Usutu virus (USUV), a mosquitoborne zoonotic virus of the genus Orthoflavivirus, was first detected in the Netherlands in 2016 (1). Eurasian blackbirds (Turdus merula) are remarkably susceptible to USUV disease; exceptional die-off among blackbirds is a hallmark of the (re)-emergence of virus circulation in a region (2,3). Information on survival of blackbirds after USUV infection and the resulting prevalence of USUV antibodies in such populations is limited, and knowledge of the longitudinal dynamics in USUV antibody responses in individual naturally infected blackbirds is lacking. We studied the presence of USUV-specific antibodies in blackbirds during the first 3 years after the initial detection of the virus in the Netherlands.

During March 2016–October 2018, as part of a broader study of zoonoses in songbirds in the Netherlands, we registered 1,181 blackbird captures; 969 unique birds were captured 1–9 times (1,4). Sampling locations were biased; of 77 sampling locations, just 4 contributed nearly 50% of all samples (Overdinkel, 52°14′N, 7°01′E; Wageningen, 51°59′N, 5°39′E; Haarzuilens, 52°08′N, 5°00′E; Eastermar, 53°11′N, 6°03′E).

We sampled healthy birds by wing vein puncture. Overall, a total of 667 serum samples from 534 unique blackbirds were tested. The samples were screened in a 2-tier system of protein microarray testing including the nonstructural 1 proteins of USUV and West Nile virus (WNV), followed by confirmation of positive IgG results based on comparative USUV and WNV neutralization (5). USUV and WNV belong to the same serogroup, causing probable cross-reactivity in serologic assays (6). Therefore, we only considered serum samples confirmed for antibodies when microarray reactivity was confirmed by comparative virus neutralization test (VNT) results showing USUV antibody titers >4-fold higher than a WNV antibody titer, or vice versa. Serum samples with a positive USUV microarray result but no conclusive confirmation by VNT were considered orthoflavivirus positive (Table). To determine the USUV seroprevalence by calendar year, we only included birds that were repeatedly sampled within the same year once, and in cases of seroconversion within a year (n = 4), we only included the positive outcome.

According to the stated criteria for confirmed infection, 3 (4.8%) of 63 unique birds were positive for USUV-specific antibodies in 2016; in 2017, the total was 21 (7.5%) of 280; and in 2018, the total was 26 (10.0%) of 237 (Table). Results showed a significant increase in seroprevalence over those years [χ²(1) = 6.01, p = 0.01; Hosmer-Lemeshow goodness-of-fit χ²(8) = 3.24, p = 0.92], with a peak in autumn 2017 (data not shown) (7). That increase is in line with observations in Austria, where seropositivity of 9.1% in blackbirds was found 2 years after the virus was first detected, and 9.6% 3 years after (8). However, in Germany, seropositivity remained low (<3.9%) in the 5 years after a massive blackbird die-off (9).

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Figure. Longitudinal results for Usutu virus (USUV) serologic testing of serum samples from naturally study of USUV antibody dynamics in naturally infected blackbirds, the Netherlands, 2016–2018. Birds were recaptured and sampled...

Starting in September 2016, the Netherlands study captured 124 blackbirds >1 time (range 2–9 times); 84 birds had serum samples taken at >1 capture. Of those birds, 17 were confirmed seropositive >1 time; 7 seroconverted during the study period, and 10 were positive at all time points (Figure, panel A). Of the remaining 67 recaptured birds, 66 remained seronegative throughout the study, whereas 1 bird seroconverted for USUV, based on microarray and USUV VNT only. No seroreversion was observed.

The longest period of seropositivity recorded was >361 days. That bird was already seropositive at first sampling and was not followed beyond 361 days. To gain insight into functional neutralizing antibody kinetics, we plotted over time the VNT titers of the 12 birds that were positive at multiple sampling points (Figure, panel B). Six birds showed waning neutralizing antibody titers in time with variable kinetics. Two birds showed no changes in titers in samples taken 33 and 42 days apart, whereas 2 other birds showed an increase in titers taken 5 and 21 days apart. Two birds showed an initial decline in VNT titers, then an increase within 41 and 113 days; corresponding cloaca swab specimens showed a similar trend (positive-negative-positive) for the presence of USUV RNA (no sequence information available). That finding might be indicative for the occurrence of reinfection in the presence of low USUV-neutralizing antibodies, whether or not by different USUV lineages; Africa-3 is the most common infecting lineage in the Netherlands, followed by Europe-3 lineage (4). Alternatively, such observations might be the result of exacerbation of a persistent infection (10). Conclusive evidence is needed from additional longitudinal studies, including experimental infections. Further insight into the longevity of USUV-specific antibodies in different bird species, the occurrence of reinfections, and the relationship between variations in the immune-status of a specific bird population and variations in the level of virus circulation will contribute to a better understanding of the added value of the assessment of public health risks based on seroecologic surveillance.

In conclusion, our study provides a unique longitudinal dataset on the kinetics of USUV-specific antibodies in naturally infected wild blackbirds. We show seropositivity in individual blackbirds for >1 year and the possible occurrence of reinfection in the presence of low-level virus neutralizing antibodies.

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