Dynamic evolution in the key honey bee pathogen deformed wing virus: Novel insights into virulence and competition using reverse genetics.

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.

Simulated vector transmission differentially influences dynamics of two viral variants of deformed wing virus in honey bees (Apis mellifera).

Understanding how vectors alter the interactions between viruses and their hosts is a fundamental question in virology and disease ecology. In honey bees, transmission of deformed wing virus (DWV) by parasitic Varroa mites has been associated with elevated disease and host mortality, and Varroa transmission has been hypothesized to lead to increased viral titres or select for more virulent variants. Here, we mimicked Varroa transmission by serially passaging a mixed population of two DWV variants, A and B, by injection through in vitro reared honey bee pupae and tracking these viral populations through five passages. The DWV-A and DWV-B variant proportions shifted dynamically through passaging, with DWV-B outcompeting DWV-A after one passage, but levels of both variants becoming equivalent by Passage 5. Sequencing analysis revealed a dominant, recombinant DWV-B strain (DWV-A derived 5' IRES region with the rest of the genome DWV-B), with low nucleotide diversity that decreased through passaging. DWV-A populations had higher nucleotide diversity compared to DWV-B, but this also decreased through passaging. Selection signatures were found across functional regions of the DWV-A and DWV-B genomes, including amino acid mutations in the putative capsid protein region. Simulated vector transmission differentially impacted two closely related viral variants which could influence viral interactions with the host, demonstrating surprising plasticity in vector-host-viral dynamics.

Research paper on abiotic factors and their influence on Ixodes ricinus activity-observations over a two-year period at several tick collection sites in Germany.

Tick-borne diseases are a public health issue. To predict vector tick abundance and activity, it is necessary to understand the driving factors for these variables. In this study, the activity of Ixodes ricinus was investigated in forest and meadow habitats in Germany with a focus on abiotic factors. Ixodes ricinus adults, nymphs and larvae were caught by flagging over a period of 2 years. Microclimatic and weather conditions were recorded at the collection sites. Statistical models were applied to describe correlations between abiotic factors and tick activity in univariable and multivariable analyses. Tick activity was observed in a broad range of air temperature between 3 and 28 °C, and air humidity varied between 35 and 95%. In general, tick activity of nymphs and larvae was higher in forest habitats than that in meadows. With the exception of a single specimen of Dermacentor reticulatus, all ticks were Ixodes ricinus, most of them nymphs (63.2% in 2009 and 75.2% in 2010). For the latter, a negative binomial mixed-effects model fitted best to the observed parameters. The modelling results showed an activity optimum between 20 and 23 °C for air temperature and between 13 and 15 °C for ground temperature. In univariable analyses, the collection site, month, season, ground and air temperature were significant factors for the number of ticks caught and for all life stages. In the multivariable analysis, temperature, season and habitat turned out to be key drivers. Ixodes ricinus positive for RNA of tick-borne encephalitis virus was only found at a single sampling site. The results of this study can be used in risk assessments and to parameterise predictive models.

Questions surrounding the taxonomic validity of the species Garlic mite-borne filamentous virus (genus Allexivirus).

Garlic mite-borne filamentous virus is one of the oldest recognized allexivirus species but, paradoxically, one with the least well studied member viruses. In this paper, we review the history of this taxon and highlight problems in designating a holotype (exemplar isolate). Analyses are presented that suggest that GarMbFV is conspecific with Garlic virus A, and therefore the former taxon should be abolished.

Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.).

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees (Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher (P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

Bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker and its effects on predatory mites in citrus groves.

The acaricidal bioactivity of an oxymatrine-based commercial formulation against Brevipalpus yothersi Baker (Acari: Tenuipalpidae), a vector mite of the Citrus leprosis virus (CiLV), and its impact on predatory mites were assessed. For this purpose, laboratory and field assays using bioacaricide concentrations ranging from 0.5 to 2.0 mg L-1 of oxymatrine were performed during the years from 2015 to 2016. Laboratory results showed that the oxymatrine-based commercial formulation does not cause deleterious effects on B. yothersi eggs; however, it causes high larval mortality. For adult females, the bioacaricide caused high acute toxicity and residual effect for at least 5 days after application. In the field, the bioacaricide exhibited high acaricidal activity against B. yothersi, with efficacy levels similar to that of synthetic acaricide spirodiclofen (48 mg L-1) until 49 days after the application. The application of the bioacaricide did not negatively affect the population levels of phytoseiid predatory mites. Therefore, our results suggest that the oxymatrine-based commercial formulation is an important tool for management of the citrus leprosis mite in citrus groves.

Four Year Surveillance of the Vector Hard Ticks for SFTS, Ganghwa-do, Republic of Korea.

The seasonal abundance of hard ticks that transmit severe fever with thrombocytopenia syndrome virus was monitored with a collection trap method every April to November during 2015-2018 and with a flagging method every July and August during 2015-2018 in Ganghwa-do (island) of Incheon Metropolitan City, Republic of Korea. This monitoring was performed in a copse, a short grass field, coniferous forest and broad-leaved forest. A total of 17,457 ticks (8,277 larvae, 4,137 nymphs, 3,389 females, and 1,654 males) of the ixodid ticks comprising 3 species (Haemaphysalis longicornis, H. flava, and Ixodes nipponensis) were collected with collection traps. Of the identified ticks, H. longicornis was the most frequently collected ticks (except larval ticks) (94.26%, 8,653/9,180 ticks (nymphs and adults)), followed by H. flava (5.71%, 524/9,180) and Ix. nipponensis (less than 0.04%, 3/9,180). The ticks collected with collecting traps were pooled and assayed for the presence of SFTS virus with negative results. In addition, for monitoring the prevalence of hard ticks, a total of 7,461 ticks (5,529 larvae, 1,272 nymphs, 469 females, and 191 males) of the ixodid ticks comprising 3 species (H. longicornis, H. flava, and Ix. nipponensis) were collected with flagging method. H. longicornis was the highest collected ticks (except larval ticks) (99.53%, 1,908/1,917 ticks (nymphs and adults)), followed by H. flava (1.15%, 22/1,917).

[Confirmation of Tick-borne encephalitis virus in an European hedgehog (Erinaceus europaeus)]. / Frühsommer-Meningoenzephalitis-Virus Nachweis beim Europäischen Igel (Erinaceus europaeus).

INTRODUCTION: European hedgehogs (Erinaceus europaeus) have a high exposure to various ticks, which could transmit pathogens with direct health significance for the host and may have zoonotic potential. Tick-borne meningoencephalitis (FSME) is an important tick-borne disease in Switzerland, caused by the tick-borne encephalitis virus. About its occurrence in the European hedgehog population is little known. The present study examined various organs, blood and ticks of 65 European hedgehogs to obtain data of FSME virus presence in this species in Switzerland. Real-time RT-PCR from the lungs, liver, spleen and kidney of 56 hedgehogs and of 114 infesting ticks (Ixodes hexagonus or Ixodes ricinus) were used for the detection of viral RNA. In addition, 19 blood samples were tested for antibodies against FSME by ELISA. FSME virus antibodies were detected for the first time in the serum of a European hedgehog. Lung and spleen tissue samples of the same animal tested also weak virus positive on RT-PCR. Clinically, the hedgehog showed neurological symptoms, although these symptoms could have originated from an other diseases. No viral RNA was detected in any of the ticks. This study could not confirm if the meningoencephalitis in the hedgehog was triggered by the FSME viral infection. Nevertheless, the simultaneous detection of antibodies and virus RNA in the same animal makes the European hedgehog a competent host of the tick-borne encephalitis virus and leads to the assumption that this species can act as a reservoir.

INTRODUCTION: En raison du nombre élevé de tiques présents chez les hérissons d'Europe (Erinaceus europaeus), ces animaux sont fortement exposés aux différents pathogènes qu'ils transmettent, pathogènes qui, en plus de l'importance directe pour la santé de l'hôte, peuvent aussi avoir un potentiel en termes de zoonose. La méningo-encéphalite à tique est, en Suisse, une maladie importante transmise par les tiques. Elle est causée par le virus de la méningo-encéphalite verno-estivale. Son occurrence chez les hérissons d'Europe est jusqu'à maintenant peu connue. Au travers de l'étude des organes, du sang et des tiques provenant de 65 hérissons européens, il devrait pour la première fois être possible de se prononcer sur la présence du virus chez cette espèce en Suisse. La détection de l'ARN viral a été effectuée au moyen d'une RT-PCR en temps réel sur les poumons, le foie, la rate et les reins de 56 hérissons ainsi que sur un total de 114 tiques dont ils étaient porteurs, appartenant aux espèces Ixodes hexagonus ou Ixodes ricinus. En outre, 19 échantillons de sang ont été testés par ELISA pour des anticorps contre le virus. Dans la présente étude, des anticorps contre le virus de l'encéphalite à tiques dans le sérum d'un hérisson européen ont pu être détectés pour la première fois. Les échantillons de poumon et de rate du même animal ont également montré une faible présence virale. Le même hérisson a présenté des symptômes neurologiques, mais ceux-ci pouvaient également être associés à d'autres maladies. On n'a démontré la présence d'ARN viral chez aucune tique. La possibilité d'une encéphalite causée par l'infection virale chez les hérissons ne peut pas être confirmée ou exclues avec cette étude. La détection simultanée des anticorps et de l'ARN viral chez le même animal fait du hérisson européen non seulement un hôte compétent du virus de l'encéphalite verno-estivale mais donne également également à penser que cette espèce pourrait servir de réservoir.

Transmission of Severe Fever with Thrombocytopenia Syndrome Virus by Haemaphysalis longicornis Ticks, China.

We demonstrate maintenance and transmission of severe fever with thrombocytopenia syndrome virus by Haemaphysalis longicornis ticks in the larva, nymph, and adult stages with dissemination in salivary gland, midgut, and ovarian tissues. The H. longicornis tick is a competent vector to transmit this virus in both transovarial and transstadial modes.

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants.

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Case report: detection of the identical virus in a patient presenting with severe fever with thrombocytopenia syndrome encephalopathy and the tick that bit her.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease. Haemophysalis longicornis ticks have been considered the vector of severe fever with thrombocytopenia syndrome virus (SFTSV). However, clear data on the transmission of SFTS from ticks to humans are limited. CASE PRESENTATION: We report an 84-year-old woman who presented with fever and altered mentality, which was confirmed as SFTS with encephalopathy by reverse-transcription polymerase chain reaction in blood and cerebrospinal fluid. The SFTSV was also identified in the tick that bit her, H. longicornis. Phylogenetic analyses indicated that the SFTSV from the patient and the tick was identical. The patient gradually recovered with treatments of corticosteroids and immunoglobulin. CONCLUSION: These findings provide further evidence of SFTS viral transmission from H. longicornis to human.

Prevalence and spatial distribution of Ixodid tick populations in the forest fringes of Western Ghats reported with human cases of Kyasanur forest disease and monkey deaths in South India.

Kyasanur forest disease (KFD) is a major tick-borne viral haemorrhagic fever caused by KFD virus (KFDV) (Flaviviridae). The disease was reported to be confined to five districts of Karnataka state India until 2011. During 2012-2016, emergence of KFD has been reported in newer areas of Karnataka and adjoining states. Therefore, survey of tick vectors was carried out in these new areas of Karnataka and adjoining states reported with monkey deaths and human cases of KFD. In all selected sites, ticks from the forest floor were collected by lint clothes using flagging method. Tick samples were tested for KFDV nucleic acid by real-time RT-PCR. A total of 4772 ticks, comprising eight species of genus Haemaphysalis and one species each of genus Amblyomma, Ixodes and Rhipicephalus was collected. Haemaphysalis spinigera, the principal vector of KFDV was the predominant tick species (59.5%) collected followed by H. turturis (8.6%). The abundance of H. spinigera ranged from 9.2 to 33.9 per man-hour in the six districts surveyed. Of 214 (4418 tick samples) pools screened by real-time RT-PCR, two pools of H. spinigera were positive for KFDV. High abundance of Haemaphysalis vectors in the six districts indicated that the districts are receptive for KFD outbreaks. KFDV was detected in the tick vectors in the new foci of the KFD. Data on tick distribution will be useful in creating KFD risk map for strengthening the ongoing preventive measures such as vaccination and supply of insect repellents to the high risk groups and intensive health education.

Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus, Spain.

Two cases of Crimean-Congo hemorrhagic fever were reported in Spain during 2016. We obtained the virus from a patient sample and characterized its full genomic sequence. Phylogenetic analysis indicated that the virus corresponds to the African genotype III, which includes viruses previously found in West and South Africa.

Bourbon Virus in Field-Collected Ticks, Missouri, USA.

Bourbon virus (BRBV) was first isolated in 2014 from a resident of Bourbon County, Kansas, USA, who died of the infection. In 2015, an ill Payne County, Oklahoma, resident tested positive for antibodies to BRBV, before fully recovering. We retrospectively tested for BRBV in 39,096 ticks from northwestern Missouri, located 240 km from Bourbon County, Kansas. We detected BRBV in 3 pools of Amblyomma americanum (L.) ticks: 1 pool of male adults and 2 pools of nymphs. Detection of BRBV in A. americanum, a species that is aggressive, feeds on humans, and is abundant in Kansas and Oklahoma, supports the premise that A. americanum is a vector of BRBV to humans. BRBV has not been detected in nonhuman vertebrates, and its natural history remains largely unknown.

Tick-Borne Encephalitis Virus in Ticks and Roe Deer, the Netherlands.

We report the presence of tick-borne encephalitis virus (TBEV) in the Netherlands. Serologic screening of roe deer found TBEV-neutralizing antibodies with a seroprevalence of 2%, and TBEV RNA was detected in 2 ticks from the same location. Enhanced surveillance and awareness among medical professionals has led to the identification of autochthonous cases.

Travel-Related Tick-Borne Encephalitis, Israel, 2006-2014.

During 2006-2014, four tick-borne encephalitis (TBE) cases occurred among Israeli travelers. We calculated TBE incidence at 321.0, 45.0, 13.2, and 7.5 cases/100,000 travelers/year of travel to Sweden, Switzerland, Austria, and Germany, respectively. TBE incidence among travelers to these destinations appears to justify TBE vaccination in accordance with World Health Organization recommendations.

Tick-Borne Encephalitis in Sheep, Romania.

Little is known about the occurrence of tick-borne encephalitis in Romania. Sheep are an infection source for humans and are useful sentinels for risk analysis. We demonstrate high antibody prevalence (15.02%) among sheep used as sentinels for this disease in 80% of the tested localities in 5 counties of northwestern Romania.

Genetic characterization of Cacipacoré virus from ticks collected in São Paulo State, Brazil.

Cacipacoré virus (CPCV) is a potential emerging virus classified in the genus Flavivirus, family Flaviviridae. In the present study, we present the genetic characterization of a CPCV isolated from ticks (Amblyomma cajennense) collected from a sick capybara (Hydrochoerus hydrochaeris) in São Paulo State, Brazil. The CPCV isolate shares the typical genomic organization of flaviviruses with 10,857 nucleotides in length and a single open reading frame of 10,284 nucleotides encoding a polyprotein of 3,427 amino acids. Phylogenetic analysis revealed that CPCV is unique, as a potentially tick-borne virus, in the Japanese encephalitis virus serogroup.

Molecular (ticks) and serological (humans) study of Crimean-Congo hemorrhagic fever virus in the Iberian Peninsula, 2013-2015.

INTRODUCTION: Crimean-Congo hemorrhagic fever (CCHF) is a viral disease, mainly transmitted through tick bite, of great importance in Public Health. In Spain, Crimean-Congo hemorrhagic fever virus (CCHFV) was detected for the first time in 2010 in Hyalomma lusitanicum ticks collected from deer in Cáceres. The aim of this study was to investigate the presence of CCHFV in ticks from Cáceres, and from other Spanish areas, and to evaluate the presence of antibodies against the virus in individuals exposed to tick bites. METHODS: A total of 2053 ticks (1333 Hyalomma marginatum, 680 H. lusitanicum and 40 Rhipicephalus bursa) were analyzed using molecular biology techniques (PCR) for CCHFV detection. The determination of specific IgG antibodies against CCHFV in 228 serum samples from humans with regular contact with ticks (at risk of acquiring the infection) was performed by indirect immunofluorescence assay. RESULTS: The CCHFV was not amplified in ticks, nor were antibodies against the virus found in the serum samples analyzed. CONCLUSION: The absence of the CCHFV in the ticks studied and the lack of antibodies against the virus in individuals exposed to tick bites would seem to suggest a low risk of acquisition of human infection by CCHFV in Spain.

Tick-borne viruses.

Tick-borne viruses (TBVs) belong to the largest biological group known as arboviruses with unique mode of transmission by blood-feeding arthropods (ticks, mosquitoes, sand flies, biting midges, etc.) to a susceptible vertebrate host. Taxonomically, it is a heterogenous group of vertebrate viruses found in several viral families. With only one exception, African swine fever virus, all TBVs have a RNA genome. To date, at least 160 tick-borne viruses are known, some of them pose a significant threat to human and animal health worldwide. Recently, a number of established TBVs has re-emerged and spread to new geographic locations due to the influence of anthropogenic activities and few available vaccines. Moreover, new emerging tick-borne diseases are constantly being reported. Major advances in molecular biotechnologies have led to discoveries of new TBVs and further genetic characterization of unclassified viruses resulting in changes in TBVs classification created by the International Committee for the Taxonomy of Viruses. Although TBVs spend over 95% of their life cycle within tick vectors and the role of ticks as vectors has been known for over 100 years, our knowledge about TBVs and molecular processes involved in the virus-tick interactions is scarce.