The treatment for mosquito-borne viral diseases such as dengue virus (DENV), zika virus (ZIKV), and chikungunya virus (CHIKV) has become difficult due to delayed diagnosis processes. In addition, sharing the same transmission media and similar symptoms at the early stage of infection of these diseases has become more critical for early diagnosis. To overcome this, a common platform that can identify the virus with high sensitivity and selectivity, even for the different serotypes, is in high demand. In this study, we have attempted an electrochemical impedimetric method to detect the ZIKV, DENV, and CHIKV using their corresponding antibody-conjugated sensor electrodes. The significance of this method is emphasized on the fabrication of a common matrix of gold–polyaniline and sulfur, nitrogen-doped graphene quantum dot nanocomposites , which have a strong impedimetric response based only on the conjugated antibody, resulting in minimum cross-reactivity for the detection of various mosquito-borne viruses, separately. As a result, four serotypes of DENV and ZIKV, and CHIKV have been detected successfully with an LOD of femtogram mL The severity of mosquito-borne diseases is a global threat caused by protozoa, viruses, or parasites, resulting in nearly 700 million illnesses and over one million deaths each year . The annAmong the various diagnosis methods, virus isolation, enzyme-linked immunosorbent assay, and reverse-transcriptase polymerase chain reaction (RT-PCR) have generally been used for detecting viral infections ,14,15. AFollowing the advancement of nanotechnologies in virus detection, many reports can be found for direct or indirect ZIKV, CHIKV, and DENV detection ,23. Amon−1 concentration, which confirms the applicability of this sensor for sensitive and rapid detection. The successful results in this study encourage the extension of this research to explore the sensor performance in multiple detection platforms for different mosquito-borne viruses in single-pot measurements in future.Inspired by our few recent reports on electrochemical sensing ,27,28,294, N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich Co., LLC . Chikungunya virus lysate (strain: Chikungunya virus), Zika virus lysate , Mouse anti-Chikungunya virus capsid protein (clone: CA980), and Mouse anti-Zika virus antibody (Clone: ID5-2-H7-G3) were purchased from The Native Antigen Company . Bm5 cells were provided by Prof. K. S. Boo  and maintained at 27 °C in Sf-900II serum-free medium  supplemented with 1% fetal bovine serum and Antibiotic–Antimycotic solution .Sodium acetate, sulfuric acid, phosphate-buffered saline (PBS) buffer, aniline, toluene, potassium hydroxide (KOH), hydrochloric acid (HCL), methanol, ethanol, citric acid anhydrous, thiourea, and acetone were purchased from Wako Pure Chemical . Bovine serum albumin (BSA), HAuClThe monoclonal anti-dengue virus envelope protein of serotype 1 (Clone E29), serotype 2 (Clone 3H5-1), serotype (Clone E1), and serotype 4 (Clone E42) were purchased from Bei Resources . For the selectivity test, influenza virus A/H1N1  was purchased from Prospec-Tany Techno Gene Ltd. , and norovirus-like particle (NoV-LP) was provided by Dr. Tian-Cheng Li .Transmission electron microscopy (TEM) images of nanocomposites were obtained using a TEM system  operated at 100 kV. Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano series . Electrochemical impedance spectroscopy (EIS) and electrochemical cyclic voltammetry (CV) were performed by using an SP-150 , which consists of a conventional three-electrode cell containing platinum wire. Saturated Ag/AgCl was used as an electrolyzer .4 as an aqueous oxidant and the polyaniline as a monomer in the organic toluene layer [3+ ions oxidized the aniline monomer to its conducting emeraldine salt polymer formation in the nanotube structure, whereas it was reduced to the nano Au0 form itself. The AuNP was therefore entrapped on the nanotube surface of the polyaniline, resulting in AuNP-PAni nanocomposites. Finally, the AuNP-PAni nanocomposites were drop-casted on the PAni-coated Au electrode for further analysis.AuNP-PAni nanocomposites were synthesized via interfacial self-oxidation–reduction polymerization with HAuClne layer . These tThe synthesis of N,S-GQDs was followed by a hydrothermal reaction system . AntibodDeposition of nanocomposites on the gold electrode produces high conductivity to perform electrochemical analysis. Initially, the polyaniline was electrochemically deposited on the gold surface using cyclic voltammetry (CV) in a three-electrode system to prepare the gold electrode. The obtained curve for CV was recorded at a scan rate of 20 mV/s in a potential range of 0–1 V for 10 cycles. After that, 15 µL of the mixed solution of Ab-N,S-GQD with AuNP-PAni was deposited by drop-casting onto the gold surface with polymerized PAni. The sulfur on N,S-GQD formed strong bonds of Au–S with AuNPs via the soft acid–soft base interaction. To minimize the reactivity of the base matrix of Au-PAni, the final sensor electrode was immersed in a solution of 0.2% BSA for blocking before using it for virus detection.The dengue virus-like particles (DENV-LPs) serotypes 1–4 were prepared according to a previously reported protocol . Briefly−1 to 1 ng mL−1 using filtered 0.1 M PBS solution. For detection, 10 µL of virus solution was dropped on the gold electrode containing the Ab-N,S-GQD@AuNP-PAni nanocomposite and incubated for 10 min at room temperature. The virus was bound with the antibody-conjugated surface of the electrode. The unbound virus was washed by dipping the electrode in PBS and then kept in the electrolytic solution. The value for charge transfer resistance (Rct) on the electrode was then measured by the potential EIS with a sinusoidal amplitude of 5 mV within a frequency range from 100 kHz to 0.1 Hz. The virus detection time using the gold electrode was about 15 min.The virus solution was diluted in a series of concentrations from 10 fg mL4 as an aqueous oxidant and the polyaniline as a monomer in the organic toluene layer. These two layers met at their interfaces; then, the aniline was oxidized to its conducting emeraldine salt polymer formation in the nanotube structure. Au3+ was reduced to Au0, entrapped on the nanotube surface. The synthesis and their TEM images are shown in AuNP-PAni nanocomposites were synthesized via interfacial self-oxidation–reduction polymerization with HAuClIt can be anticipated that the conductivity and the charge storage property of the sensor electrode should possess a high value due to the presence of a conducting surface of AuNP and PAni nanotubes. As shown in the cyclic voltammogram b, the AuCHIKV). The antibody was conjugated with the N,S-GQDs and dialyzed well before proceeding to Au-PAni-N,S-GQD formation. After that, only sensor electrodes without virus loading were recorded in impedance as the control, which was compared with different virus concentrations loaded on the electrodes. The Nyquist plots of the Au|PAni|Au-PAni-N,S-GQD electrode before and after virus loading are shown in Z′ and Z′′ represent the real and imaginary parts, respectively, of the impedance over the frequency range 100 kHz to 100 MHz with an AC amplitude of 5 mV. The plot clearly shows that the increasing pattern in impedance occurred with the increasing virus concentrations from 100 fg·mL−1 to 1 ng·mL−1. To observe the individual contributions of impedance value, all Nyquist plots were fitted with several possible equivalent circuit diagrams over frequencies ranging from 100 kHz to 100 MHz. The best-fitted diagram was applied to decipher the individual contribution of the circuit parameters, which is depicted in Rct, represents the transfer of electrons at the electrode to the electrolyte interface. The Rct values found in different concentrations of viruses were compared with their corresponding control values before addition of the virus and are plotted in Rct. The change in Rct was calculated in percentages to obtain the calibration lines. As shown in Rct and the CHIKV concentration. The limit of detection (LOD), determined by 3σ/S  [−1. The detection ability was investigated on chikungunya virus with the Au|PAni|Au-PAni-N,S-GQD electrode with anti-chikungunya antibody (Abon line) , was fou−1 CHIKV in addition to other samples of ZIKV, DENV-LP-2, Influenza virus A/H1N1, and NoV-LP . In their Nyquist plots in Rct values are deciphered from the same circuit diagram, and the percentage change has been plotted in the bar diagram in The main goal of this study was to detect different mosquito-borne viruses. These viruses also have similar surface functionalities that can affect the sensor specificity; thus, it was imperative to investigate the sensor’s selectivity, especially with ZIKV and DENV. To analyze the selectivity, the sensor electrode was tested with 10 pg mLRct values for each electrode were deciphered with the same circuit, and the calibration line has been drawn against concentration in −1, confirming the detection mechanism of EIS-based virus sensing. Similarly, the Au|PAni|Au-PAni-N,S-GQD-AbZIKV sensor electrode should possess high selectivity, as shown in the corresponding selectivity values in The detection mechanism was based on antibody–antigen interactions; therefore, it can be anticipated that the sensor should be applicable for the detection of other virused by changing the corresponding antibodies on the sensor surface. Therefore, a similar sensor was developed for Zika virus sensing with anti-Zika antibody-conjugated Au-PAni-N,S-GQD nanocomposites. The Nyquist plot and the calibration line show identical results for CHIKV, as presented in Rct values for the calibration lines in −1 for DENV-LP-1, DENV-LP-2, DENV-LP-3, and DENV-LP-4, respectively.Among the mosquito-borne virus diseases, the fatality rate of DENV infection is very high. Treatment becomes critical when secondary infection occurs by a virus serotype different from that of the initial infection . TherefoRct for clear understanding. It can be observed from DENV-2 sensor electrode, the cross-reactivity showed a 350% enhancement in DENV-LP-1 and four of the target DENV-LP-2, where the actual target of DENV-LP-2 showed a 605% enhancement. The specificity of the antibody for the DENV-LP-2 target was observed to be the most interfering in nature; therefore, the high cross-reactivity of the sensor electrode can be justified [Although the antibodies used for the sensor preparation were monoclonal, the chances of cross-reactivity were high due to the similarity of viral origin. To check the ability of this sensor to distinguish the identity of different serotypes, four similar electrodes with different antibody-conjugation were applied to verify the cross-reactivity of all four dengue serotypes. The interactions between the four types of sensor electrodes with four serotypes of DENV-LP and four controls are presented in ustified ,45,46. TRct value between the sensor electrode and the electrolyte solution exhibits a significant change, resulting in a large increase in the EIS result. Due to the usage of antibody–antigen interactions on the nanocomposites, the sensor shows excellent selectivity and minimal cross-reactivity in the presence of other viruses. In the case of DENV serotyping, where the possibility of cross-reactivity is very high, this sensor can identify the serotype in the concentration of pg mL−1, which to the best of our knowledge, is the first attempt using an electrochemical process. In recent studies, the identification of serotypes has not been investigated in DENV detection. As listed in This paper proposes an electrochemical biosensor with PAni|Au-PAni-N,S-GQD nanocomposites, combining different antibodies to detect their corresponding viruses. The AuNP-PAni nanocomposites that conjugated N,S-GQDs-Ab enhanced the electron transfer process, which improved the electrochemical response and provided a small resistive value in the electrochemical impedance spectroscopy. The Au nanoparticles (AuNPs) with well-defined and controlled shapes incorporated in the nanochain of a conducting polymer, polyaniline, have attracted increasing attention as a promising material for biosensing matrixes. On the other hand, N,S-GQDs with structural defects in N and S can present useful functionality for nanocomposite formation. In N,S-GQDs, the nitrogen atom drastically enhances the electrochemical properties of GQD. In contrast, sulfur can increase the coordinate binding with the AuNP, situated in the Au-PAni nanocomposites. The N,S-GQDs can conjugate with the antibody embedded with Au-PAni. Therefore, the Ab-N,S-GQDs@AuNP-PAni nanocomposites show excellent electroactivity in solution, and can be applied for impedimetric virus detection. After the addition of the virus, the sensor electrode can bind with the viruses due to the conjugated antibody on the electrode surface, where the −1 for CHIKV, ZIKV, DENV-LP-1, DENV-LP-2, DENV-LP-3, and DENV-LP-4, respectively. We hope that the proper development of this method for applications in disposable and multiplex systems can result in a single sensor to detect several closely related viruses in the near future.During the past few decades, several investigations have been carried out to establish a sensitive detection technique of viruses. Although few biosensors have improved virus detection in selectivity, sensitivity, and response time, practical usages are limited, especially in cases where the analytes derive from similar origins. This report has successfully developed an electrochemical biosensor with PAni|Au-PAni-N,S-GQD nanocomposites, combining different antibodies to detect their corresponding viruses. We have targeted the mosquito-borne viruses DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, and ZIKV in their detection, conjugating their corresponding antibodies on the nanocomposites. In all cases, we achieved high sensitivity, with LOD values of 22.1, 31.1, 27.4, 24.5, 41.4, and 13.3 fg mL


Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1–4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Ōkahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Māori authority over a territory and its associated treasures), we propose the name Karaka Ōkahu purepure virus in te reo Māori (the Māori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Ōkahu), and the spotted symptom  that this endemic virus appears to cause.We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country’s first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree ( Corynocarpus laevigatus) is an endemic tree occurring naturally in the coastal and lowland forests of Aotearoa New Zealand’s North Island [Karaka  Mount Albert Research Campus (MARC) in Auckland. This study looked to first identify the potential causal agent for the chlorotic spots using high throughput sequencing (HTS) approaches, to understand the genetic relationship between the novel emaravirus and other similar genus members, then to correlate the presence of symptoms and the virus, and finally to assess the distribution of the virus within a wider Auckland region. Collectively, the information was then used to endow the most appropriate name, in consultation with mana whenua (indigenous Māori authority over a territory and its associated treasures). The tentative name of the virus is Karaka Ōkahu purepure emaravirus (KŌPV) and is used hereafter.https://en.genomics.cn/ accessed on 25 May 2021) as described by Blouin et al. [Symptomatic leaf samples were collected in 2015 from a single tree at the MARC site of Plant & Food Research in Auckland, New Zealand. From symptomatic leaves, double-stranded RNA (dsRNA) was isolated and sequenced as described previously . In brien et al. .The sequencing data were assembled de novo using Trinity v. v2.3.2  and the https://www.geneious.com accessed on 25 May 2021, Biomatters, Auckland, New Zealand) by pairwise alignment to their respective RNA sequence previously generated by Illumina sequencing. To confirm the tentatively completed genome was correct, sRNA from the NGS data was mapped to the completed genome sequence for each segment by reference assembly using Geneious v6.1.8. Any discrepancies between the two sequences were analysed and the genome sequence amended based on the number of reads in the original NGS sequence and the number reads in the sRNA assembly giving the final genome (GenBank #MZ391827–MZ391831). To confirm the terminal nucleotides at each end of each RNA segment, sequences obtained from the RACE analysis of each segment were analysed using Geneious v6.1.8 software (www.ncbi.nlm.nih.gov accessed on 25 May 2021) [https://www.bioinformatics.org/sms/prot_mw.html accessed on 25 May 2021); putative N-glycosylation and O-glycosylation sites were identified using NetNGlyc 1.0 NetOGlyc 4.0, respectively [To identify similar nucleotide (nt) sequences, assembled contigs were used as query sequences using BLASTn to search the GenBank database within the National Center for Biotechnology Information website  . BLASTx ectively ,33; signectively ; transmeectively ; the preectively ; COILS wectively .The virus sequences and their predicted protein products were compared with genome and protein data of all known emaraviruses. Percent pairwise identities for the protein products of RNA 1, RNA 2 and RNA 3 are provided in the The relationship between KŌPV and other emaraviruses was investigated using phylogenetic analysis. The P1 aa sequences (polymerase or L) were used for comparison with KŌPV (listed in n = 12).Surveying and collection of samples in Auckland were performed using a 2 km × 2 km grid created in QGIS . The surIn total, 115 symptomatic and 143 asymptomatic trees were surveyed and sampled. From the symptomatic trees, a symptomatic leaf and an asymptomatic leaf were collected; from the asymptomatic trees, only an asymptomatic leaf could be collected. Total RNA was extracted from leaf samples using the CTAB method , and a mKaraka RNA was assessed using the internal primers VvNAD5 developed by Chooi et al.  to test Virus-like symptoms  superfamily (Accession cl20265). This indicates that P1 is the RdRp [The KŌPV RNA 1 is 7141 nt in length with an open reading frame (ORF) found between AUG64-66 and UUA7024-7026  that encthe RdRp . P1 is tthe RdRp . Motif Athe RdRp ,45,46. Mthe RdRp . In KŌPVthe RdRp ,49,50.RNA 2 is 1943 nt in length and contains a large ORF between AUG59-61 and UAG1856-1858 and is predicted to encode a glycoprotein (P2) of 599 aa, predicted MW = 68.23 kDa. The protein shares the highest aa identity with chrysanthemum mosaic-associated virus (ChMaV) (31.9%). Unexpectedly, the KŌPV P2 does not appear to contain any N-glycosylation or O-glycosylation sites; emaraviruses have been previously reported to contain between four and six N-glycosylation sites ,51. MoreRNA 3 is 1479 nt in length and contains an ORF between UUG42-44 and UAA963-965 that is predicted to encode a nucleocapsid protein (P3) of 307 aa, predicted MW = 34.49 kDa. The protein has 37.6% aa identity with ChMaV and contains three conserved aa regions: A (NKFVMSSNR119-127), B (NRLA173-176) and C (GVEN194-197) . Region RNA 4 is 1518 nt in length with an ORF between AUG103-105 and UAA1054-1056 that is predicted to encode a movement protein (P4) of 317 aa, predicted MW = 36.20 kDa. A signal peptide was detected between aa positions 1 and 18 using SignalP 5.0 and it is in a similar position to a signal peptide Actinidia chlorotic ringspot-associated virus (AcCRaV) . AlthougRNA 5 is 1576 nt in length with an ORF between UUG87-89 and UAA894-896 that encodes a protein with unknown function (P5) of 269 aa, predicted MW = 30.30 kDa. The aa sequence appears to share no similarity with any other viral protein nor has any conserved domains. The aa sequence has no coiled-coil domains, signal peptides for a secretory pathway or cleavage sites, and shows a poor likelihood for forming transmembrane helices. Four candidate N-glycosylation sites and ten potential O-glycosylation sites were identified using NetNGlyc 1.0 and NetOGlyc 4.0, respectively. Phylogenetic analysis of the P1 protein was undertaken to determine the relationship between KŌPV and other emaraviruses . Amino aAmino acid identities for proteins encoded by RNAs 1, 2 and 3 are provided in The distribution of KŌPV in central Auckland was investigated by surveying and sampling symptomatic and asymptomatic trees. A subset of RNA samples from 40 asymptomatic leaves from symptomatic karaka trees and 40 asymptomatic leaves from asymptomatic trees were tested with the internal primers VvNAD5 which validated the karaka RNA isolation method and the integrity of RNA for the RT-PCR (data not shown). From 258 trees sampled, 44.6% showed symptoms . The locVirus-like symptoms were identified on several karaka leaves at the Plant & Food Research MARC site in Auckland in 2015. To our knowledge, these symptoms have not been previously described in literature or transcripts of Māori oral histories. Cucumber mosaic virus was reported to infect karaka in Christchurch (South Island) showing mosaic and concentric ring markings . AdditioThe complete KŌPV genome sequence shares the fundamental prerequisites for membership of the Emaravirus genus. It has at least five segmented, negative-sense RNA segments (RNA 1–5) of which RNA 1 to 4 appear to encode an RdRp, glycoprotein, nucleocapsid and movement protein, respectively. These identifications are based on predicted protein size, and the presence of motifs and features reported previously for emaraviruses ,46. WhilKŌPV has unique genome features. The predicted glycoprotein encoded by RNA 2 does not appear to have a glycosylation site, nor the expected cleavage site. The protein predicted from RNA 4 (P4) is considered to be the putative movement protein since it shows overall similarity to the movement proteins of other emaraviruses, despite not having the expected conserved emaravirus 30 kDa movement superfamily domain ,47. InteDesignation of the Emaravirus genus into phylogroups is under constant review as new emaraviruses are discovered. The clade to which KŌPV belongs has only recently been identified ,62, highKaraka trees are widely found within Auckland, with evidence that symptomatic and asymptomatic trees appear to be evenly distributed. No obvious point of origin for the virus was observed. However, the origin could have been overlooked in this study, as only a subset of karaka trees were sampled. Our survey and sampling protocols prioritised the distribution of trees within a geographic region over the abundance of trees; therefore, regions that could potentially contain a denser abundance of symptomatic trees were accessed during this study. In addition, we avoided private property and sacred places  at Orakei Beach, Auckland) because permission to enter had not been sought. However, we did observe apparent evidence of symptomatic karaka trees in some locations. A future study focused on determining the abundance of symptomatic karaka in Auckland could provide greater insight as to a potential point of origin for the KŌPV. Indeed, future surveys should include assessments of karaka throughout New Zealand, as detailed by Atherton et al. . FurtherAnother scenario for the even distribution of KŌPV observed in Auckland is that KŌPV initially spread (perhaps by eriophyid mites) through a nursery, followed by an artificial spread by human activity to the rest of Auckland and potentially wider New Zealand through planting of the infected trees. In this study, we could not determine with confidence whether a sampled tree was the result of natural seed dispersal or was planted from nursery material. Future surveys may consider investigating methods of differentiating naturally sown trees from purposefully planted trees to assist with determining the natural viral spread. Atherton et al.  suggesteThe number of symptomatic leaves present varied greatly from one karaka tree to another. Some trees displayed a few symptomatic leaves only whereas on other trees symptoms were widely observed throughout the canopy. Karaka trees were preferentially surveyed in public spaces and then visually assessed for symptoms; however, trees with few or rudimentary foliar symptoms may have been falsely recorded as asymptomatic. The survey was carried out predominantly on foot by the same person and, as a result, tall trees could not be completely assessed without a ladder or cherry picker, for example. Use of unmanned aerial vehicles may be trialed in future to more comprehensively assess individual trees and to reach trees in more remote or inaccessible locations. Previously, emaraviruses have been found in association with eriophyid mites, including pear chlorotic leaf spot-associated virus which Kubota et al.  detectedPerhaps KŌPV represents an example of an emaravirus that has not yet gained a movement protein to confer entry into the phloem companion cells to achieve cell-to-cell movement and assist with systemic infection. Karaka could be the plant reservoir for an eriophyid mite virus that depends on uninfected mites feeding on the viruliferous chlorotic spots for transmission to its new mite host. This transmission path that severely limits the geographic spread of KŌPV would suggest that KŌPV is also not vertically transmitted by seed nor pollen, both of which require systemic infection, or to putative KŌPV host karaka gall mite offspring. An alternative hypothesis may be a more recent arrival and evolution at or near Ōkahu Bay, which is adjacent to a major port that is currently operational and historically has been home for Māori for ~900 years. Further research on the associations between KŌPV, karaka and karaka gall mite will deepen our understanding of the evolution of the virus and provide further insights into its associations within Aotearoa New Zealand.The novel emaravirus found in karaka has tentatively been named Karaka Ōkahu purepure emaravirus (KŌPV). Parts of the name are in the Māori language to reflect the importance of the endemic host plant to Māori. Many existing karaka groves in New Zealand have cultural significance as a historical food source and are therefore taonga, meaning “treasured”, creating an obligation for conservation of the trees in these groves . Ōkahu iThis present research has raised questions about KŌPV, such as where it came from and whether it is endemic to New Zealand. The phylogenetic analysis indicated that KŌPV shares a common ancestor with the rest of the emaraviruses but appears genetically distinct. A molecular clock study could be performed to determine the evolutionary age of the virus, as has been performed before for many plant RNA viruses ,68. ThisCorynocarpus laevigatus) that belongs to the Emaravirus genus. It is present in Auckland and in parts of the North Island. A te reo Māori name was chosen to reflect the importance of the endemic host plant to Māori. Karaka Ōkahu purepure virus is a novel RNA virus present within karaka (


Apis mellifera, is quasi-globally spread. This successful species acted as and, in some cases, became a maintenance host for pathogens. This systematic review collects and summarizes spillover cases having in common Apis mellifera as the mainteinance host and some of its pathogens. The reports are grouped by final host species and condition, year, and geographic area of detection and the co-occurrence in the same host. A total of eighty-one articles in the time frame 1960–2021 were included. The reported spillover cases cover a wide range of hymenopteran host species, generally living in close contact with or sharing the same environmental resources as the honey bees. They also involve non-hymenopteran arthropods, like spiders and roaches, which are either likely or unlikely to live in close proximity to honey bees. Specific studies should consider host-dependent pathogen modifications and effects on involved host species. Both the plasticity of bee pathogens and the ecological consequences of spillover suggest a holistic approach to bee health and the implementation of a One Health approach.Honey bees, and pollinators in general, play a major role in the health of ecosystems. There is a consensus about the steady decrease in pollinator populations, which raises global ecological concern. Several drivers are implicated in this threat. Among them, honey bee pathogens are transmitted to other arthropods populations, including wild and managed pollinators. The western honey bee,  Interspecific transmission may occur from a definite maintenance host (aka “reservoir”) to an incidental or non-maintenance species (aka “spillover host”). Spillover cases are crucial to pathogen dynamics ,2.In a single-host scenario, reservoirs are sufficient and pathogen replication does not need other host species . The bassensu lato, commonly indicating a multifaceted range of host shift events [Strictly speaking, spillover only occurs when the recipient species is characterized by R0 ≈ 0 . Howevert events .Pollinators are crucial to the generation of crops contributing to the human diet . These aApis mellifera is known to share pathogens with bumblebee species, including viruses, bacteria, fungi and protozoa [Nosema ceranae, Crithidia bombi, and Apicystis bombi [Pathogens and parasites are deemed drivers of this decline, together with other factors including pesticides and global warming. Nonetheless, the global picture is certainly far from complete, since data may misrepresent the actual distribution and gaps remain in our understanding of both epidemiological features and invasion dynamics of many pathogens ,23,24,25protozoa ,32,33,34is bombi . Pathogeis bombi ,23,30,36is bombi ,38,39.Interspecific transmission to arthropods sharing the same environment as honey bees may occur orofecally, via direct contact and by pollen contamination . BesidesHoney bees and other insects have natural immune defense systems against bacteria, protozoa, mites, and viruses. They include antimicrobial peptides like apidaecin, defensin, abaecin, hymenoptaecin, and lysozyme, which are regulated by the immune pathways Toll, IMD, JAK/STAT and JNK . Those dSpillover events are difficult to prove. Indeed, viral infection and replication in new hosts, which may not develop under artificial conditions, can occur in nature ,54,55. Tsensu lato reported by the literature and involving honey bee pathogens. Other arthropods were also considered as alternative hosts. The spillover cases are grouped by: (i) host species, condition and stage, (ii) geographical region and year of the report, and (iii) co-occurrence in the same host.This systematic review is intended to collect, group, and summarize the spillover cases In total, from 1960 to 2021, 81 studies investigated spillover cases of honey bee pathogens to wild and/or managed arthropods . Some ofAs shown in The geographical distribution of spillover studies present in the literature  shows a N. ceranae, SBPV and LSV resulted implicated with progressively decreasing frequency.The chord graph  shows alAethina tumida. A high number of co-infections was reported for Eucera nigrescens, Osmia bicornis and Osmia cornuta, for which 6 pathogens were found in the same individuals. Besides, the most abundant coinfecting pathogens able to co-infect the arthropods hosts were DWV, BQCV, SBV, ABPV and N. ceranae.Some individuals were found infected with multiple honey bee pathogens . The higThe results of this systematic review highlights that the case history of spillover events involving honey bee pathogens increased over the past six decades. This is consistent with the growing interest of the scientific community in understanding the underlying factors ,20,22,54V. destructor  and quasi-ubiquitous pathogens  were among the most frequently reported cases.Viruses vectored by Bumblebees, mason bees and leafcutter bees were the species in which the spillover was studied more intensely, possibly because of their use in crops and fruit pollination. The fact that some of the surveys were carried out on arthropods ranging freely in the same environment as the managed honey bees is indicative of a pathogen circulation in their common environment. Despite honey bee pathogens were detected in other arthropods, symptoms and other effects on the alternative host populations remain unknown—except for some publications reporting individual bumblebees with crippled wings and scoring positive to DWV ,69.Aethina tumida individuals, which shows the importance of other arthropods as incidental hosts. The multiple infections that were identified  virus belonging to e family . The DWVe family ,78.V. destructor [V. destructor mites [The DWV is probably the most known, spread, prevalent, and studied honey bee pathogen, often associated to structor . The DWVor mites .The impact of DWV on honey bees leads to increased interspecific transmission, reaching several species of hymenopterans and other arthropods .A. tumida, G. mellonella, Vespa spp. [Apis and non-Apis species that may act as incidental hosts [B. terrestris and B. pascuorum individuals were found with crippled wings [Bombus species [The virus was identified not only in species living in close contact with the honey bees, like spa spp. ,47,81,82al hosts ,39. DWV al hosts ,59,79, aed wings ,69. Besi species ,83,84,85 species .Cripavirus genus within the Dicistroviridae family [KBV is a non-enveloped ssRNA (+) virus belonging to the e family ,81,87. Te family ,89,90.Although the virus is considered endemic in America and New Zealand, it has been rarely reported in other regions, both in honey bees and other arthropods .Bombus spp. [Eucera spp., Anthophora spp., Osmia spp. [A. tumida [Galleria melonella, earwigs, roaches and crickets [KBV was found in various Hymenoptera species, like bus spp. ,91,92,93mia spp. , wasps, mia spp. ,91,94,95mia spp. ,91. It w. tumida ,81, Gallcrickets ,91.Apavirus genus within the Dicistroviridae family [V. destructor, where is is reported incapable to replicate [Bombus species were found infected in the United Kingdom [Bombus species as well as a wide range of other bee species [Ancistrocerus auctus, Polistes spp., V. germanica, Scolia flavifrons and Linepithema humile [ABPV is a non-enveloped virus and widespread ssRNA (+) virus belonging to e family ,96. As re family . ABPV waeplicate ,98. ABPV Kingdom . The lis species ,100,101.a humile ,54,94.Apavirus genus within the Dicistroviridae family, whose genome shows high homology to ABPV and KBV [Israeli acute paralysis virus (IAPV) is a non-enveloped ssRNA (+) virus, belonging to  and KBV ,102,103. and KBV .Apis bee species  is an icosahedral non-enveloped ssRNA(+) virus from the e family ,106. Thee family ,108.Bombus spp., E. nigriscens and O. bicornis in Kyrgyzstan, Germany and Georgia [E. nigriscens and O. bicornis species in Kyrgyzstan, Germany and Georgia scored positive for SBPV infection [Recently, it was found in wild  Georgia , in the  Georgia ,111 and  Georgia . Furthernfection   is an unclassified enveloped ssRNA (+) virus carachterized by articulate genome and association to a satellite virus (CBPSV) ,114. TheB. dalhbomii [B. impatiens and B. ruderatus [B. pauloensis [B. terrestris [X. augusti and X. nigrocinta [X. dissimilis [H. amplilobus [H. parallelum [C. vagus and F. rufa also [CBPV is capable to infect other insects. Spillover was reported in the wild in individuals of alhbomii , B. impauderatus , B. paululoensis , B. terrrrestris ,116, X. grocinta , X. dissssimilis , H. amplplilobus  and H. prallelum . Replicaufa also .Iflavirus genus and Dicistroviridae family [Apis bees (SBV is a non-enveloped ssRNA (+) virus belonging to e family ,28,119. e family . The vire family ,123,124.pis bees  and othepis bees  [38,39,5Cripavirus genus within Dicistroviridae family. As the other Dicistroviridae, BQCV is non-enveloped and ssRNA (+) virus [BQCV belongs to the +) virus ,131,132.Despite a high prevalence in adult honey bees ,134,135,A. mellifera, A. cerana indica, A. cerana japonica, A. dorsata and A. florea [BQCV is spread worldwideand affects several honey bee species and subspecies, like . florea . The ran. florea ,138,139.. florea ,130.Sinhaliviridae family and Sinaivirus genus, of which two strains have been identified so far: LSV-1 and LSV-2 [LSV is an ssRNA(+) belonging to the nd LSV-2 . The virnd LSV-2 ,141. Andrena spp. [Bombus spp., [Halictidae and Megachilidae [Apoidea superfamily so far.Cases of LSV spillover have been reported in ena spp. ,127, Bomus spp., ,112,127,AmFV is an unclassified dsDNA isolated from honey bees, whose relationship with the host and epidemiology are poorly studied. Originally, the pathogen was described as a rickettsia disease, but recently it has been recognized as a virus [ a virus ,143. Sev a virus ,145,146.Andrena spp. [Bombus spp. [Osmia spp. [A. tumida [Few spillover cases have been reported so far. They involved as alternative hosts ena spp. , Bombus bus spp. , Osmia smia spp.  and in A. tumida  (SupplemVdMLV is an unclassified ssRNA(+) virus of the Tymoviridae family. The mite V. destructor is its primary host and the virus was found in the honey bees as a likely result of the trophic activity of the parasite [VdMLV  structor . Since ites spp. , Vespulaula spp. ,130,150,velutina  and L. h. humile . Besidescapensis   protocols . The resV. destructor, are obligate parasites.The search intentionally excluded arthropods living in close contact with the honey bees that, like The article search was carried out in PubMed, Web of Science, Science Direct, Google Scholar, and Scopus scientific databases for studies aimed to assess the detection of spillover cases of honey bee pathogens. Filters were used to select articles published from January 1960 to April 2021. The last search date was 31 May 2021.Apis mellifera” OR “Honey Bee Diseases” OR “Honey Bee Virus” OR “Honey Bee Bacteria” OR “Honey Bee Microsporidia” OR “Honey Bee Protozoa” OR “Managed Bees” OR “Wild Bees” OR “Commercial Bees” OR “Artificial Infection” OR “Replicative Virus” OR “Bumblebees” OR “Colony Collapse Disorder” OR “Deformed Wing Virus” OR “Acute Bee Paralysis Virus” OR “Israeli Acute Paralysis Virus” OR “Black Queen Cell Virus” OR “Sacbrood Virus” OR “Apis mellifera Filamentous Virus” OR “Kashmir Bee Virus” OR “Slow Bee Paralysis Virus” OR “Lake Sinai Virus” OR “Varroa destructor” OR “Macula-like Virus” OR “Nosema apis” OR “Nosema ceranae” OR “Nosema bombi” OR “Spiroplasma” OR “Ascosphaera” OR “Apicystis” OR “Arthopods” OR “Entomofauna” OR “Hive Hosts” OR “Hive” OR “Free-Ranging Insect” OR “Bee Interaction” OR “Varroa destructor”. The logical operator “OR” was used to combine the descriptors.The following search strategy was designed and utilized: “Honey Bee Pathogens” OR “Bumblebee Pathogens” OR “Spillover” OR “Spill-over ” OR “Inter species transmission” OR “Inter taxa transmission” OR “Host species transmission” OR “Studies carried out both in field and laboratory conditions were selected. Besides, studies that did not assess whether the presence of the honey bee pathogens could be related to external contamination were not included. The detected active replication of honey bee viruses was also reported in the Duplicate studies were excluded. The search and screening for titles, abstracts and results were carried out independently by the authors, including all articles, letters, notes, scientific notes and communications aimed to assess a spillover case of honey bee pathogens and excluding reviews, books, book chapters and theses.The potentially eligible research articles were read and reviewed independently by the authors and the data were compared to ensure integrity and reliability.For each article included in this review, relevant information related to the authors, publication year, host species, host conditions, host stage, pathogens and prevalence were extracted. The data from the eligible studies are expressed in the This review shows that, in recent years, the frequency of recorded spillover cases of honey bee pathogens to other arthropods, including wild bees, has dramatically increased. Certainly, human movements and globalization have fostered the inflow of novel pathogenic microorganisms, often with detrimental consequences. However, it should also be considered that the analytical methods currently available give impulse to the research on bee pathology, increasing the chance to identify interspecific transmission events.A. tumida, G. melonella, V. velutina, L. humile) should be considered with extreme carefulness.The host plasticity shown by some honey bee pathogens raises ecological concern for the potential negative consequences on the pollinating entomofauna and ecosystems in general. Despite the fact that research on these pathogens has significantly improved, we have limited knowledge of their potential impact on other bees, insects, and arthropods in general and the cascade of environmental effects. Laboratory studies are not sufficient to cover this gap, for the intricate interaction of the involved biotic and abiotic factors. For the same reasons, the exploitation of these pathogens in the control of arthropods considered as pests (e.g., The tight interaction between honey bees and the other environmental components suggests a holistic approach to the study of bee diseases, including their control. Indeed, pathogens may survive in alternate hosts, generating spillback events and possibly jeopardizing the efficacy of the treatments. This emphasizes the beekeeper’s responsibility to maintain healthy colonies to benefit both their production and the environment.Spillover of honey bee pathogens may have undetected yet important repercussions on the health and functioning of an ecosystem. Health management of honey bee colonies is of high importance in this context. Honey bees and the beekeeping industry should, therefore, undertake an essential role in the One Health concept. This requires the adoption of dedicated research actions.


Glycogen storage disease (GSD) type IXb is one of the rare variants of GSDs. It is a genetically heterogeneous metabolic disorder due to deficient hepatic phosphorylase kinase activity. Diagnosis of GSD can be difficult because of overlapping manifestations. Mutation analysis of the genes related to each type of GSD is supposed to be problem-solving, however, the presence of novel mutations can be confusing. In this case report, we will describe our experience with a young girl with the diagnosis of GSD and two novel mutations related to GSD type IXb.PHKB gene. The variant in the PHKB gene was classified as pathogenic.A 3-year- old girl presented with short stature, hepatomegaly, and liver cirrhosis. No specific diagnosis was made based on laboratory data, so liver biopsy and targeted-gene sequencing (TGS) were performed to find out the specific molecular basis of her disease. It was confirmed that the patient carries two novel variants in the PHKB were identified and one of them was a pathogenic split-site mutation. In conclusion, for the first time, identification of the novel variants in this patient expands the molecular and the phenotype basis of dual variants in GSD-IXb.This is the first reported case of a dual molecular mutation of glycogen storage disease type IXb in the same patient. Two novel variants in  PHKA2 gene , namely GSD IXa, which is liver-specific with X-linked inheritance. The PHKB gene  indicating GSD IXb encodes the β subunit, and the PHKG2 gene  indicating GSD IXc encodes the γ subunit [Glycogen storage disease (GSD) type IX is caused by phosphorylase b kinase (PhK) deficiency EC 2.7.1.38), a key enzyme in glycogen degradation [.38, a ke subunit . Both ofPHKB gene mutations resulting in deficient phosphorylase kinase in both liver and muscle are very infrequent [Reports on the frequent , 6. GSD frequent . Unlike frequent –10.PHKB gene.So far, there has not been any case report of GSD type IXb from Iran. We present the first case diagnosed in our center, i.e. a child with GSD IXb, whose specific symptoms are related to a dual mutation in the A 3-year-old girl was referred to the pediatrician with hepatomegaly and developmental delay. The girl was the first child of consanguineous marriage in an Iranian family. She was delivered following a normal and term pregnancy with a birth weight of 2.95 kg, and height of 47 cm. No hypoglycemia was noted in the perinatal period and the postnatal transition.At the age of 1, she developed abdominal distension. However, abdominal ultrasound has been reported as unremarkable. During childhood, she frequently experienced morning nausea, vomiting, and lethargy.At the age of 2, she was referred to our center because of developmental delay. Biochemical lab tests revealed high hepatic transaminases , mild neutropenia (750 per microliter), microcytic hypochromic anemia , as well as high triglyceride (498 mg/dl), and cholesterol (268 mg/dl).A abdominal ultrasound revealed hepatomegaly with mild diffuse heterogeneous echogenicity of the liver parenchyma. A liver biopsy was performed, which revealed cirrhosis with severe swelling of the hepatocytes with clear cytoplasm. Portal tracts showed very mild lymphocytic infiltration Fig. . AccordiPHKB gene. In silico analysis revealed that the novel variant, c.1127-2 A > G (p.?) is pathogenic, which would have possibly damaged the splice site, and two other ones are variants of uncertain significance (VUS). However, samples from the parents were not available for the zygosity determination of these novel variants. It should be mentioned that both parents were asymptomatic.Targeted gene sequencing (TGS) with a custom-targeted Ion AmpliSeq panel was performed. The panel included 7219 amplicons covering 450 genes associated with Inborn Metabolic Diseases consisting of glycogen storage disorders genes with hepatic involvement. Sanger sequencing validated identified the variants, using an ABI Prism 3500 Genetic Analyzer . Analyses were done using an Ion Torrent 540 chip . The human genome 19 was used as the reference. Polyphen2, SIFT, and Mutation Taster were used for in silico analysis, GERP and Phastcons scores were used to evaluate the conservation of the variants. The population frequency of each variation was evaluated, using data from the gnomAD database. ACMG guidelines were used for variant interpretation . The seqPHKB gene, leading to loss of function, in a 3-year-old girl born to a consanguineous family of Iranian descent.Herein we are reporting novel homozygous variants in the PHKB, our patient who has dual variants showed a more severe short stature and liver dysfunction. To find and carefully evaluate all reported mutations and effects on the presentation of GSD IXb, we did a literature search in August 2020. To date, 23 variants were identified in PHKB gene in 18 patients with GSD-IXb (Table To the best of our knowledge, our patient is the only and the first case of dual homozygote variants in GSD-IXb, with severe liver involvement. Compared with other GSD-IXb patients with a single mutation in Molecular method confirmed a dual mutation in GSD-IXb. Co-occurrence of two different mutations in GSD subtype in one patient is exceedingly rare and has never been reported so far. Posey et al.  reportedPHKB gene were identified, one of which is pathogen. The report of these variants could expand the mutation spectrum of GSD-IXb. Dual mutations in the GSD subtype in one patient is rare; however, with the progress in molecular diagnostic methods, we may be able to identify more patients with multiple mutations in different genes, and because of that, our knowledge about inherited human diseases will be improved.In conclusion, our study describes an Iranian patient who suffered from GSD-IXb. Two novel variants in the


However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days while maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus, and no viremia or clinical signs were observed postimmunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood.The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress toward vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study, deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages IMPORTANCE African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R gene alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing that the proteins act synergistically. Importantly, the infected pigs were protected following infection with the wild-type virus that kills pigs. Ornithodoros species that inhabit warthog burrows, can be persistently infected but show few disease signs (https://www.fao.org/ag/againfo/programmes/en/empres/ASF/situation_update.html). Since there are no vaccines or targeted therapeutics currently available, control relies on implementing strict biosecurity measures and culling of infected herds.African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs and wild boar with case fatality approaching 100%. In contrast, its wildlife hosts in Africa, warthogs, bushpigs, and soft ticks of se signs . ASFV haAsfarviridae family and has a predominantly cytoplasmic replication. The virus genome contains up to 167 genes, including many that are not required for the virus to replicate in cells but have roles in interactions with the host to facilitate its survival and transmission  and pEP153R , 12. Thes effect . Interacs effect , 18. Thes effect . Interess effect . An invos effect .2+-dependent glycan-binding proteins that are involved in cell-cell adhesion playing key roles in both innate and adaptive immune responses. For example, C-type lectin receptors (CLRs) are important for recognition and capture of pathogens, as these pattern recognition receptors (PRRs) have a high affinity for their ligands, which results in internalization of the pathogens  alone was deleted from BeninΔDP148R or both genes EP153R and EP402R  were deleted simultaneously. These gene(s) were replaced by mNeonGreen under the control of VP30 promoter in the attenuated BeninΔDP148R virus (8R virus  and D. I8R virus . The expin vitro. At different days postinfection (17 50% tissue culture infective dose (TCID50)/mL between 24 and 48 h postinfection and were maintained for the remainder of the culture time. The results show that deletion of the DP148R, EP153R, EP402R, or EP153R and EP402R gene did not significantly alter the ability of the virus to replicate in culture.Porcine bone marrow cells were infected with the Benin 97/1, BeninΔDP148R, BeninΔDP148RΔEP402R, BeninΔDP148RΔEP153RΔEP402R, and BeninΔDP148RΔEP153R at a multiplicity of infection (MOI) of 0.01 to determine if deletion of the genes affected the ability of the virus to replicate fection 15, total fection 1. Virus t3 TCID50 as we used in the previously published experiments (eriments  with the4 TCID50 of the triple deletion mutant BeninΔDP148RΔEP153RΔEP402R (group B). The higher dose, compared to that of experiment 1, of 104 TCID50 was chosen, as we expected that the triple gene-deleted virus may be more attenuated by the additional deletion of the EP153R gene. An additional boost with 106 TCID50 was carried out at day 28, as measurement of cellular responses at day 21 indicated that low levels of ASFV-specific IFN-γ producing cells had been induced and we wanted to increase the chances of inducing a protective response against challenge.In experiment 2, we immunized pigs and boosted at day 21 with 105 TCID50 BeninΔDP148RΔEP153R (group C) and 105 TCID50 BeninΔDP148R (group D), respectively. The aim of these experiments was to determine the effect of the additional deletion of EP153R from the BeninΔDP148R virus. An additional aim was to determine the safety of both viruses delivered at the higher dose of 105 TCID50 compared to the previous dose of 103 TCID50.In experiments 3 and 4, we immunized and boosted pigs with 104 50% hemadsorption dose (HAD50) in 1 mL, while group B and group F were challenged at 45 dpi with 103 HAD50 in 1 mL with virulent Benin 97/1 virus (One pig in group A (A4) was euthanized at day 9 postimmunization due to a non-ASFV-specific condition. Naive, nonvaccinated pigs  served as controls for the challenge with the virulent Benin 97/1 isolate. Groups A and E were challenged intramuscularly at 42 days postimmunization (dpi) with 10/1 virus . Groups Rectal temperatures and clinical scores  were recIn group B (BeninΔDP148RΔEP153RΔEP402R), no increase in temperature or other clinical signs were observed in any of the pigs before challenge. After challenge, an increase in temperature at or above 41°C was observed in one pig at 3 days postchallenge and in the remaining pigs at day 4 postchallenge . Pigs B3In group C (BeninΔDP148RΔEP153R), moderate clinical signs were observed after immunization, comparable with those of group D (BeninΔDP148R), with increased temperatures above 40.5°C for 1 or 2 days between days 3 to 6. However, after challenge, none of the pigs from either of the groups showed any clinical signs or temperatures  and D. IIn group D (BeninΔDP148R), pigs D1, D2, and D3 had increased temperatures at day 2 postimmunization, with temperatures ranging from 40.6 to 41°C. Pigs D4 and D5 showed increased temperatures on day 4, but these dropped on the following day . No tempApart from mild lymphadenopathy, pigs in group A  immunized with BeninΔDP148RΔEP402R did not show any other relevant macroscopic lesions. In this group, pig A4 was euthanized due to welfare reasons before challenge, not displaying lesions characteristic of ASF . In grou5 to 106 genome copies per milliliter in pigs A1 and A2 and 103 in pig A3 (Levels of virus genome in blood were measured by qPCR and infectious virus by titration in porcine bone marrow cells. In group A (BeninΔDP148RΔEP402R), virus genome was first detected at day 5 postimmunization, coinciding with the onset of clinical signs, and increased to a peak of between 10n pig A3 . Virus g3.75 to 104.5 per milliliter in pigs A1 and A2. In pig A3, infectious virus was detected at a level below accurate measurement. Infectious virus was not detected by day 14 postimmunization in A1 and A3, and only very low levels were detected in pig A2 at this time. No infectious virus was detected after boost or challenge.Titration of infectious virus from blood showed a similar pattern, although detection was lower than that of genome copies . Infecti5.3 and 106.2 genome copies per milliliter. In the surviving 6 pigs, the maximum levels detected varied between ∼102 and 105.9 genome copies/mL. Levels of virus genome decreased until termination at day 20 postchallenge, becoming undetectable in 2 pigs (B5 and B8) and reduced to 102.6 in others . Only in one pig (B7), levels remained at 104.5 at termination.In group B (BeninΔDP148RΔEP153RΔEP402R), no virus genome was detected in blood from pigs before challenge . After c5.25/mL in pig B3 and 106.75/mL in pig B4 (6.75/mL) at day 7 postchallenge, reducing to 104.75/mL at the end of the experiment. The peak infectious virus after challenge in the other pigs varied between 104.25 and 105.25, except that for pig B5, which had infectious virus below the level of accurate measurement. At termination of the experiment, very low or no infectious virus was detected in these pigs.In group B, no infectious virus was detected in blood before challenge even when the volume added to titrations was increased 10-fold. After challenge, levels of infectious virus were detected at day 3 postchallenge in pigs B3 and B4 and reached levels at termination of 10n pig B4 . In the 6.7 to 107.6 genome copies/mL) at 3 days postimmunization for all pigs except C2, in which levels peaked at day 7. These values then declined gradually but persisted throughout the experiment and had decreased to between 104 to 105 genome copies/mL by the end of the study (day 60)  had higher levels of genome copies in blood (approximately 10(day 60) . Levels (day 60)  in blood6 to 106.8 genome copies per milliliter by day 4 postimmunization, and thereafter levels of genome decreased gradually to between 102 to 103.6 genome copies/mL at termination on day 60  had between 10n day 60 . Levels n day 60  also peaP < 0.01) than those for group D, and this difference persisted throughout the study . However, no significant differences were detected in levels of infectious virus comparing groups C and D.Levels of genome copies in blood at day 10 postimmunization for group C were significantly higher (7 to 108 per milliliter) were detected in all pigs from each control group .As expected, very high levels of virus genome and infectious virus  from immunized pigs to ASFV were measured at different times postimmunization by IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay. An ASFV-specific response was not detected before immunization in any group of pigs. Very high numbers of IFN-γ-producing cells  were induced in all pigs in group A before boost, which then decreased before challenge to levels ranging from 178 to 463 spots/million cells . In contAntibody responses to ASFV p72 capsid protein were measured using a commercially available blocking ELISA. As expected, an antibody response was detected in all pigs in group A by day 14 and increased after boost . In contTargeted deletions of nonessential genes from the ASFV genome have been used successfully to construct candidate live attenuated vaccine strains and to understand the role of the genes during infection of cells and pigs. A vaccine candidate with an acceptable safety profile should have reduced clinical signs and vaccine virus persistence postimmunization but retain high levels of protection.One of the potential candidates for targeted deletions represents the EP402R gene, which encodes the CD2v protein, since it is required for the binding of infected cells or extracellular viral particles to red blood cells, thus playing an important role in viral dissemination in different tissues in pigs. During the sylvatic cycle, in ticks, the binding of the virus to red blood cells probably helps retention of the virus with the blood meal and also crossing the midgut barrier . Deletio3 HAD50, virus genome levels peaked by day 5 or 6 postimmunization and gradually declined but persisted until the end of the experiment at day 59. Infectious virus also peaked at day 5 or 6 postimmunization but declined more rapidly and was not detected after day 28 postimmunization. Similar results were obtained here using a higher dose of 105 HAD50/mL of the BeninΔDP148R virus (group D) and 105 HAD50 mL of the BeninΔDP148RΔEP153R virus (group C). In both groups C and D, a moderate level of virus genome in blood was observed to peak early postimmunization, coincident with the onset of clinical signs, and gradually declined, although levels of genome of 102 to 104 per milliliter were detected at termination on day 59 or 60 postimmunization. Levels of infectious virus were similar to genome levels at 3 or 4 days postimmunization but then declined more rapidly, and no or very low levels of infections virus were present by day 39 or 40 postimmunization, a few days before challenge. These results are consistent with an early virus replication in blood coinciding with clinical signs. The gradual decline of virus levels in blood thereafter is consistent with our hypothesis that virus persists attached to the red blood cells mediated by the CD2v protein on the surface of extracellular virus particles. The more rapid decline of infectious virus in comparison to that of genome may be due to a loss in infectivity of the nonreplicating virus attached to red blood cells over time. Comparison of immunizations with 103 or 105 HAD50 of BeninΔDP148R showed that peak virus genome and infectious virus levels after immunization were similar as were clinical signs after immunization and challenge. The time to the onset of clinical signs was reduced by 1 or 2 days following immunization with the higher dose.Our approach to reduce the virus persistence and clinical signs postimmunization of pigs with a moderately attenuated ASFV isolate, BeninΔDP148R , was to In contrast to these results, from the immunizations of pigs with BeninΔDP148R and BeninΔP148RΔEP153R, deletion of the EP402R gene from the BeninΔDP148R virus resulted in a much shorter period of virus persistence in blood. Immunization of pigs with the virus BeninΔDP148RΔEP402R (group A) resulted in detection of both virus genome and infectious virus in blood only until day 14 postimmunization  7). The. The7). The group A immunized pigs still displayed moderate clinical signs postimmunization and mounted a strong immune response since all pigs were protected against challenge with the virulent virus. We were surprised that deleting the EP402R gene did not have a greater effect on reducing clinical signs postimmunization, since attenuation was observed when this gene was singly deleted from the virulent BA71 virus, another genotype I isolate . In orde23–in vitro , pigs did not show any clinical signs or viremia before challenge . Howeverin vitro . An intr6 genome copies/mL)  , with 2 in vivo. This may be mediated by the cooperation of both proteins in binding of virus particles and infected cells to red blood cells and possibly other cells. In addition, the two proteins may also act singly or synergistically to evade initial steps of the immune response. A role in immune modulation has already been shown for the EP402R protein. C-type lectin proteins have diverse roles in mediating cell-to-cell adhesion. For example, C-type lectin receptors play a crucial role in natural killer (NK) cell activity. It is tempting to speculate that EP153R may have evolved to evade NK host immune responses since NK activity was shown to correlate with protection following immunization with the attenuated NH/P68 isolate  or endpoint titers based on expression of mNeonGreen or TagRFP-T fluorescent protein markers and presented as TCID50/mL, calculated using the Spearman and Karber formula. To increase the sensitivity of virus detection, blood samples with no detectable infectious virus by titration were further tested by adding 100 μL of the sample to 6-well plates seeded with PBMs.The ASFV Benin 97/1 wild-type and BeninΔDP148R isolates have been described elsewhere . DeletioTransfer plasmids. Transfer plasmid pΔEP402R-VP72GUS was constructed by cloning amplified right and left regions of the Benin 97/1 flanking the EP402R gene into the previously published pLoxPVP72GUSLoxP vector (P vector .-GGATCCTTATTATTTTATAATTTTAAAATTGAATGGATTTTATTTTAAATATATCCATGGTGTCTAAGGGCGAAGAGCT) and a reverse primer containing an ASFV transcription termination signal (bold) (GAATTCAAAAAAAAAACTTGTACAGCTCGTCCATGCCAT). The TagRFP-T fluorescent marker was kindly provided by Chris Netherton . The amplified product was then swapped with the VP72GUS cassette of pLoxPVP72GUSLoxP vector. Using this newly produced pLoxPVP30TagRFP-TLoxP, the left and flanking regions of DP148R were then cloned to produce transfer plasmid pΔDP148R-VP30TagRFP-T.Vector pLoxPVP30TagRFP-TLoxP was generated by first amplifying the TagRFP-T gene  with a fl (bold)  and EcoRThe other transfer plasmids pΔEP153R-VP30mNG and pΔEP153RΔEP402R-VP30mNG were synthesized commercially . pΔEP153R-VP30mNG contains the left and right flanking regions of EP153R, while pΔEP153RΔEP402R-VP30mNG contains the left flanking region of EP153R and the right flanking region of EP402R. In both plasmids, between the flanking regions, a reporter gene, mNeonGreen (mNG), under the control of the ASFV VP30 promoter flanked with LoxP sites was added .Homologous recombination and virus purification. Recombinant ASFV BeninΔDP148RΔEP402R was produced in a sequential 2-step deletion method. First, BeninΔEP402R  were added to purified PBMs at an MOI of 0.01 in triplicate in 24-well plates. Cells and supernatants were collected at different times postinfection and subjected to 3 freeze-thaw cycles. Cellular debris was removed by centrifugation, and virus titers were determined by the fluorescence assay or HAD, as described above. The experiment was carried out in purified PBMs from 2 different pigs.3 TCID50 BeninΔDP148RΔEP402R and boosted by the same route with the same recombinant virus at 104 TCID50 on day 20 postimmunization. In experiment 2, 8 pigs were immunized by the intramuscular route with 104 TCID50 BeninΔDP148RΔEP153RΔEP402R and were boosted twice with the same virus at 104 TCID50 on day 21 and at 106 TCID50 on day 28 postimmunization. The additional boost with 106 TCID50 was performed following the measurement of both cellular and antibody responses at day 21, which indicated low levels of ASFV-specific antibodies and that IFN-γ-producing cells had been induced. In experiment 3, 6 pigs were immunized by the intramuscular route with 105 TCID50 BeninΔDP148RΔEP153R and boosted with the same dose on day 21 postimmunization. In experiment 4, 6 pigs were immunized by the intramuscular route with 105 TCID50 BeninΔDP148R and boosted with the same dose on day 21 postimmunization. Nonimmunized control pigs and immunized pigs were challenged by the intramuscular route with either 104  or 103  HAD50 of the virulent Benin 97/1 isolate approximately 3 weeks after the boost  were immunized by the intramuscular route with 10he boost .Daily clinical scoring was carried out to record temperatures, anorexia, behavior including lethargy, vomiting, or hemorrhagic diarrhea, respiratory distress, lameness, and hemorrhagic skin lesions . Pigs we8 to 101 copies/mL. Results were reported as log10 genome copies/mL.DNA was extracted from whole blood using MagVet universal isolation kit (Life Technologies) at different days throughout the study. Samples were assayed in duplicate for the presence of viral DNA by quantitative PCR (qPCR) on a Stratagene Mx3005P system  following a protocol modified  from usiThe level of antibody responses against ASFV-p72 in serum was measured using a commercial blocking ELISA  following the manufacturer’s instructions. The percentage of blocking (PB) was calculated using the following formula: [(negative-control OD − sample OD)/(negative-control OD − positive-control OD)] × 100, where OD is optical density. Samples were considered positive if the PB was above the cutoff value of 50% blocking.5 and 4 × 105 per well), in RPMI 1640, Glutamax (Gibco) supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The cells were then incubated overnight at 37°C in a final volume of 200 μL with 105 HAD50 of Benin 97/1, an equivalent volume of mock inoculum, or 20 μg/mL phytohemagglutinin as a positive control. Cells were lysed by incubation for 5 min in water and then washed with PBS. Following incubations with biotinylated anti-porcine IFN-γ monoclonal antibody  and streptavidin conjugated to alkaline phosphatase, AP conjugate substrate kit (Bio-Rad) was used to develop spots. The spot-forming cells were then counted using an ELISpot assay reader system . The number of spots per well was converted into the number of spots per million cells, and the mean for duplicate wells was plotted using GraphPad Prism 8 software. No cells were collected for pigs belonging to group D.Peripheral blood mononuclear cells (PBMCs) were collected at 3 different time points, including preimmunization, preboost, and prechallenge. PBMCs were purified from EDTA blood tubes using Histopaque-1083 or -1077 gradient medium. ELISpot plates were coated overnight at 4°C with 4 μg/mL IFN-γ monoclonal antibody  in 0.05 M carbonate-bicarbonate coating buffer. After incubation, plates were washed four times with phosphate-buffered saline (PBS). Cells were plated in duplicate at two different dilutions  followed by Dunnett’s multiple-comparison test was performed to evaluate the


Culicoides-borne viruses such as bluetongue, African horse sickness, and Schmallenberg virus cause major economic burdens due to animal outbreaks in Africa and their emergence in Europe and Asia. However, little is known about the role of Culicoides as vectors for zoonotic arboviruses. In this study, we identify both veterinary and zoonotic arboviruses in pools of Culicoides biting midges in South Africa, during 2012–2017. Midges were collected at six surveillance sites in three provinces and screened for Alphavirs, Flavivirus, Orthobunyavirus, and Phlebovirus genera; equine encephalosis virus (EEV); and Rhaboviridae, by reverse transcription polymerase chain reaction. In total, 66/331 (minimum infection rate (MIR) = 0.4) pools tested positive for one or more arbovirus. Orthobunyaviruses, including Shuni virus (MIR = 0.1) and EEV (MIR = 0.2) were more readily detected, while only 2/66 (MIR = 0.1) Middelburg virus and 4/66 unknown Rhabdoviridae viruses (MIR = 0.0) were detected. This study suggests Culicoides as potential vectors of both veterinary and zoonotic arboviruses detected in disease outbreaks in Africa, which may contribute to the emergence of these viruses to new regions. Culicoides midges have been implicated in the transmission of various pathogens of medical and veterinary importance [Reoviridae: Orbivirus) [Peribunyaviridae: Orthobunyavirus) into northern Europe [Culicoides midges (Diptera: Ceratopogonidae) as arbovirus vectors in establishing epidemics in animals. To date, over 50 arboviruses have been isolated from Culicoides, 45% of which have not been detected in other arthropods [Culicoides species, and as yet, little information exists on the role of Culicoides as vectors for zoonotic pathogens [portance . The unpbivirus)  followedn Europe  and the n Europe  highlighthropods . A recenPeribunyaviridae: Orthobunyavirus) which are responsible for congenital deformities in sheep and cattle and from mild febrile illness to severe disease in humans [Flaviviridae: Flavivirus) [Peribunyaviridae: Orthobunyavirus) [Nairoviridae: Nairovirus) [Phenuiviridae: Phlebovirus) [Culicoides pools, although the role of midges in the epidemiology of these viruses is unknown.Biting midges have been implicated as the primary vectors of SBV and Oropouche virus (OROV)  , SHUV (Pyavirus) , Dugbe vrovirus) , and Rifbovirus)  have alsCulicoides midges collected in three provinces in South Africa to identify zoonotic arboviruses that may contribute to disease in animals and humans in urban settings as well as at the human/livestock/wildlife interface.In this study, we screened Culicoides midges were collected monthly from September 2012 to May 2017, at six sites in the three provinces, for two consecutive nights, using 220 V Onderstepoort light traps, as previously described [Vector surveillance sites  were selescribed ,16. SubsCulicoides midges were separated from other insects using a light microscope and pooled (100 ≤ n ≤ 500), according to site and month, and homogenized using a TissueLyser™ . Following maceration, RNA was extracted using the QIAamp® Viral RNA mini kit (QIAGEN), according to the manufacturer’s instructions. During the summer months, when insect numbers were high, testing was limited to only two randomly selected pools per site per month. Whereas during winter months, insect numbers declined, therefore, only one pool was available and tested and, in some cases, no insects were collected, and no pools tested (n < 100). Pool sizes were, therefore, determined based on the number of midges collected per site, per month, with a maximum of 500 and a minimum of 100 midges per pool.Alphavirus [Flavivirus, [Orthovirus [Phlebovirus [Rhabdoviridae [Bunyaviridae RT-PCR. The One Step Bunyaviridae conventional RTPCR was used to obtain a > 500 nt gene regions for known orthobunyavirus positives. SuperScript® III/Platinum® Taq Mix  was used in combination with 20 pmol each of BUN 1 and BUN 2 primers [https://www.qiagenbioinformatics.com (accessed on 19 September 2019)) and MEGA 6.06 software (https://www.megasoftware.net (accessed on 20 September 2019)). Multiple sequence alignments were compiled using the online version of MAFFT version 7 software (http://mafft.cbrc.jp (accessed on 19 September 2019)). All assembled sequences were compared to the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 19 September 2019)). Sequences were also compared to positive controls to exclude false positives. The sequences >300 nucleotides (nt) were submitted to GenBank  were used to screen pools for arboviruses in the genera phavirus , Flavivivivirus,  Orthovirthovirus , and Phlebovirus , as wellebovirus  and Rhaboviridae . All ort primers  in a 50  primers . All SHUhttps://www.cdc.gov/epiinfo/index.html (accessed on 20 October 2019)) using Fisher’s exact test with a 95% confidence interval (CI). The minimum infection rate (MIR) was calculated, using PooledInfRate (www.cdc.gov/ncidod/dvbid/westnile/software.htm (accessed on 10 July 2021)), per location as:  × 1000). The MIR uses the assumption that a positive pool contains at least one infected specimen [Statistical analyses were done in Epi Info version 7.2.0.1  with peak activity in February and a decrease in numbers during winter months (June to August), although never zero. In total, 66/331 pools tested positive for one or more arbovirus, resulting in a MIR of 0.4  0.0–0.5)  . A totalividuals . From th0.0–0.5) . The hig0.0–0.5) .p = 0.9). The orthobunyaviruses were further identified using phylogenetic analyses described below. Shuni virus (SHUV) was the predominant orthobunyavirus  detected, while 15 other orthobunyaviruses  were also identified , and Boschkop; GAU093_14 (Sabo virus/EEV)  and EEV  were equally abundant (entified . Two Midrus/EEV) .Orthobunyavirus phylogenetic analysis based on a 152 nt sequence of the S segment identified two major clades of the Simbu serogroup, clade A (bootstrap (bs) = 100) and clade B (bs = 100)  (n = 5), Sabo virus (SABV) (n = 2), Sango virus (SANV) (n = 3), Schmallenberg virus (SBV) (n = 2), Satuperi virus (SATV) (n = 1), Ingwavuma virus (INGV) (n = 1), and two unknown viruses (s = 100) . Of thess = 100) . Further viruses . Sample  viruses .The phylogenetic analysis of a 503 nt sequence of the S segment  as well Phylogenetic analyses based on a 198 nt gene region of the partial Alphavirus NsP4 gene confirmed the two MIDV positives, with samples MN29_15 and MN36_16 clustering with samples obtained from horses in South Africa (bs = 98)  with theEquine encephalitis virus phylogenetic analysis based on the NS3 gene (Segment 10) (401 nt)  cluster Rhabdoviridae viruses  to Riverside- (RISV) and Tongilchon virus (TCHV) (bs = 99) which, in turn, was well supported as a yet unclassified monophyletic group (bs = 99). The p-distance analysis (not shown) reveals the highest nucleotide identities (86.6%) of samples MN009_15, MN23_15, and MN47_16 to RISV .Phylogenetic analysis of the L segment of three  viruses  confirmeCulicoides to detect viral RNA previously implicated in outbreaks in animals in South Africa and establish a possible association of Culicoides as vectors for the identified viruses. Midge pools, collected from 2012 to 2017, tested positive for viruses from the Alphavirus and Orthobunyavirus genera and family Rhabdoviridae as well as EEV. No Flavivirus or Phlebovirus positives were reported. The highest positivity of viruses was detected in February followed by January and March. This is typical seasonality for arboviruses in South Africa as the arbovirus season is late summer to autumn (February to May). Pools collected during winter months were small or not collected at all, resulting in no positives during July. Overwintering of viruses has, however, been demonstrated for BTV [This study used field surveillance and molecular screening of  for BTV  and AHSV for BTV  in SouthCulicoides pools as well as in malformed livestock in Israel [Culex mosquitoes [Orthobunyaviruses, specifically, SHUV had the highest prevalence across the sites with the highest minimum infection rate in Marakele National Park, while no orthbunyaviruses were detected in KNP in our collection. Phylogenetic analysis based on a 152 nt sequence of the S segment clustered the newly sequenced SHUV strains with known SHUV sequences from horses  possiblyn Israel . Antibodn Israel  and Mozan Israel , indicatn Israel . The Ingsquitoes . Since tsquitoes ,37.Additional PCR assays were successful in obtaining longer sequences for six orthobunyavirus positive samples and could confidently identify five viruses based on the S segment. One orthobunyavirus positive remained unidentified (LAP066_13). This may suggest a new or uncharacterized virus and requires further investigation including additional reference sequences. The conflicting data for sample KYA358_17 could be because the S segment is highly conserved, where the smaller sequence is very similar to various members of the broader Simbu complex with the larger, 503 nt sequence revealing higher identity to SHUV. The variability of the S segment between strains could also be due to reassortment as the multi-segmented nature of orthobunyaviruses allows for genetic exchange to increase the fitness, spread, and pathogenicity of the virus or serogroup . The inaCulicoides pools during the surveillance period suggests that the virus is present in the area and that the midges were able to ingest the virus but that they might play a less important role as a vector. Phylogenetic analyses cluster the MIDV detected in the midges with MIDV strains found in horses [Culicoides [During the six-year surveillance, MIDV was only detected twice in midge pools from KNP, despite detecting animal cases . The lown horses . This con horses . No flavlicoides , althougCulicoides midges, specifically C. imicola and Culicoides bolitinos, are the primary vectors for EEV. Cases of febrile, neurological, and respiratory disease in horses infected with EEV were also frequently detected across South Africa during the same period [Zebra burchelli) from KNP [The high prevalence of EEV is not surprising, as e period . Equine e period . No EEV from KNP , althougRhabdoviridae family were accidentally discovered after the flavivirus genus PCR produced bands of the right size on gel electrophoreses. Sanger sequence and p-distance analyses revealed uncharacterized viruses from the Rhabdoviridae family. The phylogenetic analysis of the L segment clusters these viruses with unclassified viruses (RISV and TCHV), although further sequence data is needed to characterize the virus species. The Riverside virus was first isolated in Hungary from an unknown Aedes species, while TCHV was isolated in South Korea from Culex bitaeniorhynchus [Culicoides-associated viruses such as Sweetwater Branch, Bivens Arm, and Tibrogargan virus indicates possible incidental ingestion. Another reason could be that both RISV and TCHV were only recently discovered and could be associated with Culicoides and warrant further investigation. The Culicoides-transmitted Rhabdoviridae viruses has been implicated as disease-causing agents in Australia, the Americas, Asia, and Africa [The four viruses from the rhynchus ,43. Groud Africa ,45, as wd Africa .Unfortunately, virus isolation was unsuccessful on mammalian cells (Vero and BHK) and will, in the future, be attempted on insect cells such as C6/36 or KC cell. Further attempts at next generation sequencing and virus isolation will be made to resolve the unplaced and unidentified viruses. A limiting factor could be large pool sizes resulting in decreasing detection rate, while future studies could benefit by decreasing pool sizes to <100.Orthobunyavirus MIR in Lapalala Wilderness was <1, indicating a low probability of an outbreak, a SHUV outbreak in wildlife occurred from the end of 2017 to mid-2018 [Culicoides population was sampled and more extensive surveillance could have accurately predicted the outbreak. Marakele National Park had the highest virus diversity with various orthobunyaviruses and EEV detected. In Mpumalanga, there is a strong human–livestock–wildlife interface at the collection sites. Spillovers of disease from wildlife to domestic animals have been reported in these areas [Culicoides species from subsamples collected at the various surveillance sites demonstrates the high abundance of C. imicola at all sites, supporting previous findings [C. imicola and Culicoides schultzei in areas with high infection rates could provide valuable insight into these species as vectors for emerging arboviruses in South Africa.The six vector surveillance sites were selected based on previous outbreaks of neurological disease in animals due to arbovirus infections as well as the strong humans–animal interface at these sites ,13,14,15mid-2018 ; only a se areas . In Boscse areas , SHUV [2se areas , MIDV [1findings ,50. The Culicoides midges, because oral susceptibility and transmission as well as the association of the infected arthropod with the vertebrate population in which the infection is occurring need to be demonstrated [Culicoides, however, represent a non-invasive method to determine virus presence in an area without using invasive methods such as to immobilize and bleed animals or involved human patients. Previous studies have also implicated field collected Culicoides in the spread of SHUV [Culicoides nubeculosus and the North American Culicoides sonorensis [Taking everything into consideration, detection of viral RNA by PCR does not prove vector status for nstrated . Screeni of SHUV  and succnorensis .Culicoides collected in wildlife and urban areas in South Africa. Even though vector competence was not shown, the data suggest that Culicoides may play a role in disease outbreaks warranting further investigations into their potential as vectors for the identified viruses and their capacity to spread these pathogens to new regions.This study identified viruses of medical and veterinary importance from RNA isolated from


The Research Topic was initiated in October 2019 to demonstrate how next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats-associated Cas protein (CRISPR-Cas) editing are being applied in plant virology. NGS combined with bioinformatics has changed both basic and applied research in many biological disciplines by its ability to deliver fast, inexpensive and accurate genome and transcriptome information  Utilization of NGS in research and diagnosis of plant viruses and viroids; (2) Direct targeting of specific nucleotide sites of plant viruses and viroids by CRISPR-based genome editing to enable the fast introduction of resistance; (3) Targeting sites of specific plant genes such as eIF4E gene or eIF (iso) 4E gene by CRISPR-Cas editing to develop plants resistant to viruses; (4) Application of the CRISPR-Cas systems for rapid and accurate diagnosis of plant viruses and viroids,. Sixteen articles have been included in this Research Topic, comprising four reviews and 12 research papers. Nine of the research papers were on plant viruses and three on viroids. Articles dealt with utilization of NGS in discovering and characterizing novel and known viruses and viroids, and applying CRISPR-Cas editing to plant translation factors for developing plants resistant to virus infection, and for rapid detection of RNA plant viruses.Li et al.). It was also revealed by NGS-based analyses of mRNA and sRNA of grapevine in India that 23 known viruses and viroids occur in this host . The mRNA -based approach identified more pathogens than that of the sRNA. The former approach was at the same level as that of the whole transcriptome in viral identification. Genomes of 19 viruses and viroids were characterized. Identification of three recombination events and phylogenetic analyses using characterized genomes suggested possible introduction of grapevine viruses and viroids into India from several continents through infected vegetatively propagated planting material. In kiwifruit, NGS analyses revealed high molecular diversity in Actinidia virus 1 (AcV-1) populations, with the highest sequence variation among its 12 open reading frames (ORFs) occurring at ORF1a, ORF2, and ORF3 . Different domain compositions were shown for the first-time in the viral ORF1a. In addition, molecular recombination events among AcV-1 variants were revealed.Apple russet ring and green crinkle diseases were of undetermined etiology for many years. Using NGS, other technologies, and applying Koch's postulates, it was demonstrated that one of the sequence variants of apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on fruits of infected apple trees and that a sequence variant of apple stem pitting virus causes green crinkle symptoms on an infected apple fruit. Koch's postulates were fulfilled to demonstrate the viral etiology of both apple diseases  and a large number of betaflexiviruses were discovered by NGS . NGS and phylogenetic analyses of two of the novel viruses, tentatively named camellia chlorotic ringspot viruses, suggested that they may represent a novel genus in the family Fimoviridae. The other three novel viruses belong to the genera Idaeovirus, Badnavirus, and Marafivirus. These findings may serve as a basis for better management of the above viruses in common camellia and possibly other hosts. NGS was also used for deciphering the virome of alfalfa plants which led to the characterization of several previously known but not fully described viruses as well as the identification of many novel viral pathogens, including alfalfa dwarf virus, alfalfa enamovirus, alfalfa leaf curl virus, alfalfa virus F., alfalfa ringspot-associated virus, and others . Exploring the alfalfa virome by NGS has also demonstrated the impotence of viral infections in a single plant. NGS from a mixed infection of an unknown cultivar of Veronica sp. revealed the genome sequences of two isolates of helenium virus S (HelVS) and two distinct isolates of butterbur mosaic virus (ButMV), ButMV-A and ButMV-B. A major deletion in an essential gene of ButMV-B was identified for the first time by NGS which is maintained through complementation by ButMV-A. The HelVS host range was extended to Veronica sp. and it was confirmed that the virus is a distinct species in the genus Carlavirus . The full-length genome of a novel emaravirus was identified and characterized by NGS from diseased symptomatic sycamore maple (Acer pseudoplatanus)—a tree species of significance in urban and forest areas. The virus was tentatively named maple mottle-associated virus (MaMaV). Phylogenetic and sequence analyses place MaMaV in the distinct “subgroup a” clade within the Emaravirus genus. This is the first time an emaravirus has been described from maple and fully genetically characterized . NGS also allowed the discovery and genome sequences of at least 70 negative-sense and ambisense RNA (NSR) plant viruses. These viruses belong to several genera in seven families: Ophiovirus in the family Aspiviridae; Coguvirus, Rubodvirus, and Tenuivirus in the family Phenuiviridae; Orthotospovirus in the family Tospoviridae; Emaravirus in the family Fimoviridae; Cytorhabdovirus, Betanucleorhabdovirus, Alphanucleorhabdovirus, Dichorhavirus, and Varicosavirus in the family Rhabdoviridae. It is predicted that the increasing use of NGS, not only for plant samples but also in arthropod vectors, will allow the identification of many novel NSR viruses which will be crucial to unraveling the evolution of many NSR virus clades .In diseased Adkar-Purushothama et al.). Novel circular RNAs, 357–360 nt, containing hammerhead ribozymes in both polarity strands were discovered by NGS analyses of fig tree leaves . Bioassays, however, are needed to demonstrate whether the RNAs are viroids or viral satellites. MicroRNAs (miRNAs) were identified by NGS in dwarfed citrus trees in response to infection by citrus dwarfing viroid (CDVd). The 60 miRNAs identified were conserved in stem and root tissues. Three conserved miRNAs  were significantly downregulated in the stems of CDVd-infected trees compared to the non-infected controls. These miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected sweet orange on trifoliate orange rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. These findings indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus tree growth and developmental processes which may participate in the cellular changes leading to the observed citrus tree dwarf phenotype .The 10 most abundant sequence variants of potato spindle tuber viroid (PSTVd) RG1, expressed 1–4 weeks after infecting tomato plants, were identified by NGS in the regions favoring mutations. The findings of the effect of mutations on PSTVd secondary structure and its derived small RNAs increased our knowledge of the biological role of sequence variants, PSTVd interaction with host components, stability of structures generated by mutants during the course of infection, and stabilizing viroid population dynamic as influenced by variant sequences . Similarly, site specific mutation of the tomato eIF4E1 gene by CRISPR-Cas9 successfully conferred enhanced resistance to infection by pepper mottle virus . A banana streak virus (BSV) genome sequence that integrates at a single locus into the banana B genome is known as endogenous BSV (eBSV) whereas the virus genome in the replicative form in cells is known as the episomal form. CRISPR-Cas editing was applied to banana to control BSV by inactivating the eBSV integrated into the host genome .The eukaryotic translation initiation factor Shahid et al.).NGS and CRISPR-Cas editing technologies were presented in a review article that discussed in detail the development, various applications, advantages and drawbacks, as well as potential future of both technologies in plant virology (in vitro Specific CRISPR-based Assay for Nucleic acids detection (iSCAN) was combined with reverse transcription-recombinase polymerase amplification (RT- RPA), to develop a one-pot detection assay termed iSCAN-one-pot (iSCAN-OP), for specific, rapid, and sensitive detection of plant RNA viruses . The RT-RPA pre-amplification step converts the RNA genome of the virus into dsDNA that serves as a substrate for the Cas12a cis activity. Cas12a targeting of the dsDNA triggers its collateral activity, which in turn cleaves the ssDNA reporter molecules and releases the signal. The reaction was incubated isothermally at 42°C for 20 min. The fluorescence signal as a result of trans-cleavage activity of Cas12a was measured with a commercially available P51 Molecular Fluorescence viewer using the Tecan plate reader (The e reader . This deAH wrote the editorial and prepared the final version of the editorial. HC and JR reviewed the editorial and made useful suggestions. All authors approved the editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chlamydia trachomatis, an obligate intracellular Gram-negative bacterium, is characterized by a wide range of different serotypes responsible for several local or systemic human diseases, including genital tract manifestations (D–K), trachoma (A–C), and lymphogranuloma venereum (L1–3). Among them, C. trachomatis genital infections are the most common sexually transmitted diseases of bacterial origin, with more than 130 million new cases per year worldwide [C. trachomatis genital infection is still a relevant public health problem due to the high prevalence of asymptomatic infections, in both women and men  that, untreated, may lead to chronic complications, such as pelvic inflammatory disease, ectopic pregnancy, as well as reactive arthritis and infertility [C. trachomatis in these pathologies is related to its ability to infect and reproduce within different cell types beside the epithelial cells of the genital tract, including synovial and testicular cells [enereum L–3. Amongertility . The invar cells ,5. Folloar cells . As the ar cells .C. trachomatis infection [C. trachomatis genital infection. Peculiar cervico-vaginal microbial signatures were also described in C. trachomatis-positive pregnant women, or in women after C. trachomatis treatment [To date, it is of utmost importance to shed light on the pathogenetic mechanisms underlying host–Chlamydia interaction and influencing the clinical outcomes of chlamydial-mediated genital diseases. Over the course of the last decades, as for example, the injection of chlamydial virulence factors in host cells via the type-3 secretion system, the escape from the endocytic pathway via chlamydial Incs proteins, have been described as mechanisms responsible for chlamydial adhesion, invasion and intracellular survival . In recenfection . Indeed,nfection . Furtherreatment ,11.C. trachomatis-positive patients, a higher involvement of the biosynthesis of chorismate, [C. trachomatis.Despite all of this important evidence, the wealth of information from sequencing data is still largely underexploited. It has recently been hypothesized that it is possible to predict metabolic profiles based solely on the sequencing data from a microbial community, favored also by the large amount of metabolomic data deposited in public databases . In thisrismate, , a precuC. trachomatis genital infection. Indeed, their application may generate novel, testable hypotheses that will bolster and accelerate future mechanistic studies, as well as the discovery of novel potential targets for drug development. Many critical questions still remain unanswered, such as the double-edged interaction between C. trachomatis and the resident microbiota, as well as the nature of the host susceptibility or resistance to this pathogen and the development of innate or adaptive immune responses. Therefore, this Special Issue will serve as a collection of the most up-to-date progress in the field.Consequently, all these advanced bioinformatic approaches might be of great help for a deeper understanding of the pathogenetic pathways underlying