Culicoides-Specific Fitness Increase of Vesicular Stomatitis Virus in Insect-to-Insect Infections.

Vesicular stomatitis virus (VSV) is an arthropod-borne virus affecting livestock. In the United States, sporadic outbreaks result in significant economic losses. During epizootics, Culicoides biting midges are biological vectors and key to the geographic expansion of outbreaks. Additionally, Culicoides may play a role in VSV overwintering because females and males are capable of highly efficient venereal transmission, despite their relatively low virus titers. We hypothesized that VSV propagated within a midge has increased fitness for subsequent midge infections. To evaluate the potential host-specific fitness increase, we propagated three viral isolates of VSV in porcine skin fibroblasts and Culicoides cell lines. We then evaluated the viral infection dynamics of the different cell-source groups in Culicoides sonorensis. Our results indicate that both mammalian- and insect-derived VSV replicate well in midges inoculated via intrathoracic injection, thereby bypassing the midgut barriers. However, when the virus was required to infect and escape the midgut barrier to disseminate after oral acquisition, the insect-derived viruses had significantly higher titers, infection, and dissemination rates than mammalian-derived viruses. Our research suggests that VSV replication in Culicoides cells increases viral fitness, facilitating midge-to-midge transmission and subsequent replication, and further highlights the significance of Culicoides midges in VSV maintenance and transmission dynamics.

Untangling an insect's virome from its endogenous viral elements.

BACKGROUND: Insects are an important reservoir of viral biodiversity, but the vast majority of viruses associated with insects have not been discovered. Recent studies have employed high-throughput RNA sequencing, which has led to rapid advances in our understanding of insect viral diversity. However, insect genomes frequently contain transcribed endogenous viral elements (EVEs) with significant homology to exogenous viruses, complicating the use of RNAseq for viral discovery. METHODS: In this study, we used a multi-pronged sequencing approach to study the virome of an important agricultural pest and prolific vector of plant pathogens, the potato aphid Macrosiphum euphorbiae. We first used rRNA-depleted RNAseq to characterize the microbes found in individual insects. We then used PCR screening to measure the frequency of two heritable viruses in a local aphid population. Lastly, we generated a quality draft genome assembly for M. euphorbiae using Illumina-corrected Nanopore sequencing to identify transcriptionally active EVEs in the host genome. RESULTS: We found reads from two insect-specific viruses (a Flavivirus and an Ambidensovirus) in our RNAseq data, as well as a parasitoid virus (Bracovirus), a plant pathogenic virus (Tombusvirus), and two phages (Acinetobacter and APSE). However, our genome assembly showed that part of the 'virome' of this insect can be attributed to EVEs in the host genome. CONCLUSION: Our work shows that EVEs have led to the misidentification of aphid viruses from RNAseq data, and we argue that this is a widespread challenge for the study of viral diversity in insects.

microRNA Expression Dynamics in Culicoides sonorensis Biting Midges Following Blood-Feeding.

Culicoides sonorensis midges vector multiple livestock arboviruses, resulting in significant economic losses worldwide. Due to the tight association between virus transmission, blood feeding, and egg development, understanding midge physiology is paramount to limiting pathogen transmission. Previous studies have demonstrated the importance of small non-coding RNAs (ncRNAs), specifically microRNAs (miRNAs), in multiple aspects of vector physiology. These small ncRNAs regulate gene expression at the post-transcriptional level and display differential expression during pathogen infection. Due to the lack of annotated miRNAs in the biting midge and associated expression profiles, we used small RNA-Seq and miRDeep2 analyses to determine the Culicoides miRNAs in whole females and midgut tissues in response to blood feeding. Our analyses revealed 76 miRNAs within C. sonorensis composed of 73 orthologous and three candidate novel miRNAs, as well as conserved miRNA clusters. miRNA conservation suggests an interesting evolutionary relationship between miRNA expression and hematophagy in the infraorder Culicomorpha. We also identified multiple blood meal-regulated and tissue-enriched miRNAs. Lastly, we further identified miRNAs with expression patterns potentially associated with virus infection by probing publicly available datasets. Together, our data provide a foundation for future ncRNA work to untangle the dynamics of gene regulation associated with midge physiology.

Why do viruses make aphids winged?

Aphids are hosts to diverse viruses and are important vectors of plant pathogens. The spread of viruses is heavily influenced by aphid movement and behaviour. Consequently, wing plasticity (where individuals can be winged or wingless depending on environmental conditions) is an important factor in the spread of aphid-associated viruses. We review several fascinating systems where aphid-vectored plant viruses interact with aphid wing plasticity, both indirectly by manipulating plant physiology and directly through molecular interactions with plasticity pathways. We also cover recent examples where aphid-specific viruses and endogenous viral elements within aphid genomes influence wing formation. We discuss why unrelated viruses with different transmission modes have convergently evolved to manipulate wing formation in aphids and whether this is advantageous for both host and virus. We argue that interactions with viruses are likely shaping the evolution of wing plasticity within and across aphid species, and we discuss the potential importance of these findings for aphid biocontrol.

Los áfidos albergan diversos virus y son vectores de importantes patógenos de plantas. La propagación de virus está fuertemente influenciada por el movimiento y el comportamiento de los áfidos. En consecuencia, la plasticidad de las alas (en la cual algunos individuos desarrollan alas dependiendo de las condiciones ambientales) es un factor importante en la propagación viral asociada a los áfidos. En este documento revisamos varios ejemplos fascinantes en los que virus de plantas transmitidos por áfidos interactúan con la plasticidad fenotípica de las alas, indirectamente manipulando la fisiología de la planta y directamente a través de interacciones moleculares con los mecanismos de plasticidad fenotípica del áfido. También describimos ejemplos recientes que demuestran como algunos virus específicos de áfidos y elementos virales endógenos localizados en los genomas de áfidos influyen en la formación de alas. Últimamente, discutimos por qué virus no relacionados con diferentes modos de transmisión han evolucionado convergentemente para manipular la formación de alas en áfidos y si este fenómeno es beneficioso para el insecto y el virus. Nosotros objetamos que las interacciones con virus están probablemente influenciando la evolución intra- e interespecífica de la plasticidad de las alas en áfidos, y discutimos el potencial de estos hallazgos para el control biológico de los áfidos.

Comparison of Endemic and Epidemic Vesicular Stomatitis Virus Lineages in Culicoides sonorensis Midges.

Vesicular stomatitis virus (VSV) primarily infects livestock and is transmitted by direct contact and vectored by Culicoides midges (Diptera: Ceratopogonidae). Endemic to Central and South America, specific VSV lineages spread northward out of endemic regions of Mexico and into the U.S. sporadically every five to ten years. In 2012, a monophyletic epidemic lineage 1.1 successfully spread northward into the U.S. In contrast, the closest endemic ancestor, lineage 1.2, remained circulating exclusively in endemic regions in Mexico. It is not clear what roles virus-animal interactions and/or virus-vector interactions play in the ability of specific viral lineages to escape endemic regions in Mexico and successfully cause outbreaks in the U.S., nor the genetic basis for such incursions. Whole-genome sequencing of epidemic VSV 1.1 and endemic VSV 1.2 revealed significant differences in just seven amino acids. Previous studies in swine showed that VSV 1.1 was more virulent than VSV 1.2. Here, we compared the efficiency of these two viral lineages to infect the vector Culicoides sonorensis (Wirth and Jones) and disseminate to salivary glands for subsequent transmission. Our results showed that midges orally infected with the epidemic VSV 1.1 lineage had significantly higher infection dissemination rates compared to those infected with the endemic VSV 1.2 lineage. Thus, in addition to affecting virus-animal interactions, as seen with higher virulence in pigs, small genetic changes may also affect virus-vector interactions, contributing to the ability of specific viral lineages to escape endemic regions via vector-borne transmission.

Effect of Constant Temperatures on Culicoides sonorensis Midge Physiology and Vesicular Stomatitis Virus Infection.

Culicoides midges play an important role in vesicular stomatitis virus (VSV) transmission to US livestock. After VSV-blood feeding, blood digestion followed by oviposition occurs while ingested virus particles replicate and disseminate to salivary glands for transmission during subsequent blood-feeding events. Changes to environmental temperature may alter the feeding-oviposition-refeeding cycles, midge survival, VSV infection, and overall vector capacity. However, the heterothermic midge may respond rapidly to environmental changes by adjusting their thermal behavior to resting in areas closer to their physiological range. Here we investigated the effects of four constant environmental temperatures (20, 25, 30, and 35 °C) on C. sonorensis survival, oviposition, and VSV infection, as well as resting thermal preferences after blood-feeding. We found that most midges preferred to rest in areas at 25-30 °C. These two constant temperatures (25 and 30 °C) allowed an intermediate fitness performance, with a 66% survival probability by day 10 and oviposition cycles occurring every 2-3 days. Additionally, VSV infection rates in bodies and heads with salivary glands were higher than in midges held at 20 °C and 35 °C. Our results provide insight into the implications of temperature on VSV-Culicoides interactions and confirm that the range of temperature preferred by midges can benefit both the vector and the arbovirus.

Surveillance along the Rio Grande during the 2020 Vesicular Stomatitis Outbreak Reveals Spatio-Temporal Dynamics of and Viral RNA Detection in Black Flies.

Vesicular stomatitis virus (VSV) emerges periodically from its focus of endemic transmission in southern Mexico to cause epizootics in livestock in the US. The ecology of VSV involves a diverse, but largely undefined, repertoire of potential reservoir hosts and invertebrate vectors. As part of a larger program to decipher VSV transmission, we conducted a study of the spatiotemporal dynamics of Simulium black flies, a known vector of VSV, along the Rio Grande in southern New Mexico, USA from March to December 2020. Serendipitously, the index case of VSV-Indiana (VSIV) in the USA in 2020 occurred at a central point of our study. Black flies appeared soon after the release of the Rio Grande's water from an upstream dam in March 2020. Two-month and one-year lagged precipitation, maximum temperature, and vegetation greenness, measured as Normalized Difference Vegetation Index (NDVI), were associated with increased black fly abundance. We detected VSIV RNA in 11 pools comprising five black fly species using rRT-PCR; five pools yielded a VSIV sequence. To our knowledge, this is the first detection of VSV in the western US from vectors that were not collected on premises with infected domestic animals.

Detection of Vesicular Stomatitis Virus Indiana from Insects Collected during the 2020 Outbreak in Kansas, USA.

Vesicular stomatitis (VS) is a reportable viral disease which affects horses, cattle, and pigs in the Americas. Outbreaks of vesicular stomatitis virus New Jersey serotype (VSV-NJ) in the United States typically occur on a 5-10-year cycle, usually affecting western and southwestern states. In 2019-2020, an outbreak of VSV Indiana serotype (VSV-IN) extended eastward into the states of Kansas and Missouri for the first time in several decades, leading to 101 confirmed premises in Kansas and 37 confirmed premises in Missouri. In order to investigate which vector species contributed to the outbreak in Kansas, we conducted insect surveillance at two farms that experienced confirmed VSV-positive cases, one each in Riley County and Franklin County. Centers for Disease Control and Prevention miniature light traps were used to collect biting flies on the premises. Two genera of known VSV vectors, Culicoides biting midges and Simulium black flies, were identified to species, pooled by species, sex, reproductive status, and collection site, and tested for the presence of VSV-IN RNA by RT-qPCR. In total, eight positive pools were detected from Culicoides sonorensis (1), Culicoides stellifer (3), Culicoides variipennis (1), and Simulium meridionale (3). The C. sonorensis- and C. variipennis-positive pools were from nulliparous individuals, possibly indicating transovarial or venereal transmission as the source of virus. This is the first report of VSV-IN in field caught C. stellifer and the first report of either serotype in S. meridionale near outbreak premises. These results improve our understanding of the role midges and black flies play in VSV epidemiology in the United States and broadens the scope of vector species for targeted surveillance and control.

Blood Meals With Active and Heat-Inactivated Serum Modifies the Gene Expression and Microbiome of Aedes albopictus.

The Asian "tiger mosquito" Aedes albopictus is currently the most widely distributed disease-transmitting mosquito in the world. Its geographical expansion has also allowed the expansion of multiple arboviruses like dengue, Zika, and chikungunya, to higher latitudes. Due to the enormous risk to global public health caused by mosquitoes species vectors of human disease, and the challenges in slowing their expansion, it is necessary to develop new and environmentally friendly vector control strategies. Among these, host-associated microbiome-based strategies have emerged as promising options. In this study, we performed an RNA-seq analysis on dissected abdomens of Ae. albopictus females from Manhattan, KS, United States fed with sugar and human blood containing either normal or heat-inactivated serum, to evaluate the effect of heat inactivation on gene expression, the bacteriome transcripts and the RNA virome of this mosquito species. Our results showed at least 600 genes with modified expression profile when mosquitoes were fed with normal vs. heat-inactivated-containing blood. These genes were mainly involved in immunity, oxidative stress, lipid metabolism, and oogenesis. Also, we observed bacteriome changes with an increase in transcripts of Actinobacteria, Rhodospirillaceae, and Anaplasmataceae at 6 h post-feeding. We also found that feeding with normal blood seems to particularly influence Wolbachia metabolism, demonstrated by a significant increase in transcripts of this bacteria in mosquitoes fed with blood containing normal serum. However, no differences were observed in the virome core of this mosquito population. These results suggest that heat and further inactivation of complement proteins in human serum may have profound effect on mosquito and microbiome metabolism, which could influence interpretation of the pathogen-host interaction findings when using this type of reagents specially when measuring the effect of Wolbachia in vector competence.

Impacts of Infectious Dose, Feeding Behavior, and Age of Culicoides sonorensis Biting Midges on Infection Dynamics of Vesicular Stomatitis Virus.

Culicoides sonorensis biting midges are biological vectors of vesicular stomatitis virus (VSV) in the U.S. Yet, little is known regarding the amount of ingested virus required to infect midges, nor how their feeding behavior or age affects viral replication and vector competence. We determined the minimum infectious dose of VSV-New Jersey for C. sonorensis midges and examined the effects of multiple blood-feeding cycles and age at the time of virus acquisition on infection dynamics. A minimum dose of 3.2 logs of virus/mL of blood resulted in midgut infections, and 5.2 logs/mL resulted in a disseminated infection to salivary glands. For blood-feeding behavior studies, ingestion of one or two non-infectious blood meals (BM) after a VSV infectious blood meal (VSV-BM) resulted in higher whole-body virus titers than midges receiving only the single infectious VSV-BM. Interestingly, this infection enhancement was not seen when a non-infectious BM preceded the infectious VSV-BM. Lastly, increased midge age at the time of infection correlated to increased whole-body virus titers. This research highlights the epidemiological implications of infectious doses, vector feeding behaviors, and vector age on VSV infection dynamics to estimate the risk of transmission by Culicoides midges more precisely.

Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins.

Aedes aegypti is the primary mosquito vector of several human arboviruses, including the dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help identify novel targets useful in designing effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human red blood cells reconstituted with either heat-inactivated (IB) or normal plasma (NB). The RNA-seq based transcript expression of IB and NB mosquitoes was compared against sugar-fed (SF) mosquitoes. In in vitro experiments, we treated Aag2 cells with a recombinant version of complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24 h. The transcript expression analysis revealed that human complement proteins modulate approximately 2300 transcripts involved in multiple biological functions, including immunity. We also found 161 upregulated and 168 downregulated transcripts differentially expressed when human complement protein C3 (hC3) and human complement protein C5a (hC5a) treated cells were compared to the control untreated cells. We conclude that active human complement induces significant changes to the transcriptome of Ae. aegypti mosquitoes, which may influence the physiology of these arthropods.

Venereal Transmission of Vesicular Stomatitis Virus by Culicoides sonorensis Midges.

Culicoides sonorensis biting midges are well-known agricultural pests and transmission vectors of arboviruses such as vesicular stomatitis virus (VSV). The epidemiology of VSV is complex and encompasses a broad range of vertebrate hosts, multiple routes of transmission, and diverse vector species. In temperate regions, viruses can overwinter in the absence of infected animals through unknown mechanisms, to reoccur the next year. Non-conventional routes for VSV vector transmission may help explain viral maintenance in midge populations during inter-epidemic periods and times of adverse conditions for bite transmission. In this study, we examined whether VSV could be transmitted venereally between male and female midges. Our results showed that VSV-infected females could venereally transmit virus to uninfected naïve males at a rate as high as 76.3% (RT-qPCR), 31.6% (virus isolation) during the third gonotrophic cycle. Additionally, VSV-infected males could venereally transmit virus to uninfected naïve females at a rate as high as 76.6% (RT-qPCR), 49.2% (virus isolation). Immunofluorescent staining of micro-dissected reproductive organs, immunochemical staining of midge histological sections, examination of internal reproductive organ morphology, and observations of mating behaviors were used to determine relevant anatomical sites for virus location and to hypothesize the potential mechanism for VSV transmission in C. sonorensis midges through copulation.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus.

BACKGROUND: Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1-24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25-27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called 'differential virus replication' in vitro. METHODS: Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into 'corrections' of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. CONCLUSION: Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Vesicular Stomatitis Virus Transmission: A Comparison of Incriminated Vectors.

Vesicular stomatitis (VS) is a viral disease of veterinary importance, enzootic in tropical and subtropical regions of the Americas. In the U.S., VS produces devastating economic losses, particularly in the southwestern states where the outbreaks display an occurrence pattern of 10-year intervals. To date, the mechanisms of the geographic spread and maintenance cycles during epizootics remain unclear. This is due, in part, to the fact that VS epidemiology has a complex of variables to consider, including a broad range of vertebrate hosts, multiple routes of transmission, and an extensive diversity of suspected vector species acting as both mechanical and biological vectors. Infection and viral progression within vector species are highly influenced by virus serotype, as well as environmental factors, including temperature and seasonality; however, the mechanisms of viral transmission, including non-conventional pathways, are yet to be fully studied. Here, we review VS epidemiology and transmission mechanisms, with comparisons of transmission evidence for the four most incriminated hematophagous dipteran taxa: Aedes mosquitoes, Lutzomyia sand flies, Simulium black flies, and Culicoides biting midges.

Serosurvey of Human Antibodies Recognizing Aedes aegypti D7 Salivary Proteins in Colombia.

BACKGROUND: Dengue is one of the most geographically significant mosquito-borne viral diseases transmitted by Aedes mosquitoes. During blood feeding, mosquitoes deposit salivary proteins that induce antibody responses. These can be related to the intensity of exposure to bites. Some mosquito salivary proteins, such as D7 proteins, are known as potent allergens. The antibody response to D7 proteins can be used as a marker to evaluate the risk of exposure and disease transmission and provide critical information for understanding the dynamics of vector-host interactions. METHODS: The study was conducted at the Los Patios Hospital, Cucuta, Norte de Santander, Colombia. A total of 63 participants were enrolled in the study. Participants were categorized into three disease status groups, age groups, and socioeconomic strata. The level of IgG antibodies against D7 Aedes proteins was determined by ELISA. We used a statistical approach to determine if there is an association between antibody levels and factors such as age, living conditions, and dengue virus (DENV) infection. RESULTS: We found that IgG antibodies against D7 proteins were higher in non-DENV infected individuals in comparison to DENV-infected participants. Also, the age factor showed a significant positive correlation with IgG antibodies against D7 proteins, and the living conditions (socioeconomic stratification), in people aged 20 years or older, are a statistically significant factor in the variability of IgG antibodies against D7 proteins. CONCLUSION: This pilot study represents the first approximation to elucidate any correlation between the antibody response against mosquito D7 salivary proteins and its correlation with age, living conditions, and DENV infection in a dengue endemic area.

Mosquito species (Diptera, Culicidae) in three ecosystems from the Colombian Andes: identification through DNA barcoding and adult morphology.

Colombia, one of the world's megadiverse countries, has a highly diverse mosquito fauna and a high prevalence of mosquito-borne diseases. In order to provide relevant information about the diversity and taxonomy of mosquito species in Colombia and to test the usefulness of DNA barcodes, mosquito species collected at different elevations in the departments of Antioquia and Caldas were identified combining adult morphology and barcode sequences. A total of 22 mosquito species from eight genera were identified using these combined techniques. We generated 77 barcode sequences with 16 species submitted as new country records for public databases. We examined the usefulness of DNA barcodes to discriminate mosquito species from the Neotropics by compiling 1,292 sequences from a total of 133 species and using the tree-based methods of neighbor-joining and maximum likelihood. Both methodologies provided similar results by resolving 105 species of mosquitoes separated into distinct clusters. This study shows the importance of combining classic morphological methodologies with molecular tools to accurately identify mosquitoes from Colombia.

Updated list of the mosquitoes of Colombia (Diptera: Culicidae).

BACKGROUND: A revised list of the mosquitoes (Diptera: Culicidae) known to occur in Colombia is presented. A total of 324 species from 28 genera of Culicidae are included. The species names are organized in alphabetical order according to the current generic and subgeneric classification, along with their authorship. The list is compiled in order to support mosquito research in Colombia. NEW INFORMATION: Our systematic review and literature survey found, by 16 February 2015, 13 records of culicid species previously overlooked by mosquito catalogs for Colombia: Anophelescostai da Fonseca & da Silva Ramos, 1939, An.fluminensis Root, 1927, An.malefactor Dyar & Knab, 1907, An.shannoni Davis, 1931, An.vargasi Galbadón, Cova García & Lopez, 1941, Culexmesodenticulatus Galindo & Mendez, 1961, Haemagoguscapricornii Lutz, 1904, Isostomyiaespini (Martini, 1914), Johnbelkinialeucopus (Dyar & Knab, 1906), Mansoniaindubitans Dyar & Shannon, 1925, Psorophorasaeva Dyar & Knab, 1906, Sabethesglaucodaemon (Dyar & Shannon, 1925), and Wyeomyiaintonca Dyar & Knab, 1909. Moreover, Wyeomyia (Dendromyia) luteoventralis Theobald, 1901 is recorded for Colombia for the first time. This work provides important insights into mosquito diversity in Colombia, using the current nomenclature and phylogenetic rankings.