Prevalence of West Nile virus and Usutu virus serum antibodies in Dutch bird ringers

West Nile virus (WNV) is a mosquito-borne zoonosis which is endemic-epidemic in
Europe where cases are reported in southern, eastern and western countries. WNV
is transmitted in an enzootic cycle between mosquitoes and birds which act as
amplifying hosts. Mammals can become infected from the bite of an infected
mosquito but are considered dead-end hosts [4]. About 80% of human infections
are asymptomatic while 20% of cases develop West Nile fever (WNF) presenting
symptoms such as headache and muscle pain. Severe disease progressions are rare
(<1%) and include syndromes of meningitis, encephalitis, and acute flaccid
paralysis/poliomyelitis. Older people and immunocompromised persons are at
higher risk of developing West Nile neuroinvasive disease (WNND) [3]. In 2019,
Germany reported the first autochthonous human cases of WNV. In 2020, as of 29
October, 12 human infections have been reported in the country. Five regions
reported locally-acquired human cases of WNV for the first time (regions of
Barnim, Ostprignitz-Ruppin, Saalekreis Halle (Saale) and Meissen) [5]. Since
July 2020, virus circulation in birds has been documented in the country in a
similar area as in 2019 (north-eastern regions) [6].
Usutu virus (USUV) is a flavivirus closely related to WNV, belonging to the
Japanese encephalitis virus (JEV) sero-complex. Human USUV infections have been
reported in Europe and Africa. USUV infection in humans seems to be often
asymptomatic or leading to mild clinical illness [7]. Although clinical
manifestation of USUV are rather similar to WNV, the first are far less common.
According to literature, as of 2019, 25 human cases of USUV infection have been
described [8].
In Europe, few cases of neurological complications have been reported in Italy,
Croatia and France. In addition, USUV infections among healthy blood donors
have been reported in Italy, Germany and Austria [9].
In 2018, USUV-RNA and -antibodies have been found in blood donor screenings in
The Netherlands [10]. USUV has been circulating extensively in birds in the
Netherlands at least since 2016 [11][12].

In August 2020, a swab testing positive for WNV was collected from a live
common whitethroat bird in Haarzuilens (Utrecht). This was the first time that
a bird is found to be positive for WNV in The Netherlands. Previous tests in
the same bird in 2019 and May 2020 were negative, indicating local transmission
[1]. Enhanced mosquito and bird surveillance were immediately implemented to
determine the extent of the introduction and to assess the risk of transmission
to humans and animals. As of 15 October 2020, WNV has also been found in five
mosquito pools collected in August and September and three additional birds
have been found positive for WNV.
In addition, on 12 October 2020, one case of West Nile virus infection has been
detected in a man with no recent travel history outside of The Netherlands. The
transmission likely occurred in the Utrecht region and this is the first time
that a locally-acquired human case of WNV infection has been reported in The
Netherlands [2]. Up to 22nd of October, six additional cases have been
identified, one of which from the region Gelderland-Midden.
Among the collected and caught birds in 2020, at least five dead birds and one
live bird from different locations in The Netherlands, tested positive for
USUV. Moreover, USUV has been found in one mosquito pool in Haarzuilens.

In The Netherlands, birds are caught and sampled to detect possible
introduction and spread of zoonotic arboviruses. Wild birds are randomly
captured using different trapping methods and sampled as part of a wider study
on the presence of zoonotic viruses in the country. Recapturing and resampling
of ringed birds occurs frequently during the breeding season. In addition,
mortality in captive and wild birds is reported through a citizen science based
alerting system. Dead birds are collected and autopsy is performed for research
[1].

Bird catching activities occur in the bird*s natural habitat and bird ringers
are intensively exposed to mosquito bites at the same time of day mosquitos are
active (i.e. dusk and down). This makes them more susceptible to infection than
other people in the area. In addition, some of them are also actively involved
in sampling of birds and are therefore in direct contact with blood and other
avian fluids. Although mosquito*s bite is the most common route of
transmission, WNV infections following occupational exposure have been also
reported in literature [13][14].

For these reasons, in this study, we aim to determine the prevalence of WNV and
USUV antibodies in serum specimens collected from bird ringers in The
Netherlands. This will allow us to investigate the potential human exposure in
areas where positive WNV and/or USUV birds have been found, and also to explore
potential WNV and/or USUV exposure in bird ringers from other regions not only
as a control group but also as part of early warning activities.

Primary objective: To determine the seroprevalence of WNV and USUV antibodies
in serum samples from bird ringers in The Netherlands.

This is a prospective cross-sectional observational study. In November, bird
ringers who carried out their activities in The Netherlands during spring,
summer and autumn 2020 will be invited to participate in this study. After
informed consent, a single blood sample of 4 mL will be drawn at the local
huisartsenlaboratorium.
In addition, participants will be asked to fill out a questionnaire to provide
information on demographic data, exposure at the bird ringing sites, travel
history, previous vaccinations against arboviruses such as tick-borne
encephalitis (TBE), yellow fever (YF) and Japanese encephalitis (JE), previous
infections with flaviviruses, and possible complaints compatible with WNF or
WNVD.
Collected serum samples will be tested in the Laboratory for Virology at
Erasmus University Medical Center for the presence of WNV and USUV specific IgM
and/or IgG. Samples testing positive will be tested by virus neutralisation
assays for antibodies to WNV and USUV.

The study has no personal benefit for the participants. The participants will
be informed about their serostatus for WNV and USUV. The risks associated with
participation is limited to limited pain and possible bruising following the
venapuncture.