Experimental inoculation of house sparrows (Passer domesticus) with buggy creek virus.

We performed experimental inoculations of house sparrows (Passer domesticus) with Buggy Creek virus (BCRV), a poorly known alphavirus (Togaviridae) vectored primarily by the swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) that is an ectoparasite of the cliff swallow (Petrochelidon pyrrhonota) and house sparrow. Viremias were detected by plaque assay in two of six birds on days 1-3 postinoculation; viremia was highest on day 2. Viral RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in blood of six of 12 birds ranging from day 1 to day 15 postinoculation. Infectious BCRV was detected in nasopharyngeal swab samples from two birds by plaque assay. Three control birds that were housed with viremic individuals showed evidence of BCRV RNA in blood (by RT-PCR), suggesting possible bird-to-bird transmission of this virus. Viral RNA also was detected by RT-PCR in brain and skin tissue of six birds on necropsy at the end of the 16-day experiment. Introduced house sparrows are apparently a competent amplifying host for BCRV, and their presence year-round at cliff swallow colonies may facilitate persistence of the virus locally, especially when cliff swallows abandon a site temporarily. The findings that BCRV can be shed orally, that it persists in bird skin, and that control birds could apparently be infected by conspecifics suggest that this virus may be transmitted from bird to bird in the crowded conditions of many cliff swallow colonies.

Ecological correlates of buggy creek virus infection in Oeciacus vicarius, southwestern Nebraska, 2004.

Buggy Creek virus (family Togaviridae, genus Alphavirus, BCRV) is an alphavirus within the western equine encephalitis virus complex whose primary vector is the swallow bug, Oeciacus vicarius Horvath (Hemiptera: Cimicidae), an ectoparasite of the colonially nesting cliff swallow, Petrochelidon pyrrhonota, that is also a frequent host for the virus. We investigated ecological correlates of BCRV infection in 100-bug pools at 14 different swallow colony sites in southwestern Nebraska from summer 2004, by using plaque assay on Vero cells to identify cytopathic virus and reverse transcription-polymerase chain reaction to identify noncytopathic viral RNA. We found 26.7% of swallow bug pools positive for BCRV, with 15.6% showing cytopathic ("infectious") virus and 11.0% noncytopathic ("noninfectious") viral RNA. The prevalence of cytopathic BCRV increased with cliff swallow colony size in the current year; the percentage of noncytopathic samples at a site did not vary with colony size in the current year but increased with the previous year's colony size at a site. Active colony sites (those used by swallows) had higher percentages of cytopathic BCRV in bug pools than at inactive colony sites, but the reverse held for noncytopathic viral RNA. Nests that were occupied by birds at some time in the season had more pools with cytopathic BCRV than did inactive nests. Colonies used by birds for the first or second time had less virus in bugs than did sites that had had a longer history of bird use. The percentage of pools with BCRV was affected by whether bugs were clustering at nest entrances or distributed elsewhere on a nest. The prevalence of cytopathic samples decreased at inactive colony sites and increased at active sites over the course of the summer, whereas the reverse pattern held for noncytopathic samples. Noncytopathic bug pools seem to reflect infection patterns from a previous year. The results suggest that the birds play an important role in amplification of the virus and that the spatial foci of BCRV occurrence can be predicted based on characteristics of cliff swallow colonies and the cimicid bugs that are associated with them.

Phylogenetic analysis of Buggy Creek virus: evidence for multiple clades in the Western Great Plains, United States of America.

We present the first detailed phylogenetic analysis of Buggy Creek virus (BCRV), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (Oeciacus vicarius) vector and cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) as the principal avian hosts. Nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire PE2 gene, were determined for 33 BCRV isolates taken from swallow bugs at cliff swallow colonies in Nebraska and Colorado in the summer of 2001 and were compared with the corresponding region of BCRV isolates collected from Oklahoma in the 1980s. We also analyzed isolates of the closely related Fort Morgan virus (FMV) collected from Colorado in the 1970s. Phylogenetic analysis indicated that BCRV falls into the western equine encephalomyelitis complex of alphaviruses, in agreement with antigenic results and a previous alphavirus phylogeny based on the E1 coding region. We found four distinct BCRV/FMV clades, one each unique to Nebraska, Colorado, and Oklahoma and one containing isolates from both Nebraska and Colorado. BCRV isolates within the two clades from Nebraska showed 5.7 to 6.2% nucleotide divergence and 0.7 to 1.9% amino acid divergence, and within these clades, we found multiple subclades. Nebraska subclades tended to be confined to one or a few cliff swallow colonies that were close to each other in space, although in some cases, near-identical isolates were detected at sites up to 123 km apart. Viral gene flow occurs when cliff swallows move (bugs) between colony sites, and the genetic structure of BCRV may reflect the limited dispersal abilities of its insect vector.