Screening and identification of persistent viruses in cell lines derived from medically important arthropods.

Arthropod-derived cell lines serve as crucial tools for studying arthropod-borne viruses (arboviruses). However, it has recently come to light that certain cell lines harbor persistent infections of arthropod-specific viruses, which do not cause any apparent cytopathic effects. Moreover, some of these persistent viral infections either inhibit or promote the growth of arboviruses. Therefore, it is of utmost importance to identify the presence of such persistent viruses and understand their impact on arboviral infections. In this study, we conducted a comprehensive virome analysis of several arthropod-derived cell lines, including mosquito-derived NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, CCL-126 cells, and tick-derived IDE8 cells, along with flesh fly-derived NIH-Sape-4 cells. The aim was to determine if these cells were infected with persistent viruses. The results revealed the presence of 15 persistent viruses in NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, and IDE8 cells. Among these, 11 were already known arthropod-specific viruses, while the remaining 4 were novel viruses belonging to Orthophasmavirus, Rhabdoviridae, Totiviridae, and Bunyavirales. In contrast, CCL-126 and NIH-Sape-4 cells appeared to be free of viral infections. This study provides valuable insights into the diversity and latency of arthropod-specific viruses within arthropod-derived cell lines. Further investigations are required to explore persistent viral infections in other arthropod-derived cell cultures and their effects on arbovirus replication. Understanding these factors will enhance the accuracy and reliability of experimental data obtained using these cell lines.

First detection of Lake Sinai virus in honeybees (Apis mellifera) and wild arthropods in Japan.

Lake Sinai virus (LSV), an RNA virus, is suspected to be associated with poor health in honeybees (Apis mellifera). We examined LSV in 26 specimens of healthy honeybees and 44 specimens of wild arthropods in the Gifu Prefecture, Japan. LSV was found more frequently in honeybee specimens (11/26, 42.3%) than in wild arthropod specimens (1/44, 2.3%) (P<0.01). Phylogenetic and nucleotide sequence analysis revealed two lineages: LSV3 in honeybees, and LSV4 in both honeybees and wild arthropods. To our knowledge, this is the first report of LSV prevalence in honeybees and wild arthropods in Japan.

Virome of Bat-Infesting Arthropods: Highly Divergent Viruses in Different Vectors.

Bats are reservoirs of important zoonotic viruses like Nipah and SARS viruses. However, whether the blood-sucking arthropods on the body surface of bats also carry these viruses and the relationship between viruses carried by the blood-sucking arthropods and viruses carried by bats have not been reported. This study collected 686 blood-sucking arthropods on the body surface of bats from Yunnan Province, China, between 2012 and 2015, and they included wingless bat flies, bat flies, ticks, mites, and fleas. The viruses carried by these arthropods were analyzed using a meta-transcriptomic approach, and 144 highly diverse positive-sense single-stranded RNA, negative-sense single-stranded RNA, and double-stranded RNA viruses were found, of which 138 were potentially new viruses. These viruses were classified into 14 different virus families or orders, including Bunyavirales, Mononegavirales, Reoviridae, and Picornavirales. Further analyses found that Bunyavirales were the most abundant virus group (84% of total virus RNA) in ticks, whereas narnaviruses were the most abundant (52 to 92%) in the bat flies and wingless bat flies libraries, followed by solemoviruses (1 to 29%) and reoviruses (0 to 43%). These viruses were highly structured based on the arthropod types. It is worth noting that no bat-borne zoonotic viruses were found in the virome of bat-infesting arthropod, seemingly not supporting that bat surface arthropods are vectors of zoonotic viruses carried by bats. IMPORTANCE Bats are reservoirs of many important viral pathogens. To evaluate whether bat-parasitic blood-sucking arthropods participate in the circulation of these important viruses, it is necessary to conduct unbiased virome studies on these arthropods. We evaluated five types of blood-sucking parasitic arthropods on the surface of bats in Yunnan, China, and identified a variety of viruses, some of which had high prevalence and abundance levels, although there is limited overlap in virome between distant arthropods. While most of the virome discovered here is potentially arthropod-specific viruses, we identified three possible arboviruses, including one orthobunyavirus and two vesiculoviruses (family Rhabdoviridae), suggesting bat-parasitic arthropods carry viruses with risk of spillage, which warrants further study.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Citizen science data opens multiple avenues for iridovirus research and prompts first detection of Invertebrate iridescent virus 31 in Australia.

From citizen science data we report the first records of blue to violet-colored oniscideans (Oniscidea: Isopoda), indicating potential invertebrate iridescent virus (IIV; Betairidovirinae: Iridoviridae) infection: in Africa, South America, and Oceania; and of the new hosts Armadillidium nasatum and Balloniscus sellowii. DNA sequencing of indigo Porcellio scaber confirms the presence of Invertebrate iridescent virus 31 in Australia. Beyond the Oniscidea, new, putative IIV hosts are identified: hoverfly pupae (Eristalinae), a tortrix moth larva (Phaecasiophora niveiguttana), and a millipede (Harpaphe haydeniana). In addition, the purported positive correlation between virion diameter and wavelength of iridescence is analyzed qualitatively for the first time.

Persistent Joint Pain Following Arthropod Virus Infections.

PURPOSE OF REVIEW: Persistent joint pain is a common manifestation of arthropod-borne viral infections and can cause long-term disability. We review the epidemiology, pathophysiology, diagnosis, and management of arthritogenic alphavirus infection. RECENT FINDINGS: The global re-emergence of alphaviral outbreaks has led to an increase in virus-induced arthralgia and arthritis. Alphaviruses, including Chikungunya, O'nyong'nyong, Sindbis, Barmah Forest, Ross River, and Mayaro viruses, are associated with acute and/or chronic rheumatic symptoms. Identification of Mxra8 as a viral entry receptor in the alphaviral replication pathway creates opportunities for treatment and prevention. Recent evidence suggesting virus does not persist in synovial fluid during chronic chikungunya infection indicates that immunomodulators may be given safely. The etiology of persistent joint pain after alphavirus infection is still poorly understood. New diagnostic tools along and evidence-based treatment could significantly improve morbidity and long-term disability.

Retrospective evaluation of vector-borne pathogens in cats living in Germany (2012-2020).

BACKGROUND: Blood-feeding arthropods can transmit parasitic, bacterial, or viral pathogens to domestic animals and wildlife. Vector-borne infections are gaining significance because of increasing travel and import of pets from abroad as well as the changing climate in Europe. The main objective of this study was to assess the percentage of cats with positive test results for selected vector-borne pathogens in Germany and explore any possible association of such results with time spent abroad. METHODS: This retrospective study included test results from cats included in the "Feline Travel Profile" established by the LABOKLIN laboratory at the request of veterinarians in Germany between April 2012 and March 2020. This diagnostic panel includes the direct detection of Hepatozoon spp. and Dirofilaria spp. via PCR as well as indirect detection assays (IFAT) for Ehrlichia spp. and Leishmania spp. The panel was expanded to include an IFAT for Rickettsia spp. from July 2015 onwards. RESULTS: A total of 624 cats were tested using the "Feline Travel Profile." Serum for indirect detection assays was available for all 624 cats; EDTA samples for direct detection methods were available from 618 cats. Positive test results were as follows: Ehrlichia spp. IFAT 73 out of 624 (12%), Leishmania spp. IFAT 22 out of 624 (4%), Hepatozoon spp. PCR 53 out of 618 (9%), Dirofilaria spp. PCR 1 out of 618 cats (0.2%), and Rickettsia spp. IFAT 52 out of 467 cats (11%) tested from July 2015 onwards. Three cats had positive test results for more than one pathogen before 2015. After testing for Rickettsia spp. was included in 2015, 19 cats had positive test results for more than one pathogen (Rickettsia spp. were involved in 14 out of these 19 cats). CONCLUSIONS: At least one pathogen could be detected in 175 out of 624 cats (28%) via indirect and/or direct detection methods. Four percent had positive test results for more than one pathogen. These data emphasize the importance of considering the above-mentioned vector-borne infections as potential differential diagnoses in clinically symptomatic cats.

Depicting the RNA Virome of Hematophagous Arthropods from Belgrade, Serbia.

Hematophagous arthropods are important vectors for zoonotic pathogens. To date, a huge number of viruses have been identified in these arthropods, with a considerable proportion of them being human pathogens. However, the viromes of hematophagous arthropods are still largely unresearched. In this study, a number of arthropods were collected from Belgrade, Serbia including mosquitoes, ticks and bedbugs. The viromes of these arthropods were identified and characterized using Illumina MiSeq sequencing. In total, 21 viruses belonging to 11 families were characterized, with 11 of them representing novel species. These results may contribute to our knowledge of RNA viruses in arthropods and the discovery of novel human pathogens.

Understanding the Mechanisms Underlying Host Restriction of Insect-Specific Viruses.

Arthropod-borne viruses contribute significantly to global mortality and morbidity in humans and animals. These viruses are mainly transmitted between susceptible vertebrate hosts by hematophagous arthropod vectors, especially mosquitoes. Recently, there has been substantial attention for a novel group of viruses, referred to as insect-specific viruses (ISVs) which are exclusively maintained in mosquito populations. Recent discoveries of novel insect-specific viruses over the past years generated a great interest not only in their potential use as vaccine and diagnostic platforms but also as novel biological control agents due to their ability to modulate arbovirus transmission. While arboviruses infect both vertebrate and invertebrate hosts, the replication of insect-specific viruses is restricted in vertebrates at multiple stages of virus replication. The vertebrate restriction factors include the genetic elements of ISVs (structural and non-structural genes and the untranslated terminal regions), vertebrate host factors (agonists and antagonists), and the temperature-dependent microenvironment. A better understanding of these bottlenecks is thus warranted. In this review, we explore these factors and the complex interplay between ISVs and their hosts contributing to this host restriction phenomenon.

Family Level Phylogenies Reveal Relationships of Plant Viruses within the Order Bunyavirales.

Bunyavirales are negative-sense segmented RNA viruses infecting arthropods, protozoans, plants, and animals. This study examines the phylogenetic relationships of plant viruses within this order, many of which are recently classified species. Comprehensive phylogenetic analyses of the viral RNA dependent RNA polymerase (RdRp), precursor glycoprotein (preGP), the nucleocapsid (N) proteins point toward common progenitor viruses. The RdRp of Fimoviridae and Tospoviridae show a close evolutional relationship while the preGP of Fimoviridae and Phenuiviridae show a closed relationship. The N proteins of Fimoviridae were closer to the Phasmaviridae, the Tospoviridae were close to some Phenuiviridae members and the Peribunyaviridae. The plant viral movement proteins of species within the Tospoviridae and Phenuiviridae were more closely related to each other than to members of the Fimoviridae. Interestingly, distal ends of 3' and 5' untranslated regions of species within the Fimoviridae shared similarity to arthropod and vertebrate infecting members of the Cruliviridae and Peribunyaviridae compared to other plant virus families. Co-phylogeny analysis of the plant infecting viruses indicates that duplication and host switching were more common than co-divergence with a host species.

Serologic Evidence of Arthropod-Borne Virus Infections in Wild and Captive Ruminants in Ontario, Canada.

Arthropod-borne viruses (arboviruses) are globally widespread, and their transmission cycles typically involve numerous vertebrate species. Serologic testing of animal hosts can provide a routine surveillance approach to monitoring animal disease systems, can provide a surveillance alternative to arthropod testing and human case reports, and may augment knowledge of epizootiology. Wild and captive ruminants represent good candidate sentinels to track geographic distribution and prevalence of select arboviruses. They often are geographically widespread and abundant, inhabit areas shared by humans and domestic animals, and are readily fed on by various hematophagous arthropod vectors. Ontario, Canada, is home to high densities of coexisting humans, livestock, and wild cervids, as well as growing numbers of arthropod vectors because of the effects of climate change. We collected blood samples from 349 livestock (cattle/sheep) and 217 cervids (wild/farmed/zoo) in Ontario (2016-2019) to assess for antibodies to zoonotic and agriculturally important arboviruses. Livestock sera were tested for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). Sera from cervids were tested for antibodies to BTV, EHDV, West Nile virus (WNV), eastern equine encephalitis virus (EEEV), Powassan virus (POWV), and heartland virus (HRTV). Fifteen (9.0%) cattle were seropositive for EHDV-serotype 2. Nine (4.2%) cervids were seropositive for arboviruses; three confirmed as WNV, three as EEEV, and one as POWV. All animals were seronegative for BTV and HRTV. These results reveal low seroprevalence of important agricultural, wildlife, and zoonotic pathogens and underline the need for continued surveillance in this and other regions in the face of changing environmental conditions.

Low Incidence and Mortality from SARS-CoV-2 in Southern Europe. Proposal of a hypothesis for Arthropod borne Herd immunity.

SARS-CoV-2 incidence and mortality in Europe have shown wide variation. Northern Italy in particular the Lombardy region, north-eastern French regions, Switzerland and Belgium were amongst the hardest hit, while the central and southern Italian regions, all the Balkan countries from Slovenia to Greece and the Islands of Malta and Cyprus had much fewer cases and deaths per capita, and deaths per number of cases. Differences in public health measures, and health care delivery, in the author's opinion, can only partly explain the difference. The geographical distribution of Phlebotomus sand-flies and the relative distribution of arthropod borne diseases Leishmaniasis and Phlebovirus infections especially the Sicilian Sandfly fever group corresponds to most areas of low prevalence of SARS-CoV-2. A hypothesis is proposed whereby repeated arthropod or sandfly vector infection of humans by novel viruses of zoonotic origins carrying bat or mammalian RNA/DNA, such as phleboviruses may have resulted in the development of an effective evolutionary immune response to most novel zoonotic viruses such as SARS-CoV-2 by means of survival of the fittest possibly over many generations. This process probably ran in parallel and concurrent with the progressive evolution of novel coronaviruses which spread from one mammalian species to another. Other possible, but less likely mechanisms for the role of sandfly meals within a much shorter time frame may have led to, (i) previous exposure and infection of humans with the SARS-Cov-2 virus itself, or a closely related corona virus in the previous decades, or (ii) exposure of human populations to parts coronavirus protein namely either S or more likely N protein carried mechanically by arthropods, but without clinical disease causing direct immunity or (iii) by causing infection with other arthropod borne viruses which could carry bat DNA/RNA and have similar functional proteins resulting in an immediate cross-reactive immune response rather than by natural selection. The Evidence possibly supporting or disputing this hypothesis is reviewed, however the major problem with the hypothesis is that to date no coronavirus has ever been isolated from arthropods. Such a hypothesis can only be supported by research investigating the possible biological relationship of arthropods and coronaviruses where paradoxically they may be promoting immunity rather than disease.

[Transmission of plant and vertebrate viruses by arthropods]. / La transmission des virus de plantes et de vertébrés par arthropodes.

Many plant and vertebrate viruses use mobile vectors to be transmitted between hosts. These vectors, mainly arthropods, acquire or inoculate the virus by feeding on plant extract or vertebrate blood. Several virus transmission modes have been characterized based on the tight interactions between the virus and the vector. Some viruses are internalized into cells and migrate through different tissues and organs before being released. In the vector, the virus can replicate in some cases. Other viruses are retained, specifically or non-specifically, on the vector mouthparts. Acquiring knowledge on the molecular mechanisms of virus transmission by arthropods consists in studying (i) virus receptors in the vectors, (ii) the mode of virus uptake into vector cells, (iii) virus localization and transport in the vector, and (iv) viral determinants required for transmission. This review, although non exhaustive, presents a state-of-the-art of plant and vertebrate virus transmission by arthropods, notably by pointing to their similarities and differences.

Revisiting an old friend: new findings in alphavirus structure and assembly.

Alphaviruses are transmitted by an arthropod vector to a vertebrate host. The disease pathologies, cellular environments, immune responses, and host factors are very different in these organisms. Yet, the virus is able to infect, replicate, and assemble into new particles in these two animals using one set of genetic instructions. The balance between conserved mechanisms and unique strategies during virus assembly is critical for fitness of the virus. In this review, we discuss new findings in receptor binding, polyprotein topology, nucleocapsid core formation, and particle budding that have emerged in the last five years and share opinions on how these new findings might answer some questions regarding alphavirus structure and assembly.

Nudivirus Remnants in the Genomes of Arthropods.

Endogenous viral elements (EVEs), derived from all major types of viruses, have been discovered in many eukaryotic genomes, representing "fossil records" of past viral infections. The endogenization of nudiviruses has been reported in several insects, leading to the question of whether genomic integration is a common phenomenon for these viruses. In this study, genomic assemblies of insects and other arthropods were analyzed to identify endogenous sequences related to Nudiviridae. A total of 359 nudivirus-like genes were identified in 43 species belonging to different groups; however, none of these genes were detected in the known hosts of nudiviruses. A large proportion of the putative EVEs identified in this study encode intact open reading frames or are transcribed as mRNAs, suggesting that they result from recent endogenization of nudiviruses. Phylogenetic analyses of the identified EVEs and inspections of their flanking regions indicated that integration of nudiviruses has occurred recurrently during the evolution of arthropods. This is the first report of a comprehensive screening for nudivirus-derived EVEs in arthropod genomes. The results of this study demonstrated that a large variety of arthropods, especially hemipteran and hymenopteran insects, have previously been or are still infected by nudiviruses. These findings have greatly extended the host range of Nudiviridae and provide new insights into viral diversity, evolution, and host-virus interactions.

Genetic Strain Diversity of Multi-Host RNA Viruses that Infect a Wide Range of Pollinators and Associates is Shaped by Geographic Origins.

Emerging viruses have caused concerns about pollinator population declines, as multi-host RNA viruses may pose a health threat to pollinators and associated arthropods. In order to understand the ecology and impact these viruses have, we studied their host range and determined to what extent host and spatial variation affect strain diversity. Firstly, we used RT-PCR to screen pollinators and associates, including honey bees (Apis mellifera) and invasive Argentine ants (Linepithema humile), for virus presence and replication. We tested for the black queen cell virus (BQCV), deformed wing virus (DWV), and Kashmir bee virus (KBV) that were initially detected in bees, and the two recently discovered Linepithema humile bunya-like virus 1 (LhuBLV1) and Moku virus (MKV). DWV, KBV, and MKV were detected and replicated in a wide range of hosts and commonly co-infected hymenopterans. Secondly, we placed KBV and DWV in a global phylogeny with sequences from various countries and hosts to determine the association of geographic origin and host with shared ancestry. Both phylogenies showed strong geographic rather than host-specific clustering, suggesting frequent inter-species virus transmission. Transmission routes between hosts are largely unknown. Nonetheless, avoiding the introduction of non-native species and diseased pollinators appears important to limit spill overs and disease emergence.

The discovery of segmented flaviviruses: implications for viral emergence.

In the last five years, several novel segmented RNA viruses have been discovered in ticks, mosquitoes, or other arthropods, and two viruses, including Jingmen tick virus and Alongshan virus, are associated with human febrile illness in northeastern China. The viral genome includes four or five segments, two of which are genetically derived from unsegmented flaviviruses. Phylogenetic analysis reveals that segmented flaviviruses belong to a separate Jingmenvirus group distinctive from the genera of Flavivirus, Hepacivirus, Pestivirus, and Pegivirus in the Flaviviridae family. Tick-borne segmented flaviviruses are closely related to mammalian isolates from humans, monkeys and voles, showing their important significance of public health. Future studies should be focused on disease surveillance, epidemiology, animal infection model and reverse genetics of these emerging segmented flaviviruses.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals.

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.

ICTV Virus Taxonomy Profile: Peribunyaviridae.

Peribunyaviruses are enveloped and possess three distinct, single-stranded, negative-sense RNA segments comprising 11.2-12.5 kb in total. The family includes globally distributed viruses in the genera Orthobunyavirus, Herbevirus, Pacuvirus and Shangavirus. Most viruses are maintained in geographically-restricted vertebrate-arthropod transmission cycles that can include transovarial transmission from arthropod dam to offspring. Others are arthropod-specific. Arthropods can be persistently infected. Human infection occurs through blood feeding by an infected vector arthropod. Infections can result in a diversity of human and veterinary clinical outcomes in a strain-specific manner. Segment reassortment is evident between some peribunyaviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Peribunyaviridae, which is available at ictv.global/report/peribunyaviridae.

Effects of Arbovirus Multi-Host Life Cycles on Dinucleotide and Codon Usage Patterns.

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.