Amy T. Moore

An enzootic vector-borne virus is amplified at epizootic levels by an invasive avian host

Determining the effect of an invasive species on enzootic pathogen dynamics is critical for understanding both human epidemics and wildlife epizootics. Theoretical models suggest that when a naive species enters an established host–parasite system, the new host may either reduce (‘dilute’) or increase (‘spillback’) pathogen transmission to native hosts. There are few empirical data to evaluate these possibilities, especially for animal pathogens. Buggy Creek virus (BCRV) is an arthropod-borne alphavirus that is enzootically transmitted by the swallow bug (Oeciacus vicarius) to colonially nesting cliff swallows (Petrochelidon pyrrhonota). In western Nebraska, introduced house sparrows (Passer domesticus) invaded cliff swallow colonies approximately 40 years ago and were exposed to BCRV. We evaluated how the addition of house sparrows to this host–parasite system affected the prevalence and amplification of a bird-associated BCRV lineage. The infection prevalence in house sparrows was eight times that of cliff swallows. Nestling house sparrows in mixed-species colonies were significantly less likely to be infected than sparrows in single-species colonies. Infected house sparrows circulated BCRV at higher viraemia titres than cliff swallows. BCRV detected in bug vectors at a site was positively associated with virus prevalence in house sparrows but not with virus prevalence in cliff swallows. The addition of a highly susceptible invasive host species has led to perennial BCRV epizootics at cliff swallow colony sites. The native cliff swallow host confers a dilution advantage to invasive sparrow hosts in mixed colonies, while at the same sites house sparrows may increase the likelihood that swallows become infected.

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

Abstract 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.

Phylogeographical structure and evolutionary history of two Buggy Creek virus lineages in the western Great Plains of North America

Buggy Creek virus (BCRV) is an unusual arbovirus within the western equine encephalitis complex of alphaviruses. Associated with cimicid swallow bugs (Oeciacus vicarius) as its vector and the cliff swallow (Petrochelidon pyrrhonota) and house sparrow (Passer domesticus) as its amplifying hosts, this virus is found primarily in the western Great Plains of North America at spatially discrete swallow nesting colonies. For 342 isolates collected in Oklahoma, Nebraska, Colorado and North Dakota, from 1974 to 2007, we sequenced a 2076 bp region of the 26S subgenomic RNA structural glycoprotein coding region, and analysed phylogenetic relationships, rates of evolution, demographical histories and temporal genetic structure of the two BCRV lineages found in the Great Plains. The two lineages showed distinct phylogeographical structure: one lineage was found in the southern Great Plains and the other in the northern Great Plains, and both occurred in Nebraska and Colorado. Within each lineage, there was additional latitudinal division into three distinct sublineages. One lineage is showing a long-term population decline. In comparing sequences taken from the same sites 8-30 years apart, in one case one lineage had been replaced by the other, and in the other cases there was little evidence of the same haplotypes persisting over time. The evolutionary rate of BCRV is in the order of 1.6-3.6x10(-4) substitutions per site per year, similar to that estimated for other temperate-latitude alphaviruses. The phylogeography and evolution of BCRV could be better understood once we determine the nature of the ecological differences between the lineages.

Host and vector movement affects genetic diversity and spatial structure of Buggy Creek virus (Togaviridae)

Determining the degree of genetic variability and spatial structure of arthropod‐borne viruses (arboviruses) may help in identifying where strains that potentially cause epidemics or epizootics occur. Genetic diversity in arboviruses is assumed to reflect relative mobility of their vertebrate hosts (and invertebrate vectors), with highly mobile hosts such as birds leading to genetic similarity of viruses over large areas. There are no empirical studies that have directly related host or vector movement to virus genetic diversity and spatial structure. Using the entire E2 glycoprotein‐coding region of 377 Buggy Creek virus isolates taken from cimicid swallow bugs (Oeciacus vicarius), the principal invertebrate vector for this virus, we show that genetic diversity between sampling sites could be predicted by the extent of movement by transient cliff swallows (Petrochelidon pyrrhonota) between nesting colonies where the virus and vectors occur. Pairwise FST values between colony sites declined significantly with increasing likelihood of a swallow moving between those sites per 2‐day interval during the summer nesting season. Sites with more bird movement between them had virus more similar genetically than did pairs of sites with limited or no bird movement. For one virus lineage, Buggy Creek virus showed greater haplotype and nucleotide diversity at sites that had high probabilities of birds moving into or through them during the summer; these sites likely accumulated haplotypes by virtue of frequent virus introductions by birds. Cliff swallows probably move Buggy Creek virus by transporting infected bugs on their feet. The results provide the first empirical demonstration that genetic structure of an arbovirus is strongly associated with host/vector movement, and suggest caution in assuming that bird‐dispersed arboviruses always have low genetic differentiation across different sites.

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.

Isolation of Buggy Creek Virus (Togaviridae: Alphavirus) From Field-Collected Eggs of Oeciacus vicarius (Hemiptera: Cimicidae)

ABSTRACT Alphaviruses (Togaviridae) rarely have been found to be vertically transmitted from female arthropods to their progeny. We report two isolations of Buggy Creek virus (BCRV), an ecologically unusual alphavirus related to western equine encephalomyelitis virus, from field-collected eggs of cimicid swallow bugs (Oeciacus vicarius Horvath), the principal vector for BCRV. Ten percent of egg pools were positive for BCRV, and we estimated minimum infection rates to be 1.03 infected eggs per 1,000 tested. The results show potential vertical transmission of BCRV, represent one of the few isolations of any alphavirus from eggs or larvae of insects in the field, and are the first report of any virus in the eggs of cimicid bedbugs. The specialized ecological niche of BCRV in swallow bugs and at cliff swallow (Petrochelidon pyrrhonota Vieillot) nesting sites may promote vertical transmission of this virus.

Ecological divergence of two sympatric lineages of Buggy Creek virus, an arbovirus associated with birds

Most arthropod-borne viruses (arboviruses) show distinct serological subtypes or evolutionary lineages, with the evolution of different strains often assumed to reflect differences in ecological selection pressures. Buggy Creek virus (BCRV) is an unusual RNA virus (Togaviridae, Alphavirus) that is associated primarily with a cimicid swallow bug (Oeciacus vicarius) as its vector and the Cliff Swallow (Petrochelidon pyrrhonota) and the introduced House Sparrow (Passer domesticus) as its amplifying hosts. There are two sympatric lineages of BCRV (lineages A and B) that differ from each other by > 6% at the nucleotide level. Analysis of 385 BCRV isolates all collected from bug vectors at a study site in southwestern Nebraska, USA, showed that the lineages differed in their peak times of seasonal occurrence within a summer. Lineage A was more likely to be found at recently established colonies, at those in culverts (rather than on highway bridges), and at those with invasive House Sparrows, and in bugs on the outsides of nests. Genetic diversity of lineage A increased with bird colony size and at sites with House Sparrows, while that of lineage B decreased with colony size and was unaffected by House Sparrows. Lineage A was more cytopathic on mammalian cells than was lineage B. These two lineages have apparently diverged in their transmission dynamics, with lineage A possibly more dependent on birds and lineage B perhaps more a bug virus. The long-standing association between Cliff Swallows and BCRV may have selected for immunological resistance to the virus by swallows and thus promoted the evolution of the more bug-adapted lineage B. In contrast, the recent arrival of the introduced House Sparrow and its high competence as a BCRV amplifying host may be favoring the more bird-dependent lineage A.

No Evidence for Spring Re-introduction of an Arbovirus by Cliff Swallows

Abstract We sampled 100 Cliff Swallows (Petrochelidon pyrrhonota), just after arrival in Nebraska breeding areas, to ascertain if migrating birds re-introduce Buggy Creek virus (BCRV; Togaviridae) to north-temperate localities in spring. Most birds sampled were previously banded and were known to have used parasite-free nesting colonies in past summers and/or were seronegative to BCRV; thus, they were unlikely to have been previously exposed to the virus in their breeding areas. None of the birds had evidence of viral RNA in blood, as measured by RT-PCR. These results are consistent with other studies that have shown little evidence that migratory birds re-introduce arboviruses to temperate localities between years.

Persistence of Buggy Creek Virus (Togaviridae, Alphavirus) for Two Years in Unfed Swallow Bugs (Hemiptera: Cimicidae: Oeciacus vicarius)

ABSTRACT Alphaviruses (Togaviridae) have rarely been found to persist for long in the adult insects that serve as their vectors. The ectoparasitic swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius Horvath), the vector for Buggy Creek virus (BCRV; Togaviridae, Alphavirus), lives year-round in the mud nests of its host, the cliff swallow (Petrochelidon pyrrhonota Vieillot). We measured the prevalence of BCRV in swallow bugs at sites with cliff swallows present and at the same sites after cliff swallows had been absent for 2 yr. We collected bugs directly from cliff swallow nests in the field and screened bug pools with BCRV-specific real-time-polymerase chain reaction (RT-PCR) and plaque assay. At two colony sites last occupied by birds 2 yr earlier, we found 12.5 and 55.6% of bug pools positive for BCRV RNA by RT-PCR, Infection rates (per 1,000 bugs) for these sites were 1.32 and 7.39. RNA prevalence in the unfed bugs was not significantly different from that in fed bugs 2 yr earlier at the same sites. The RNA-positive samples from unfed bugs failed to yield cytopathic BCRV by Vero-cell plaque assay. However, viral RNA concentrations did not differ between unfed bugs and bugs at active sites, and over 84% of positive bug pools were cytopathic to Vero cells 4–5 wk later, after cliff swallows moved into one of the colony sites. These data demonstrate the persistence of potentially infectious BCRV in unfed swallow bugs for at least 2 yr in nature.

Prevalence of Buggy Creek virus (Togaviridae: Alphavirus) in insect vectors increases over time in the presence of an invasive avian host.

Invasive species can disrupt natural disease dynamics by altering pathogen transmission among native hosts and vectors. The relatively recent occupancy of cliff swallow (Petrochelidon pyrrhonota) nesting colonies in western Nebraska by introduced European house sparrows (Passer domesticus) has led to yearly increases in the prevalence of an endemic arbovirus, Buggy Creek virus (BCRV), in its native swallow bug (Oeciacus vicarius) vector at sites containing both the invasive sparrow host and the native swallow host. At sites without the invasive host, no long-term changes in prevalence have occurred. The percentage of BCRV isolates exhibiting cytopathicity in Vero-cell culture assays increased significantly with year at sites with sparrows but not at swallow-only sites, suggesting that the virus is becoming more virulent to vertebrates in the presence of the invasive host. Increased BCRV prevalence in bug vectors at mixed-species colonies may reflect high virus replication rates in house sparrow hosts, resulting in frequent virus transmission between sparrows and swallow bugs. This case represents a rare empirical example of a pathogen effectively switching to an invasive host, documented in the early phases of the hosts arrival in a specialized ecosystem and illustrating how an invasive species can promote long-term changes in host-parasite transmission dynamics.