In 1999 WNV, was introduced into the United States where it caused severe epidemic of meningoencephalitis in New York City
In 1999 WNV, was introduced into the United States where it caused severe epidemic of meningoencephalitis in New York City. important to improve public health awareness about the disease these viruses may cause. [1,2]. WNV was first isolated in 1937 in the West Nile region of Uganda from a patient suffering a mild febrile illness [3,4]. Over the past decades, the virus became endemic in Europe, Africa, Asia, the Middle East and Australia  and it is now well recognized as a human [1,4,5,6], equine [7,8], and avian neuropathogen [1,4,9,10]. In 1999 WNV, was introduced into the United States where it caused severe epidemic of meningoencephalitis in New York City. Subsequently, it spread throughout Canada, Central America and the Caribbean . The occurrence of WNV was confirmed in 20 countries of the European Union, including close neighbors of Poland [1,12]. Previous serological surveys in Poland showed low seroprevalence of WNV antibodies in wild birds, horses and humans [3,12,13,14]. It is rather surprising then that results of recent study in humans indicate high exposure to WNV . USUV was first isolated in 1959 in South Africa from mosquitoes . In Europe, it was first isolated in Austria, in 2001, after a significant die-off of Rabbit Polyclonal to Tyrosine Hydroxylase Eurasian blackbirds. Subsequently, it has been isolated throughout Europe from countries such as Hungary, Switzerland, Spain, Italy, Germany and Belgium . Serological evidence of USUV presence in birds was also found in England, Czech Republic, Spain, Switzerland, Germany, Italy and Greece. In countries neighboring Poland, such as Germany and Czech Republic, the recurrence of the virus suggests persistence of the transmission cycle in the affected areas, possibly through overwintering mosquitoes . In Poland, antibodies to USUV were found in 2008 in a seagull (411) were bled on different farms throughout Poland. They were aged 1C28 years old, and included mares (251), stallions (96) and geldings (64) representing polish half-bred (74), Arabian (42), thoroughbred (39), Hucul pony (19) AngloCArabian (15), Malopolski (26), Wielkopolski (32), Orexin 2 Receptor Agonist Silesian (22), polish coldblood (37) and Fjord half-bred (11) horses. In 94 Orexin 2 Receptor Agonist cases, the breed was unknown. None of the horses were vaccinated against WNV. Although no clinical cases were reported in sampled horses, clinical disease in past is not excluded, because neurological symptoms in horses in Poland are usually diagnosed as equine herpesvirus type 1 (EHV-1) infection or Wobbler syndrome and WNV or USUV infection is not considered [36,37]. 2.3. Virus Isolation and Identification Individual tissue samples from birds were processed using a previously described procedure  and inoculated separately (50 L per well, 8 replicates per sample) into different cell lines: rabbit kidney cells (RK-13, ATCC, Manassas, VA, USA, No CCL-37 TM) and green monkey kidney (Vero, ATCC, No CCL-81 TM) seeded in 96-well polystyrene plates the day before inoculation. Plates were incubated at 37 C/5% CO2 and observed daily for the development of cytopathic effect up to 10 days for up to five blind passages. Simultaneously,s virus isolation was attempted in embryonated chicken eggs (ECE). Supernatant from cell cultures and allantoic liquid were harvested and tested by hemagglutination assay and a flavivirus generic RT-PCR, using primers FU1 and CFD2 . 2.4. Serology Horse and bird sera were tested for the presence of virus neutralizing antibodies to WNV and USUV using Orexin 2 Receptor Agonist microneutralization procedure as described before . USUV reference isolate SA-AR1776 (South Africa, 1959), Vero passage 4, was used for the USUV Orexin 2 Receptor Agonist neutralization test. The antibody titers were expressed as the reciprocal of the serum dilution inhibiting 100% of viral cytopathic effect. Serum samples with a virus neutralization titer of 1 1:10 were considered positive. IgM serology was performed using the ID Screen West Nile IgM Capture ELISA for equines (IDVet, Grabels, France). 2.5. Statistical Analysis All statistical analysis was conducted in PQStat version 1.6.1 (PQ Stat Software, Pozna, Poland), at a significance level of 5% using the following tests: chi-square and the Spearman rank correlation coefficient (2), 1:20 (1), 1:40 (43), 1:80 (1), 1:160 (1) 1:320 (5), 1:640 (4), and 1:1280 (5). No WNV IgM antibodies were detected in a subset of 44 horses with confirmed neutralizing antibodies. Among 411 horse serum samples, 115 (27.98%) were positive for neutralizing antibodies to Usutu virus (Figure 1, Table 2). Sixty-seven of serologically positive horses (58.26%) did not travel outside Polish borders. The travelling horses were in Germany (25 horses), Hungary (6), France (4), Slovakia (4), Austria (3), Italy (3), and the Netherlands (3). WNV neutralizing antibodies titers in horses.