Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic we re conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, includin g 5 of 13 ducks (38%), 1 of I goose, 19 of 52 chickens (37%), 1 of 1 peahen , and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 2 6) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46) . Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted fo r 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obt ained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97 -50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained fro m a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenet ic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest th e existence of two large lineages of viruses. Bootstrap analysis of the nuc leotide alignment-indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Al geria, and west, central, and east Africa, all of the European isolates, th ose from France and Romania, an Israeli isolate, and an isolate of KUN viru s from Australia. The nucleotide sequence of RO97-50 was identical to the s equence of a WN virus isolate obtained from Cx. neavei mosquitoes from Sene gal and Cx. univittatus mosquitoes from Kenya. The phylogenetic analyses we re compatible with the introduction of virus into Romania by birds migratin g from sub-Saharan Africa, to northern Africa, and into southern Europe.