West Nile virus seroprevalence rates of various avian species

The house sparrow ( Passer domesticus ) is a common and abundant amplifying host of West Nile virus (WNV) and many survive infection and develop humoral immunity. We experimentally inoculated house sparrows with WNV and monitored duration and protection of resulting antibodies. Neutralizing antibody titers remained relatively constant for ≥ 36 months ( N = 42) and provided sterilizing immunity for up to 36 months post-inoculation in 98.6% of individuals ( N = 72). These results imply that immune house sparrows are protected from WNV infection for multiple transmission seasons. Additionally, individuals experiencing WNV-associated mortality reached significantly higher peak viremia titers than survivors, and mortality during acute infection was significantly higher in caged versus free-flight sparrows. A better understanding of the long-term immunity and mortality rates in birds is valuable in interpreting serosurveillance and diagnostic data and modeling transmission and disease dynamics. 865 WNV IMMUNITY IN SPARROWS Sample collection and preparation. After initial inoculation, all but 14 sparrows were housed free-flight within rooms. These 14 sparrows (seven naturally immune and seven nonimmune) were caged and bled 0.1 mL via jugular venipuncture from 1 to 6 days PI and then released into the room with the remainder of the sparrows. All sparrows were caught by hand-held nets and bled 0.2 mL via jugular venipuncture at 1, 6, 12, 18, 24, 30, and 36 months PI. At 6 months PI the 21 naturally immune sparrows that had been inoculated 6 months prior were bled and euthanized. Challenge experiments (i.e., re-inoculation or secondary exposure) occurred at 6, 12, 24, and 36 months PI. Sparrows were placed into cages for several days and then needleinoculated subcutaneously with 2,500–3,500 PFU of WNV strain NY99-4132. After challenge inoculation (or initial inoculation for non-immune controls), blood samples were collected from 1–7 and on 14 days PI, when birds were euthanized. Blood samples were either added to BA1 with 20% fetal bovine serum (FBS) in cryovials for an approximate 1:10 serum dilution (for viremia analysis) or dispensed undiluted into serum separator tubes (for antibody analysis). Blood samples were held at room temperature for 20–30 minutes for coagulation, centrifuged for 10 minutes at 6,000 × G and sera frozen to −80°C (diluted samples) or for 3 minutes at 12,000 × G and sera frozen to −20°C (undiluted samples). Sparrows that died or were euthanized as a result of morbidity < 10 days PI, any non-immune controls that succumbed during the study, and eight non-immune controls euthanized at 14 days PI were necropsied, at which time oropharyngeal swabs, spleen, kidney, heart, and brain were collected and placed in 1 mL BA1 with 20% FBS (tissues were weighed for a 10% suspension). Tissues were processed as previously described 17 and tested for WNV by plaque assay. These birds were considered to have experienced acute WNV-associated mortality if WNV was isolated from multiple tissues. Vero cell plaque assay and plaque reduction neutralization test. Sera collected from 1 to 7 days PI, as well as oral swabs and tissue homogenates from birds dying < 10 days PI, were tested for infectious WNV by Vero cell plaque assay as previously described. 18 Representative plaques were confirmed as WNV through reisolation and testing by VecTest WNV Antigen Assay (Medical Analysis Systems, Camarillo, CA) as previously described. 17 The detection thresholds for WNV were 10 1.7 PFU/mL serum and 10 0.7 PFU/swab or mL of tissue homogenate. Sera were tested for neutralizing antibodies to WNV using the plaque reduction neutralization test (PRNT) 19 with the same WNV strain as for inoculation of sparrows. Sera that neutralized ≤ 60% of WNV PFU were considered negative for antibodies, whereas sera that neutralized > 90% were considered positive (no serum samples neutralized between 60–90% of viral plaques). Antibody positive serum samples were serially diluted 2-fold and tested in duplicate to determine reciprocal endpoint 90% neutralization (PRNT 90 ) titers. Anamnestic antibody responses to challenge were considered significant when a ≥ 4-fold increase in PRNT 90 titer was observed within ~2–4 weeks of challenge. Mathematical and statistical analyses. To assess the variation in PRNT 90 titers of all sparrows alive for at least two consecutive time points, the multiple-fold change in titer for each individual at a given time point and the one immediately following was represented by a numerical value (e.g., −2 for a 2-fold decrease, 0 for no change in titer, 2 for a 2-fold increase). These values were averaged among all individuals to determine average changes in titer at each time period ( Table 1 ). This analysis avoided eliminating individuals that were not present throughout all time points. A χ 2 test (α = 0.05) was used to compare mortality rates (as proportions) of caged sparrows that were frequently captured and sampled with those of free-flight sparrows not handled after inoculation. Peak viremia titers in log 10 PFU/mL serum (the dependent variable) were analyzed as a function of disposition (death versus survival; the fixed variable) using general Figure 1. Timeline of West Nile virus experimental inoculation of three experimental groups of house sparrows. * Antibody (Ab) titer indicates when serum samples were titrated to determine WNV PRNT 90 antibody titers. † All birds were bled at 1 month postinoculation to confirm seroconversion and assess antibody titers.