[First detection of Rift Valley Fever Virus among Culex pipiens in Tahoua, Niger]. / Détection du virus de la fièvre de la vallée du Rift chez Culex pipiens à Tahoua au Niger.

Background: The Rift Valley Fever (RVF) is an arbovirus disease responsible of regular epizootics and epidemics in sub-Saharan Africa and Arabian Peninsula. In 2016, Niger experienced its first outbreak of RVF in Tahoua region, which resulted in high consequences in animal and human health. The aim of this study was to investigate on the RVFV circulation among potential vectors of the disease. Methods: This was a cross-sectional survey carried out in Tahoua and Agadez regions in August 2021. Adult mosquitoes were collected by using the morning spray in human dwellings and the CDC light trap methods. After morphological identification, viral RNA was extracted. The RNA was extracted by using QIAamp Viral RNA Mini Kit (Qiagen). The RVFV detection was performed by using the qRT-PCR method. Results: A total of 2487 insects (1978 mosquitoes, 509 sandflies and 251 biting midges) were identified and divided into three families (Culicidae, Psychodidae and Ceratopogonidae). The Culicidae family composed of the Culex genus being the most abundant with a predominance of Cx.pipiens (31.88%; n = 793) followed by Mansonia sp (21.51%; n = 535), Anophelesgambiae s.l. (8.44%; n = 210), An. pharoensis (0.72%; n = 18), An. rufipes (0.48%; n = 12), Cx. quinquefasciatus (6.39%; n = 159), the Psychodidae with sandflies (20.46%; n = 509), and the Ceratopogonidae with Culicoides genus (10.09%; n = 251). The qRT-PCR carried out on a sample of mosquitoes (N = 96) highlighted that one individual of Cx.pipiens was found positive to RVFV. This specimen was from Tassara locality (Tahoua) and collected by CDC Light Trap method. Conclusion: This study reveals for the first time the circulation of RVFV among Cx.pipiens in Niger and highlights the possible vectorial role of this vector in the disease transmission. Further investigations should be carried out to identify the biological and ecological determinants that support the maintenance of the virus in this area in order to guide control interventions.

Beet curly top virus affects vector biology: the first transcriptome analysis of the beet leafhopper.

Curly top disease, caused by beet curly top virus (BCTV), is among the most serious viral diseases affecting sugar beets in western USA. The virus is exclusively transmitted by the beet leafhopper (BLH, Circulifer tenellus) in a circulative and non-propagative manner. Despite the growing knowledge on virus-vector interactions, our understanding of the molecular interactions between BCTV and BLH is hampered by limited information regarding the virus impact on the vector and the lack of genomic and transcriptomic resources for BLH. This study unveils the significant impact of BCTV on both the performance and transcriptome response of BLHs. Viruliferous BLHs had higher fecundity than non-viruliferous counterparts, which was evident by upregulation of differentially expressed transcripts (DETs) associated with development, viability and fertility of germline and embryos in viruliferous insects. Conversely, most DETs associated with muscle movement and locomotor activities were downregulated in viruliferous insects, implying potential behavioural modifications by BCTV. Additionally, a great proportion of DETs related to innate immunity and detoxification were upregulated in viruliferous insects. Viral infection also induced notable alterations in primary metabolisms, including energy metabolism, namely glucosidases, lipid digestion and transport, and protein degradation, along with other cellular functions, particularly in chromatin remodelling and DNA repair. This study represents the first comprehensive transcriptome analysis for BLH. The presented findings provide new insights into the multifaceted effects of viral infection on various biological processes in BLH, offering a foundation for future investigations into the complex virus-vector relationship and potential management strategies for curly top disease.

Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

Dynamical analysis of a stochastic maize streak virus epidemic model with logarithmic Ornstein-Uhlenbeck process.

To describe the transmission dynamics of maize streak virus infection, in the paper, we first formulate a stochastic maize streak virus infection model, in which the stochastic fluctuations are depicted by a logarithmic Ornstein-Uhlenbeck process. This approach is reasonable to simulate the random impacts of main parameters both from the biological significance and the mathematical perspective. Then we investigate the detailed dynamics of the stochastic system, including the existence and uniqueness of the global solution, the existence of a stationary distribution, the exponential extinction of the infected maize and infected leafhopper vector. Especially, by solving the five-dimensional algebraic equations corresponding to the stochastic system, we obtain the specific expression of the probability density function near the quasi-endemic equilibrium of the stochastic system, which provides valuable insights into the stationary distribution. Finally, the model is discretized using the Milstein higher-order numerical method to illustrate our theoretical results numerically. Our findings provide a groundwork for better methods of preventing the spread of this type of virus.

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

Genetic variations and gene expression profiles of Rice Black-streaked dwarf virus (RBSDV) in different host plants and insect vectors: insights from RNA-Seq analysis.

Rice black-streaked dwarf virus (RBSDV) is an etiological agent of a destructive disease infecting some economically important crops from the Gramineae family in Asia. While RBSDV causes high yield losses, genetic characteristics of replicative viral populations have not been investigated within different host plants and insect vectors. Herein, eleven publicly available RNA-Seq datasets from Chinese RBSDV-infected rice, maize, and viruliferous planthopper (Laodelphax striatellus) were obtained from the NCBI database. The patterns of SNP and RNA expression profiles of expected RBSDV populations were analyzed by CLC Workbench 20 and Geneious Prime software. These analyses discovered 2,646 mutations with codon changes in RBSDV whole transcriptome and forty-seven co-mutated hotspots with high variant frequency within the crucial regions of S5-1, S5-2, S6, S7-1, S7-2, S9, and S10 open reading frames (ORFs) which are responsible for some virulence and host range functions. Moreover, three joint mutations are located on the three-dimensional protein of P9-1. The infected RBSDV-susceptible rice cultivar KTWYJ3 and indigenous planthopper datasets showed more co-mutated hotspot numbers than others. Our analyses showed the expression patterns of viral genomic fragments varied depending on the host type. Unlike planthopper, S5-1, S2, S6, and S9-1 ORFs, respectively had the greatest read numbers in host plants; and S5-2, S9-2, and S7-2 were expressed in the lowest level. These findings underscore virus/host complexes are effective in the genetic variations and gene expression profiles of plant viruses. Our analysis revealed no evidence of recombination events. Interestingly, the negative selection was observed at 12 RBSDV ORFs, except for position 1015 in the P1 protein, where a positive selection was detected. The research highlights the potential of SRA datasets for analysis of the virus cycle and enhances our understanding of RBSDV's genetic diversity and host specificity.

Tomato yellow leaf curl virus manipulates Bemisia tabaci, MEAM1 both directly and indirectly through changes in visual and volatile cues.

The sweetpotato whitefly, Bemisia tabaci MEAM1, is one of the most devastating pests of row-crop vegetables worldwide, damaging crops directly through feeding and indirectly through the transmission of many different viruses, including the geminivirus Tomato yellow leaf curl virus (TYLCV). Y-tube olfactometer tests were conducted at different stages of TYLCV infection in tomatoes to understand how TYLCV affects B. tabaci behavior. We also recorded changes in tomato hosts' color and volatile profiles using color spectrophotometry and gas chromatography-mass spectrometry (GC-MS). We found that the infection status of B. tabaci and the infection stage of TYLCV influenced host selection, with uninfected whiteflies showing a preference for TYLCV-infected hosts, especially during the late stages of infection. Viruliferous B. tabaci attraction to visual targets significantly differed from non-viruliferous B. tabaci. Late-stage infected hosts had larger surface areas reflecting yellow-green wavelengths and higher emissions of methyl salicylate in their volatile profiles. These findings shed new light on several critical mechanisms involved in the viral manipulation of an insect vector and its economically important host.

Arboviruses antagonize insect Toll antiviral immune signaling to facilitate the coexistence of viruses with their vectors.

Many plant arboviruses are persistently transmitted by piercing-sucking insect vectors. However, it remains largely unknown how conserved insect Toll immune response exerts antiviral activity and how plant viruses antagonize it to facilitate persistent viral transmission. Here, we discover that southern rice black-streaked dwarf virus (SRBSDV), a devastating planthopper-transmitted rice reovirus, activates the upstream Toll receptors expression but suppresses the downstream MyD88-Dorsal-defensin cascade, resulting in the attenuation of insect Toll immune response. Toll pathway-induced the small antibacterial peptide defensin directly interacts with viral major outer capsid protein P10 and thus binds to viral particles, finally blocking effective viral infection in planthopper vector. Furthermore, viral tubular protein P7-1 directly interacts with and promotes RING E3 ubiquitin ligase-mediated ubiquitinated degradation of Toll pathway adaptor protein MyD88 through the 26 proteasome pathway, finally suppressing antiviral defensin production. This virus-mediated attenuation of Toll antiviral immune response to express antiviral defensin ensures persistent virus infection without causing evident fitness costs for the insects. E3 ubiquitin ligase also is directly involved in the assembly of virus-induced tubules constructed by P7-1 to facilitate viral spread in planthopper vector, thereby acting as a pro-viral factor. Together, we uncover a previously unknown mechanism used by plant arboviruses to suppress Toll immune response through the ubiquitinated degradation of the conserved adaptor protein MyD88, thereby facilitating the coexistence of arboviruses with their vectors in nature.

Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Tomato yellow leaf curl virus: Characteristics, influence, and regulation mechanism.

Tomato yellow leaf curl virus (TYLCV), a DNA virus belonging to the genus Begomovirus, significantly impedes the growth and development of numerous host plants, including tomatoes and peppers. Due to its rapid mutation rate and frequent recombination events, achieving complete control of TYLCV proves exceptionally challenging. Consequently, identifying resistance mechanisms become crucial for safeguarding host plants from TYLCV-induced damage. This review article delves into the global distribution, dispersal patterns, and defining characteristics of TYLCV. Moreover, the intricate interplay between TYLCV and various influencing factors, such as insect vectors, susceptible host plants, and abiotic stresses, plays a pivotal role in plant-TYLCV interactions. The review offers an updated perspective on recent investigations focused on plant response mechanisms to TYLCV infection, including the intricate relationship between TYLCV, whiteflies, and regulatory factors. This comprehensive analysis aims to establish a foundation for future research endeavors exploring the molecular mechanisms underlying TYLCV infection and the development of plant resistance through breeding programs.

African swine fever virus DNA is present in non-biting flies collected from outbreak farms in Romania.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA. METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA). RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12. CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

Orthotospovirus iridimaculaflavi (iris yellow spot virus): An emerging threat to onion cultivation and its transmission by Thrips tabaci in India.

The yellow spot disease caused by the virus species Orthotospovirus iridimaculaflavi (Iris yellow spot virus-IYSV), belonging to the genus Orthotospovirus, the family Tospoviridae, order Bunyavirales and transmitted by Thrips tabaci Lindeman. At present, emerging as a major threat in onion (Allium cepa) in Tamil Nadu, India. The yellow spot disease incidence was found to be 53-73 % in six districts out of eight major onion-growing districts surveyed in Tamil Nadu during 2021-2023. Among the onion cultivars surveyed, the cultivar CO 5 was the most susceptible to IYSV. The population of thrips was nearly 5-9/plant during vegetative and flowering stages. The thrips infestation was 34-60 %. The tospovirus involved was confirmed as IYSV through DAS-ELISA, followed by molecular confirmation through RT-PCR using the nucleocapsid (N) gene. The predominant thrips species present in onion crops throughout the growing seasons was confirmed as Thrips tabaci based on the nucleotide sequence of the MtCOI gene. The mechanical inoculation of IYSV in different hosts viz., Vigna unguiculata, Gomphrena globosa, Chenopodium amaranticolor, Chenopodium quinoa and Nicotiana benthamiana resulted in chlorotic and necrotic lesion symptoms. The electron microscopic studies with partially purified sap from onion lesions revealed the presence of spherical to pleomorphic particles measuring 100-230 nm diameter. The transmission of IYSV was successful with viruliferous adult Thrips tabaci in cowpea (Cv. CO7), which matured from 1st instar larva fed on infected cowpea leaves (24 h AAP). Small brown necrotic symptoms were produced on inoculated plants after an interval of four weeks. The settling preference of non-viruliferous and viruliferous T. tabaci towards healthy and infected onion leaves resulted in the increased preference of non-viruliferous thrips towards infected (onion-61.33 % and viruliferous thrips towards healthy onion leaves (75.33 %). The study isolates shared 99-100 % identity at a nucleotide and amino acid level with Indian isolates of IYSV in the N gene. The multiple alignment of the amino acid sequence of the N gene of IYSV isolates collected from different locations and IYSV isolates from the database revealed amino acid substitution in the isolate ITPR4. All the IYSV isolates from India exhibited characteristic amino acid substitution of serine at the 6th position in the place of threonine in the isolates from Australia, Japan and USA. The phylogenetic analysis revealed the monophyletic origin of the IYSV isolates in India.

Seasonal change and influence of environmental variables on host-seeking activity of the biting midge Culicoides sonorensis at a southern California dairy, USA.

BACKGROUND: As a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk. METHODS: The diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fisher's exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity. RESULTS: Host-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking. CONCLUSIONS: This study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.

Visceral Leishmaniasis-Human Immunodeficiency Virus-Coinfected Patients Are Highly Infectious to Sandflies in an Endemic Area in India.

In an area endemic with Indian visceral leishmaniasis (VL), we performed direct xenodiagnosis to evaluate the transmission of Leishmania donovani from patients with VL-human immunodeficiency virus (HIV) coinfection to the vector sandflies, Phlebotomus argentipes. Fourteen patients with confirmed VL-HIV coinfection, with a median parasitemia of 42 205 parasite genome/mL of blood, were exposed to 732 laboratory-reared pathogen-free female P argentipes sandflies on their lower arms and legs. Microscopy revealed that 16.66% (122/732) of blood-fed flies were xenodiagnosis positive. Notably, 93% (13/14) of the VL-HIV group infected the flies, as confirmed by quantitative polymerase chain reaction and/or microscopy, and were 3 times more infectious than those who had VL without HIV.

Genetic diversity in vector populations influences the transmission efficiency of an important plant virus.

The transmission efficiency of aphid-vectored plant viruses can differ between aphid populations. Intra-species diversity (genetic variation, endosymbionts) is a key determinant of aphid phenotype; however, the extent to which intra-species diversity contributes towards variation in virus transmission efficiency is unclear. Here, we use multiple populations of two key aphid species that vector barley yellow dwarf virus (BYDV) strain PAV (BYDV-PAV), the grain aphid (Sitobion avenae) and the bird cherry-oat aphid (Rhopalosiphum padi), and examine how diversity in vector populations influences virus transmission efficiency. We use Illumina sequencing to characterize genetic and endosymbiont variation in multiple Si. avenae and Rh. padi populations and conduct BYDV-PAV transmission experiments to identify links between intra-species diversity in the vector and virus transmission efficiency. We observe limited variation in the transmission efficiency of Si. avenae, with transmission efficiency consistently low for this species. However, for Rh. padi, we observe a range of transmission efficiencies and show that BYDV transmission efficiency is influenced by genetic diversity within the vector, identifying 542 single nucleotide polymorphisms that potentially contribute towards variable transmission efficiency in Rh. padi. Our results represent an important advancement in our understanding of the relationship between genetic diversity, vector-virus interactions, and virus transmission efficiency.

Virus-vectoring thrips regulate the excessive multiplication of tomato spotted wilt virus using their antiviral immune responses.

The tomato spotted wilt virus (TSWV) is a member of the Tospoviridae family and has an negative/ambisense single-stranded RNA genome. Frankliniella occidentalis and F. intonsa are known to be dominant pests in Capsicum annuum (hot pepper) and can cause damage to the plant either directly by feeding, or indirectly by transmitting TSWV in a persistent and propagative manner, resulting in serious economic damage. This study compared the immune responses of two different thrips species against TSWV infection by transcriptome analysis, which then allowed the assessment of antiviral responses using RNA interference (RNAi). Both adult thrips shared about 90 % of the transcripts in non-viruliferous conditions. Most signal components of the immune pathways were shared by these two thrips species, and their expression levels fluctuated differentially in response to TSWV infection at early immature stages. The functional assays using RNAi treatments indicated that the Toll and JAK/STAT pathways were associated with the antiviral responses, but the IMD pathway was not. The upregulation of dorsal switch protein one supported its physiological role in recognizing TSWV infection and triggering the eicosanoid biosynthetic pathway, which mediates melanization and apoptosis in thrips. In addition, the signal components of the RNAi pathways fluctuated highly after TSWV infection. Individual RNAi treatments specific to the antiviral signalling and response components led to significant increases in the TSWV amount in the thrips, causing virus-induced mortality. These findings suggest that immune signalling pathways leading to antiviral responses are operating in the thrips to regulate TSWV litres to prevent a fatal viral overload. This study also indicates the differential antiviral responses between the TSWV-transmitting F. occidentalis and F. intonsa.

Blowflies are potential vector for avian influenza virus at enzootic area in Japan.

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

Establishment and partial characterisation of a new cell line derived from adult tissues of the tsetse fly Glossina morsitans morsitans.

BACKGROUND: Insect cell lines play a vital role in many aspects of research on disease vectors and agricultural pests. The tsetse fly Glossina morsitans morsitans is an important vector of salivarian trypanosomes in sub-Saharan Africa and, as such, is a major constraint on human health and agricultural development in the region. METHODS: Here, we report establishment and partial characterisation of a cell line, GMA/LULS61, derived from tissues of adult female G. m. morsitans. GMA/LULS61 cells, grown at 28 °C in L-15 (Leibovitz) medium supplemented with foetal bovine serum and tryptose phosphate broth, have been taken through 23 passages to date and can be split 1:1 at 2-week intervals. Karyotyping at passage 17 revealed a predominantly haploid chromosome complement. Species origin and absence of contaminating bacteria were confirmed by PCR amplification and sequencing of fragments of the COI gene and pan-bacterial 16S rRNA gene respectively. However, PCR screening of RNA extracted from GMA/LULS61 cells confirmed presence of the recently described Glossina morsitans morsitans iflavirus and Glossina morsitans morsitans negevirus, but absence of Glossina pallipides salivary gland hypertrophy virus. GMA/LULS61 cells supported infection and growth of 6/7 different insect-derived strains of the intracellular bacterial symbiont Wolbachia. CONCLUSIONS: The GMA/LULS61 cell line has potential for application in a variety of studies investigating the biology of G. m. morsitans and its associated pathogenic and symbiotic microorganisms.

Culicoides Midge Abundance across Years: Modeling Inter-Annual Variation for an Avian Feeder and a Candidate Vector of Hemorrhagic Diseases in Farmed Wildlife.

(1) Background: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are orbiviruses that cause hemorrhagic disease (HD) with significant economic and population health impacts on domestic livestock and wildlife. In the United States, white-tailed deer (Odocoileus virginianus) are particularly susceptible to these viruses and are a frequent blood meal host for various species of Culicoides biting midges (Diptera: Ceratopogonidae) that transmit orbiviruses. The species of Culicoides that transmit EHDV and BTV vary between regions, and larval habitats can differ widely between vector species. Understanding how midges are distributed across landscapes can inform HD virus transmission risk on a local scale, allowing for improved animal management plans to avoid suspected high-risk areas or target these areas for insecticide control. (2) Methods: We used occupancy modeling to estimate the abundance of gravid (egg-laden) and parous (most likely to transmit the virus) females of two putative vector species, C. stellifer and C. venustus, and one species, C. haematopotus, that was not considered a putative vector. We developed a universal model to determine habitat preferences, then mapped a predicted weekly midge abundance during the HD transmission seasons in 2015 (July-October) and 2016 (May-October) in Florida. (3) Results: We found differences in habitat preferences and spatial distribution between the parous and gravid states for C. haematopotus and C. stellifer. Gravid midges preferred areas close to water on the border of well and poorly drained soil. They also preferred mixed bottomland hardwood habitats, whereas parous midges appeared less selective of habitat. (4) Conclusions: If C. stellifer is confirmed as an EHDV vector in this region, the distinct spatial and abundance patterns between species and physiological states suggest that the HD risk is non-random across the study area.

Transcriptomic Investigation of the Virus Spectrum Carried by Midges in Border Areas of Yunnan Province.

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (-ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases.