Formica exsecta viruses, along with ORF predictions, and functional annotation of genes. The Formica exsecta virus-4  is a newly discovered negative-sense single-stranded RNA virus representing the first identified member of order Mononegavirales in ants, whereas the Formica exsecta virus-1 , and the Formica exsecta virus-2  are positive single-stranded RNA viruses initially identified (but not characterized) in our earlier study. The new virus FeV4 was found by re-analyzing data from a study published earlier. The Formica exsecta virus-4 genome is 9,866 bp in size, with an overall G + C content of 44.92%, and containing five predicted open reading frames (ORFs). Our bioinformatics analysis indicates that gaps are absent and the ORFs are complete, which based on our comparative genomics analysis suggests that the genomes are complete. Following the characterization, we validate virus infection for FeV1, FeV2 and FeV4 for the first time in field-collected worker ants. Some colonies were infected by multiple viruses, and the viruses were observed to infect all castes, and multiple life stages of workers and queens. Finally, highly similar viruses were expressed in adult workers and queens of six other Formica species: F. fusca, F. pressilabris, F. pratensis, F. aquilonia, F. truncorum and F. cinerea. This research indicates that viruses can be shared between ant species, but further studies on viral transmission are needed to understand viral infection pathways.We present the genome organization and molecular characterization of the three  Dicistroviridae and Iflaviridae families, which belong to the order Picornavirales (positive sense single strand RNA viruses). Virus infection by Dicistroviridae has been reported in the ant species Nylanderia pubens , disease symptoms have been studied at length  high virus loads has been suggested to negatively impact colony function, such as reduced brood production, decline in foraging efficiency, and death of the colony , and present the primary genome structure and organization of a third virus (tentatively named FeV4) discovered using the same data. In addition, we construct a phylogeny at the family level for all three viruses, validate the presence of the viruses from new field collections, and demonstrate the presence of viral RNA of three viruses from different castes and life stages in F. exsecta. Finally, we report on the presence and levels of viral RNA of these viruses in the adult worker and queen castes of six other Formica species: F. fusca, F. pressilabris, F. pratensis, F. aquilonia, F. truncorum and F. cinerea.The aim of this study was to identify and characterize ant-infecting viruses. Our study species sian ant . In 2013sian ant . From thSAMN02046301 –SAMN02046306; Formica exsecta at six localities within a range of 50 km2 on the Hanko Peninsula, and the islands outside the Tvärminne zoological station, SW Finland. Screening and annotation of the viruses from these genomic resources was done using RNAseq data published by F. exsecta population at the Tvärminne zoological station   , to testexsecta) ), and unexsecta) , and hadplicates . We thenSamtools .F. exsecta (http://www.premierbiosoft.com/netprimer/) for primer melting temperature, molecular weight, GC%, and secondary structures. After initial PCR amplification tests, we obtained a single primer pair for each virus, that amplified consistently , 2 µl Forward + Reverse primer mix (5 µM each), 3 µl MQ water, 1µl cDNA (dilution 1:2). The PCR program was 3 min at 95 °C, 5 cycles of 10 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C, followed by 39 cycles of 10 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C, and final extension for 10 min at 95 °C. Agarose gel electrophoresis was used to check the expected PCR product size after which the final PCR products were purified using Ampure  and sequenced using Sanger methods on ABI 3730 DNA analyzer .http://opal.biology.gatech.edu/GeneMark/) software for open reading frame (ORF) prediction, which uses specific models for predicting viral genomes (http://linux1.softberry.com/berry.phtml??topic=virus&group=%20programs&subgroup=gfin?dv%20)  , and by n?dv%20)  to detern?dv%20) .Dicistroviridae (17 genomes), Iflaviridae (23 genomes), and the insect infecting Mononegavirales (12 genomes) from the GenBank database and added those discovered from the six Formica ant transcriptomes . Alignments were carried out at the nucleotide level for FeV1 & FeV2 and protein level for FeV4 with the software MAFFT . A similar comparison was not done for the FeV4-like sequences, as the fragment was much longer, and encompassed almost the entire gene. Although some FeV-like sequences were very short, they nonetheless give an indication of the phylogenetic affiliation of these fragments. Figures were created using FIGTREE version 1.2 . We aligned all reads from each library to the virus genomes, extracted the aligned regions and counted the reads. To avoid counting misaligned reads, we made sure at least 5 pair-end reads mapped to the virus genomes with >= 25 mapping quality. This read count was used to calculate RPKM values (Reads per kilobase of genome per million reads mapped) for each virus across the different castes and ages. The number of individuals per library varied from 4 to 30. Thus, the RPKM values were adjusted for the number of individuals in each library, and plotted in heat maps. To assess the levels of viral RNA in different castes in closely-related species, we performed the same analyses on the individual queen and worker libraries of F. pressilabris, F. fusca, F. cinerea, F. aquilonia, F. truncorum, F. pratensis and F. exsecta from the To investigate the occurrence of viral infections across castes, and development in F. exsecta. Of these, two (FeV1 and FeV2) were reported earlier , KF500002 , MF287670 . The average genome coverage was 6900X for virus FeV1, 3020X for FeV2 and 2651X for FeV4, in the F. exsecta transcriptome  . Its genDicistroviridae family, with the closest similarity to the Kashmir bee virus (KBV). The FeV1 and KBV are about 75% identical across the entire genome (with 97% genome coverage), with considerable differences between them in the 3′ non-translated region (23% nucleotide identity), 5′ non-translated region (67% nucleotide identity), in the helicase, and 3C-protease domains of the non-structural polyprotein (60% amino acid identity), and in a 110 amino acid stretch of the structural polyprotein (50% amino acid identity).A blast search to GenBank showed that the FeV1 shows highest similarity to viruses from the Formica species (mature workers and queens) revealed FeV1-like sequences in F. pressilabris, F. fusca, F. cinerea, F. aquilonia, F. truncorum. The sequences were similar over a length of 704 to 6,070 bp, Formica species notwithstanding, the maximum likelihood phylogeny for FeV1 shows 100% bootstrap support, indicating a common ancestor of FeV1 and FeV1-like sequences in Formica  showing non-negligible levels of FeV1-like fragments . The nucleotide level similarity with these viruses was low (only 10–20% genome coverage), but the protein level similarity was high. This suggests that FeV2 also shares genomic architecture with all three viruses, (ORF: 90% coverage and 40% identity).FeV2 showed the closest nucleotide similarities with the deformed wing virus , the Kakugo virus , and the F. pressilabris, F. fusca, F. cinerea, and F. truncorum . The FeV2-like sequences from F. fusca, and F. truncorum were only partially assembled, as good quality sequencing data was unavailable for the transcriptome assembly process. The phylogeny for FeV2 virus contained 23 genomes of the Iflaviridae family, and suggest that the FeV2 and FeV2-like fragments comprise a monophyletic taxon with very strong (100%) bootstrap support, and that they cluster with other social insect viruses  with 100% bootstrap support  of ale data . FeV2-li samples .Mononegavirales. FeV4 blast searches showed similarities  to the conserved domain mononeg RNA pol (pfam_00946), which is mainly present in Mononegavirales, an order of non-segmented negative stand viruses  with a nucleotide composition of 31.16% A, 23.92% U, 22.94% C, 21.98% G . The FeV viruses , such asF. exsecta, a fragment with 80% nucleotide identity (87% protein level) to FeV4 was found only in F. fusca . They exhibit differences in genome organization; FeV1 is dicistronic, FeV2 is monocistronic, whereas FeV4 is pentacistronic. FeV1 and FeV2 show similarity in sequence and genome organization to many positive-strand RNA viruses that infect ants and honey bees  viruses , and one new negative-sense single stranded RNA (-ssRNA) virus (FeV4), in the ant ney bees . FeV4 isDicistroviridae, Iflaviridae, Picornaviridae, Marnaviridae and Secoviridae family. The linear, positive sense, ssRNA genome is 7 to 12.5 kb with a viral genome-linked protein (VPg) covalently linked at the 5′ end and a 3′ polyA tract. The third virus, FeV4, has a genome similar to Mononegavirales, but contains unique elements, and may represent a new family of ssRNA viruses. The genome composition of the FeV1 and Hel-Pro-RdRp order, with the di-cistronic structural proteins at the 3′-end of the genome, rather than at the 5′ end, are characteristic to the members of the order Picornavirales , but given that FeV4 differs from these in nucleotide/amino acid sequence identity, and has a UTR/IGR, previously not reported for Mononegavirales, it may represent a previously unknown family of -ssRNA viruses.We also found a new negative-sense single stranded (-ssRNA) virus (FeV4), with a genome organization consistent with the order oviridae . We founFormica exsecta viruses, from five other ant species of the genus Formica . Of these, the FeV2-like sequences found in F. pressilabris and F. cinerea clustered with, were similar in length, and showed high sequence identity to FeV2. Most likely. they represent closely related forms of the FeV2 virus but their status needs to be confirmed. The FeV1-like, and FeV4-like sequences showed reasonable (80–98%) sequence identity with their respective FeV counterparts, but the sequences were short, which precludes firm conclusions. They nonetheless clustered with their respective FeV counterparts in F. exsecta with nearly 100% bootstrap support, and the analysis based on short fragments produced a similar topology. Overall, the tree topology of the phylogeny is similar to the one constructed using only complete genome sequences from a previous study (We found sequence fragments (FeV-like), with similarity to one of the three us study . HoweverDicistroviridae have wide host-ranges, and are capable of infecting several insect orders, such as Hymenoptera, Diptera, Hemiptera, Lepidoptera, and Orthoptera , to which FeV1 belongs, are indeed often expressed in all life stages, even in eggs  are known to be transmitted by Varroa mites , and developmental stages , are infected by Iflaviridae viruses  , but only in mature individuals of ey bees) . AlthougF. exsecta samples obtained by the RNAseq data generated in this study, and the sequence data generated in Formica RNASeq data sets generated by Viral RNA levels differed considerably between the F. exsecta viruses  suggest that the genome assemblies of all three viruses belong to the “coding complete” category proposed for virus genome quality standards (The use of meta-transcriptome data (bulk RNA-Seq) is a powerful approach to characterize viromes, and is key to the recent rapid increase of published virus genomes . The smatandards . To fit tandards , furthertandards . The FeVS. invicta and KBV in the honeybee, commonly persist within brood and adults as non-symptomatic infections , but the underlying cause has not been investigated. To date, no close relatives to FeV4 have been reported from social insects, and in Mononegavirales disease symptoms are mostly unknown, with the exception of the Sigma virus that causes vulnerability to CO2 exposure in Drosophila melanogaster  which is new to science, with a genetic makeup never described before in any virus found in ants. We further analyzed the prevalence of three viruses  in different life stages and castes of  studies , demonst10.7717/peerj.6216/supp-1Table S1Formica exsecta are given together with a number of individuals pooled in each library along with number source colonies in parentheses .The number of individuals pooled in each of the totals of 14 libraries sequenced by Institute for Molecular Medicine Finland (FIMM) or Beijing Genomics Institute (BGI). Caste and developmental stage of Click here for additional data file.10.7717/peerj.6216/supp-2Table S2Biosample details of raw sequencing data. Each biosample includes information on sequencing location, developmental stage, sample name and Sequence read archive (SRA) ID.Click here for additional data file.10.7717/peerj.6216/supp-3Table S3Genes and primer sequences used for RT-PCR verification of viruses.Click here for additional data file.10.7717/peerj.6216/supp-4Table S4All virus genome sequences used for the phylogenetic analysis of FeV1, FeV2, and FeV4, with detailed information on virus family, virus name, sequence length (bp), and NCBI ID.Click here for additional data file.10.7717/peerj.6216/supp-5Table S5RT-PCR validation on the original cDNA pools in which the virus was detected, and on 14 colonies collected in July 2013. In 5 colonies no viruses were found, in one colony two viruses were found.Click here for additional data file.10.7717/peerj.6216/supp-6Table S6F. exsecta.Raw RPKM and LN-transformed (lnx+1) values , obtained using either different developmental stages of pooled individuals  or a mixture of these stages  of Click here for additional data file.10.7717/peerj.6216/supp-7Table S7Formica species (Raw data from Raw RPKM and LN-transformed (lnx+1) values  for seven Click here for additional data file.10.7717/peerj.6216/supp-8Figure S1Discistroviridae family viruses. (B) the FeV2 phylogenetic tree as derived from a 375 bp region similar in length to FeV2-like sequences and a few Iflaviridae family viruses.Phylogenetic assignment of FeV1 and FeV2 using only short fragment data. (A) the FeV1 phylogenetic tree as derived from a 325 bp region similar in length to the FeV1-like sequences, and a few Click here for additional data file.


Cryptococcus gattii molecular type VGII is one of the causes of cryptococcosis, a severe fungal disease that is acquired from the environment and affects many host species . In Australia, disease caused by C. gattii VGII is largely confined to western and central northern parts of the country, with sporadic cases reported in eastern Australia. We investigated an unusual case cluster of cryptococcosis, caused predominantly by C. gattii VGII, in a group of captive koalas in eastern Australia. This research identified that the movements of koalas between wildlife parks, including an initial transfer of a koala from Western Australia, introduced and subsequently spread C. gattii VGII in this captive environment. The spread of this pathogen by koalas could also impact other species, and these findings are significant in the implications they have for the management of koala transfers and captive environments. Cryptococcus gattii molecular type VGII is one of the etiologic agents of cryptococcosis, a systemic mycosis affecting a wide range of host species. Koalas (Phascolarctos cinereus) exhibit a comparatively high prevalence of cryptococcosis  and nasal colonization, particularly in captivity. In Australia, disease associated with C. gattii VGII is typically confined to Western Australia and the Northern Territory , occupying an enigmatic ecologic niche. A cluster of cryptococcosis in captive koalas in eastern Australia , caused predominantly by C. gattii VGII, was investigated by surveying for subclinical disease, culturing koala nasal swabs and environmental samples, and genotyping cryptococcal isolates. URA5 restriction fragment length polymorphism analysis, multilocus sequence typing (MLST), and whole-genome sequencing (WGS) provided supportive evidence that the transfer of koalas from Western Australia and subsequently between several facilities in Queensland spread VGII into uncontaminated environments and environments in which C. gattii VGI was endemic. MLST identified VGII isolates as predominantly sequence type 7, while WGS further confirmed a limited genomic diversity and revealed a basal relationship with isolates from Western Australia. We hypothesize that this represents a founder effect following the introduction of a koala from Western Australia. Our findings suggest a possible competitive advantage for C. gattii VGII over VGI in the context of this captive koala environment. The ability of koalas to seed C. gattii VGII into new environments has implications for the management of captive populations and movements of koalas between zoos.IMPORTANCECryptococcus gattii molecular type VGII is one of the causes of cryptococcosis, a severe fungal disease that is acquired from the environment and affects many host species . In Australia, disease caused by C. gattii VGII is largely confined to western and central northern parts of the country, with sporadic cases reported in eastern Australia. We investigated an unusual case cluster of cryptococcosis, caused predominantly by C. gattii VGII, in a group of captive koalas in eastern Australia. This research identified that the movements of koalas between wildlife parks, including an initial transfer of a koala from Western Australia, introduced and subsequently spread C. gattii VGII in this captive environment. The spread of this pathogen by koalas could also impact other species, and these findings are significant in the implications they have for the management of koala transfers and captive environments. Cryptococcus gattii species complex comprises a group of environmentally acquired fungal pathogens that, along with the Cryptococcus neoformans species complex, are the etiologic agents of cryptococcosis  analysis molecular types (VGI to VGIV and VNI to VNIV) also used, when known. This disease affects a wide range of host species, from mammals to birds and reptiles  and the Northern Territory (NT)    , 6–8. In Canada) –12, whileported) , 13, 14.Phascolarctos cinereus) appear particularly prone to developing cryptococcosis caused by C. gattii, with clinical disease, asymptomatic antigenemia , and nasal colonization well documented among captive individuals   –27. Otheispersal –34, espeevidence .C. gattii VGII was observed in captive koalas in eastern Australia, with five confirmed clinical cases over a 4-year period, including three mortalities  determining the environmental burden of C. gattii and the prevalence of cryptococcal nasal colonization in the koalas housed at parks 1 to 3, (ii) using molecular epidemiologic tools to characterize environmental, colonizing, and disease strains from parks 1 to 4, and (iii) assessing the prevalence of subclinical disease (asymptomatic antigenemia) among the koalas domiciled in parks 1 to 3.A case cluster of cryptococcosis largely attributed to talities , and a ftalities . The invconazole ). This sCryptococcal nasal colonization was identified in 14 of 44 (32%) koalas on at least one occasion . Of the In 2015, 14% (1/7), 14% (3/22), and 45% (5/11) of koalas were positive for nasal colonization at parks 1, 2, and 3, respectively. The single positive individual located at park 1 had recently been transferred from park 2. In 2017, following the closure of park 2 and transfer of all koalas to parks 1 and 3, 19% (3/16) and 33% (5/15) of koalas were positive at parks 1 and 3, respectively.Cryptococcus spp. (colonies exhibiting the brown color effect were observed). At park 2, 86% (6/7) of enclosures were positive, while at park 3, 60% (3/5) were positive  of all enclosures cultured positive for positive . No encl in 2015 . Of the Cryptococcus spp., comprising two enclosures at park 1 and three at park 3. This meant that at parks 1 and 3, Cryptococcus spp. were cultured from 15% (2/13) and 30% (3/10) of enclosures, respectively  of all enclosures tested positive for ectively . The degectively  to 2017  of all eCryptococcal antigenemia was detected in 20 of 58 (34%) koalas on at least one occasion during this study, with two testing positive by a lateral flow assay (LFA) only , and the remaining 18 were positive using both tests . ConfirmNasal colonization results were available from 16/20 koalas with cryptococcal antigenemia . Of these 16 koalas, 9 were negative for nasal colonization on all occasions tested, while 7 were positive on at least one occasion .Cryptococcus species strains were obtained, with 5 disease-associated, 33 nasal colonizing, and 33 environmental isolates. C. gattii VGI accounted for 39/71 isolates . C. gattii VGI was not isolated from any samples collected  at park 1 . All 32 C. gattii VGII isolates from parks 1 to 3 were determined to be mating type α.A total of 71 t park 1 . C. gattC. gattii VGII isolates (of the same sequence type [ST]) were obtained from a single nasal swab from a koala recently transferred from park 2. In 2017, seven isolates were obtained, with C. gattii VGI accounting for two nasal colonizing isolates from one koala and C. gattii VGII accounting for one nasal colonizing isolate and four environmental isolates. Both colonized koalas had been transferred from park 2.At park 1, in 2015, two C. gattii VGII. Three koalas were colonized by C. gattii, with C. gattii VGI in one and VGII in the remaining two. Among the six positive enclosures at this park, 2/6 contained only C. gattii VGII, 2/6 contained only C. gattii VGI, and the remaining 2/6 had a mixed burden of both C. gattii VGI and VGII , while C. gattii VGI was identified as the etiologic agent in a single case. All isolates collected from park 3 in 2015 were identified as C. gattii VGI—this included nasal-colonizing isolates from four koalas and environmental isolates from two enclosures. In 2017, five koalas were colonized by C. gattii and either exhibited a C. gattii VGI burden (2/5), a C. gattii VGII burden (1/5), or mixed colonization by both VGI and VGII (2/5). All koalas with any C. gattii VGII colonization had a history of relocation from park 2. Among the three enclosures identified as positive for C. gattii in 2017, two contained C. gattii VGI and one contained C. gattii VGII  previously collected from park 4 were identified for comparative inclusion in this study. Mating type α accounted for 33/34 isolates; a single mating type a environmental isolate was identified.A total of 34 C. gattii VGII isolates from parks 1 to 3 identified in this study, 30/32 were sequence type (ST) 7. The remaining two isolates were novel to the C. gattii multilocus sequence typing (MLST) database and were classified as ST 539; these were both nasal-colonizing isolates collected at the same time from a koala residing at park 2. Allele differences were observed only at the URA5 locus, with two unique allele types identified (one for ST 7 and one for ST 539).Among the 32 GPD1, LAC1, and URA5, whereas three allele types each were observed at CAP59, IGS1, PLB1, and SOD1.At park 4, 30/34 isolates were ST 7. The remainder were ST 38 and ST 48, accounting for one and three isolates, respectively. Allele type differences were observed at all seven MLST loci. Two unique allele types each were observed at the C. gattii VGIIb clade with ST 7. The isolates from park 4, however, exhibited greater diversity, with ST 48 grouping closer to the C. gattii VGII standard strain (WM 178) than to ST 7 strains  data separated the isolates into two major clades, with isolates from all parks present in both clades . An overA phylogenetic tree with additional relevant genomes from veterinary and environmental isolates displays the same overall topology with two major clades of VGIIb . The isoC. gattii VGII. The detection of an unusually high prevalence in an area where C. gattii VGII is considered to be nonendemic and the documented transfer of a single koala from a wildlife park in Western Australia where C. gattii VGII is endemic provided the opportunity to investigate what happens after C. gattii VGII is introduced into (i) an environment with no C. gattii (enclosures in park 1) or (ii) an environment with a moderate presence of C. gattii VGI (park 3). Our findings suggest that C. gattii VGII can indeed colonize a new environment , be amplified and further dispersed by the presence of captive koalas, and compete successfully with an existing population of C. gattii VGI. This study also offered some insight into the effectiveness of environmental decontamination as a management tool to prevent cryptococcosis in captive scenarios.This study provides evidence in support of the notion that koalas can seed environments with C. gattii VGII was made particularly apparent by its absence in the environment at parks 1 and 3 in 2015 and subsequent presence in 2017. This presumably was the result of the transfer of koalas from park 2, a facility with abundant environmental C. gattii VGII, to parks 1 and 3 in 2016. At park 1 in 2015, the only place we were able to detect C. gattii VGII was in the nasal cavity of one translocated koala from park 2; VGII was not detected in the environment. Although koalas were transferred between all three facilities prior to 2016, this was sporadic, typically involved just one individual at a time, and was predominantly between parks 2 and 3. Presumably by chance, this left park 1 as a low environmental presence zone for C. gattii, despite the potential amplifying effect of the captive koala population. Such low environmental presence in captive environments has been described previously  were diagnosed prior to the closure of park 2, it seems likely that this was a key site for the ongoing distribution of cases even prior to the major transfer event.In 2016, approximately 20 koalas from park 2 were transferred to parks 1 and 3, with roughly 10 individuals going to each of the two facilities, in short succession. This provided an opportunity for a mass introduction of  endemic , provideC. gattii VGII is difficult to find in natural environments in Australia, even in areas of endemicity    , 26, 37. species , 26, 38.history) . It theror study . Given tC. gattii VGII population in these three facilities is almost exclusively ST 7, which was the most common type found in a prior study of Australian isolates   . The samr nasal) . The exaail beds ) or froml growth .C. gattii may be dispersed by anthropogenic disturbances   (Given the evidence that  Canada) , one faclonized) .C. neoformans yeasts in pigeon droppings within 30 min of contact time at a concentration of 0.062%, while rapid killing was observed at a concentration of 0.5% .The current Australian requirement for koalas prior to travel or export is to test only for cryptococcal antigenemia . In the eatments . Thus, pC. gattii VGII population in eastern Australia. The relative environmental competitiveness of C. gattii VGI and VGII remains uncertain, but our findings could suggest an increased fitness of VGII in this scenario. Confirmation of the capacity of koalas to translocate C. gattii to previously uncontaminated environments, and provision of compelling new evidence that this extends to C. gattii VGII, has implications for the prevention and management of potential outbreaks in Australia.This study offers valuable insights into the management of captive koala cryptococcosis and the composition of the C. gattii VGII strains isolated previously from the environment and nasal cavities of koalas in park 4  in the vicinity of Cairns, Queensland  and one near Perth, WA  . Koalas C. gattii during this study. This included the changing of all koala perches every 6 months (more often if possible) and a decontamination protocol, enacted every 3 months , during a “rest period” for each enclosure. This protocol included manual cleaning by scrubbing and hosing all surfaces with detergents followed by the dousing of the enclosure in a quaternary ammonium disinfectant  used at the manufacturer’s recommended concentration for fungal disinfection and utilizing a 30-min contact period. After this, enclosures were thoroughly rinsed with water, allowed to dry, and then eventually returned to use. Prior to this protocol, the facilities changed the perches less frequently (approximately every 12 months), and while the floors and walls of each enclosure were often scrubbed, less attention was paid to the furniture  unless there was visible soiling.The enclosure environment, including furniture and leaf browse, was similar across parks 1 to 3. All three parks also had similar protocols in place, enacted in March 2015, for the management of environmental Nasal swabs from parks 1 to 3 were submitted to Veterinary Pathology Diagnostic Services (VPDS), The University of Sydney, for culture. A sterile, moistened cotton-tipped swab was inserted into the nasal vestibule on both sides and rotated gently, as per methods of prior studies , 18. ThiSamples were collected by moistening a sterile swab with sterile saline and running the tip thoroughly over the surface of perches and enclosure furniture, similar to previously described methods . In DeceCryptococcus spp.) were observed. If no growth was observed by 7 to 10 days, the plates were considered negative and discarded. Positive samples were classified according to the number of cryptococcal colonies counted on the agar plates as exhibiting either a low (1 to 10 colonies), moderate (11 to 100 colonies), or heavy (>100 colonies) extent of growth. A minimum of one cryptococcal colony from each positive plate was subcultured onto Sabouraud’s agar and incubated at 37°C for isolation and DNA extraction.All swabs  were initially cultured on Staib’s bird seed agar containing antibiotics (penicillin and gentamicin) by rolling the swabs gently across the agar. Plates were incubated at 27°C and examined at least once daily for 7 to 10 days. Samples were considered positive if yeast-like colonies exhibiting the brown color effect . If the LFA was positive, an LCAT  was performed to confirm the result and establish a reciprocal antigen titer . Both prURA5 gene was then performed, with the resulting product undergoing RFLP analysis and comparison to known standards  as described previously  as described previously  was then used to assign allele types and STs. C. gattii VGI isolates did not proceed to MLST analysis.MLST of all . gattii . This inC. gattii standard strains were included for out grouping: WM 179 (VGI), WM 178 (VGII), WM 175 (VGIII), and WM 779 (VGIV).Concatenated MLST sequences were aligned (MUSCLE), and a maximum likelihood phylogenetic analysis was performed  were selected for WGS, including all four available disease isolates. Three isolates from park 4 (of the same multilocus sequence type as isolates from parks 1 to 3) were also included for comparative purposes , making a total of 17 isolates.A representative group of 14 DNA for WGS was extracted using the Quick-DNA Fungal/Bacterial Miniprep kit  according to the manufacturer’s instructions. Genomic DNA then was fragmented using a Q800R2 sonicator  to approximately 500 bp, and genome libraries were prepared for paired-end sequencing using the NEBNext Ultra II DNA Library Prep kit  and quantified using the SequalPrep Normalization Plate kit . Libraries were pooled and sequenced at 2 × 150 bp on a NextSeq .C. gattii VGIIb genomes, 17 from the present study and 2 additional C. gattii VGIIb Australian genomes for comparison  were included in data analysis. Reads were trimmed of adapter sequence and low-quality bases with Trimmomatic v0.32 (Read data from 19 ic v0.32 . Parametde novo assembly created with read data from isolate WM 18.93 using SPAdes (v3.10.1)   with “cav3.10.1)  as the rv3.10.1)  as the SC. gattii VGIIb isolates . As an extended phylogeny, we also identified whole-genome SNP differences across 32 genomes of VGIIb veterinary and environmental isolates  following the same procedure described above . WGS data are available as BioProject PRJNA524387. All C. gattii VGII isolates collected for this study are available in the Medical Mycology Culture Collection at The Westmead Institute for Medical Research in Westmead, New South Wales, Australia . Strains are subsequently ordered by sequence type number, then culture collection number. Isolates in boldface font were included in whole-genome analyses. Download Table S1, PDF file, 0.3 MB.List of Copyright © 2019 Schmertmann et al.2019Schmertmann et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00216-19.2TABLE S2Cryptococcus gattii VGIIb in Table S2, PDF file, 0.2 MB.List of all genomes included in the phylogenetic analysis of Copyright © 2019 Schmertmann et al.2019Schmertmann et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


The 1999 New York City outbreak of West Nile virus (WNV) was associated with a high incidence of West Nile virus neuroinvasive disease (WNVND) where the outcomes for these patients were very poor. We describe a case of West Nile virus neuroinvasive disease (WNVND) characterized by acute flaccid quadriplegia with a favorable outcome in Winnipeg, Manitoba, Canada. Flavivirus and family Flaviviridae, is transmitted by Culex spp. mosquitoes .The first case of WNV was documented in Uganda in 1937, and the first case report of West Nile virus neuroinvasive disease (WNVND) was traced to an outbreak in Israel in 1951 . Five deWest Nile virus is the most common arboviral disease that causes 95% of West Nile virus neuroinvasive diseases (WNVNDs); the incidence is 0.41 in 100,000 and the case fatality rate is up to 9% between 1999 and 2017 in the United States of America , 14.West Nile virus disease (WNVD) is known to be symptomatic in minority of cases up to 20%, where these cases present in a form of febrile illness called West Nile fever (WNF) that may mimic flu-like symptoms. About 1% of these cases developed West Nile virus neuroinvasive disease (WNVND), which carry various ranges of outcomes including complete recovery and various minor or major residual neurological defects or mortality. The long-term outcomes are not always directly correlated with the severity of WNVND at the presentation , 13, 15.In the absence of vaccine or proven therapy for West Nile virus diseases (WNVDs) to date, WNVND treatment remains supportive. The polyclonal intravenous immunoglobulin (IVIG), interferons, ribavirin, and steroids have been tried without proven benefit , 16, 17.This case represents, to our knowledge, the first description of life-threatening acute flaccid quadriplegia due to WNV in this region of Canada. The patient's incomplete recovery from the WNV-mediated Guillain–Barré Syndrome (GBS) required a lengthy period of hospitalization and rehabilitation. He was treated with two doses of IVIG consistent with recommendations . This ca


Zika virus, an arthropod-borne flavivirus pathogen in humans, is unusual because it can be sexually transmitted and can be shed for prolonged periods in semen. We report viral shedding in vaginal secretions for up to 6 months, indicating the potential for sexual and vertical transmission by infected women. Zika virus had been considered relatively benign, but during the Americas epidemic, several new pathogenic features were identified: association with severe birth defects in infants born to women infected during pregnancy, transmission by sexual contact, prolonged shedding in semen, and Guillain-Barré syndrome  at a public health center and Hospital Escuela Oscar Danilo Rosales Argüello in León, Nicaragua. We enrolled 5 women in a prospective cohort to characterize duration of viral shedding in the genital tract. We collected blood by venipuncture, and each woman provided urine and saliva samples.During October 2016–November 2017, we recruited women http://www.bd.com). Vaginal swabs were self-collected by the woman, who inserted the swab ≈5 cm into the vagina, left it in place for 5 minutes, removed it, and placed it into swab transport media. Women did not provide samples during menstruation to avoid contamination with blood. Study staff transported the swab specimens at 4°C within 3 hours to the Universidad Nacional Autónoma de Nicaragua-León microbiology laboratory, where it was stored at 4°C for up to 4 months until RNA extraction.We tested whole blood, urine, and saliva from each woman for Zika virus RNA at enrollment with quantitative reverse transcription PCR (qRT-PCR). If we detected Zika virus RNA in any fluid, we asked women to provide blood, urine, saliva, and vaginal secretions at 7, 14, 21, 28, 60, 90, and 180 days postonset (dpo) of initial symptoms. We collected vaginal fluid using the BBL Culture Swab Collection and Transport System  according to manufacturer instructions. We detected Zika virus RNA by qPCR  on an ABI 7500 RT-PCR system (Applied Biosystems) using published primers that detect all known Zika virus genotypes (Zika1087/1108FAM/1163c) and those that are specific to the Asian genotype (Zika4481/4507cFAM/4552c) (t) <38 in either reaction.To elute Zika virus RNA, we incubated vaginal swabs in 140 μL of phosphate-buffered saline at room temperature for 10 min. We extracted Zika virus RNA from the eluent using the QIAamp Viral RNA Mini Kit  and University of North Carolina at Chapel Hill (protocol no. 16–0541). All subjects provided written informed consent at enrollment.>1 sample of vaginal secretions. The median age of these participants was 25 years (range 18–54 years). Four women were pregnant; gestational age at enrollment was 1–8 months. Infants of all 4 women were healthy at birth with no obvious congenital anomalies. The 54-year-old subject had hypertension; no other participants reported chronic medical conditions. Symptoms started a median of 4 days before enrollment (range 1–6 days).Five women with acute Zika virus infection provided >1 of the primer sets, and 3 were positive by both. Neither sample collected at 7 dpo was qRT-PCR positive, but 2/3 (67%) were Zika virus positive at 14 dpo, 3/4 (75%) at 21 dpo, 1/3 (33%) at 28 dpo, 2/2 (100%) at 60 dpo, 1/2 (50%) at 90 dpo, and 1/2 (50%) at 180 dpo. Three women had only 1 vaginal fluid sample positive for Zika virus by qRT-PCR; 1 had 2 of 3 positive; and 1 woman, who shed through 180 dpo, had Zika virus in 5 of 6 samples tested  were Zika virus positive by qRT-PCR with s tested .<6 months has crucial implications for couples planning pregnancy and for sexual transmission. In contrast to our results, in a population-based study of patients with symptomatic acute Zika virus infection followed for 6 months, only 1 of 50 women had Zika virus detectable in vaginal secretions, in a single specimen collected 3 dpo (Detection of Zika virus RNA in vaginal secretions 60–180 days after the onset of symptoms is a novel observation; the presence of Zika virus in vaginal secretions for Most of our subjects were pregnant at enrollment; pregnant women are known to shed Zika virus in blood for up to 3 times longer than nonpregnant women, possibly because of fetal infection (Our findings have implications for future pregnancies. Persistent Zika virus shedding in the reproductive tract could be a source of fetal infection. Vaginal shedding of Zika virus could result in ascending infection, as occurs with vaginal cervical herpesvirus 2 infections (>18 articles; however, there is evidence of infectious Zika virus particles in vaginal fluids (Our findings also suggest that sexual transmission of Zika virus from women to their sexual partners may be possible for up to 6 months after infection. Infectivity studies are warranted to investigate whether Zika virus shed in vagina secretions is capable of replication and infection. Sexually acquired Zika virus, mainly by male-to-female transmission, has been reported in


We report a case of enterovirus C105 infection in an 11-year-old girl with lower respiratory tract symptoms that was identified through the Respiratory Virus Surveillance System, which covers 30 sentinel hospitals in all 16 districts of Beijing, China. The presence of this virus strain in China confirmed its geographically wide distribution.  The Beijing Center for Disease Prevention and Control established the Respiratory Virus Surveillance System (RVSS) in 2014. The RVSS tracks patients with respiratory tract infections and pneumonia in 30 sentinel hospitals throughout Beijing. The RVSS is an active system, designed to alert for future outbreaks of respiratory infections (RTIs). To study enterovirus infections, we tested 24,093 clinical specimens  from patients with RTIs that were reported through RVSS during June 2014–December 2017. RVSS classifies persons <14 years of age as children and those We screened all samples using real-time PCR for influenza virus, parainfluenza virus types 1–4, respiratory syncytial virus, coronaviruses , metapneumovirus, adenovirus, bocavirus, and enteroviruses . The patient was an 11-year-old girl with no underlying disease who was brought to the outpatient clinic of the Beijing Children’s Hospital on May 23, 2016, with an 8-day history of fever (highest temperature 38.7°C), coughing, and difficulty breathing. Blood tests in the clinic showed total leukocyte count 1.41 × 1010 cells/L; neutrophils, 79.2%; lymphocytes, 12.1%; total platelet count, 5.2 × 1011/L; and hemoglobin, 130 g/L. Chest radiographs showed thickness or turbulence in the texture in both lungs, which was diagnosed as pneumonia. She received supportive treatment and received antimicrobial drugs empirically before being sent home the same day. She was not hospitalized during her illness. According to a follow-up survey, she recovered 14 days later. We detected no other respiratory pathogens in this patient.We further genotyped enterovirus-positive samples with primers sequentially targeting the viral protein (VP) 1 region (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the amplicon (Beijing-R2759) showed that the sequences had 98% identity with the USA/OK/2014–19362 strain (GenBank accession no. KX276189.1) and 91.4% with the reference prototype genotype . Phylogenetic analysis of the VP1 gene performed with MEGA version 6.06 software (http://www.megasoftware.net) showed that Beijing-R2759 belonged to genotype EV-C105.BLAST analysis (https://www.dnastar.com). The genome of Beijing-R2759 (GenBank accession no. MH229997) was 7,316 nt, including 6,618 nt in open reading frame. The EV-C105 polyprotein sequence for this strain shares 96.6%–99.4% amino acid identity with 8 of the EV-C105 sequences in GenBank: 96.6% with accession no. KM880097 (Burundi); 96.8% with accession no. KM880096 (Burundi); 97.7% with accession no. JX393302 (Peru); 98.5% with accession no. JX514943 (Republic of the Congo); 98.6% with accession no. KM880098 ; 98.8% with accession no. KM880099 (Romania); 99.3% with accession no. KX276189 (United States); and 99.4% with accession no. KM880100 . To further characterize this virus strain, we amplified the genome sequence directly from the nasopharyngeal swab sample using reverse transcription PCR with overlapping primers; we sequenced each amplicon 4 times using the Sanger method. We assembled sequences using Lasergene version 5.01  and EV-C105 strain 34S . In this study, we grouped Beijing-R2759 with the strain obtained from the United States in 2014 (The full length of the Beijing-R2759 VP1 gene was 888 nt. The deduced amino acid sequence in VP1 had 94.9%–100% identity with those from Italy, Peru, Republic of the Congo, and the United States. Alignment results analysis of VP1 aa sequences showed differences between the strains isolated in this study (Met in 2014 . We obseOur report confirms that the distribution of EV-C105 is geographically wider than previously believed. A greater awareness of EV-C105 may enable improved detection of this virus (


Apis mellifera L. queens is a widely employed technique used in honeybee breeding that enables the effective control of mating. However, drone semen represents a potential source of honeybee viruses. In this study, 43 semen doses collected from apparently healthy drones, and consequently used in instrumental insemination, were analysed using PCR or RT-PCR to detect the presence of viral genome of 11 honeybee viruses. In 91% of samples, viral infection was detected. The survey revealed genomes of five viruses, namely Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Sacbrood virus (SBV), and A. mellifera filamentous virus (AmFV) in 84%, 19%, 14%, 2%, and 67% of samples, respectively. Single infection (30% of samples) as well as multiple infection (61% of samples) of two, three or four pathogens were also evaluated. To the best of our knowledge, this is the first study describing the presence of the BQCV and SBV genome sequence in drone ejaculate. Phylogenetic analysis of BQCV partial helicase gene sequence revealed the high similarity of nucleotide sequence of described Czech strains, which varied from 91.4% to 99.6%. The findings of our study indicate the possibility of venereal transmission of BQCV and SBV.Instrumental insemination of  Apis mellifera queens started in the 1920s [Instrumental insemination of he 1920s . As honehe 1920s , the usehe 1920s ,5. Naturhe 1920s ,7,8,9. Hhe 1920s ,11. Anothe 1920s . Actuallhe 1920s . Moreovehe 1920s . Queen hhe 1920s . Regardlhe 1920s ,17,18,19he 1920s . HoweverPaenibacillus larvae, the causative agent of American foulbrood ; the presence of ectoparazitic mite Varroa destructor was also monitored. Colony conditions were evaluated by the beekeeper based on direct observation. All semen doses included in the survey were prepared using a protocol for instrumental insemination [In 2016 and 2017, a total of 43 pooled semen doses of Austrian (2), Czech (33), Hungarian (3), and German (5) origin were collected. Each dose contained ejaculate obtained from 10 drones per colony. Active drones aged two weeks were collected from flight cages placed in apparently healthy colonies showing no signs of overt viral infection. Moreover, the sampled colonies were regularly tested for the presence of mination . BrieflyBoth DNA and RNA were extracted simultaneously using magnetic beads. Chemagic Viral DNA/RNA Kit  was employed according to the slightly modified manufacturer’s protocol. In short, immediately before nucleic acid extraction, the semen samples were homogenised directly in a lysis buffer supplied with the extraction kit in the presence of Garnet Beads 0.70 mm  by vortexing (3500 rpm/2 min). Homogenates were centrifugated for 1 min at 13,000 rpm and 200 μL of the supernatants were mixed with 30 μL of magnetic beads. Subsequently, the manufacturer’s protocol was continued. The obtained nucleic acids were stored at −80 °C until further use.Varroa destructor macula-like virus (VdMLV), Big Sioux River virus (BSRV), Lake Sinai virus (LSV), Chronic bee paralysis virus (CBPV), and Apis mellifera filamentous virus (AmFV). The specificity of detection primers was verified on our own field isolates of honeybee viruses. The PCR products were examined by electrophoreses in a 1.5% agarose gel stained with Midori Green stain  and visualized by ultraviolet transillumination. Selected PCR products of employed diagnostic assays were submitted to sequencing  and the obtained sequences were analysed with the use of MEGA version 7 [For the detection of RNA viruses, a random primed reverse transcription (RT) was carried out with the use of ProtoScript® II First Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. Target viral sequences were detected by PCR with specific primers employing Aptamer Hot Start Master Mix  of samples. Single infection  as well as multiple infection  of two, three or four pathogens were also evaluated. The results including pathogen combinations are summarized in n = 4/43) of samples were free of all tested viruses. Detailed description of detected viral pathogens in individual samples and summary of multiple infections are described in In total, 43 pooled drone semen samples were surveyed for the presence of 11 honeybee viruses. The screening revealed genomes of five viruses, namely DWV, ABPV, BQCV, SBV, and n = 1/43) was confirmed). This sequence of SBV partial polyprotein gene (nt position 240–668 referring to the complete genome sequence HM237361) was aligned with all partial European sequences of SBV and also with several Asian complete sequences of SBV available in the GenBank  were compared to the available BQCV sequences see . PhylogeBank see . The phyBank see B showed Varroa destructor [AmFV in 91% (n = 39/43) of the ejaculate doses tested. Similarly, sequences of viral nucleic acid of DWV, ABPV, IAPV, and AmFV were detected in the semen of apparently healthy drones [Dicistroviridae), one of the honeybee viruses most often detected with laboratory techniques in adult honeybees worldwide [n = 5/43) of semen doses obtained from apparently healthy drones and subsequent use of the sperm for instrumental insemination of a bee queen could imply future risk to the colony health via sold queen. However, in order to assess the effect of insemination with the contaminated ejaculate, the life story of these colonies will have to be monitored. Interestingly, one ejaculate sample was found to be positive for SBV, which is a causative agent of sacbrood disease. The virus is transmitted horizontally to young larvae through food contaminated by glandular secretion of latently infected nurse bees. The highest prevalence of sacbrood disease is observed during early spring, probably due to cold stress induced by fluctuating temperatures [Apis cerana) capable of causing huge losses in the Asian apiculture industry [Only limited data exist on the effect of stressors on health and quality of reproductive casts of honeybees and the available studies are historically focused on queens. Currently, drone health and quality are presented as vital aspects of reproduction management . Howeverstructor . In the y drones ,17,18,19y drones ,38, and  91% n = /43 of thy drones  when theorldwide , has notorldwide . The preorldwide . Althougorldwide . BQCV inorldwide . Thus, teratures . Althougeratures ,49,50,51eratures . On the industry . SBV wasindustry . Since Sindustry , the impindustry ,47,48.In conclusion, we demonstrated the presence of BQCV and SBV partial genome sequences in semen doses used in instrumental insemination of honeybee queens. Thus, our results indicate the possibility of venereal transmission of those two important honeybee pathogens. The use of ejaculate obtained from infected drones could represent an effective model of BQCV’s transmission and the virological analysis of the collected semen testing could help to maintain the high quality of produced queen bees.


Neohydatothrips variabilis), and two additional secondary vectors, tobacco thrips (Frankliniella fusca) and eastern flower thrips (F. tritici). This study was undertaken to elucidate the association between virus acquisition  and transmission efficiency  in the primary vector, N. variabilis, and to examine the mechanisms of vector competence by analyzing the effect of AAP  on virus infection in various tissues. In addition, we examined virus infection in tissues of the two secondary vectors. We found a significant effect of virus acquisition on transmission efficiency, transmission rate post 6 and 48 h AAP was significantly lower than 12 and 24 h AAP. Our analysis did not reveal a correlation between virus transmission rate and virus RNA in corresponding N. variabilis adults. On the contrary, N. variabilis adults harboring higher accumulation of the virus (>104) resulted in lower transmission rates. Analysis of SVNV infection in the tissues revealed the presence of the virus in the foregut, midgut , tubular salivary glands and principal salivary glands (PSG) of adults of all three vector species, however, the frequency of infected tissues was highest in N. variabilis followed by F. fusca and F. tritici. The frequency of SVNV infection in individual tissues specifically the salivary glands was lowest after 6 h AAP compared to 12 and 24 h AAP. This finding is in agreement with the transmission assays, where significantly lower virus transmission rate was observed post 6 h AAP. In addition, N. variabilis adults with high PSG infection (12 and 24 h AAP) were likely to have high percentage of foregut and midgut region 2 infection. Overall, results from the transmission assays and immunolabeling experiments suggest that shorter AAP results in reduced virus infection in the various tissues especially PSG, which are important determinants of vector competence in SVNV-thrips interaction.Soybean vein necrosis virus (SVNV) is a newly discovered species of tospovirus infecting soybean plants that is transmitted by the primary vector, soybean thrips ( Glycine max (L.) Merr.] is an important crop throughout the world due to its versatility as a food source along with applications in various industries such as textiles and biofuels. The United States is the global leader in soybean production with approximately 83 million acres planted and an estimated 117 million metric tons produced in 2017  and was first identified in Tennessee in 2008 , groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV). Frankliniella occidentalis transmitted all four tospoviruses with different efficiencies. Frankliniella schultzei transmitted TCSV, GRSV, and CSNV, but T. tabaci and T. plami could not transmit any of the abovementioned viruses . Transmission efficiencies can also vary within thrips populations or biotypes of the same species  was recognized as the primary vector of SVNV  and eastern flower thrips (F. tritici) are able to transmit SVNV albeit at a lower efficiency  compared to the primary vector, soybean thrips (72%)  . This waeviously .N. variabilis, (2) understand mechanisms of vector competency by examining the effect of AAP  on virus infection in the tissues of adult N. variabilis and (3) compare virus infection in the tissues all three vector species, N. variabilis, F. tritici, and F. fusca. Virus accumulation in adult insects was determined by absolute quantification method using Reverse Transcriptase-quantitative PCR (RT-qPCR) and transmission of virus to leaf tissues was determined by SVNV-specific double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Virus infection was determined by immunolabeling with antibody against SVNV-NP and imaged using confocal florescence microscopy.There has been progress in understanding various aspects of tospoviruses , but manSoybean variety Asgrow AG3334  plants were grown in 15.24 cm pots at 25 ± 1°C with 60–70% relative humidity and a photoperiod of 16:8 (L:D) hours in an Environmental Control Room with IntellusUltra controller . For plant maintenance, soybeans were fertilized weekly with Peters water-soluble fertilizer  at a rate of 3 g/l.N. variabilis originally collected from several different soybean fields located in Purdue Agricultural Centers (PAC) throughout Indiana were maintained in 45.72 × 45.72 × 76.20 cm thrips-proof insect cages  on soybean varAG3334. Despite several attempts, we could not transmit SVNV by mechanical-inoculation from which acquisition assays could be conducted. Other studies reported having similar difficulty  in modified 50 ml Falcon tubes with a water source at the bottom and lids fitted with thrips-proof mesh for ventilation. Both F. tritici and F. fusca had higher fecundity on red bean compared to soybean, hence we used red bean for routine colony maintenance to obtain sufficiently large population needed for molecular analysis and confocal microscopy experiments. However, previous research in our lab showed that F. tritici and F. fusca can feed and reproduce on soybean albeit not as successfully as N. variabilis  hours. Disease-free, V3 stage of soybean plants were alternately added into SVNV-infected colony at 4–5 week intervals, allowing inoculation by viruliferous soybean thrips for 3–4 weeks in 45.72 × 45.72 × 76.20 cm thrips-proof cage at which time they were used as viral source in experiments. Plants were routinely checked for symptom development which typically took 3 weeks. Leaf tissues were harvested from symptomatic plants and infection was confirmed using SVNV-specific double-antibody sandwich enzyme-linked immunosorbent assay, DAS-ELISA kit .SVNV was maintained in the greenhouse on soybean plants with continual transmission by N. variabilis, up to 24 h old, were placed on detached SVNV-infected soybean leaves for either 6, 12, 24, or 4 8h of AAP in 15 cm diameter Petri-dishes with lids fitted with thrips-proof mesh for ventilation. The petioles of infected detached soybean leaf were inserted through a hole made in the Parafilm cover on the top of a 1.7 ml microcentrifuge tubes, this was done to keep the soybean leaves fresh during the acquisition assay. All systemically infected leaves used for AAP were confirmed to be positive with SVNV. After varying duration of AAP, the cohort of larvae were transferred to non-infected/healthy soybean leaves sealed in Petri-dishes and reared to adulthood. The transmission efficiencies of thrips were assessed by inoculating a single 1.5 cm-diameter healthy soybean leaf disk with an individual thrips, 24 h post-adult eclosion. The soybean leaf disk was placed on a piece of filter paper partially inserted in 1000 μl micropipette tip, which was sealed off at one end and filled with water. Insects were allowed 24, 48, and 72 h of IAP, after which time insects were collected in 1.7 ml microcentrifuge tubes and flash frozen in liquid nitrogen  and three IAP durations . Given the logistics of performing a complete factorial, we focused each experiment on a specific AAP duration and the cohort of thrips post-AAP were divided into three subsamples for the three inoculation treatments: 24, 48, and 72 h IAP, respectively. Briefly, a cohort of 50 first instars of nitrogen . Virus l NP-gene . Leaf diN. variabilis obtained from the acquisition and transmission assay described above using the Chelex 100  method . To quantify SVNV levels from samples, a standard curve was generated using a plasmid containing SVNV-NP gene at a known concentration as per TM TA vector (2817 bp) using One Shot TOP10 chemically competent Escherichia coli . Plasmid DNA was extracted using Qiagen Miniprep Kit following manufacturer’s protocol  and sequenced. The nucleotide sequence was 100% identical to the target sequences deposited in GenBank (GU722322). The SVNV-NP sequence is deposited in NCBI GenBank under accession number MG190869. The concentration of the plasmid DNA containing the SVNV-NP gene was determined using a NanoDrop 1000 . The total plasmid size of 3056 bp was estimated by adding 239 bp of product size expected from the NP primers to 2817 bp of plasmid. The mass of the plasmid containing the NP gene was calculated using the following formula (-21g/bp). Ten-fold dilutions series, ranging from 7.54 × 108 copies/μl to 7.54 × 101 copies/μl, were prepared in RNase-free water for obtaining a standard curve. The qPCR Mastermix for SYBR Green®  was used according to manufacturer’s instructions. Each reaction contained 5.0 μl of SYBR Green Taq, 0.5 μl of F primer, 0.5 μl of R primer, 2.0 μl of deionized water, and 2.0 μl of cDNA template. The qPCR was run on a CFX Connect®  thermocycler, and the mean threshold cycles (Cq) values were calculated by the CFX ManagerTM Software Version 3.1. The cycling conditions used were: 95°C for 2 min; followed by 40 cycles of 95°C for 10 s, 55°C for 30 s, and then the final melt curve, starting at 65°C increasing to 95°C, in increments of 0.5°C every 5 s. Each reaction plate contained a negative and positive control and standard curve along with insect samples, which were performed in duplicate. The number of SVNV RNA molecule copies in each PCR reaction was estimated by interpolating the normalized NP Cq value against the standard curve. A normalization coefficient (Nc) was used for normalization of each NP Cq value, which was calculated from the internal control gene, Cytochrome C Oxidase Subunit I (COI), using the equation: Nc = 1 - (inCq - MinCq)/MinCq., where inCq is the Cq value of the internal control gene for a sample and MinCq is the mean inCq for all samples  method . A total formula , where m samples . The pri samples . The tot samples .N. variabilis fed on SVNV-infected leaves for 6, 12, or 24 h AAP were reared on healthy soybean leaves until adulthood as described previously. Fifty F. tritici and F. fusca first instar larvae were allowed a 24 h AAP on infected soybean tissues as results from acquisition assays with N. variablis found no difference in virus acquisition rate post 12 and 24 h AAP  using a B-2A filter and a 60X oil-immersion objective with variable scan zoom. Images were exported using Olympus FV10-ASW Version 3.1 and further processed with ImageJ Version 1.45. The pinhole aperture was fixed at ×2.0 μm for all three channels. The laser intensity and sensitivity of light absorption for specific wavelengths were set as follow: a 405 nm laser diode at 30% of sensitivity and 30% of laser intensity for detecting blue DAPI fluorescence; a 473 nm laser diode at 45% of sensitivity and 60% of laser intensity for detecting green FITC fluorescence; and a 559 nm laser diode at 45% of sensitivity and 55% of laser intensity for detecting red Alexa Fluor 594. Z-stacks were taken for every image for all three channels with an automatic calculated optimum of 1.00 μm per slide. The depth of each insect tissue for z-stack imaging was manually adjusted and set to the highest and lowest points of the observed feature, which were determined when one of the extremes was barely perceptible. Gain settings for green channel were fixed as mentioned above to compare the intensity of SVNV signal between images. Gain for the other two channels was adjusted accordingly to obtain the best detail of nuclei and actin in each image.SVNV accumulation in guts and salivary glands were examined by Olympus FluoviewTo quantify SVNV infection in the PSG, Image J software  was used10 copies in corresponding insects. Analyses of percentage of SVNV-positive tissues by immunolabeling was performed using Chi-square test. A one-way ANOVA was performed to determine differences in the MIS in N. variabilis PSG after 6, 12, and 24 h AAP, and between MIS in F. fusca and F. tritici PSG. All statistical analyses were performed using Minitab® Version 16 .Virus transmission (ELISA absorbance ratios) was treated as a binomial response (positive or negative), and differences were estimated by logistic regression analysis by using Proc GENMOD with logit link function in SAS 9.4  as per S2 = 24.85; df = 3; P < 0.0001), but not IAP  on virus transmission  in leaf disks using ELISA (P = 0.001), 6 and 24 h AAP (P = 0.001), 12 and 48 h AAP (P = 0.008), and 24 and 48 h AAP (P = 0.007). No difference in transmission rate was observed between 6 and 48 h AAP (P = 0.267) and 12 and 24 h AAP (P = 0.860). Interestingly, transmission rate was low at 6 and 48 h AAP despite the relatively high accumulation of virus in insect tissues . SVNV transmission occurred at varying levels of virus RNA accumulation in insect tissues ranging from > 102 to ≤ 106  (There was a significant effect of AAP (χng ELISA . The tra tissues . No corrto ≤ 106 . The higo ≤ 106) .N. variabilis after 6, 12, and 24 h AAP was detected using indirect immunofluorescence technique with primary antibody against SVNV-NP and FITC fluorophore-conjugated secondary antibody targeting the primary antibody and imaged using confocal fluorescent microscopy. Thrips fed on SVNV-free soybean leaves were used as negative control , with high frequency of FG infection (>50%) at 12 and 24 h AAP. Foregut infection was only observed when MG1 was infected, indeed there was a significant association between FG and MG1 infection at 6-h AAP , but not at 12 and 24 h AAP . Among three sections of midgut , the highest percentage of infection was observed in MG1 followed by MG2 and MG3 , MG2 , TSG  and PSG  compared to 12 h AAP  and PSG . The percentage of SVNV-infected tissues were similar between 12 and 24 h AAP . It was not uncommon that SVNV infection was not detectable in PSG at 6 h AAP  and 6 (26%) were considered SVNV-positive, respectively. The percentage of infection of each tissue for F. fusca and F. tritici is summarized in F. fusca and F. tritici except for MG3 in F. tritici . The extremely low infection rate of PSG in F. tritici (9%) very likely contributed to their inefficiency for virus transmission. The MIS in PSG of F. tritici was lower than F. fusca after 24 h AAP but the difference was not significant (P = 0.822)  .F. occidentalis that transmitted TSWV efficiently harbored higher titers of the virus compared to T. tabaci that failed to transmit the virus. Another study reported a positive correlation between transmission efficiency of thrips and TSWV titer in host plants  had similar transmission abilities like those harboring less virus titer (102). Similar result was reported for southern rice black-streaked dwarf virus accumulation in Sogatella furcifera adults  technique. Moreover, quantification of virus titer in salivary glands could be more informative because infection of the salivary glands is a requirement for virus transmission to occur. Nevertheless, knowledge of viral titer in the vector is a useful indicator to manage virus epidemics in the field.The viral load or number of virus RNA in transmitting adult thrips is considered a key determinant of virus transmission. For example, t plants . Rotenbea adults . One posa adults . Previoual titer . It is pN. variabilis. We found that the MG had highest virus infection compared to other tissues. In adults that had high percentage of PSG infection (12 and 24 h AAP), the first region of the midgut (MG1) had slightly greater percentage of infection than that observed in the second and third regions of the midgut . It has been reported that midgut regions have distinct morphological, histological, and genetic properties , midgut (MG), tubular salivary gland (TSG) and principal salivary gland (PSG) of adult operties , which mle cells . Hence, le cells . Our rest thrips . OverallN. variabilis. Our analysis revealed that most N. variabilis adults became infected after AAP of 6, 12, or 24 h with the infection found in all organs being studied. Interestingly, the length of AAP did not affect virus infection in MG1, however, significantly lower infection was observed in FG, MG2, and PSG post 6 h AAP. In a previous study, there was no effect of AAP on MG infection in adult F. occidentalis, however, the percentage of infected vesicular muscles cells was lower in thrips fed on infected plants for 3 h than those which had fed for 16 h AAP  in individual thrips using confocal fluorescence microcopy. However, the frequency of infected tissues was lower in F. fusca and F. tritici compared to the primary vector, N. variabilis. These results are in concordance with our previously published work where we found presence of non-structural NSs proteins in adult F. fusca and F. tritici after feeding on virus-infected material, which confirmed virus replication in these insects (N. variabilis, F. fusca, and F. tritici was similar except for lack of virus infection in MG3 of F. tritici. SVNV was also detected in TSG and PSG of both secondary vectors, which supports the theory that TSG serve as potential route for tospoviruses to infest PSG in thrips vectors. Our data showed that SVNV is able to infect PSG of F. tritici after 24 h AAP on virus-infected material. A previous study did not detect TSWV-NSs protein in PSG, ligaments, TSG, and hindgut of F. tritici (F. tritici. Overall, results of our study identified similarities and differences in virus transmission mechanisms compared to the well-studied TSWV-F. occidentalis system. There appears to be distinct associations between thrips species and their ability to transmit tospoviruses (Two secondary vector species insects . The pat tritici . Unlike oviruses , hence iPN conceived and designed the experiments, analyzed the data, and wrote the manuscript. JH performed the experiments, analyzed the data, and wrote a draft of the manuscript. VN analyzed the data and revised the manuscript. I-CY provided training for use of the confocal microscope. All authors read and approved the final manuscript.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.


Phlebovirus positive pool in Vero cells, RT-PCR and transmission electron microscopy (TEM).High throughput sequencing (HTS) boosted the discovery of novel viruses and new variants of known viruses. Here we investigated the presence of viruses in 12 pools of sand flies captured in three climatic periods in RAPELD grids at Rio Claro, Chapada dos Guimarães and at Pirizal, North Pantanal, Mato Grosso State, Midwestern Brazil by HTS, viral isolation of a putative Lutzomyia (Lutzomyia) longipalpis sand flies captured in the transitional climatic period in North Pantanal showed a tripartite genomic sequence of a putative novel Phlebovirus belonging to the phlebotomus fever serogroup. Phylogenetic analysis revealed this virus is closely related and share a common ancestor with phleboviruses included in the same clade: Chagres, Urucuri and Uriurana virus. RNA-dependent RNA polymerase (RdRP) presented 60%, 59% and 58% of amino-acid (aa) similarity with these phleboviruses, respectively. Similarity of Nucleoprotein and NSs protein codified by ambissense strategy of segment S was of 49% and 37%, respectively, with the proteins of the closest phlebovirus, Uriurana virus. Glycoproteins  and NSm protein presented 49% and 48% aa similarity with Chagres and Uriurana virus, respectively. Uriurana virus was isolated from sand flies in Brazilian Amazon and Urucuri from rodents in Utinga forest, Pará State. Chagres virus is an arbovirus responsible for outbreaks of febrile illness in Panama. This phlebovirus was isolated in Vero cells, confirmed by TEM and RT-PCR for the L segment of the virus, and named Viola phlebovirus.One pool containing three Phlebovirus from phlebotomus fever serogroup, named Viola phlebovirus.HTS, viral isolation, RT-PCR and TEM showed the presence of one virus in sand flies from North Pantanal with identity to a putative novel The online version of this article (10.1186/s13071-018-2985-3) contains supplementary material, which is available to authorized users. Metagenomic studies based on high-throughput sequencing (HTS) have increased the discovery of new viral species and novel variants of known viruses, as well as contributed to viral ecology and evolution studies , 2.The importance of arbovirus transmitted diseases dramatically increased in the past few decades, since emerging arboviruses lead to the occurrence of large viral epidemics in tropical developing regions of the world, including Brazil. Although arboviruses present worldwide distribution, viral species distribution varies among geographical regions. Notably, higher incidence has been observed in tropical areas around the globe , 4.Lutzomyia are highly anthropophilic, occur in a wide geographical distribution and constitute important vectors of human infections in the Americas.Phlebotomines  are medically important insects popularly known as sand flies, involved in the transmission of bacteria, protozoan and arboviruses to humans and animals , 6. At lRhabdoviridae (genus Vesiculovirus), Phenuiviridae (genus Phlebovirus) and Reoviridae (genus Orbivirus). Sporadic reports of human cases indicate these infections are incidental in South America, associated mainly with recreational or occupational incursions into tropical sylvatic forests. These viruses are relatively common in Brazil, since at least 20 phleboviruses were discovered in the Brazilian Amazon region between 1954 and 1994 .2 (2 μM), DNTP mix (0.2 μM), 2.5 U of HotStart DNA polymerase  and ultrapure water in 50 μl reactions was amplified at 94 °C for 1 min, 30 cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min, and a final extension of 72 °C for 2 min. PCR products (460 bp) were purified with 20% PEG and sequenced. At 5 days post-infection, the supernatant of Vero cells infected with Viola virus passage 5 was centrifuged at 159,000× g for 75 min using 3 ml of 25% sucrose cushion in polyallomer ultracentrifuge tubes . The pellet was resuspended in PBS-1X and prepared for transmission electron microscopy (TEM) by negative staining [After reverse transcription  with 100 U of GoScript , a PCR containing the same primers at 1 μM, reaction buffer, MgCln = 86; 81.2%), transitional  and dry  periods, 38 (35.8%) in the CGNP and 68 (64.2%) at North Pantanal, comprised three pools of Lutzomyia sp., one of Lutzomyia (Lutzomyia) longipalpis, two of Nyssomyia whitmani, one of Evandromyia (Aldamyia) evandroi, one of Ev. (Ald.) carmelinoi, one of Lu.  sherlocki, one of Ev. (Eva.) wilsoni and two of Brumptomyia sp. .Specimens of Illumina sequencing generated 9,308,876,090 reads and 516,344 contigs from 12 pools. BLASTx analysis of the contigs against a RefSeq viral database resulted in 1198(0.23%) viral hits.Rhabdoviridae (genus Vesiculovirus) and  Flaviviridae (genus Flavivirus)  generated hits with less than 100 bp with some viral families. Three pools of sand flies captured in North Pantanal in the three climatic periods presented contigs of 43–57 nt with 40–47% of identity with members of the families Lu. longipalpis females and males captured in the transitional period in Pirizal, North Pantanal presented the genome of a tripartite segmented virus belonging to phlebotomus fever serogroup, genus Phlebovirus, family Phenuiviridae, order Bunyavirales.One pool containing extracts of three specimens of The partial genome sequence of this virus showed a low similarity with other species classified in the same genus and therefore, represents a putative new viral species that was named Viola phlebovirus.After the identification through HTS, this virus was isolated in Vero cells in the fourth passage and confirmed by RT-PCR using L segment specific oligonucleotides and nucleotide sequencing, and also by visualizing the viral particles in the supernatant after TEM Fig. .Fig. 2RTThe L segment presents 6341 nucleotides (nt) in length and encodes the RNA-dependent RNA polymerase (RdRp)  Fig. . The maiThe M segment is 4442 nt in length and encodes a polyprotein  processed into: (i) NSm protein , characteristic of phlebotomus fever serogroup; (ii) the G1 glycoprotein ; and (iii) the G2 glycoprotein  Fig. . TransmeThe partial S segment is 1732 nt in length and encodes the nucleoprotein (N) and a nonstructural (NSs) protein in an ambisense coding strategy Fig. . The N pPhenuiviridae, genus Phlebovirus, phlebotomus fever serogroup, clustering with Urucuri virus, Changres virus and Uriurana virus  showed similarity of 61% and 49%, respectively  in the Amazon and in the Utinga Forest, State of Pará, respectively [Viola virus was more closely related to phleboviruses from phlebotomus fever serogroup isolated from sand flies (Uriurana virus) and rodents ectively , and alsectively .In addition, viral isolation of Viola virus in mammalian cell lines (Vero cells) and the presence of NSs and NSm proteins indicate that it is not an insect-specific virus (ISV) and might represent a virus that infect vertebrates.Phlebovirus (Viola phlebovirus) in Lu. longipalpis and its initial characterization contributes to our knowledge about viral diversity in arthropods from sylvatic areas of North Pantanal. Phylogeny revealed proximity with viruses causing disease in humans, rodents and isolated from sand flies belonging to phlebotomus fever serogroup. Isolation of Viola virus in mammalian cells indicates this virus is not an ISV and represents a novel species with no known vertebrate host. Therefore, future studies involving Viola phlebovirus may address its importance for public or veterinary health. This is the first study reporting a new species of virus infecting arthropods through HTS and able to replicate in Vero cells in Mato Grosso, Midwestern Brazil.This detection and isolation of a putative new Additional file 1:Figure S1. Pairwise amino acid comparision of Viola phlebovirus M segment sequences with all other M segment sequences of  members of the genus Phlebovirus available in the GenBank database. (PNG 62 kb)


Oryctolagus cuniculus). Lessons from those attempts , a cyprinid teleost fish that is the major vertebrate pest in Australian inland waterways . However, for most of the world, common carp are an important food source, and among the most farmed fish globally  and rabbit hemorrhagic disease virus (RHDV), partly address these questions  or the next generation(s) of virus as essential complementary broad-scale controls for CyHV-3  is likely to be much less efficient in transmission of CyHV-3. Secondly, we hypothesize that transmissibility will be favored by the evolution of low virulence strains of CyHV-3 that allow survival of many infected carp, with subsequent regular recrudescence of infection in, and direct transmission from, these survivors during breeding aggregations (McColl et al., Another important lesson from work on rabbit biocontrol with RHDV was that an unrecognized, cross-reactive and avirulent virus in the targeted pest species may confer protection from a biocontrol virus (McColl et al., Given the range in the severity of CyHV-3 outbreaks around the world in recent decades (Hedrick et al., not be a good idea. Consistent with this approach, we have already addressed some of the misconceptions and misunderstandings discussed in this review. However, all of these issues may become important as we continue to address the safety and efficacy of CyHV-3 as a potential biocontrol agent in Australia.We have always been aware that the release of an exotic virus into Australia's waterways, if it were to occur, would be a contentious event. Our aim from the outset has been to find reasons why release of CyHV-3 would By openly addressing these issues, we hope to curtail ill-informed discussion about the virus without ever discouraging specific, evidence-based criticisms of the proposed use of CyHV-3.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Bats are speculated to be reservoirs of several emerging viruses including coronaviruses (CoVs) that cause serious disease in humans and agricultural animals. These include CoVs that cause severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), porcine epidemic diarrhea (PED) and severe acute diarrhea syndrome (SADS). Bats that are naturally infected or experimentally infected do not demonstrate clinical signs of disease. These observations have allowed researchers to speculate that bats are the likely reservoirs or ancestral hosts for several CoVs. In this review, we follow the CoV outbreaks that are speculated to have originated in bats. We review studies that have allowed researchers to identify unique adaptation in bats that may allow them to harbor CoVs without severe disease. We speculate about future studies that are critical to identify how bats can harbor multiple strains of CoVs and factors that enable these viruses to “jump” from bats to other mammals. We hope that this review will enable readers to identify gaps in knowledge that currently exist and initiate a dialogue amongst bat researchers to share resources to overcome present limitations. Bats are an ancient and diverse group of ecologically important mammals, constituting almost a quarter of all mammalian diversity and inhabiting every continent except Antarctica. More than 1300 species of bats belong to the order Chiroptera and are further classified into two suborders, Yinpterochiroptera and Yangochiroptera ,3. The YIn this mini-review, we focus on the role of bats as reservoir hosts for important human and animal coronaviruses. We discuss the evidence of coronavirus spillover from bats, how bat ecological niches may contribute to spillover and the need to further explore bat-coronavirus interactions using viruses that have been naturally detected in bats.Coronaviridae) is the largest in the order Nidovirales. Coronaviridae consists of two subfamilies, Letovirinae and Orthocoronavirinae. Within the subfamily Orthocoronavirinae, there are four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. Alphacoronaviruses and betacoronaviruses are found in mammals, whereas gammacoronaviruses and deltacoronaviruses are primarily found in birds [The coronavirus family , transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and more recently, swine acute diarrhea syndrome coronavirus (SADS-CoV). PEDV led to a 3.21% decrease in the U.S. pig crop between September 2012 and August 2014  and IBV Many of these human and animal coronaviruses appear to have origins in a variety of bat species. With the advent of next generation sequencing technology and increased surveillance of wild animal species, an enormous number of novel coronaviruses have been identified. To date, over 200 novel coronaviruses have been identified in bats and approximately 35% of the bat virome sequenced to date is composed of coronaviruses [SARS-CoV emerged in humans in 2002 and efficient human to human transmission resulted in a global SARS epidemic which lasted 8 months . InitialMERS-CoV emerged in Saudi Arabia in 2012 and continues to cause human disease with a case fatality rate of 35% . DromedaPerimyotis subflavus and was found to be a recent common ancestor of HCoV-NL63 with an estimated divergence of ~550 years ago [In addition to emerging highly pathogenic coronaviruses, human coronaviruses that cause the common cold are also thought to have their origins in bats. HCoV-NL63 was first identified in a pediatric patient with bronchiolitis in 2004, but since then it has come to be appreciated that the virus causes approximately 1–9% of the common colds each year and has most likely circulated in humans for centuries with worldwide distribution ,50,51. Aears ago . HCoV-NLears ago , furtherears ago . HCoV-22ears ago ,55. In 2ears ago . Full geears ago . By analears ago . Interesears ago . Seroposears ago . These dears ago . Thus itScotophilus bat coronavirus (BtCoV) 512/2005 than to other known alphacoronaviruses, such as transmissible gastroenteritis coronavirus (TGEV) and HCoV-229E and HCoV-NL63, in phylogeny as well as genome organization [Eptesicus fuscus) [Porcine epidemic diarrhea (PED) was recognized as an enteric disease in pigs in the United Kingdom as early as 1971. PEDV was detected in Belgium in 1978 . The fulnization . This finization ,62. Multnization . An effenization , no clin fuscus) . PEDV reMore recently, a novel HKU2-related bat coronavirus, SADS-CoV has been shown to cause fatal enteric disease in pigs. Zhou et al. identified SADS-CoV as the causative agent for a large-scale outbreak of fatal disease in pigs in China that caused the death of 24,693 piglets across four farms. SADS-CoV-like viruses with 96–98% similarity to SADS-CoV were detected in 9.8% of the bats that were sampled in this region [Rousettus aegyptiacus bats due to the biannual birthing cycle. This pulse of virus positive bats correlates with an increased incidence of human spillover events [R. aegyptiacus bats was confirmed in a controlled experimental setting [Understanding how bats maintain a virus within a population is important for predicting spillover transmission events. For many viruses with known or suspected bat reservoirs, spillover transmission events typically occur within a defined time frame and location, which corresponds with higher than normal virus levels in the bat reservoir host. The reason for these “pulses” of virus within the reservoir host population are not clear, but proposed theories and evidence supporting these theories have been reviewed by Plowright et al. . In the r events . Horizon setting . Further setting .Pteropus scapulatus) [Myotis lucifugus) with White-Nose Syndrome causing fungus (Pseudogymnoascus destructans) leads to an increase in replication of a persistently infecting coronavirus in these bats [Studies with Hendra virus have shown that reproductive and nutritional stress can increase the levels of virus in little red flying foxes (pulatus) . The incese bats .Cardioderma cor, Ediolon helvum, Epomophorus labiatuc, Hipposideros sp., Miniopterus minor, Otomops martiensseni, Rhinolophus hildebrandtii, Rhinolophus sp., and Triaenops afer. The phylogenetic analysis of these novel CoVs found a number of cross-species transmission events, although the majority of these events appeared to be transient spillover events [Hipposideros bats and HCoV-NL63-like viruses found in Triaenops afer bats, where the gene encoding for the spike protein is more closely related to the HCoV-229E virus [A recent study by Anthony et al. evaluated the global diversity of coronaviruses in almost 20,000 animals and humans. During the course of this study, they found that the diversity of coronaviruses was highly associated with the diversity of bat species and this diversity separated into 3 distinct geographical regions, which mirrored the distribution of different species of bats. The authors report particular associations between bat families and viral sub-clades that suggest co-evolution . A surver events . The recr events , could l9E virus . Further9E virus ,23. In tAlthough bats are known to harbor a wide variety of coronaviruses, the mechanisms for virus spillover into humans or livestock are widely unknown. There is evidence that there are seasonal fluctuations in virus replication ,75, howeBats are known to harbor a wide range of viruses including many that are highly pathogenic in humans. Research to determine the mechanisms by which bats limit disease following virus infection is a relatively new field and can be difficult due to a lack of reagents and the need to develop appropriate in vitro and in vivo systems. Even with these limitations, a variety of studies have been performed that evaluate the bat immune response to virus infection at the genomics level, in vitro using cell culture systems, and performing experimental infections in vivo. Of note, very few of these studies are focused on coronavirus infections in bats and are rather centered around henipavirus and filovirus infections. Future studies evaluating the virus-host interactions of bats and coronaviruses, particularly with bat CoV isolates are important in determining why bats serve as important reservoirs for CoVs and how they control infection to limit severe pathological consequences.Pteropus alecto (black flying fox), Rousettus aegyptiacus (Egyptian rousette), and Artibeus jamaicensis (Jamaican fruit bat). Papenfuss et al. were the first to sequence the P. alecto transcriptome and identified approximately 500 genes  that encode immune-related proteins [R. aegyptiacus and A. jamaicensis [P. alecto [Multiple studies have elucidated unique adaptations in the antiviral responses of bat cells. The primary bat species being used to study the bat immune response to virus infections in vitro and in vivo are proteins . A similaicensis ,78. Thisaicensis ,77. Furt. alecto ,80,81.P. alecto has a contracted type I IFN locus [P. vampyrus, M. lucifugus, and R. aegyptiacus have expanded the number of type I IFN genes [P. alecto cells constitutively express three different IFNα genes [R. aegyptiacus do not show constitutive expression of IFNα [IFNAR1 and IFNAR2, as well as a variety of interferon-stimulated genes are upregulated in these bat cells compared to human cells [A large amount of interest in bat immune responses has focused specifically on the interferon response. Genomic analysis of the interferon loci has shown species-specific evolution in which FN locus  whereas FN genes ,84. It hNα genes  whereas an cells . The molR. aegyptiacus bat cell line and that this inhibition is dependent on the viral protein VP35 [Although it appears that bats have many of the genes that are important for responding to virus infection, how this response compares between human and bat cells is just beginning to be examined. RNA sensing and subsequent antiviral responses in bat cells have been studied using viruses known to induce an interferon response, such as Sendai virus or Newcastle disease virus or by transfecting a synthetic surrogate of viral double stranded RNA (poly(I:C)) ,88,89,90ein VP35 .Coronavirus accessory proteins are dispensable for replication but they play an important role in pathogenesis and virus fitness under the natural environment of a host ,91. MultIn addition to studying the role of CoV proteins in antagonizing the antiviral response in bat cells compared to other mammalian cell lines, it is also important to determine how CoVs isolated from bats compare to those isolated from humans. Coronavirus accessory genes have co-evolved with their natural host for optimum functionality  and thusThe vast majority of studies evaluating the bat host response to virus infection has been performed in cell lines. However, there is a great need to understand what happens during a virus infection in bats in vivo. The ability to study these questions is a daunting task and requires specialized facilities and staff, appropriate species selection especially for CoVs, and generating the necessary reagents. Because of these limitations, only a handful of studies have been performed looking at the in vivo response of bats to virus infection. In fact, there are only two published studies in which experimental infections in bats using CoVs was performed.Cynopterus brachyotis), they administered intestinal samples orally to Leschenault rousette bats (Rousettus leschenaulti). Virus could be detected by quantitative real-time PCR (qPCR) on 2 to 5 days after infection and there was an increase in viral RNA while no clinical disease was observed. Based on these data, the authors reported that this bat CoV replicates in Leschenault rousette bats; however, they were not able to isolate live virus [The first study was performed in an attempt to rescue a bat CoV isolate. Watanabe et al. detected CoVs in 57.1% of insectivorous bats and 55.6% of frugivorous bats; however, they were unable to culture the virus in vitro. To propagate CoVs detected in a lesser dog-faced fruit bat (ve virus . This stArtibeus jamaicensis) with MERS-CoV/EMC2012. The authors detected virus shedding in the respiratory and intestinal tracts for 9 days. Although the bats showed evidence of virus replication, no overt signs of disease were observed. A moderate and transient induction of the innate immune response was seen, but there were no signs of inflammation. Based on their observations, the authors reported that Jamaican fruit bats support the replication of MERS-CoV and thus, bats could be potential ancestral hosts of MERS-CoV [The second study aimed to determine if bats could be infected with MERS-CoV and, if so, what the host response looks like. Munster et al. infected ten Jamaican fruit bats . Widagdo et al. mapped the tissue distribution of DPP4 in multiple bat species to identify the differences in tissue tropism of MERS-CoV. In their study, the authors report that DPP4 in insectivorous bats is primarily detected in the gastro-intestinal (GI) tract and kidneys, whereas frugivorous bats express DPP4 in the respiratory and GI tracts . Other sThe ability of bats to harbor several different coronaviruses may seem like a mystery, but the same is true for rodents. Although bats harbor more zoonotic viruses per species, rodents harbor a larger total number of zoonotic viruses . After tPseudogymnoascus destructans) increases CoV replication in M. lucifugus [A recent study demonstrated that secondary infection with the White-nose syndrome fungus (ucifugus . This stEcological and epidemiological studies to identify landscape changes and human practices that could enable a coronavirus to spillover from bats are also necessary . Such stAs the human population expands and societal changes occur, human contact with wildlife will continue to increase. This increases the risk that emerging zoonotic viruses, including CoVs, pose to human and animal health. Surveillance combined with scientific studies to better understand zoonotic CoVs and spillover will enable us to stay a step ahead of the next epidemic.