Important Viruses Research Articles

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5' noncoding region of IBV, respectively, and a fluorescent quantitative RT-PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT-PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations.

Dicer-2 mutations in Aedes aegypti cells lead to a diminished antiviral function against Rift Valley fever virus and Bunyamwera virus infection.

Mosquitoes are known to transmit different arthropod-borne viruses belonging to various virus families. The exogenous small interfering RNA pathway plays an important role in the mosquito defence against such virus infections, with Dicer-2 (Dcr2) as one of the key proteins that initiates the cleavage of viral dsRNAs into 21 nt long virus-derived small interfering RNAs. Previous data identified the importance of various motifs in Dcr2 for its small interfering RNA (siRNA)-mediated antiviral activity. However, all these data focus on positive-strand RNA viruses, although negative-strand RNA viruses, like Bunyaviricetes, include several important mosquito-borne viruses. Here, we aim to investigate the importance of different domains of Dcr2 for antiviral activity against viruses of the Bunyaviricetes. For this, we used the Aedes aegypti-derived Dcr2 knock-out cell line Aag2-AF319 to study the importance of the helicase, RNase III and PIWI-Argonaute-Zwille domains of Dcr2 on the antiviral activity of two viruses belonging to different families of the Bunyaviricetes: the Rift Valley fever virus (RVFV) vaccine strain MP12 (Phenuiviridae, Phlebovirus) and the Bunyamwera orthobunyavirus (BUNV; Peribunyaviridae, Orthobunyavirus). All three domains were determined to be critical for the antiviral activity against both RVFV and BUNV. Interestingly, one specific mutation in the helicase domain (KN) did not result in a loss of antiviral activity for RVFV, but for BUNV, despite losing the ability to produce 21 nt siRNAs.

Enhanced Recovery and Detection of Highly Infectious Animal Disease Viruses by Virus Capture Using Nanotrap® Microbiome A Particles.

This study reports the use of Nanotrap® Microbiome A Particles (NMAPs) to capture and concentrate viruses from diluted suspensions to improve their recovery and sensitivity to detection by real-time PCR/RT-PCR (qPCR/RT-qPCR). Five highly infectious animal disease viruses including goatpox virus (GTPV), sheeppox virus (SPPV), lumpy skin disease virus (LSDV), peste des petits ruminants virus (PPRV), and African swine fever virus (ASFV) were used in this study. After capture, the viruses remained viable and recoverable by virus isolation (VI) using susceptible cell lines. To assess efficacy of recovery, the viruses were serially diluted in phosphate-buffered saline (PBS) or Eagle's Minimum Essential Medium (EMEM) and then subjected to virus capture using NMAPs. The NMAPs and the captured viruses were clarified on a magnetic stand, reconstituted in PBS or EMEM, and analyzed separately by VI and virus-specific qPCR/RT-qPCR. The PCR results showed up to a 100-fold increase in the sensitivity of detection of the viruses following virus capture compared to the untreated viruses from the same dilutions. Experimental and clinical samples were subjected to virus capture using NMAPs and analyzed by PCR to determine diagnostic sensitivity (DSe) that was comparable (100%) to that determined using untreated (-NMAPs) samples. NMAPs were also used to capture spiked viruses from EDTA whole blood (EWB). Virus capture from EWB was partially blocked, most likely by hemoglobin (HMB), which also binds NMAPs and outcompetes the viruses. The effect of HMB could be removed by either dilution (in PBS) or using HemogloBind™ (Biotech Support Group; Monmouth Junction, NJ, USA), which specifically binds and precipitates HMB. Enhanced recovery and detection of viruses using NMAPs can be applicable to other highly pathogenic animal viruses of agricultural importance.

PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

Changes in use of multiplex respiratory panel testing during the COVID-19 pandemic.

COVID-19 changed the epidemiology of community-acquired respiratory viruses. We explored patterns of respiratory viral testing to understand which tests are most clinically useful in the postpandemic era. We conducted a retrospective observational study of discharge data from PINC-AI (formerly Premier), a large administrative database. Use of multiplex nucleic acid amplification respiratory panels in acute care, including small (2-5 targets), medium (6-11), and large panels (>11), were compared between the early pandemic (03/2020-10/2020), late pandemic (11/2020-4/2021), and prepandemic respiratory season (11/2019 - 02/2020) using ANOVA. A median of 160.5 facilities contributed testing data per quarter (IQR 155.5-169.5). Prepandemic, facilities averaged 103 respiratory panels monthly (sd 138), including 79 large (sd 126), 7 medium (sd 31), and 16 small panels (sd 73). Relative to prepandemic, utilization decreased during the early pandemic (62 panels monthly/facility; sd 112) but returned to the prepandemic baseline by the late pandemic (107 panels monthly/facility; sd 211). Relative to prepandemic, late pandemic testing involved more small panel use (58 monthly/facility, sd 156) and less large panel use (47 monthly/facility, sd 116). Comparisons among periods demonstrated significant differences in overall testing (P < 0.0001), large panel use (P < 0.0001), and small panel use (P < 0.0001). Postpandemic, clinical use of respiratory panel testing shifted from predominantly large panels to predominantly small panels. Factors driving this change may include resource availability, costs, and the clinical utility of targeting important pathogenic viruses instead of testing "for everything."

Honey bee viruses in the yellow-legged hornet Vespa velutina (Lepelieter 1836): Prevalence, loads, and detection of replicative DWV and LSV forms

Apiaries in Galicia, northwestern Spain, are currently facing the invasive alien species Vespa velutina, which is well established in the region. The pressure on honey bee colonies is high, resulting in both economic and ecological losses. Honey bee colonies also face the challenge of viruses, which are becoming increasingly diverse. In recent years, honey bee viruses have been spreading across taxonomic groups beyond Apoidea, infecting the Vespoidea superfamily. This cross-species spillover has raised concerns in the scientific community due to the potential risk of viruses spreading in ecosystems. Currently, there is a lack of knowledge on this topic, and further research is needed to address this issue. This study employed qPCR and sequencing to investigate the prevalence, loads, and presence of replicative forms of important honey bee viruses in V. velutina individuals collected from 11 apiaries in Galicia. All V. velutina individuals tested positive for DWV, BQCV, AKI complex (ABPV, KBV, and IAPV), or LSV but not for CBPV. DWV showed the highest prevalence (97.0 %) and loads, with both DWV-A (67.4 %) and DWV-B (32.6 %) being detected. The AKI complex (46.3 %) and LSV (43.3 %) were also common, whereas BQCV (11.9 %) was rarer. LSV is detected for the first time in V. velutina. LSV-2 was the dominant strain (82.1 %), and two less frequent (17.9 %) unknown strains were also detected. All 44 screened V. velutina samples carried the replicative form of DWV, and six of these also carried the replicative form of LSV, raising for the first time the possibility of co-infection in the hornet. The detection of honey bee viruses in V. velutina, and the ability of these viruses to spread to other species, may indicate a potential risk of spillover in the apiaries.

Surveillance of human adenoviruses in water environments: Assessing the suitability of a locally developed quenching of unincorporated amplification signal reporters-loop-mediated isothermal amplification assay

The human adenovirus (HAdV) has shown greater environmental persistence, more water treatment resistance than bacteria, and cause infection even at low concentrations. HAdV causes gastrointestinal illnesses and is abundant in various environmental samples such as groundwater, surface water, recreational water, and drinking water. The detection of these pathogens calls for a more practical and affordable approach. A recent technology based on the quenching of unincorporated amplification signal reporters (QUASR) was adapted for the detection of human adenoviruses. This technology allows for non-inhibitory, single-step DNA detection in a closed tube. The QUASR-(loop-mediated isothermal amplification (LAMP) assay was previously optimized and was tested for its applicability to detect enteric HAdV in two areas where people can unintentionally be exposed to contaminated water. A total of 203 water samples were collected and tested using both real-time PCR and QUASR-LAMP assays. Results showed a higher positivity rate of 78.82 % (160/203) for QUASR-LAMP compared to qPCR with only 58.62 % (119/203). The sensitivity and specificity rates for QUASR-LAMP were calculated at 86.55 % and 32.14 %, respectively, when compared to qPCR. The QUASR-LAMP assay's ability to detect target analytes even at low concentrations can be attributed to its increased diagnostic sensitivity but lower specificity since there were samples that were positive in PCR but negative in the QUASR-LAMP assay. However, this characteristic does not diminish its utility as a valuable tool for the detection of HAdV. In fact, this attribute enhances its advantages in situations with constrained space and instrumentation requirements, making it suitable for rapid surveillance of important viruses. Finally, the capacity of the QUASR-LAMP assay to differentiate between positive and negative samples at a defined endpoint is highly beneficial for laboratory technicians who possess limited molecular biology expertise or experience. The QUASR-LAMP platform has demonstrated its usefulness as a diagnostic tool for surveillance of enteric adenoviruses in water sources.

Evidence of circulating recombinants between deformed wing virus and Varroa destructor virus-1 in honey bee colonies in Türkiye.

Deformed wing virus (DWV), which is an important honey bee virus transmitted by Varroa destructor (V. destructor), causes colony losses in honey bee colonies. This study aimed to investigate the prevalence and genetic diversity of DWV in honey bees in Türkiye and to determine the role of V. destructor in the transmission of the genetic variants of DWV. Honey bee samples were collected from 62 apiaries, by simple random sampling, during March 2022 and April 2023. The presence of V. destructor in collected bee samples was examined using a stereo microscope. Real-time RT-PCR was used for the detection of DWV-A and DWV-B (Varroa destructor virus-1 (VDV-1)) viruses. Genetic characterisation of the positive samples was conducted by sequencing polyprotein genomic region. Considering the V. destructor infestation rate of 3% as relevant, out of the 62 apiaries examined, 17 (27.4%) were positive. However, DWV-A and VDV-1 specific RNA was not detected in V. destructor samples. VDV-1 specific RNA was detected in 6.5% (4/62) of the apiaries, whereas DWV-A was not detected in the sampled apiaries. Phylogenetic analysis showed that isolates detected in this study were located in a separate cluster from previously characterised DWV-A and VDV-1 isolates. According to RDP4 and GARD analyses, DWV-VDV-1 recombination breakpoints were detected in field isolates. To the best our knowledge, this is the first report of the presence of VDV-1-DWV recombinants in Türkiye. Further studies are needed to determine the impact of VDV-1-DWV recombinants and their virological and antigenic properties.

A numbers game: mosquito-based arbovirus surveillance in two distinct geographic regions of Latin America.

Aedes mosquitoes, as vectors of medically important arthropod-borne viruses (arboviruses), constitute a major public health threat that requires entomological and epidemiological surveillance to guide vector control programs to prevent and reduce disease transmission. In this study, we present the collaborative effort of 1 year of Aedes ageypti (Linnaeus, 1762) mosquito-based arbovirus surveillance in 2 geographically distinct regions of Latin America (Nicaragua and Ecuador). Adult female mosquitoes were collected using backpack aspirators in over 2,800 randomly selected households (Nicaragua, Ecuador) and 100 key sites (Nicaragua) from 8 distinct communities (Nicaragua: 2, Ecuador: 6). A total of 1,358 mosquito female pools were processed for RNA extraction and viral RNA detection using real-time reverse transcription-polymerase chain reaction. Ten positive dengue virus (DENV) pools were detected (3 in Nicaragua and 7 in Ecuador), all of which were found during the rainy season and matched the serotypes found in humans (Nicaragua: DENV-1 and DENV-4; Ecuador: DENV-2). Infection rates ranged from 1.13 to 23.13, with the Nicaraguan communities having the lowest infection rates. Our results demonstrate the feasibility of detecting DENV-infected Aedes mosquitoes in low-resource settings and underscore the need for targeted mosquito arbovirus sampling and testing, providing valuable insights for future surveillance programs in the Latin American region.

VP3 protein of Senecavirus A promotes viral IRES-driven translation and attenuates innate immunity by specifically relocalizing hnRNPA2B1.

Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

Optimization of iron flocculation for enrichment of CyHV-2 in aquaculture water

Cyprinid herpesvirus 2 (CyHV-2) is an important infectious virus endangering the farmed Carassius species industry. The enrichment and identification of CyHV-2 in the water is a vital means of early warning diagnostics, which is helpful to aquatic diseases prevention and control. In this study, we employed and optimized iron flocculation to enrich the concentrations of CyHV-2 in aquaculture water. Different Fe3+ concentrations (0.5, 1.0, and 10.0 mg/L), various membrane filters (mixed cellulose ester, glass fiber, nylon microporous filter membrane), different elution buffers containing reductants (ascorbate, citrate, DL-malate, and their combinations with EDTA or TE buffer) were compared to check the recovery yield of CyHV-2. Viral genome quantitation was determined using quantitative real-time PCR. The results showed that Fe3+ concentration of 1.0 mg/L, the glass fiber and citrate-TE were determined to have high efficiency and stable recovery yield. To evaluate infectious CyHV-2 viral titres, crucian carps (Carassius auratus) were intraperitoneally injected with CyHV-2. The mortality rate was 100 % at 9 days postinoculation (dpi) with the 96.7 % detection of CyHV-2 in the pooled tissues. The histopathological phenotype showed necrotic cell death in the liver, spleen and kidney. The CyHV-2 genome was ranging from 101.1 to 107.5 copies/μl in the deceased fish, and 101.4 to 103.0 copies/μl in the feeding water at various stages from latent infection to onset of disease. The field samples from Huzhou, Zhejiang Province on June 4th and 7th had higher detection rates, 87.5 % and 100 % respectively, corresponding to higher CyHV-2 detected in the aquaculture water. CyHV-2 viral load in cultured waters tends to increase prior to fish death, which provides a new indicator for the prediction of fish diseases. In summary, we have established a unified operational standard for enrichment of CyHV-2 in the aqueous environment by iron flocculation.

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA.

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

3D Film-Like Nanozyme with a Synergistic Amplification Effect for the Ultrasensitive Immunochromatographic Detection of Respiratory Viruses.

Greatly improving the sensitivity and detection range of lateral flow immunoassays (LFAs) by at least 100 times without using additional instruments remains challenging. Herein, we develop a three-dimensional (3D) film-like nanozyme (GO-Pt30-AuPt5) by ordered assembly of one layer of 30 nm Pt nanoparticles (NPs) and one layer of small Au@Pt satellites (5 nm) onto a two-dimensional (2D) graphene oxide (GO) nanofilm, in which GO greatly increased the interface area and stability of the nanozyme whereas Pt and Au@Pt NPs synergistically enhanced colorimetric/catalytic activities. The grafting of outer Au@Pt satellites converted the 2D nanofilm into a 3D flexible nanozyme with numerous catalytic sites for enzymatic deposition signal amplification and binding sites for target capture. The introduction of GO-Pt30-AuPt5 into multiplex LFA achieved the ultrasensitive and simultaneous detection of two important respiratory viruses with sensitivity of 1 pg/mL level, which was about 100 times higher than that without signal enrichment and at least 20 and 1900 times higher than those of traditional enzyme-linked immunosorbent assay and AuNP-based LFA, respectively. The clinical utility of the proposed assay was validated through the diagnosis of 49 real clinical respiratory tract specimens. Our proposed LFA shows great potential for the ultrasensitive screening of pathogens in the field.

Viromes of arthropod parasites and their hosts: The case of bats and bat ectoparasites

Blood feeding ectoparasites of bats have been found to contain insect-specific and vertebrate-infecting viruses of agricultural and medical importance. While it is plausible that some of these are of bat origin, those would be sourced either from the bat exterior or their blood meal. Bats, in addition to their regular diets, consume numerous ectoparasites during grooming. All microbes on and in the ectoparasites would then be introduced into the bat gut upon ingestion of the ectoparasites. To investigate the potential impact of bat ectoparasite viromes on the gut viral microbiome of bats, we compared virus sequences from bats and their blood feeding ectoparasites collected from Yunnan Province, China. Although all the co-occurring viruses were bacteriophages, we observed that bats contained a larger set of viruses than their ectoparasites, and that the set of predicted viruses present in the bats were more diverse than those present in bat ectoparasites. Our analysis suggests that despite a heavy influx of ectoparasites into the digestive tract of bats through consumption, there are only few co-occurring/shared viruses between bats and their ectoparasites, and that these ectoparasites may not be a major driver of bat virome diversity. Our findings provide necessary preliminary data for the evaluation of bat ectoparasites as a potential source of bat infecting viruses.

Identification of an intrinsically disordered region (IDR) in arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

Modelling bluetongue and African horse sickness vector (Culicoides spp.) distribution in the Western Cape in South Africa using random forest machine learning.

Culicoides biting midges exhibit a global spatial distribution and are the main vectors of several viruses of veterinary importance, including bluetongue (BT) and African horse sickness (AHS). Many environmental and anthropological factors contribute to their ability to live in a variety of habitats, which have the potential to change over the years as the climate changes. Therefore, as new habitats emerge, the risk for new introductions of these diseases of interest to occur increases. The aim of this study was to model distributions for two primary vectors for BT and AHS (Culicoides imicola and Culicoides bolitinos) using random forest (RF) machine learning and explore the relative importance of environmental and anthropological factors in a region of South Africa with frequent AHS and BT outbreaks. Culicoides capture data were collected between 1996 and 2022 across 171 different capture locations in the Western Cape. Predictor variables included climate-related variables (temperature, precipitation, humidity), environment-related variables (normalised difference vegetation index-NDVI, soil moisture) and farm-related variables (livestock densities). Random forest (RF) models were developed to explore the spatial distributions of C. imicola, C. bolitinos and a merged species map, where both competent vectors were combined. The maps were then compared to interpolation maps using the same capture data as well as historical locations of BT and AHS outbreaks. Overall, the RF models performed well with 75.02%, 61.6% and 74.01% variance explained for C. imicola, C. bolitinos and merged species models respectively. Cattle density was the most important predictor for C. imicola and water vapour pressure the most important for C. bolitinos. Compared to interpolation maps, the RF models had higher predictive power throughout most of the year when species were modelled individually; however, when merged, the interpolation maps performed better in all seasons except winter. Finally, midge densities did not show any conclusive correlation with BT or AHS outbreaks. This study yielded novel insight into the spatial abundance and drivers of abundance of competent vectors of BT and AHS. It also provided valuable data to inform mathematical models exploring disease outbreaks so that Culicoides-transmitted diseases in South Africa can be further analysed.

Exploring Bemisia tabaci Middle East-Asia Minor I and Mediterranean Cryptic Species Relationship with Cowpea Mild Mottle Virus and Their Dynamics in Soybean Fields.

Cowpea mild mottle virus (CPMMV, genus Carlavirus, family Betaflexividae) is an economically important virus infecting soybeans in Brazil, where it was initially identified in 1983. CPMMV is transmitted by the whitefly, Bemisia tabaci, and occasionally by seeds. Over the last three decades, the most invasive B. tabaci Middle East-Asia Minor 1 (MEAM1), and lately the Mediterranean (MED) cryptic species, have replaced the indigenous species in Brazil, with MEAM1 being predominant. In this study, we investigated the transmission properties of CPMMV by MEAM1 and MED, and their distribution in major soybean-growing areas in São Paulo State. Our results from transmission assays with a single insect revealed that MED is a more efficient vector compared to MEAM1, transmitting the virus within a two-minute inoculation access period. B. tabaci MEAM1 is still the predominant whitefly species in São Paulo State, but MED was also identified in different places, mainly in mixed infestations with MEAM1. Some areas transitioned to a predominance of MED over the three years, while others, where MED had previously been detected, showed a reduction in the insects during the same period. Understanding the transmission dynamics of CPMMV and the distribution of its vectors is crucial for implementing effective management strategies to control the virus spread and protect soybean crops. Further research into the mechanisms driving the shifts in whitefly species dominance and CPMMV distribution will be essential for sustaining soybean production in Brazil.

Prevalence and molecular analysis of some important viruses in honey bee colonies in Türkiye: the status of multiple infections.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

Evidence of Grapevine enamovirus 1 Infecting Grapevine (Vitis vinifera L.) in Canada.

Grapevine enamovirus 1 (GEV1) belongs to the genus Enamovirus, in the family Solemoviridae. It has been reported from several countries infecting grapevines including Brazil (Silva et al. 2017), China (Ren et al. 2021) and France (Hily et al. 2022). To assess the prevalence and diversity of economically important grapevine viruses in nine Canadian vineyards, total RNA and double-stranded RNA (dsRNA) (Fall et al. 2020) were extracted from 30 and 100 composite samples respectively, with each consisting of five vines of the same cultivars. The cultivars included in this study are Frontenac noir (n=34), Vidal (n=32), Marquette (n=33), Riesling (n=31), and Pinot noir (n=31). The total RNA and dsRNA samples were subsequently multiplexed and diagnosed by high-throughput sequencing (HTS) on NovaSeq (600 S4 PE100) and MiSeq (2 × 250 cycle PE) respectively. From NovaSeq and MiSeq sequencing, an average of 410,000 to 1.3 million reads/sample were obtained, respectively, with mapped viral reads representing 10.92% to 12.48% of the total reads. After sequence quality was verified using Trimmomatic v.0.40 (Bolger et al. 2014), the clean sequences were screened against all possible viruses in the databases using the Virtool (Rott et al. 2017) and VirFind virus detection pipelines (Ho and Tzanetakis 2014). GEV1 was detected in clean sequences from two, three, and two leaf samples of cultivars 'Marquette' 'Riesling' and 'Frontenac noir' respectively. Six of the seven HTS-assembled GEV1 genomes were partial, ranging from 4,523 to 6,000 nucleotide (nt) with genome coverage varying from 71% to 89%. Only one 6,314 nt long assembled contig (Accession No. OR021829), represented a nearly complete genome, being only 53 and 3 nt shorter than Sd-CG (MT536978) at 5' and 3' untranslated regions (UTR), respectively. Isolate 3- Riesling-CAN (OR021829) shares 90.56 to 94.19% nt identities with several GEV1isolates at 96-99% of query coverage. Phylogenetically, OR021829 is closer to GEV1 isolates from France and China (Figure S1). To validate the HTS results, the developed primer pair SetF and Set1R (Silva et al., 2017) was used for RT-PCR detection. The amplicons from all seven HTS-positive samples were sequenced using Sanger sequencing, confirming the presence of GEV-1 in three studied grape cultivars in Canadian vineyards. Symptoms associated with the specific GEV1-infected vines could not be explained as composite samples were used. Each of the combined samples HTS library also tested positive for at least one of the known grape virus/viroids, namely grapevine leafroll associated-virus -3, grapevine pinot gris virus, grapevine rupestris stem pitting-associated virus, Marafivirus syrahense grapevine Syrah virus-1 and hop stunt viroid. To our knowledge, this is the first report of GEV1 being detected in grapevines in Canada, or in any North American vineyard. GEV1 is a relatively new virus, and its biology remains largely unknown. Based on this sequence new GEV1 primers can be developed to know the genetic variability among GEV-1 and improve the detection of this virus in vineyards.

Interrelation of the spatial and genetic structure of tick-borne encephalitis virus, its reservoir host (Myodes glareolus), and its vector (Ixodes ricinus) in a natural focus area.

Tick-borne encephalitis (TBE) virus is considered the medically most important arthropod-borne virus in Europe. Although TBE is endemic throughout central Europe, ticks and rodents determine its maintenance in small, difficult-to-assess, natural foci. We investigated the interrelation between the population genetics of the main TBE virus (TBEV) vector tick (Ixodes ricinus), the most important reservoir host (Myodes glareolus, syn. Clethrionomys glareolus), and TBEV. Rodents and ticks were sampled on 15 sites within an exploratory study area, which has been screened regularly for TBEV occurrence in ticks for more than 10 years. On all 15 sites, ticks and bank voles were sampled, screened for TBEV presence via serology and RT-PCR, and genetically examined. Moreover, TBEV isolates derived from these analyses were sequenced. In long-term TBEV foci bank vole populations show extraordinary genetic constitutions, leading to a particular population structure, whereas ticks revealed a panmictic genetic structure overall sampling sites. Landscape genetics and habitat connectivity modeling (analysis of isolation by resistance) showed no landscape-related barriers explaining the genetic structure of the bank vole populations. The results suggest that bank voles do not simply serve as TBEV reservoirs, but their genetic composition appears to have a significant influence on establishing and maintaining long-term natural TBEV foci, whereas the genetic structure of TBEV's main vector I. ricinus does not play an important role in the sustainability of long-term TBEV foci. A thorough investigation of how and to which extent TBEV and M. glareolus genetics are associated is needed to further unravel the underlying mechanisms.