Genomic stability of La Crosse virus during vertical and horizontal transmission

Summary We have used ribonuclease T 1 oligonucleotide fingerprint analysis to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following ingestion and replication) from the natural host,Aedes triseriatus. Genomic changes were not detected during transovarial passage of the virus through two generations of mosquitoes, nor were changes detected in the genomes of virus isolated from suckling mice that had been fed upon by second generation transovarially-infected mosquitoes. These results demonstrate that despite the well-documented phenomena of rapid nucleotide change in RNA virus genomes under various conditions, the La Crosse virus genome can remain stable during transovarial transmission in the insect host and during transfer between the insect and vertebrate hosts. The evolutionary implications of these results are discussed.     

Persistent West Nile virus infection in the house sparrow (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

Long-term persistence of West Nile virus (WNV) infection within vertebrate reservoir hosts is a potential mechanism for overwintering of this (and other) arbovirus(es) at temperate latitudes. The house sparrow (Passer domesticus), an established amplifying host for WNV and other arboviruses, was used as a model to confirm chronicity of WNV infection in passerine birds and to evaluate the feasibility of two overwintering mechanisms: blood-borne infection of arthropod vectors (recrudescence) and oral infection of vertebrate reservoir hosts (ingestion of infected tissues through predation). WNV-inoculated sparrows were monitored for persistent infection for up to 2 years. Infectious virus persisted in tissues through 43 days, but not in sera beyond 6 days. Viral RNA persisted in tissues through 65 days. Chronicity of WNV infection in some tissues, but not blood, supports the predation mechanism of WNV overwintering, but not recrudescence. RNA persistence impacts interpretation and etiologic determination of avian mortality.     

A novel genotype of beak and feather disease virus in budgerigars (<Emphasis Type="Italic">Melopsittacus undulatus</Emphasis>)

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence ¹¹²R/K-R-Q-K/R-R-F¹¹⁷ in the F₀ cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.     

Experimental exposure of swallow bugs (Hemiptera: Cimicidae) to West Nile virus

The cliff swallow (Petrochelidon pyrrhonota, Vieillot) could potentially play an important role in the transmission of West Nile virus (WNV) because of its breeding ecology, reservoir competence status, and potentially high natural exposure rates. These birds nest in colonies and their nests are occupied year round by swallow bugs (Oeciacus vicarius, Hovarth), hematophagus ectoparasites that feed primarily on cliff swallows. Swallow bugs are most likely exposed to WNV while feeding on infectious blood of cliff swallows and, thus, if competent vectors, could contribute to initiation and maintenance of seasonal WNV transmission. In addition, swallow bugs remain within nests year round and, therefore, if persistently infected and competent vectors, they could provide an overwintering mechanism for WNV. We tested the hypothesis that swallow bugs become infected with WNV through direct abdominal inoculation or ingestion of infectious blood meals. We observed that swallow bugs did not maintain or amplify WNV, and infectious virus titers within bugs declined over 15 d postexposure. These results suggest that swallow bugs may not be competent vectors of WNV, and therefore are unlikely to play a significant role in transmission.     

Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran

Neospora caninum is an intracellular protozoan parasite with a wide range of intermediate bird hosts. There is little information describing the prevalence and genetic characterization of N. caninum in bird hosts worldwide and in Iran. In this study, a total of 217 brain samples of house sparrow (Passer domesticus) were examined for N. caninum presence by nested polymerase chain reaction targeting the Nc-5 gene. N. caninum DNA was detected in 3.68% (8/217) of sparrows. Sequencing of the Nc5 genomic DNA revealed 97–99% of similarity with N. caninum sequences deposited in Genbank. To our knowledge, this study is the first molecular evidence of N. caninum DNA in bird hosts in Iran. The results of this study highlight the role of the house sparrow (Passer domesticus) in maintaining and spreading N. caninum infection to canines in the feral and domestic environment.     

Comparative infectiousness of three passerine bird species after experimental inoculation with Mycoplasma gallisepticum

Mycoplasma gallisepticum has been isolated from various species of free-living birds, and we therefore tested the hypothesis that bird species other than the main host, the house finch (Carpodacus mexicanus), could play a role in the epidemiology of the infection. We compared the disease course in the house finch, American goldfinch (Carduelis tristis) and house sparrow (Passer domesticus) after inoculation into the conjunctival sac with M. gallisepticum, and also the degree to which the three species were infectious to other naïve house finches. Severity of clinical signs was least in house sparrows, intermediate in American goldfinch and the highest in house finch. House sparrows were only mildly infectious to naïve house finches for a short time, whereas American goldfinches remained infectious for up to 49 days post inoculation, although by then there were no physical signs of disease. We conclude that since American goldfinches can be infectious without showing any conjunctivitis, and since they often make long-distance movements, they might play an as yet unsuspected but important role in M. gallisepticum dynamics in house finches.     

Occurrence of haemosporidian parasites in the paddyfield warbler, <Emphasis Type="Italic">Acrocephalus agricola</Emphasis> (Passeriformes,Sylviidae)

The blood parasite diversity was studied in paddyfield warblers (Acrocephalus agricola) breeding in NE Bulgaria, SW Russia and S. Kazakhstan. Nine cytochrome b gene lineages were recorded, 4 belonging to Haemoproteus spp. and 5 to Plasmodium spp. The overall prevalence of haemosporidians was 33.3%. The composition of parasites varied geographically, with six lineages recorded in Russia, five lineages in Bulgaria and two lineages in Kazakhstan. Two lineages are described for the first time, i.e. ACAGR1 (belonging to Plasmodium sp. and recorded from a single bird in Russia) and ACAGR2 (belonging to Haemoproteus sp., recorded from Bulgaria and Russia). The latter lineage is the most widespread parasite in the Bulgarian population, scarce in Russia and absent in Kazakhstan. It is supposed that ACAGR2 has originated from the widespread lineage ACDUM1 differing from it by a single nucleotide. One lineage only (ACDUM2) occurs in all the three populations studied and is a nonspecific parasite known from various passerines. Six of the registered lineages have been found in a single population of A. agricola and also represent non-specific parasites occurring in a wide range of passerine birds. Their records in A. agricola may indicate the high transmission rate of these parasites in the habitats where this host co-occurs with other passerines. The variation of the composition of the haemosporidian parasite communities through the breeding range of A. agricola makes up heterogeneous selection pressures that may drive intraspecific variation in important life-history traits.     

Shedding and serologic responses following primary and secondary inoculation of house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with low-pathogenicity avian influenza virus

Waterfowl and shorebirds are well-recognized natural reservoirs of low-pathogenicity avian influenza viruses (LPAIV); however, little is known about the role of passerines in avian influenza virus ecology. Passerines are abundant, widespread, and commonly come into contact with free-ranging birds as well as captive game birds and poultry. We inoculated and subsequently challenged house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with wild-bird origin LPAIV H3N8 to evaluate their potential role in transmission. Oropharyngeal shedding was short lived, and was detected in more starlings (97.2%) than sparrows (47.2%; n=36 of each). Cloacal shedding was rare in both species (8.3%; n=36 of each) and no cage-mate transmission occurred. Infectious LPAIV was cultured from oropharyngeal and cloacal swabs and gastrointestinal and respiratory tissues from both species. Seroconversion was detected as early as 3 days post inoculation (d.p.i.) (16.7% of sparrows and 0% of starlings; n=6 each); 50% of these individuals seroconverted by 5 d.p.i., and nearly all birds (97%; n=35) seroconverted by 28 d.p.i. In general, pre-existing homologous immunity led to reduced shedding and increased antibody levels within 7 days of challenge. Limited shedding and lack of cage-mate transmission suggest that passerines are not significant reservoirs of LPAIV, although species differences apparently exist. Passerines readily and consistently seroconverted to LPAIV, and therefore inclusion of passerines in epidemiological studies of influenza outbreaks in wildlife and domestic animals may provide further insight into the potential involvement of passerines in avian influenza virus transmission ecology.     

Unravelling the evolution of the head lice and body lice of humans

Recent studies of mitochondrial genes of the head and body lice of humans indicate that present-day lice comprise two lineages that diverged before the evolution of modern humans. To test if this was a locus-specific phenomenon, we studied two nuclear genes, elongation factor-1α (EF-1α) and small subunit ribosomal RNA (ssu rRNA). Our ssu rRNA phylogeny was concordant with the phylogenies from mitochondrial genes, but the EF-1α phylogeny was not concordant either with the mitochondrial phylogenies or with the ssu rRNA phylogeny. So both nuclear (ssu rRNA) and mitochondrial data indicate that there are two lineages of lice: one lineage with head lice only (H-only lineage) the other lineage with head and body lice (H+B lineage). Thus, body lice apparently evolved from just one of the two main lineages of lice. However, the date of divergence and geographical origins of the two lineages are controversial. Kittler et al. (Curr Biol 13:1414–1417, 2003; Curr Biol 14:2309, 2004) proposed that these two lineages diverged 0.77 mya, whereas Reed et al. (PLoS Biol 2:e340, 2004) proposed that they diverged 1.18 mya and suggested that one of the lineages, the H-only lineage, evolved in the New World on Homo erectus. We discuss this hypothesis in light of our results from ssu rRNA.Electronic Supplementary Material Supplementary material is available for this article at     

Haemosporidian parasites of a European passerine wintering in South Asia: diversity, mixed infections and effect on host condition

We studied haemosporidian parasites in the scarlet rosefinch Carpodacus erythrinus in a small isolated semicolony during an eight-year period using molecular methods of parasite detection. The scarlet rosefinch is an interesting model of parasite host species. It winters in South Asia which represents a rare exception among European passerines. Males express yellow to red carotenoid-based plumage ornament which is a good predictor of male reproductive success. In 240 blood samples originating from 199 adult individuals, the total parasite prevalence reached 60 %. Prevalence varied among years from 36 to 81 % in Haemoproteus, 8 to 22 % in Plasmodium, and 0 to 14 % in Leucocytozoon. Twenty parasite lineages were detected (Haemoproteus: 5 lineages, Plasmodium: 10 lineages, and Leucocytozoon: 5 lineages). Among them, the Haemoproteus ROFI2 lineage, which is a host-specific parasite lineage of the scarlet rosefinch, was the most frequently found. Parasite lineages showed varying degree of lineage specificity. While Haemoproteus lineages detected in the scarlet rosefinch have relatively narrow host breadth restricted mainly to Fringillidae family, Leucocytozoon and Plasmodium lineages generally showed wider host range. The presence of some parasite lineages hitherto detected in sedentary European passerines (SISKIN1, CCF3, BT2) or in Culicoides biting midges at the same locality (ROFI1) suggest local transmission. On the contrary, lineages LK05 and FANTAIL1 that were previously reported exclusively from Asian hosts imply parasite transmission at the scarlet rosefinch wintering sites in South Asia. Mixed infections were found in 17 % of infected samples and comprised mainly the most frequent lineages. The pattern of concomitant infections seemed to be rather random and matched expected levels based on lineage frequencies. Between-year comparisons revealed that in a majority of the repeatedly captured individual hosts the infection status remained unchanged (individuals stayed uninfected or possessed the same parasite lineages). However, 16 gains and 8 losses of lineages were also reported. We have not found any effect of haemosporidians on male carotenoid ornament expression or host body mass.     

Virulence genes,antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain

The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified: one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons.     

Genetic analysis of hantaviruses and their rodent hosts in central-south China

Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs.     

Phylogeny of Thogoto Virus

Thogoto virus is a tick-borne member of the family Orthomyxoviridae. Previously, based on the similarity in antigenic relationship by cross-neutralization test, all virus strains were concluded to have derived from the same origin. In this study, we obtained partial gene sequences of 4 genes (PB1-like protein, PA-like protein, glycoprotein, and nucleoprotein) of 8 Thogoto virus strains isolated in Africa, Asia, and Europe and studied the genetic variation and phylogeny. Unrooted phylogenetic trees created by both neighbor-joining and maximum likelihood methods based on nucleotide and amino acid sequences for 4 genes were mostly similar and revealed two lineages, Euro-Asian and African. Intra-lineage nucleotide sequence variation was greater in the Euro-Asian lineage than in the African lineage for all 4 genes. Furthermore, for the strains of Euro-Asian lineage, variations for two genes associated with RNA-dependent RNA polymerase activities were greater than those for glycoprotein or nucleoprotein gene, based on both nucleotide and amino acid sequence differences as well as on synonymous and nonsynonymous differences, indicating greater mutation rates for the polymerase activity genes in these strains.     

Molecular identification of bloodmeals from biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark

Culicoides vectors are critical to the survival and transmission of bluetongue virus as infection only occurs in areas or regions where competent vectors are present. The success of Culicoides biting midges as vectors is mainly related to their vast population sizes and to their means of dispersal. Their choice of host for blood feeding is sparsely described. The aim of the present study was to establish methods for the identification of bloodmeal hosts and determine the identity and diversity of bloodmeals of vertebrate hosts from wild-caught biting midges near livestock farms. The study includes some of the most common and abundant species of biting midges in Denmark: Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus. We collected 8,378 biting midges including nine species of Culicoides of which blood-fed specimens were found from six species. We identified 251 blood engorged biting midges, and hosts were identified in 115 of 125 analysed specimens (90%). Cow, roe deer, horse, mallard and wood pigeon were identified as hosts. The most abundant host species was cow, which constituted 73.9% of the total identified bloodmeals, but the common wood pigeon was found with a frequency as high as 18.3%. In conclusion, the molecular methods applied were proven useful in identifying bloodmeal hosts from different Culicoides species. The results indicate that Culicoides species are opportunistic in their choice of bloodmeal host with a preference for cattle when present, which is important to have in mind for epidemiologist when making predictive models. Accordingly, the results of this study will add useful parameters for modelling bluetongue virus transmission and in the development of veterinary contingency plans.     

Homogeneity of Powassan virus populations in naturally infected Ixodes scapularis

Powassan virus (POWV, Flaviviridae: Flavivirus) is the sole North American member of the tick-borne encephalitis complex and consists of two distinct lineages that are maintained in ecologically discrete enzootic transmission cycles. The underlying genetic mechanisms that lead to niche partitioning in arboviruses are poorly understood. Therefore, intra- and interhost genetic diversity was analyzed to determine if POWV exists as a quasispecies in nature and quantify selective pressures within and between hosts. In contrast to previous reports for West Nile virus (WNV), significant intrahost genetic diversity was not observed. However, pN (0.238) and d_N/d_S ratios (0.092) for interhost diversity were similar to those of WNV. Combined, these data suggest that purifying selection and/or population bottlenecks constrain quasispecies diversity within ticks. These same selective and stochastic mechanisms appear to drive minor sequence changes between ticks. Moreover, Powassan virus populations seem not to be structured as quasispecies in naturally infected adult deer ticks.     

Genetic diversity of avian blood parasites in SE Europe: Cytochrome <Emphasis Type="Italic">b</Emphasis> lineages of the genera <Emphasis Type="Italic">Plasmodium</Emphasis> and <Emphasis Type="Italic">Haemoproteus</Emphasis> (Haemosporida) from Bulgaria

We used a nested PCR protocol to examine the genetic diversity of cytochrome b (cyt b) lineages from blood parasites of the genera Plasmodium and Haemoproteus in birds in Bulgaria. In total, 460 birds of 43 species and 14 families (mostly passerines) were examined for the presence of infections. Of them, 267 were recognised as infected with haemosporidian parasites. Mixed infections were recorded in 24 individuals (9%). Besides the 24 individuals with mix infections, 114 (43%) were positive for Plasmodium spp. and 129 (48%) for Haemoproteus spp. We identified 52 genetic lineages of haemosporidian parasites: 38 of Haemoproteus and 14 of Plasmodium. Twelve new cyt b lineages of Haemoproteus were recorded; they occurred in the following hosts: grey-faced woodpecker (Picus canus), golden oriole (Oriolus oriolus), jay (Garrulus glandarius), barred warbler (Sylvia nisoria), song thrush (Turdus philomelos), spotted flycatcher (Muscicapa striata), spanish sparrow (Passer hispaniolensis), hawfinch (Coccothraustes coccothraustes), and cirl bunting (Emberiza cirlus). We also detected 22 new host records for previously known lineages. The most common lineage was SGS1 (Plasmodium relictum), which had a total prevalence of 14% and occurred in 8 host species belonging to 5 families. Three of the cyt b lineages of genus Haemoproteus (DURB1, DURB2 and SYNIS2) showed more than 5% divergence from all described morphologically lineages. These lineages probably represent at least 2 different morphospecies which remains to be identified.     

Trypanosoma cruzi-triatomine associations and the presence of mixed infections in single triatomine bugs in Paraná state, Brazil

Eighteen strains of Trypanosoma cruzi isolated from two species of triatomines in the state of Paraná, Brazil, were characterized molecularly using three strategies: RAPD (randomly amplified polymorphic DNA) with four primers, analysis of the D7 polymorphic region of the 24Sα rDNA, and RFLP (restriction fragment length polymorphism) of region 5′ of the mitochondrial gene COII (cytochrome oxidase subunit 2). The phenogram constructed with the RAPD data showed that only three strains isolated from Panstrongylus megistus collected in the Municipality of Arapongas were grouped together in a sub-branch. None of the other 15 strains could be clustered according to triatomine species or geographical origin. The strains were grouped with the T. cruzi I reference sample, indicating closer association with the sylvatic transmission cycle of T. cruzi in the state of Paraná. However, analyses of the rDNA and COII gene polymorphisms revealed the presence of populations from both T. cruzi I and II major lineages. In half of the analyzed triatomines, we found parasites from both lineages coinfecting the same bugs. Of these, most (6/9) were isolated from Triatoma sordida, and 3/9 from Panstrongylus megistus. These results contribute to a better comprehension of the ecoepidemiology of Chagas’ disease in Paraná, and raise questions about the role of studies of polyclonal population dynamics for controlling the transmission of T. cruzi to humans in this region.     

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, <Emphasis Type="Italic">Falco naumanni</Emphasis>

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.     

Polymerase chain reaction-based identification of <Emphasis Type="Italic">Plasmodium</Emphasis> (<Emphasis Type="Italic">Huffia</Emphasis>) <Emphasis Type="Italic">elongatum</Emphasis>, with remarks on species identity of haemosporidian lineages deposited in GenBank

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.