blood meal. (Heparinized blood was obtained from chickens held in a mosquito-proof aviary at the Arbovirus Field Station in Bakersfield and confirmed to be free from anti-WNV antibodies by plaque reduction neutralization assay (PRNT) [31]). First, the titer of all viruses was adjusted to 8 log₁₀ PFU/mL in DMEM and then a final concentration of 7 log₁₀ PFU/mL was obtained by an additional 1:10 dilution in heparinized chicken blood on ice. Mosquitoes were starved for 24 hours before being offered the infectious blood meal using a Hemotek membrane system (Discovery Workshops, Accrington, Lancashire, UK) for one hour in the dark at a blood meal temperature of 37C. Following blood meal exposure, mosquitoes were anesthetized with carbon dioxide and all engorged females were transferred into a fresh container. Aliquots of blood meals and five fully engorged females from each group were frozen at -80°C immediately after virus exposure and later analyzed by plaque assay titration and qRT-PCR to determine the titer and RNA copy numbers in the inocula (Table 2).

To account for relative differences in exposure to differential doses of competing viruses at the start of competition, we calculated a ratio of RNA input by dividing the RNA copy number from each of the wildtype viruses by the number of COAV997-5nt RNA copies per inoculum. Using this ratio, results were then normalized before analysis. This was also calculated for singly infected groups to facilitate side-by-side comparison of results (Tables 1 and 2).

We selected the HOFI as a model avian host, because it is a moderately susceptible and a biologically relevant host of WNV that is abundant in North America and adapts easily to captivity and laboratory conditions. Using grain-baited traps, hatching-year HOFIs were collected in Kern County, CA during early summer 2010 and transferred to a mosquito-proof aviary at the Arbovirus Field Station in Bakersfield. All birds were administered 0.2mg/mL of chlorotetracycline (Fort Dodge, Overland Park, KS) in drinking water for 14 days. Serum from all birds was screened for antibodies against WNV, SLEV and WEEV with an immunosorbent assay [32] to exclude seropositive animals from the study. A total of 47 birds were transferred to the California Animal Health & Food Safety Laboratory (CAHFS) where they were housed in groups of six in individual cages within negative pressure HEPA-filtered Horsfall-Bauer units. Birds were allowed a two-week cage adaptation prior to infection during which absence of anti-WNV neutralizing antibodies (nAb) was confirmed in serum by PRNT₉₀. All birds tested nAb-negative except for HOFI 631 (Table 3) which was moved to the mock-infected group prior to infection. HOFI 631 also showed detectable WNV RNA in the kidney homogenate (Fig 4H), suggesting that it was naturally infected with WNV but that viral RNA persisted after the viremia subsided before the animal was captured [33].

All animals were observed daily for clinical signs such as lethargy, ataxia, abnormal body posture, tremor and changes in plumage appearance. Cloacal body temperatures were recorded starting one day prior to inoculation and every day thereafter using a disinfected digital thermocouple (Fisher Scientific, USA) placed 1 cm into the cloaca prior to any other manipulations of the animal. During each blood draw, a total of 100 μL of blood was collected from each animal with a 28-gauge needle by jugular venipuncture between 1–7 days post infection (dpi) and upon termination of the study at 14 dpi. Blood was diluted in 450μL of DMEM containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, USA), 100 U/mL penicillin, 0.1 g/mL streptomycin and 1.25 μg/mL amphotericin B (Life Technologies, Gibco, Carlsbad, CA) leading to a final approximate 1:10 dilution of serum. Birds were humanely euthanized by carbon dioxide inhalation and equally sized specimens of spleen, kidney, right pectorial muscle, liver, brain and heart were collected from all animals during necropsy. Sera and tissues were stored at -80°C until analysis by plaque assay titration, PRNT₉₀ and qRT-PCR. On 11 dpi mock-infected HOFI 682 suffered a leg fracture and was humanely euthanized (Fig 2).

A laboratory-adapted colony of Cx. tarsalis was used for all vector competition studies. Mosquitoes originated from the Kern National Wildlife Refuge in California and were reared under standard insectary conditions at 22°C and a photoperiod of 16:8 (L:D) hours at the Arbovirus Field Station in Bakersfield, CA. Mosquitoes were transferred to the biosafety level 3 insectary at the University of California in Davis prior infection.

After infectious blood meal exposure, engorged females were provided daily with a 10% sucrose solution and held for 14 days at 26C, 12:12 (L:D) hours and approximately 60% humidity. On day 14 all surviving females were counted (Table 2) and anesthetized using triethylamine (Fisher Scientific, USA). Expectorant was collected from each mosquito by placing the proboscis into a glass capillary tube filled with a 1:1 5% sucrose and 50% FBS solution. After approximately 10 minutes the proboscis was removed and content expelled by centrifugation into 250μL of DMEM containing 10% FBS, 100 U/mL penicillin, 0.1 g/mL streptomycin and 1.25 μg/mL amphotericin B (Life Technologies, Gibco, Carlsbad, CA). The legs were separated from the body using sterile technique and placed into cryovials containing two 5mm glass beads and 600μL of the above DMEM solution. Expectorant, legs and bodies were stored at -80°C until further processing.

The number of infectious virus particles in the mosquito expectorant was frequently below the qRT-PCR detection limit of 250 RNA copies (Fig 5) [24]. To maximize data on vector transmission as a key factor of WNV fitness, we passaged all expectorant samples once in Vero cells with subsequent quantification of RNA copies in addition to direct analysis of expectorant samples by qRT-PCR. Despite the fact that three rounds of replication in Vero cells previously did not affect the fitness of COAV997-IC and COAV997-5nt [24], we excluded any bias from cell culture passaging by using only qualitative data from passaged expectorant samples as to whether one, both or neither of the viruses were detected in the supernatant supporting the presence or absence of infectious virus particles in the original sample.

Bird tissues were added to 1mL and mosquito bodies and legs to 600μL of diluent and two 5 mm glass beads for homogenization on a Tissue Lyser (Qiagen, USA). Samples were triturated twice for two minutes at 24 Hz frequency and the homogenate clarified by centrifugation.

Infectious viral titers in bird sera and in mosquito bodies and expectorant samples were quantified by plaque assay titration [26]. Briefly, serially tenfold diluted samples were allowed to absorb on confluent Vero cell monolayers for 1 hour at 37°C and 5% CO₂. An agarose-based overlay was added (nutrient medium, 0.5% agarose, 3% bicarbonate) and after 48 hours of incubation at 37°C and 5% CO₂ a second overlay containing additional 3% neutral red was applied. The following day visible plaques were enumerated and the titer determined as log₁₀ PFU/mL (Figs 1 and 5)

Sera taken prior to inoculation and all end point serum samples (14 dpi or last available serum sample before death) were tested by plaque reduction neutralization assay using a 90% neutralization cut-off (PRNT₉₀) [31]. Briefly, all sera were inactivated at 56°C for 30 minutes and diluted 1:2 in virus solution containing 100 PFU (CA-04 isolate) making a final serum dilution of 1:20. After incubation of the serum-virus mixture for 1 hour at 37°C and 5% CO₂, confluent Vero cell monolayers were inoculated and incubated for an additional hour at 37°C and 5% CO₂. The same double-overlay system was used as described above for plaque assay titration. Sera producing a >90% reduction of 50–100 PFU were considered positive for nAbs (Table 3).

Viral RNA was extracted utilizing a MagMAX magnetic particle processor and MagMAX-96 Viral RNA isolation Kit (Applied Biosystems, USA) according to the manufacturer’s instructions. The allele-specific qRT-PCR for quantitative detection and distinction between COAV997-5nt and wildtype viruses was previously described [24]. Briefly, specific reverse primers WNV.TAQ.WT.2464.R and WNV.TAQ.5nt.2467.R-alt were designed to bind the wildtype sequence (CTCTCC) and COAV997-5nt (TTGAGT) at their 3’-ends, respectively. A total of 10uL viral RNA was mixed with 0.5 μL of WN.2417-2431.FAM (100 nM), 0.05 μL WNV.TAQ.2393.F (25 nM), 12.5 μL TaqMan One-Step RT-PCR Master Mix 2X (ABI, USA), 0.6 μL of TaqMan One-Step RT-PCR RNase Inhibitor, and 1.3 μL of DEPC-treated water. For specific wildtype and COAV997-5nt detection, 0.05 μL of either WNV.TAQ.WT.2464.R (25 nM) or WNV.TAQ.5nt.2467.R-alt were added to two separate master mixes. Samples and standards were run in duplicate with a final volume of 25 μL per reaction and amplified in 50 cycles in singleplex format concurrently on two 7900HT Fast Real-Time PCR Systems (ABI, USA). Quantification of RNA copies was accomplished using standard curves from plasmid-derived in vitro transcribed RNA of NY99 and NY99-5nt [24]. RNA copies for COAV997-5nt were also determined in samples from COAV997-IC, NY99-IC and CA-04 singly infected groups and reciprocally to rule out cross-reactivity and to extend qRT-PCR validation.

One- and two-way ANOVAs were used for comparison between groups (Graph Pad Prism software, version 5) to determine statistical significance of RNA copies, viral titers and body temperatures between singly and within competitively infected groups (p <0.05). If significant differences were found, a Tukey-Kramer multiple comparison test was used for post-hoc analysis between means with α = 0.05.