Nepovirus subgroup C.A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus The online version of this article (10.1186/s12985-019-1181-1) contains supplementary material, which is available to authorized users. Nepovirus in the subfamily Comovirinae of the Secoviridae family possess a bipartite genome consisting of two positive ssRNAs with a 5′ viral protein genome-linked (VPg) and a 3′ poly(A) tail [Secoviridae known to have a single CP [Viruses from the genus (A) tail . The RNA(A) tail , 2. Nepoingle CP . Nepoviringle CP , 3. Seedingle CP , 2. In hCarum carvi L.) is an aromatic biennial plant in the Apiaceae family [Nicotiana benthamiana and chlorotic local lesions were observed on inoculated leaves followed by systemic chlorosis and necrosis. Symptom recovery was not observed. The virus was provisionally named “caraway yellows virus” (CawYV).Caraway  revealed the presence of isomeric virus particles of about 30 nm in diameter in preparations made from the original infected caraway sample Fig. b, indica EM Fig. c. This hN. benthamiana using Double-RNA Viral dsRNA Extraction Mini Kit for Plant Tissue (iNtRON) following the manufacturer’s instructions. The extracted dsRNA was sent for library preparation and high throughput sequencing (HTS) at Eurofins GATC Biotech GmbH. The dsRNA was fragmented, strand specific cDNA was synthesized using random primers (the dsRNA was denatured at 99 °C for 2 min), followed by adapter ligation and adapter specific PCR amplification then sequencing on Illumina NovaSeq 6000 platform (2 × 150).To obtain the full genome of CawYV, double stranded RNA (dsRNA) was extracted from infected Using Geneious Prime v. 2019.0.4), the raw reads 15,468,416) were quality trimmed, filtered, normalized, and error corrected followed by de novo assembly using Geneious assembler (Medium sensitivity/Fast setting). 36,634 contigs of lengths between 100 and 23,141 nt were generated. A BLASTn search of the contigs against a local database for viruses and viroids downloaded from NCBI showed that two contigs of 7180 and 6341 nt had 72% identity  to peach rosette mosaic virus (PRMV) and 79%  to blueberry latent spherical virus (BLSV), respectively. The 5′ ends of both RNA segments were confirmed using RNA ligase-mediated amplification of cDNA ends (RLM-RACE) [,416 were9.0.4, th27WX11LX21LXE) was located at aa residues 438–502. The typical NTP-binding helicase motif A (GX4GKS), motif B (D), and motif C (N) were found at aa 752–759, 803, and 852, respectively. A serine protease motif was found at aa 1280–1449 (HX40EX106SGX8GX5GXHX2G) of the CawYV RNA1 polyprotein sequence  but was described for some members of genus Nepovirus subgroup C i.e., BLSV, CLRV, PRMV and soybean latent spherical virus (SLSV). The RNA-dependent RNA polymerase (RdRp) core domain was located at aa 1774–1880 (DX4DX56GX3TX3NX33GDD). Pairwise analysis of the protease-polymerase (Pro-Pol) region aa sequences showed a closest identity to PRMV Pro-Pol with 80.1%  encoding a polyprotein of 2213 aa in length. Pairwise comparisons of nt and aa sequences of this ORF to its homologues of the other nepoviruses were performed using ClustalW . The resCawYV-RNA2 contains an open reading frame (nt position 95 to 5116) encoding a polyprotein of 1673 aa in length. Pairwise comparisons of RNA2-ORF nt and aa sequences to the homologues of other nepoviruses showed the highest similarity with PRMV with 31% nt and 21.3% aa identities  [Each of the CawYV polypeptides is predicted to be proteolytically cleaved into putative peptides by the virus-encoded protease. Sequence alignment of all nepovirus ORF aa sequences suggest a putative proteolytic cleavage sites at dipeptides (H/S). This potential cleavage site was not identified before in the entally) . The H/SNepovirus subgroup C  based on the aa alignments of the Pro-Pol and the CP regions were additional evidence for the relatedness of CawYV to the 6 based oApiaceae family. The serological cross-reactivity is well known for members of the same genus in the family Secoviridae [Nepovirus. Further studies are needed to investigate the natural mode of transmission and the biological characteristics of CawYV.The International Committee on Taxonomy of Viruses (ICTV) suggests the following criteria for species demarcation : distincoviridae . This mioviridae  but are Additional file 1:Table S1. List of the primers used for caraway yellows virus 5′ and 3′ ends confirmation. (DOCX 18 kb)


Flaviviruses are globally distributed pathogens causing millions of human infections every year. Flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. Mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response. Flaviviridae family, includes many pathogens causing severe human disease, ranging from mild fever to encephalitis and hemorrhagic fever. There are more than 70 viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (DENV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV) and ticks , Langat virus (LGTV), Kyasanur forest disease virus (KFDV), Omsk hemorrhagic fever virus (OHFV), Powassan virus (POWV), and Louping-ill virus (LIV)) . It. It48]. settings .Although not much is known about POWV pathogenesis, recent studies in mice have found that tick saliva was important to enhance POWV transmission and the outcome of disease . Furtherhttps://www. CDC.gov). There are no antiviral treatments or vaccines available against POWV.During the last 10 years there has been an increase of POWV in the USA with approximately 100 reported cases ,55. The Ixodes ricinus . Th. Th17]. nfection . Muskratnfection . Secretinfection . The exanfection . OHFV manfection . No antiHemaphysalis but other tick genera have also been shown to be able to transmit KFDV [KFDV is only found in India , althougmit KFDV ,65. Durimit KFDV . The secmit KFDV , with camit KFDV ,65. In 1mit KFDV ,68,69. Tmit KFDV .TBFVs are enveloped viruses around 50 nm in diameter. The envelope carries two surface proteins, the envelope (E) protein and the membrane (M) protein. The latter is derived from a precursor protein, prM. The nucleocapsid (NC) lies inside the viral envelope and consists of multiple copies of capsid (C) protein and the viral genome. The TBFV genomes are single stranded, positive-sense RNA of approximately 11,000 nucleotides. It has a 5′-cap with a single open reading frame (ORF). The ORF is flanked by 5′ and 3′ untranslated regions (UTRs). The viral protein is encoded by the ORF as a single polyprotein, which is co- and post-translationally cleaved by cellular and viral proteases into individual viral proteins . The polyprotein is arranged in the order 5′-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′ ,71.The first step of virus replication starts when the virus binds to its receptor and is taken up into the cell by receptor-mediated endocytosis. The attachment is mediated by viral E protein and entry receptor on the host cell. The entry receptor for TBFVs has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding and entry . Once thIn arthropods, such as mosquitos and ticks, RNA interferences (RNAi) are the most important antiviral defense and tick cells have been shown to mount RNAi responses against LGTV and TBEV . ProteinThe innate immune response provides the first line of defense against viral infections. This defense includes physical and chemical barriers such as the skin and mucous membranes and the acidity of the stomach. They serve as an initial barrier that protects the host from infection. Once these barriers have been breached, the innate immune response relies on a set of germline-encoded receptors, known as pathogen recognition receptors (PRRs), which recognize pathogen specific molecular patterns that are sensed as a danger signal by the host cell ,96. EngaPeromyscus lecuopus, which are known as a natural host of the virus [In 1957 Isaacs and Lindenmann discovered IFN as a secreted factor that interfered with viral replication . In the he virus . Similarhe virus , and IFNhe virus .Three distinct classes of IFN, type I (IFN α and β), type II (IFNγ), and type III IFN (IFNλ) have been described. The expression of the IFNλ-receptors and IFNγ are restricted, but the type I IFNs can be expressed by most cell types and their receptor, the interferon-α/β receptor (IFNAR) is expressed on all nucleated cells . There aThe host cell detects invading pathogens through PRRs, which recognize foreign molecular patterns that are generated during infection. The particular PRR involved in the recognition of the pathogen depends on the infecting virus . In flavUpon recognition, the different PRR will recruit distinct adaptor molecules. TLR3 recruits TIR-domain-containing adapter-inducing interferon-β (TRIF) , TLR7 anAlthough the innate antiviral response has been well-studied in mosquito-borne flavivirus infection, the responses to TBFVs are less investigated, and most studies have used TBEV which therefore will be the main focus of the remainder of this review.−/− mice had lower induction of IFNβ within the olfactory bulb despite higher viral burdens [In TBEV infection, IFNβ induction has been shown to correlate with amount of intracellular viral RNA, and the IPS-1 pathway has been shown to protect mice from lethal LGTV infection and prolong survival after TBEV infection ,128. Abs burdens ,83. Inte burdens . SG cont burdens . SG are  burdens . Thus, SLittle is known about the role of the TLR7/8-Myd88 pathway in tick-borne flavivirus infection; however, TLR7 was shown to suppress LGTV replication within neurons of the brain although it did not affect pathogenesis . What weTaken together, studies so far have demonstrated the importance of the RIG-I-like-IPS-1 pathway in tick-borne flavivirus infection, whereas the role of the TLR pathways remains unclear.After secretion, type I IFN signals in an autocrine and paracrine manner by binding to the heterodimeric IFNAR, which consists of two subunits, IFNAR1 and IFNAR2 . These s−/− mice survived the infection, demonstrating that the IFNαs can compensate for loss of IFNβ in LGTV infection. Furthermore, IFNAR was shown to be a critical determinant of LGTV tropism as LGTV RNA was found in all organs in the absence of IFNAR, whereas in WT, only low viral burdens can be detected in the olfactory bulb [The IFN response in vivo in mice is very important to protect mice from lethal infection with LGTV. Mice lacking IFNAR all succumbed within 6 days of infection, whereas 80% of WT mice (6–8-week-old) survived the infection. Interestingly, all IFNβory bulb ,128,141.ory bulb ,100.Oas1b, which is often lacking in inbred mice strains, confers strain dependent resistance against neurovirulence from far eastern TBEV [Within the CNS, LGTV mainly infected neurons ,128, whiern TBEV . Two othThe TRIM proteins are a family of proteins able to mediate antiviral activity against many different viruses . The rodflaviviridae, viperin has been shown to inhibit several members, such as WNV [Viperin is highly conserved in evolution and was first identified as an IFN-inducible protein with antiviral activity against human cytomegalovirus in 2001 . Over thh as WNV , DENV [1h as WNV , ZIKV [1h as WNV , hepatith as WNV , and TBEh as WNV ,149,150.h as WNV ,164,165 h as WNV , and a hh as WNV . Although as WNV . Viperinh as WNV ,149,150.h as WNV . The Fe/h as WNV , and bash as WNV ,169,170.h as WNV . GBF1 ish as WNV ,173,174 Although viperin has been demonstrated to be antiviral active against many different viruses in vitro, few studies have investigated viperin’s role in vivo. In TBFV infection, viperin was shown to control LGTV dissemination and replication in the brain after intraperitoneal administration, and viperin promoted survival after intracranial infection . InteresTaken together, viperin has been shown to be an ISG that strongly inhibits tick-borne flaviviruses in vivo and in vitro. This strong antiviral effect on TBEV is mediated by targeting the virus at multiple steps of the life cycle. Interestingly, viperin targeting the synthesis of plus-sense RNA is dependent on the SAM domain or the C-terminal domain, while the N-terminal domain of viperin is responsible for interfering with virus assembly and release.In order to establish an infection, a pathogen needs to overcome or evade the innate immune response. To breach the very first line of defense, the skin—TBFVs use the tick to deliver the virus through the skin via tick saliva. Furthermore, tick saliva contains immunomodulatory compounds that enhance viral transmission and dissemination ,176. AltFlaviviridae family is the antagonism of IFNAR signaling carried out by NS5; this mechanism is conserved between several mosquito and tick-borne flaviviruses [The most conserved inhibition of the type I IFN system within the iviruses ,180,181.iviruses . Similariviruses . Knockdoiviruses . Furtheriviruses . PEPD isiviruses ,182. In iviruses .During TBFV infection, a subgenomic noncoding RNA is formed, called subgenomic flavivirus RNA (sfRNA) ,184,185.Even though recent studies have shed light on the role of innate immunity during tick-borne flavivirus infection, much remains unknown. For example, the role of the TLR-TRIF and TLR-MyD88 pathway in pathogenesis and viral recognition. Furthermore, most studies were performed using LGTV and TBEV, and although they are closely related to POWV, LIV, KFDV, and OHFV, their interactions with the innate immune response might differ. Although IFN has been shown to strongly control viral tropism and pathogenesis of tick-borne flaviviruses, few antiviral ISGs have been identified. Viperin has been shown to be the most important ISG in cortical neurons, however, other ISGs that target TBEV expressed in astrocytes and granular cell neurons are yet to be identified and very little is known about the early response against the hemorrhagic TBFVs. We also know very little about how the innate immune response regulates the neuroinvasion of neurotropic TBFV, and the specific interactions between the tick vector and the different viruses.


Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin  and the cobalamin lower ligand  in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.Cobalamin (vitamin B Classifair Fig. . The syn1%) Fig. . When evProteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota) were associated with all 12 representative cobalamin biosynthesis genes . The number of genera with the potential for complete cobalamin synthesis showed a relationship with soil type  and the cobalamin transporter protein coding gene (btuB) at the genus level across soil metagenomes, we observed mutual exclusion (permutation test p < 0.001) of these two gene complements for both rare biosphere members  were also surveyed among all 155 soil metagenomes to evaluate potential demand for cobalamin by genera that either produce (with enzymes encoded by cob/cbi and bluB genes) or transport (with transporter encoded by btuB gene) this cofactor  accounted for over 70% of the total rpoB-encoding community  than the encoded potential for cobalamin production and transport  extracts . The presence of DMB in soil samples has been reported in a grove soil and creek bank soil [cbi/cob gene groups . Within the cobalamin pool , we observed OH-B12 as the dominant cobalamin form  based on its concentration in the total pool (sum of both water-leachable and non-water-leachable)  also dominated in the non-water-leachable pool that contained an average of 82.4 ± 31.8% of the total extractable DMB ligand. The average concentration of DMB was higher than that of total cobalamin Fig. . In one ank soil , and thep < 0.01), richness , and Shannon indices . Soil extracted DNA concentrations, a rough estimate of biomass, correlated positively with total cobalamin concentration .We tested for links between microbial diversity, DNA concentration, and cobalamin concentrations in the 40 soil samples analyzed for cobalamin chemistry  [Thaumarchaeota in exponential phase can be orders of magnitude higher than for members of the Proteobacteria [Thaumarchaeota may be a more important source of soil cobalamin than gene counts alone would suggest [Proteobacteria dominated cobalamin synthesis gene affiliations, this was not necessarily the phylum that contributed most to cobalamin biosynthesis because the genes may not be functional and there is no fixed relationship between gene presence and cellular quotas for cobalamin.Based on previous surveys of putative cobalamin-producing phyla , 19, Proh marine  and soilronments , 19, butsamples) , 19, thasamples) , and the suggest . SimilarThaumarchaeota demonstrated that when a genome contains genes necessary for corrin biosynthesis, it is likely to also have genes for DMB biosynthesis and activation [Sphingomonas, an abundant genus, affiliated with >20% of soil btuB and cob/cbi/bluB genes in some soil metagenomes. However, when retrieving representative genomes and examining them at the species level, we noticed either a lack of an annotated btuB gene in the genomes or a btuB gene with a low annotation score, whereas cobalamin synthesis genes are present in different species, suggesting that individual species probably synthesize or transport cobalamin, but not both. A caveat is that taxonomic assignment for a functional gene requires increased resolution at lower taxonomic ranks, in this case at the species level, to avoid masking by a higher rank. Horizontal gene transfer can also complicate taxonomic classification, which might cause a cobalamin biosynthesis gene to be attributed to the wrong species, and consequently overestimate certain genera.Our results show that, collectively, rare biosphere (<0.1% relative abundance) and intermediate abundance (0.1–5.0% relative abundance) genera are equally likely to be cobalamin producers (~50%) but the most abundant soil genera (>5% relative abundance) generally lack genes coding for cobalamin synthesis Table . Becausetivation . AlthougbluB gene codes for the aerobic pathway for DMB synthesis, and is only reported for Gram-negative bacteria and members of the Thaumarchaeota [bzaABCDE genes [Rhodobacter sphaeroides, has been shown to remodel pseudocobalamin with amidohydrolase (CbiZ) and cobinamide-phosphate synthase (CbiB) [In addition to measuring cobalamin concentrations in soils, we measured DMB, which has no other known function in cells beyond serving as the lower ligand of cobalamin. We found relatively high concentrations of DMB compared with cobalamin. Group C cobalamin biosynthesis gene abundances were higher than group A and B cobalamin biosynthesis gene abundances Fig. , suggestrchaeota , 48, 59,e (CbiB) , 60. It e (CbiB) , or thatNitrospira spp., which are diverse and abundant nitrite-oxidizing bacteria [Candidatus Nitrospira defluvii genome [Nitrospira sequences were associated with ~0.5% of all cobalamin biosynthesis genes across 155 soil metagenomes  alone accounted for ~90% of all microbial community members, almost eightfold more abundant than microbial cobalamin producers. Indeed, Giovannoni proposed that cofactor production by a subset of microbes in a community could be “the most widespread and important example of metabolic outsourcing” [Cobalamin may be exuded into the soil matrix by living cells or released by cell lysis. In either case, cobalamin producers may play a role that is consistent with the Black Queen hypothesis . Becauseourcing” . In the ourcing” , 71–73. 2-cobalamin) was recently discovered in a marine ammonia-oxidizing archaea isolated from an oxygen deficient zone and other low oxygen water samples [2BL; data not shown). Differences in cobalamin patterns between terrestrial and marine habitats might underpin distinct cobalamin usage mechanisms and consequently metabolisms among microorganisms.As novel cobalamin analogs are being discovered, it is likely that our understanding of how this cofactor relates to soil microbial ecology will continue to expand. For example, nitrocobalamin  [btuB [metH, mutA, and rsmB [cob/cbi, bluB, btuB, metH, mutA, and rsmB genes were retrieved from TIGRFAM [rpoB, from FunGene [We analyzed 155 soil metagenomes available from the Metagenomics RAST Server  –76. The B) [btuB , 77, wasand rsmB  were inc TIGRFAM  or Pfam  TIGRFAM , and the FunGene . Metagen FunGene . The det2BL) soils covering wide range of soil biomes across Canada [rare Charitable Research Reserve, Cambridge, Ontario) [Soil samples for cobalamin and DMB measurements  , and a pOntario) . Both waOntario) . DetaileSoil DNA was used for microbial community size estimation in these soils, and was extracted according to the manufacturer protocols with the PowerSoil DNA Isolation Kit , and quantified with a Qubit 2.0 fluorometer .vegan (V 2.4.3) package in R. In order to test the relationship between cobalamin-producing and consuming taxa in the soil metagenomes studied, Spearman correlation coefficients between the relative abundance of cob/cbi/bluB and btuB genes were calculated and compared to the coefficient through 105 permutations to determine significance values. The phyloseq (V 1.14.0) [Soil metagenome data were transformed into sample-based relative abundance  before statistical analysis. All statistical analyses were carried out using R (V 3.2.3). Analysis of variance (ANOVA) was performed to test the effect of environmental factors on gene abundance or cobalamin concentration, followed by post-hoc Tukey HSD test. A Shapiro–Wilk normality test was used to check normality assumption prior to correlation analysis. Spearman’s rank correlations were employed to compare cobalamin concentrations and microbial biomass, and a simple linear regression was used to test for a significant linear relationship. The influence of soil types on cobalamin-producing phyla composition was evaluated by permutational multivariate analysis of variance (adonis) with the  1.14.0)  R packagMerged supplemental materialSupplemental material S1Supplemental material S2Supplemental material S3


Marmota flaviventer) and test how these effects vary with age and between two contrasting environments. We provide evidence for cumulative long‐term effects: females that had both reproduced frequently and weaned large litters on average in previous years had decreased current reproduction probability. We found no evidence for short‐term reproductive costs between reproductive bouts. However, females weaned larger litters when they had weaned larger litters on average in previous years and had lower current reproduction probability when their previous reproductive success was low. Together these results suggest that, alongside persistent among‐individual variation, long‐term reproductive history affects current reproductive success.The cumulative cost of reproduction hypothesis predicts that reproductive costs accumulate over an individual's reproductive life span. While short‐term costs have been extensively explored, the prevalence of cumulative long‐term costs and the circumstances under which such costs occur alongside or instead of short‐term costs, are far from clear. Indeed, few studies have simultaneously tested for both short‐term and cumulative long‐term reproductive costs in natural populations. Even in mammals, comparatively little is known about cumulative effects of previous reproduction, especially in species with high variation in offspring numbers, where costs could vary among successful reproductive events. Here, we quantify effects of previous short‐term and cumulative long‐term reproduction on current reproduction probability and litter size in wild female yellow‐bellied marmots ( HoweverWhen there are short‐term costs of reproduction, a reduction in current reproductive success is expected following a successful reproductive event in the previous year Stearns, . When thLarus occidentalis, Pyle, Nur, Sydeman, & Emslie, Parus montanus, Orell & Belda, Pyrrhocorax pyrrhocorax, Reid, Bignal, Bignal, McCracken, & Monaghan, Uria aalge, Reed et al., Parus major, Bouwhuis, Charmantier, Verhulst, & Sheldon, Bison bison, Green & Rothstein, Mirounga angustirostris, Sydeman, Huber, Emslie, Ribic, & Nur, Cervus elaphus, Nussey, Kruuk, Donald, Fowlie, & Clutton‐Brock, Ovis canadensis, Bérubé, Festa‐Bianchet, & Jorgenson, Dama dama, McElligott & Haydon 2000; red deer, C. elaphus, Moyes et al., Oreamnos americanus, Panagakis, Hamel, & Côté, Gorilla gorilla, Robbins, Robbins, Gerald‐Steklis, & Steklis, Orcinus orca, Ward, Parsons, Holmes, Balcomb, & Ford, Arctocephalus gazella, Arnould & Duck, Tamiasciurus hudsonicus, McAdam, Boutin, Sykes, & Humphries, In birds, long‐term reproductive costs on both future reproduction and survival appear to be common , a polytocous, hibernating rodent , over a 5 km stretch of the Upper East River Valley, Colorado. The study area includes two distinct regions: “up‐valley” and “down‐valley”. Marmot movement between the two regions is rare, and individuals included in our analyses lived either up‐valley or down‐valley throughout their entire lives. The “up‐valley” region is at approximately 165 m higher elevation than “down‐valley” and is characterized by later snowmelt, and hence later onset of vegetation growth and marmot emergence from hibernation  = 0.57; r(Nr.pups) = 0.48; Figure We defined and used three metrics of previous reproduction: one quantifying short‐term reproduction and two quantifying cumulative long‐term reproduction. To quantify short‐term reproduction, females were initially classified as having reproduced last year or not . Due to collinearity issues between the number of reproductive events and number of weaned offspring in previous years , are isometrically related . The first estimated a female's probability to reproduce in the current year, using a binomial distribution with logit link. The second estimated the weaned litter size in the current year given that a female reproduced, using a Poisson distribution with log link.Fixed effects in both models included the short‐term previous reproduction variable RLY, the two long‐term previous reproduction variables PRF and PALS, second‐degree polynomial age effects (hereafter: “linear and quadratic age”), age at first reproduction (AFR), valley (a two‐level factor: up vs. down), the number of mature daughters living in the same colony as the mother, and interactions between those variables.Specifically, to test the hypotheses that females incurred short‐term and/or cumulative long‐term costs of previous reproduction on current reproduction, we fitted the short‐term (RLY) and long‐term (PRF and PALS) previous reproduction variables in both models. To test whether costs are only expressed when individuals have both reproduced frequently and weaned large litters on average, we fitted a two‐way interaction between the two long‐term previous reproduction variables (PRF and PALS).To test the hypothesis that reproductive costs increase in older individuals, we fitted two‐way interactions between linear and quadratic age and all three previous reproduction variables . To test the hypothesis that costs differ between environments, we fitted two‐way interactions between valley and all three previous reproduction variables.(PRF) = 0.20; r(PALS) = 0.09), because females only reach sexual maturity at 2 years or older.Since age at first reproduction (AFR) affects the number of potentially available reproductive seasons over which costs may accumulate, we also fitted age at first weaned litter to account for differences in onset of reproduction. Further, large matriline sizes have previously been found to negatively affect female yellow‐bellied marmot reproductive success  and with completely known reproductive histories from age at sexual maturity to death. To avoid selective disappearance biases in the data structure, all individuals from nonextinct cohorts were excluded, with the exception of two nearly extinct cohorts . To allow for simultaneous testing of short‐term and cumulative long‐term effects of previous reproduction on current reproduction, analyses were further restricted to females with at least three years of reproductive activity . Every female therefore had at least two years of previous reproductive activity.To account for nonindependence of repeated measures, random individual identity, year, and cohort effects were also fitted. We found little evidence for overdispersion of current reproduction probability , and little evidence of underdispersion of current litter size (ratio = 0.8), thus it was not necessary to take them into account.p > 0.05) were backwards eliminated as they may bias other estimates  as fixed effects in mixed models of the same trait  can lead to biases in model parameters by violating model assumptions of independence. That is because the random intercept is not statistically independent of the lagged dependent variable as it directly affects it  instead of RLY Tables  and S2. Another factor to consider is that maternal body mass in spring affects current reproduction, but conversely reproduction also affects body mass: lactating females allocate resources to their pups and start accumulating fat reserves later than nonreproducing females  ranged from 0.1 to 1.0  on either current reproduction probability Table  or curreThere were cumulative, long‐term effects of previous reproduction on current reproduction. Specifically, there was a significant interactive effect of the two cumulative reproductive variables (PRF and PALS) on current reproduction probability Table . FemalesThere was also a significant main effect of PALS on current litter size Table , showingThere were no effects of age or valley on current reproduction probability or litter size Tables  and 2. F4The importance of cumulative long‐term reproductive costs alongside short‐term costs, and how such costs vary with age and under different environmental conditions, is little explored in wild polytocous mammals. Although we did not find any evidence for short‐term effects of reproduction from one year to the next, we present evidence of cumulative long‐term effects of previous reproduction on current reproduction in a natural population of yellow‐bellied marmots. Reproduction probability decreased in females with both high previous reproductive frequencies and high average litter sizes. However, individuals with higher average litter sizes in previous years also weaned larger litters in the current year. Our results thus support both cumulative reproductive costs and persistent among‐individual differences in reproductive success  similarly found no effects of successfully weaning a litter in the previous year on current reproduction probability and survival  and Olympic marmots (Marmota olympus), in relation to previous weaning and previous parturition success, respectively , suggesting cumulative reproductive costs. If lactation was the major reproductive cost and was independent of litter size, only frequent reproduction may lead to detectable costs. A study in bison, a monotocous species, suggested that maternal allocation per se may be limited , but not the amount allocated if a female does reproduce , for example, offspring in larger litters had lower weaning weights that offspring from smaller litters  had decreased current reproduction probabilities. Again, this is consistent with the idea that previous weaning experience indicates persistent among‐individual differences in acquisition and utilization of resources, as a result of which some females consistently have low values for reproductive traits. Individuals with smaller litters and high reproductive frequencies  could also be expected to be less successful. However, our results do not support this expectation: generally, individuals appear to have either intermediate or low values for both cumulative reproductive measures, and individuals that reproduce often but wean small litters (high‐low) or reproduce less often but wean larger litters (low‐high) have higher current reproduction probabilities than either of the other two groups . Mothers can reduce allocation to their young through decreased milk production (Fite et al., A decrease in reproductive success with age in individuals with high previous reproductive frequencies or litter sizes would suggest that manifestation of cumulative reproductive costs is partly age‐dependent. For example, a study in female northern elephant seals showed reproductive success to decrease with previous reproductive frequency, but only in individuals aged between 11 and 15 years (Sydeman et al., Our study is in line with Hamel et al.’s prediction , in thatAs we did not test for reproductive costs on survival, it is unclear whether reproductive costs on current reproduction were relatively easier to detect than reproductive costs on survival, and this requires further investigation. Finally, transgenerational studies would be interesting to elucidate whether cumulative reproductive costs are transferred to the offspring, reflected as decreases in offspring fitness components.None declared.S.B.K., J.G.A.M., and J.M.R. designed the study. S.B.K. analyzed the data and wrote the manuscript, with contributions from all authors.https://doi.org/10.5061/dryad.9mk47dg.Data for this study have been archived in the Dryad Digital Repository  Click here for additional data file.


Tupaia belangeri) has been proposed as an alternative laboratory animal to primates in biomedical research in recent years. However, characteristics of the tree shrew gut virome remain unclear. In this study, the metagenomic analysis method was used to identify the features of gut virome from fecal samples of this animal. Results showed that 5.80% of sequence reads in the libraries exhibited significant similarity to sequences deposited in the viral reference database , and these reads were further classified into three major orders: Caudovirales (58.0%), Picornavirales (16.0%), and Herpesvirales (6.0%). Siphoviridae (46.0%), Myoviridae (45.0%), and Podoviridae (8.0%) comprised most Caudovirales. Picornaviridae (99.9%) and Herpesviridae (99.0%) were the primary families of Picornavirales and Herpesvirales, respectively. According to the host types and nucleic acid classifications, all of the related viruses in this study were divided into bacterial phage (61.83%), animal-specific virus (34.50%), plant-specific virus (0.09%), insect-specific virus (0.08%) and other viruses (3.50%). The dsDNA virus accounted for 51.13% of the total, followed by ssRNA (33.51%) and ssDNA virus (15.36%). This study provides an initial understanding of the community structure of the gut virome of tree shrew and a baseline for future tree shrew virus investigation.The tree shrew ( Tupaia belangeri) belongs to the family Tupaiidae, order Scandentia, which has a wide distribution in South Asia, Southeast Asia and Southwest China  of the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College.Caudovirales , Picornavirales , and Herpesvirales  (Siphoviridae (46.0%), Myoviridae (45.0%), and Podoviridae (8.0%) comprised most Caudovirales. Picornaviridae (99.9%) and Herpesviridae (99.0%) were the primary families of Picornavirales and Herpesvirales, respectively, as shown in Microviridae (47%), unclassified (36%), Phycodnaviridae (3%), Mimiviridae (3%), Anelloviridae (3%), Inoviridae (2%), Polyomaviridae (1%) and others (5%)  were associated with viruses through comparisons of reads against the NCBI nonredundant nucleotide databases, reference viral protein databases and ACLAME database. These reads were further classified into three major orders, s, 6.0%) . At the ers (5%) . UnknownSiphoviridae (composition ratio 27.07%), followed by Myoviridae (26.03%), Picornaviridae (16.22%), and Microviridae (9.11%) , Cardiovirus (4.92%), Cosavirus (4.73%), and Tunalikevirus (3.84%) had a high relative abundance at the genus level. At the species level, high relative abundances were found for Cercopithecine herpesvirus (composition ratio 6.0%) and African bat icavirus A (5.52%); all others belonged to bacterial phages  . The topl phages . All thePicornavirales, ssDNA viruses (Circovirus), and dsDNA viruses (Adenovirus and Herpesvirus). In addition, some of the results were related to insect viruses in Densoviridae and Iridoviridae and plant viruses in Caulimoviridae and Potyviridae.According to the host types and nucleic acid classifications, all of the related viruses in this study were divided into bacterial phages (61.83%), animal-specific viruses (34.50%), plant-specific viruses (0.09%), insect-specific viruses (0.08%) and other viruses (3.50%) . The dou3’UTR gene of adenovirus. The results showed that the 3’UTR of adenovirus in this study had 93% similarity to the newly described tree shrew adenovirus A (GenBank: AF258784.1)  .Cercopithecine herpesvirus 5 showed 93% similarity with reference NC_012783, ranging from 63,701 to 64,006 positions (306 bp) of the genome, and referred to the UL48 (accession: FJ483968) gene. The second most similar virus with this contig was Cercopithecine herpesvirus 5 strain Colburn (accession: FJ483969), which showed 92% similarity, ranging from 63,916 to 64,221 positions of the genome. These two gene with contigs composed a cluster in Stealth virus) formed two other groups, as shown in the pink and gray areas of Theilovirus showed 92% similarity with Theiler’s murine encephalomyelitis virus (TMEV) (accession: X56019), Theiler's encephalomyelitis virus (accession: U32924), and Theiler’s murine encephalomyelitis (accession: M20562). These viruses constituted a cluster (blue area) in Theiler’s encephalomyelitis virus clustered in two groups in African bat icavirus A and Cosavirus JMY-2014, only one BLAST result was obtained. The contig of the tree shrew showed 83% similarity with African bat icavirus A (accession: KP100644), ranging from 4,995 to 5,223 positions of the genome, and several transitions or transversions were found by alignment analysis, as shown in Cosavirus JMY-2014 (accession: KM516909), ranging from 4,171 to 4,320 positions of the genome, and only 14 single base substitutions were discovered, as shown in For the perspective phylogenetic analysis, the coverage of generated contigs of top virus species were above 80% for each reference genomes, and the details of the alignments were shown in The viral metagenomics method has been employed to identify both commensal viruses and viral pathogens successfully in recent years and has the potential to detect most viruses through sequence similarity searches , 25. DueDensovirinae, Iridoviridae) and plant viral sequences  reflected the diet of rodents. They also noted the presence of plant viruses, such as Virgaviridae, in the virome of the rodents’ feces. Similar results could be found in our study because insect- or plant-specific viruses were identified, which also reflected the eating habits or life cycles of tree shrew. Furthermore, the phylogenetic analysis of tree shrew contigs or genes showed high similarity with reference viruses, such as Cercopithecine herpesvirus 5, Theilovirus, African bat icavirus A, Cosavirus JMY-2014 or adenovirus, possibly reflecting real infectants of the animal.A large number of studies have been performed on animal viromes, both in wild animals and in domestic animals , 26–27. Microviridae, Podoviridae, Myoviridae and Siphoviridae. Carding et al. [Siphoviridae, Myoviridae and Podoviridae. For the non-human primates, D’arc et al. [Siphoviridae, Myoviridae and Podoviridae) represented 67.5 and 68% of the total annotated reads in SIVgor-infected and uninfected individuals, respectively. Specifically, the Siphoviridae family was more frequent in SIVgor-infected individuals compared with uninfected individuals, while two other bacteriophage families were more frequent in uninfected individuals. Liu et al. [Hofer et al.  showed 8g et al.  reviewedc et al.  evaluateu et al.  performeSiphoviridae, reflected the distribution characters of tree shrew gut virome. In addition, we also found some specific viruses in the gut virome of tree shrew. Approximately 15.36% of the virome was detected as the ssDNA virus, the dominant virus; for example, Mischivirus, a pathogen that may cause human disease, was also found in the body of a bat [Orthopoxvirus, under the family Poxviridae, uses vertebrates, including mammals and humans, and arthropods as natural hosts [Alphapolyomavirus, under the family of Polyomaviridae, may infect humans and other mammals [Polyomaviridae is a family of viruses whose natural hosts are primarily mammals and birds. Some members of the family, such as Merkel cell polyomavirus and raccoon polyomavirus, are oncoviruses known to cause tumors or cancers in their natural hosts [Mastadenovirus, under the family of Adenoviridae, has human, mammal, and vertebrate natural hosts. Diseases associated with this genus included respiratory, gastrointestinal and eye infections, among others [Singapore grouper iridovirus, under the family Iridoviridae [Bacteriophages have biomedical importance because they can transmit genes to their bacterial hosts, conferring increased pathogenicity, antibiotic resistance, and new metabolic capacity. Previous studies have shown that siphophage fragments were the most common fragments observed in published metagenomic libraries. In particular, siphophages constituted 44% of phage sequences in the sediment library . Virusesof a bat . For dsDal hosts . Disease mammals . Polyomaal hosts . The thig others . Furtheroviridae  and cateOne major limitation of this study was pooling samples from 50 tree shrews to generate a single metagenome. The primary purpose of this research was to characterize the stool virome of tree shrew, and it would have been more informative to show variability of viruses detected and their proportional abundances across individuals, and to test whether virome composition correlated with host parameters . This would be the future investigation of tree shrew gut virome for our further study.S1 FigCercopithecine herpesvirus 5.A. The diagram of reads mapping to reference Theilovirus.B. The diagram of reads mapping to reference African bat icavirus A.C. The diagram of reads mapping to reference Cosavirus JMY-2014.D. The diagram of reads mapping to reference (TIF)Click here for additional data file.


Culicoides, EHDVs circulate wherever competent vectors and susceptible ruminant host populations co-exist. The impact of variation in the level and duration of EHDV viremia in white-tailed deer (Odocoileus virginianus) on Culicoides infection prevalence is not well characterized. Here we examined how infection prevalence in a confirmed North American vector of EHDV-2 (Culicoides sonorensis) varies in response to fluctuations in deer viremia. To accomplish this, five white-tailed deer were experimentally infected with EHDV-2 and colonized C. sonorensis were allowed to feed on deer at 3, 5, 7, 10, 12, 14, 18, and 24 days post infection (dpi). Viremia profiles in deer were determined by virus isolation and titration at the same time points. Blood-fed Culicoides were assayed for virus after a 10-day incubation (27 °C) period. We found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified four infected midge samples  after feeding on one deer 18 and 24 dpi, when viremia was no longer detectable by virus isolation. The ability of ruminants with low-titer viremia to serve as a source of EHDV for blood-feeding Culicoides should be explored further to better understand its potential epidemiological significance.Epizootic hemorrhagic disease viruses (EHDVs) are arboviral pathogens of white-tailed deer and other wild and domestic ruminants in North America. Transmitted by various species of  Orbivirus: Reoviridae), the causative agent of epizootic hemorrhagic disease (EHD), is transmitted by biting midges of the genus Culicoides to a wide range of wild and domestic ruminants. The EHDV serogroup is comprised of seven serotypes worldwide [Culicoides vector–ruminant host cycle [Culicoides sonorensis is the only confirmed vector of EHDV [Culicoides species are likely involved in transmission [Odocoileus virginianus) are the most severely affected ruminant hosts, suffering significant morbidity and mortality [Epizootic hemorrhagic disease virus (EHDV)  were used and were 3–4 days post-emergence at the time of feeding. All animal procedures were approved by the Institutional Animal Care and Use Committee at Kansas State University (protocol #3438).Six hand-raised white-tailed deer were obtained from the Whitehall Deer Research Facility  and transported to the Large Animal Research Center . The fawns were housed indoors and were seven months old at the time of inoculation. Laboratory-reared Culicoides sonorensis cell line; USDA-ARS) [6.2 tissue culture infective doses (TCID50)/mL as determined by virus titration using CPAE cells in a 96 well format as described in [The EHDV-2 isolate used for inoculation was originally isolated at the Southeastern Cooperative Wildlife Disease Study from the spleen of a free-ranging WTD (CC12-304) from Coffey County, Kansas, during a 2012 EHD outbreak. The virus was originally isolated on cattle pulmonary artery endothelial (CPAE) cells , passaged once in baby hamster kidney (BHK) cells (ATCC), and then to CuVaW8A (CuVa) cells (SDA-ARS) ,19. The ribed in  and endp6.5 TCID50) by a combination of subcutaneous and intradermal injections (0.05–0.1 mL per injection) in the cervical and inguinal regions. The negative control deer was similarly administered a sham inoculum. Each animal was visually monitored for clinical signs of disease twice daily. At 0, 3, 5, 7, 10, 12, 14, 18, and 24 dpi, deer were sedated for physical examination, rectal temperature, blood collection, and Culicoides feeding. Deer were sedated with intramuscular xylazine  and sedation was reversed with slow intravenous injection of tolazoline . Blood in sodium citrate was used for virus isolation and titration. Serum from additive-free blood tubes was used for serology. All EHDV-infected animals were euthanized at 24 dpi by intravenous injection of sodium pentobarbital (1 mL/5 kg).Five deer were inoculated with 2 mL of virus stock  and RNA extracted using a QIAamp Viral RNA Mini kit  according to manufacturer instructions. Virus isolates were confirmed by RT-PCR using previously published primers [To evaluate the diagnostic utility of various cell lines for EHDV, virus isolation from deer blood was performed on CPAE cells, BHK cells, and CuVa cells. All attempts for virus isolation and titration from blood were performed on the day of collection using methods similar to those previously described . Briefly primers . Virus t primers  and endp primers . Serotyp primers  except fg. For virus isolation, 100 µL of supernatant were inoculated onto BHK cell monolayers in a 24-well  or 12-well plate format (pooled midges). Plates were incubated for 7 days in 5% CO2 at 34 °C and wells were monitored daily for CPE. After 7 days, samples were passaged by aspirating 100 µL of cells and supernatant from the plate and inoculating them onto fresh BHK monolayers for a second 7-day culture in 5% CO2 at 37 °C [Individual or pooled midges  were manually homogenized in microcentrifuge tubes using sterile pestles before being sonicated for 15 s in a sonicating water bath . Homogenized midges were then centrifuged at 4 °C for 12 min at 1500× at 37 °C .50/midge) were determined [2.3 TCID50/midge. To calculate and graph mean virus titers, a value of 101.15 TCID50/midge was assigned to samples that were CPE positive, but titer was below the limit of detection. Our approach assumes all values between 102.3 TCID50/midge and zero could be present in these cases and their average titer could be as high as half of the limit of detection [2.7 TCID50/midge to be potentially competent as previous work with bluetongue virus (BTV)—an orbivirus closely related to EHDV—has demonstrated that C. sonorensis midges reaching these titers are capable of efficient virus transmission to susceptible hosts [2.7 TCID50/midge corresponded with the dissemination of virus from the mid-gut to organs including the salivary glands [For all samples where CPE was observed on the second passage, the original sample was titrated for virus using BHK cells as previously described  and endptermined . The minetection . We consle hosts . Furthery glands .10 EHDV titers were compared over time using linear mixed models with deer as a random effect. The percentage of midges with positive virus isolation results, and with titers 102.7 TCID50, were compared over time using mixed logistic regression models with deer as a random effect. Pairwise comparisons of different time points were performed using the Bonferroni procedure to limit the type I error probability to 5% over all comparisons. All tests assumed a two-sided alternative hypothesis, and p < 0.05 was considered statistically significant. Analyses were performed using commercially available statistical software .Log3.98 TCID50/mL at 5 dpi (3.7 to 106 TCID50/mL (p < 0.001). Compared to 3, 5, and 7 dpi, the mean titers on 12 and 14 dpi were significantly lower. The mean titer on 10 dpi was intermediate, and did not differ significantly from the mean titer on any other day. EHDV-2 was not detected in any deer after 14 dpi.All five inoculated deer had a detectable viremia and developed mild to moderate clinical disease. Mild clinical signs were observed in deer 2, 3, 4, and 6, and included elevated body temperature, hyperemia of oral mucosa and conjunctiva, and erythema of non-haired regions: ear pinnae, perineum, nares, periorbital zones. In addition to the above clinical signs, deer 5 exhibited moderate clinical disease, which included depression, lethargy, and lameness. The lameness observed in deer 5 occurred at 11–15 dpi and was characterized by a reluctance to stand and a hunched posture with rigid forelimb extension while attempting to walk. Small hemorrhages in the oral mucosa were occasionally observed on 10 dpi . The baseline body temperature was 39.1 °C, determined by –3 and 0 dpi recordings for all deer in the study. Peak elevations in body temperature occurred on 5 dpi for deer 6 (+1.2 °C) and 7 dpi for deer 2 (+0.2 °C), 3 (+0.8 °C), 4 (+1.3 °C), and 5 (+0.9 °C). Viremia profiles for individual deer are presented in at 5 dpi  while peCID50/mL . There wVirus isolation results varied over the course of infection depending on the cell line used . There wNeutralizing antibodies were first detected in all deer on day 7 and all had an antibody titer of 160 or higher by 12 dpi. Neutralizing antibodies were not detected at any time point in the sham inoculated negative control.Midge infection prevalence, calculated as the number of midges that were virus isolation positive out of the total number of midges tested, varied over the course of viremia and between individual deer in the trial. Virus was rarely isolated from midges that were sampled prior to the 10-day incubation period. We successfully isolated virus from 6 out of 265 (2.3%) of these midges (five from deer 5 at 7 dpi and one from deer 3 at 7 dpi). All six cases resulted in titers below the threshold of detection. All analyses omitted these midges and focused solely on midges that underwent the 10-day incubation period. Midge virus isolation results and titers are summarized in p < 0.001). Compared to 5 dpi, the proportion of midges with positive virus isolation results was significantly lower at 3, 10, 14, and 18 dpi. EHDV-2 was not detected in midges at 12 dpi. There was also a significant relationship between the percentage of midges with a positive virus isolation result and deer EHDV titers (p < 0.001). For every one-log10 increase in a deer’s EHDV titer, the odds that midges feeding on that deer would have a positive virus isolation result were 2.6 times higher .There was a significant difference in the proportion of midges with positive virus isolation results over time  and ranged from 101.15 TCID50/midge to 103.54 TCID50/midge. There was also a significant difference in the proportion of midges with an EHDV titer ≥ 102.7 TCID50 over time (p < 0.001). High percentages of infected midges from 5 and 7 dpi were determined to be potentially competent vectors, 68.2% and 80%, respectively (2.3 TCID50/midge). Mean titers on 5 and 7 dpi were significantly higher than the mean titer at 14 dpi, but no other pairwise comparisons between days were statistically significant . For every one-log10 increase in a deer’s EHDV titer, the EHDV titer in midges feeding on that deer increased by an average of 0.49 log10 units . Likewise, there was a statistically significant correlation between the percentage of midges with an EHDV titer ≥ 102.7 TCID50 and deer EHDV titers (P < 0.001). For every one-log10 increase in a deer’s EHDV titer, the odds that midges feeding on that deer would have an EHDV titer ≥ 102.7 TCID50 were 3.7 times higher .Days 5 and 7 post infection produced the most virus positive midges, accounting for 69 out of 85 (81.2%) of the total infected midges processed over the course of the study. In addition to midge infection prevalence derived from virus isolation data, we determined midge viral titers and the proportion of infected midges at each time point that reached titers ≥ 10r EHDV-2 . Mean viectively . In factectively . Mean minificant . Mean min = 5) that fed on deer 5 at 24 dpi. Incidentally, deer 5 was the deer that reached the highest EHDV-2 titer (peak 106 TCID50/mL) in our trial.Although no virus was isolated from the blood of any deer on 18 dpi, we were able to isolate viruses from 3 midges that fed on deer 5 at 18 dpi and from a pool of midges (Culicoides cells (KC cells) have previously been shown to outperform other culture systems [Clinical signs of EHD observed in this study were generally mild. However, moderate clinical disease was observed in deer 5, with prominent lethargy and lameness present 11–15 dpi. Interestingly, this animal had the highest peak blood virus titer, and there is often a correlation between peak viremia and severity of clinical disease during experimental infections. An unexpected feature of the observed viremia profiles was the relatively short duration. While significant individual variation exists, deer infected with EHDV often have a prolonged viremia lasting as long as 59 days . All dee systems , althoug systems . ViremiaOur results indicate that EHDV-2 midge infection prevalence tracks fluctuations in white-tailed deer viremia. We found that increases in the blood EHDV titer of deer significantly increased the likelihood that midges would successfully acquire EHDV, as measured through positive virus isolation. Despite the variation in viremia profiles between individual deer, the highest proportion of virus-positive midges observed for each deer tended to coincide with peak viremia, typically 5 to 7 dpi, and correspondingly decreased as blood virus titer decreased as deer recovered from infection.C. sonorensis with virus titers ≥ 102.7 TCID50/midge potentially competent vectors, a value extrapolated from BTV studies in domestic sheep that showed C. sonorensis reaching titers of ≥ 102.7 TCID50/midge were capable of infecting susceptible hosts [C. sonorensis were expected to achieve vector competence at any given time point examined [C. sonorensis is strongly influenced by the viral load of the deer they feed on. As expected, the percentages of competent C. sonorensis peaked with peak blood virus titer in deer, with more than half of EHDV-2-infected midges considered competent at 5 and 7 dpi. Not only were the highest infection prevalences observed on these days, but large percentages of those infected midges were potentially competent. No other time point yielded as many infected or potentially competent C. sonorensis in comparison. In fact, no potentially competent midges were found after 10 dpi at which time there was only one potentially competent midge. These results suggest that WTD are the most infectious to C. sonorensis during peak viremia, relatively soon after first becoming infected.To evaluate the potential for EHDV-positive midges to transmit EHDV, we also determined the virus titers of individual positive midges. Once ingested by a midge, the virus must overcome a variety of barriers to infect and eventually escape the midgut and disseminate to the salivary glands . While tle hosts . Becauseexamined . Our resC. sonorensis with EHDV has produced similar results, with deer viremias <104.0 TCID50/mL typically resulting in low infection prevalence [2.3 TCID50/mL yielding no infected midges [C. sonorensis varies over the course of viremia in WTD as a response to fluctuations in viral titer. Midges are most likely to acquire EHDV-2 and become competent vectors when feeding on deer at peak viremias. Though not conclusive, our results indicate that feeding on EHDV-infected deer with low blood virus titers results in inefficient host-to-vector transmission of EHDV-2.While we were able to isolate EHDV-2 from midges that fed on deer as far along as 18 and 24 dpi, in the case of deer 5, these infection prevalences were very low. Previous work involving the infection of evalence ,17 and td midges . It was Culicoides. The recovery of virus from Culicoides after feeding on deer 5 on 18 and 24 dpi was unexpected, as this animal did not have a detectable viremia by virus isolation at these time points. However, similar findings have occurred in sheep infected with BTV, a closely related orbivirus [Culicoides is an interesting finding and should be explored further to better understand its potential significance. Considering the low number of virus positive midges detected at these time later time points, our findings suggest that low titer EHDV viremias in WTD result in extremely inefficient host-to-vector transmission. However, attack rates on white-tailed deer by Culicoides have been shown to be extremely high in nature, with one study regularly recovering >10,000 C. debilipalpis per morning from individual deer during peak midge season [The ability of WTD to sustain low-titer EHDV viremias for prolonged periods suggests these animals are a potential source of EHDV for feeding rbivirus . The abie season . Thus, e


The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during  RNA viruses replicate with a high mutation rate, giving them the ability to generate mutations that might be beneficial under different selection pressures. Any perturbation of the accumulation of mutations has been shown to result in severe attenuation of the virus. However, the mechanism of this attenuation is still unclear. Mosquito-borne viruses, which are mostly RNA viruses, undergo repeated bottleneck events during the transmission cycle of the virus. Using a model system of Venezuelan equine encephalitis virus (VEEV) we investigated the effect of perturbing the rate of mutations in both a mosquito and vertebrate host. We show that any perturbation of the mutation frequency, either by increasing or decreasing the overall mutation frequency as well as altering the specific frequency of mutations results in attenuation. Our results also suggest that RNA viruses may use stop codon accumulation as a means of purifying out less-fit genomes by targeting them for destruction. These changes in the genome result in viruses unable to overcome the bottlenecks in the mosquito vector and to a lesser extent in the vertebrate host. Overall, our study shows that any change in the accumulation of mutations from wild-type increases the likelihood of an unsuccessful transmission cycle. RNA viruses comprise a diverse group of viruses, which exhibit high genome plasticity due to a high rate of mutation. This results in the generation of a closely related cloud of viral variants known as a ‘quasispecies’ or viral swarm . The genBottlenecks are present in all transmission cycles as viruses move from one tissue to another. Studies using marked clones have shown that bottlenecks are more common than were previously thought , 6, 8, ain vivo, but replicate similarly to wild-type in vitro [Virologists have come to understand that any perturbation of virus diversity has adverse consequences for the virus population as a whole. Almost all viruses that have been demonstrated to have either higher or lower diversity are attenuated compared to the wild-type in vitro –14. Thesin vitro .Culex taeniopus has demonstrated the presence of bottlenecks during normal transmission and infection of the mosquito vector. VEEV is a New World encephalitic alphavirus endemic to parts of Central and South America, and is transmitted between mosquitoes and small rodents. Although often misdiagnosed as dengue fever due to the constant circulation in the same geographic area, VEEV is thought to cause tens of thousands of human infections annually [Cx. taeniopus).Previous work with Venezuelan equine encephalitis virus (VEEV) in the mosquito vector annually . ImportaPrevious work with the vaccine strain of VEEV, TC-83, demonstrated that three mutations in the RdRp collectively contributed to a decrease in virus fidelity, resulting in attenuation in a vertebrate model . To deteResistance to nucleoside analogues has been one of the tools for determining the presence of a fidelity mutation, as high fidelity viruses are less likely to incorporate mutagens, while low-fidelity viruses should be more susceptible , 17, 18.To determine if these inserted mutations altered virus fidelity, each RdRp mutant along with the wild-type virus were used to infect the vertebrate cell lines, MRC-5 and Vero, and the mosquito cell line U4.4 at an MOI of 0.1. At 24 hours post infection, the viruses were harvested and the viral RNA extracted and sequenced using Illumina sequencing to determine the amount of diversity within each virus population . The G7RTo determine the stability of the mutations and the phenotype associated with them, the wild-type and RdRp mutant viruses were passaged five times using Vero cells. Following the fifth passage, the growth kinetics and sensitivity to 5FU assays were repeated with passage 5 virus . There w-4, whereas the G7R mutant has a SI of 9x10-6, and the 3x and 4x mutants have a SI of 1x 10−7 and 7x10-7, respectively.To verify the mechanism of attenuation of the RdRp mutants, stocks of the electroporated viruses were titered to determine the number of viable viral particles, as well as the number of RNA genomes produced as determined by real-time RT-PCR (i.e. to determine the specific infectivity (SI) of the viruses). All four viruses produced equivalent amounts of genome copies to the wild-type, which shows that the rate of genome replication was not altered as a result of the mutations . Howeverin silico nascent genome was translated to determine the number of additional stop codons generated compared to the wild-type 68U201. Although it was expected that the number of stop codons would increase, the model indicates that the alteration in base changes actually leads to a reduced number of stop codons compared to the wild-type 68U201 virus    (S1 Tably ANOVA) . Translay ANOVA) .RdRp mutant and wild-type viruses were inoculated into mice to determine the effects of the RdRp mutations on virulence. There was a reduction in mortality and increase in time to death for the RdRp mutants compared to the wild-type 68U201 in adult mice . All of Cx. taeniopus mosquitoes, there was a significant reduction in the number of disseminated infections compared to the wild-type 68U201 strain , and days 4, 8, and 12 . The number of marked viruses present in each sample was determined using a real-time assay that allowed each marked virus to be identified.To test how altered mutation frequencies affected the ability of the virus to traverse a bottleneck, a series of silent mutations were inserted into the G7R, 3x and 4x RdRp mutants, generating 8 neutral marked viruses of each RdRp mutant. These marked viruses were previously described in Forrester et al. 2012 , and use5 PFU/ml of the G7R marked viruses, none of the mosquitoes had virus dissemination outside of the midgut/body , blood and brain were chosen for sampling because the LN is the primary site of replication, the blood is the means for transmission and the brain indicates the severity of the pathogenesis. Compared to the wild-type infection, there was a significant reduction in the number of marked viruses in the LN and the brain for all three RdRp mutants , whereasin vivo. It also increases the potential for the virus to continue its transmission cycle. Virus diversity is thought to be even more important for arboviruses, which infect a two-host system, as the virus must infect both a vertebrate host and a mosquito vector. It is well known that the virus undergoes numerous bottlenecks, or reductions in viral numbers, when going from host to host [Viral diversity is an important factor affecting RNA virulence and transmission. Generation of viral diversity allows the virus to effectively move between cells and cell types  to host , 20. Thi to host . A more in vivo [Previous work with fidelity mutants has demonstrated that they are attenuated in vivo , 15, 22.in vivo –27. Specin vivo . To testin vivo with 1-to-stop mutants [in silico approach, a computer model was used to determine if there was a change from the wild-type in the number of stop codons. All RdRp mutants showed a reduction in the number of predicted stop codons. Given that nascent genomes with additional stop codons are likely targeted for destruction, the absence of stop codons as a result of specific mutations likely results in a larger number of less-fit genomes . Thus, replication in the G7R, 3x and 4x mutants results in more genomes that contain mutations that result in lower fitness, that if a stop codon had been inserted would otherwise be purified out during translation or packaging. From our data, it appears that attenuation of these viruses is a result of changing the frequency of specific mutations as a result of incorporating the specific RdRp mutants. Further evidence for this can be found in the reduced SI compared to the wild-type for the RdRp mutants, suggesting that there is an increase in the number of less fit genomes produced. Given the large discrepancy in the SI it is unlikely that these stop codons are the only mechanism leading to attenuation, but it is more likely a combination of factors. However, this work does raise interesting possibilities about the evolutionary pressures for specific mutation frequencies in viruses. This intriguing hypothesis will need to be evaluated experimentally both in vivo and in vitro.As the phenotype of fidelity was not stable through 5 passages, other reasons for the attenuation were investigated. The data showed that there were changes in the mutation spectrum for each RdRp mutant, and given the recent work showing attenuation of Coxsackie B3 virus and Influenza A virus  mutants , it was Cx. taeniopus mosquitoes were infected with the RdRp mutants as well as the wild-type virus, there was no significant decrease in the percentage of mosquitoes positive for virus in the body between the wild-type and mutant virus infections. However, there was a significant decrease in the legs/wings, and saliva. This indicates that changes in mutation frequency decrease dissemination within the mosquito vector. Only at the higher dose was virus detected in the legs/wings or saliva, and mosquitoes exposed to a higher dose also contained more clones than mosquitoes receiving a lower dose. When combined, these results support the hypothesis that any alteration in the generation of viral diversity results in an impaired ability to overcome bottlenecks imposed during dissemination through the mosquito compared to the wild-type virus. Although, it must be stated that some samples from the legs/wings and bodies may have been below the limit of detection, 101 PFU, for real-time analysis.When in vivo as mice infected with mutant viruses demonstrated increased time to death and increased survival rates compared to the wild-type virus 68U201. Interestingly, some mice infected with the attenuated mutants lost as much as 30% of their body weight but were able to recover. A possible explanation for this is that host immune systems are able to respond more effectively to these attenuated viruses once infection has been established, something not seen in the wild-type infection. This is not the first time this has been observed, as evidence for an improved immune response has been reported for low fidelity variants. The presence of lower fidelity resulted in an increased generation of degenerate virus particles that stimulate RIG-I, MDA5 and dendritic cell maturation [This attenuation was recapitulated turation –27, 29, This work has demonstrated that the RdRp mutants described here have a reduced ability to traverse bottlenecks during infection of vertebrate hosts and mosquito vectors. There was a tentative association showing that the G7R RdRp mutant, which is a putative low fidelity phenotype, was less sensitive to bottlenecks compared to the 3x and 4x RdRp mutants, which showed putative high fidelity phenotype. However, all mutants were more sensitive to bottlenecks than the wild-type counterpart. This study has demonstrated that there are significant costs to altering the finely tuned balance of viral diversity, which has usually been conceived as a random accumulation of mutations , 15, 30.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols (0209068 and 1309038) were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch.African green monkey kidney (Vero), Baby Hamster Kidney (BHK), and human lung fibroblast (MRC-5) cells were obtained from the American Type Culture Collection  and maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and gentamycin (100 U/ml) in a 37°C, 5% CO2 incubator. U4.4, cells were maintained in Mitsuhashi and Maramorosch media supplemented with 20% FBS, 2% Sodium Bicarbonate (7.5%) and gentamycin (100 U/ml) in a 30°C CO2 incubator. Viruses were rescued from the enzootic subtype IE VEEV strain 68U201 as described previously and without further passage . The wilFour mutations identified to cause altered fidelity in the VEEV vaccine strain TC-83 were cloned into the 68U201 backbone . Virus was harvested by removing the entirety of the medium. Harvested virus was supplemented with FBS to a final concentration of 20%, and clarified by centrifugation at 3000rpm for 5 min. The supernatant was then removed and stored at -80°C. Viral titers were determined by standard plaque assays .Rescued viruses were passaged 5 times on Vero cells to ensure genetic stability. Viruses were harvested 2 days post-infection and diluted for a MOI of 0.01 before infection of Vero cells in the subsequent passage. Following passage 5, viral suspensions from passage 1 and 5 were placed into TRIzol  and RNA extracted using the ZR Viral RNA Kit  as per the manufacturer’s protocol. cDNA was produced using the Superscript III First Round Synthesis kit  following the manufacturer's instructions. cDNA was amplified for sequencing using the Phusion enzyme  in a 50ml volume following the manufacturer's instructions using primer sets that covered the entire genome. Products were sequenced on the ABI 3700 sequencer  to confirm the sequence of the passaged virus. RNA extractions were also sent for RNA sequencing (see below).Rescued viruses from passage 1 and 5 were tested for susceptibility to 5’ fluorouracil in triplicate. Cells were pretreated with 0, 10, 25, 50, 100, 200, or 300 μg/ml of 5’ fluorouracil for 2 hours. The cells were then infected with 0.01 MOI of the RdRp mutants or wildtype 68U201. Medium containing the pretreatment amount of 5’ fluorouracil was added to the infected cells 1 hour post-infection. Virus was harvested 24 hours post-infection and titrated in duplicate using standard plaque assays.Rescued viruses were used to test for specific infectivity of the virus. Standard plaque assays were carried out in triplicated as described above. RNA was also extracted using a QIAamp Viral RNA Mini kit according to the manufacturer’s protocol. Real-time RT-PCR was performed using primers against the nsP1 using primers , and against the nsP3 using primers . Real-time RT-PCR was carried out using iTaq Universal SYBR Green Mix (Bio-Rad) and iScript Reverse Transcriptase (Bio-Rad), with single steps of 10 min at 50°C and 3 min at 95°C, followed by cycling at 15 s at 95°C, and 30 s at 57°C for 45 cycles, and a melt curve from 55–95°C (+0.5°C/cycle).10 PFU of virus. For survival studies, mice were bled on days 1, 2 and 3 to determine the level of viremia. The resulting blood samples were diluted 1:10 in DMEM supplemented with 10% FBS and stored at -80°C. Mice were weighed daily and monitored until they exhibited signs of paralysis, at which point they were sacrificed  were infected subcutaneously with 3 logcrificed .10 PFU of virus. Each mouse was perfused using PBS and the brain, heart, lung, liver, spleen, kidney and lymph node were harvested. In a second round of experiments, mice were infected with the RdRp mutants that contained the marked clones, and were again sampled daily (n = 3). Each mouse was subjected to cardiac puncture to collect serum, and the brain and brachial lymph nodes were also harvested. All samples were tested for viral presence and titer by cytopathic effect (CPE) assay and standard plaque assay on Vero cells.Mouse infections were also carried out using the set of marked clones. To determine the number of potential bottlenecks in a wild-type infection, mice were sampled daily (n = 3) following subcutaneous injection with 3 logMice used for oral mosquito exposure were injected via the tail vein with a mixture of all 8 clones in equal concentrations to generate an artificial viremia of known content. The mice were anaesthetized with ketamine/xylazine and then bled approximately 5 minutes post injection to estimate viremia titers before being presented to mosquitoes. Mosquitoes were allowed to feed on the mice for up to 45 minutes. Following a terminal blood draw, mice were sacrificed without gaining consciousness. Mouse manipulations were approved by the UTMB Institutional Animal Care and Use Committee.C. taeniopus mosquitoes  were sugar-starved for at least 16 hours, then allowed to feed for one hour on mice injected via the tail vein to generate viremia of predictable content, as described above. After one hour, engorged mosquitoes were incubated at 27°C and provided 10% sucrose ad libitum. At selected time points mosquitoes were chilled, their legs and wings removed, and then individuals were allowed to salivate for 45 minutes into a capillary tube containing FBS. The midguts were then dissected, and the remaining carcass was held separately. Midguts were cut in half and the residual blood was washed out using PBS. All mosquito tissues were placed into DMEM supplemented with 10% FBS, pen/strep and Fungizone .Cohorts of g for 10 minutes. Saliva samples were subjected to centrifugation at 6636 x g for 10 minutes prior to processing. All samples were tested for the presence of virus by CPE assay. Positive samples were stored at -80°C for subsequent analysis. For mosquito saliva samples, supernatants positive for CPE were used in a real-time RT-PCR assay, as the inconsistency in the amount of virus expectorated from the mosquito [All mosquito and vertebrate tissues were resuspended in DMEM supplemented with 10% FBS and gentamycin (for mosquito tissues Fungizone (Sigma-Aldrich)) was also added) and homogenized at 26 hz for 5 minutes, then subjected to centrifugation at 38206 x mosquito  would haT 1-Step kit (ABI) as per the manufacturer’s instructions in a 10 μl reaction. The primers and probes were identical to those used in previously published work [5−101 pfu/ml of the individual clones were used to create standard curves.Virus suspensions were placed into TRIzol  and RNA extracted using the ZR Viral RNA Kit  as per the manufacturer’s protocol. Real time RT-PCR was carried out using the ABI 7900HT Fast Real-Time PCR system . Each reaction was performed using the TaqMan RNA-to-Ched work . Each prViral RNA (0.05–1.7 mg) was fragmented by incubation at 94°C for 8 minutes in 19.5 μl of fragmentation buffer . First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Samplec Preparation kit as per the manufacturer’s protocol. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation as per the manufacturer’s protocol. Paired-end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 as per the manufacturer’s protocol.All reads were assembled using a pipeline previously described  and wereStatistical analyses were carried out using GraphPad Prism and details are found in the text.in silico the replication of (either 1m or 1bn) RNA strands for each variant, counting the number of individual strands produced that include new codons with either TAG, TAA, or TGA. For each variant, raw RNA data was obtained from the sequence of the infectious clone used to produce the virus. The algorithm begins at position 46 and reads each codon sequentially up to position 7538, skipping the opal stop at 5696. Reading continues at position 7546 and continues to 11338, this corresponds to the open reading frame for VEEV strain 68U201. Each base within each codon has a likelihood of mutation of 0.0001; if mutation occurs, the base changes randomly, according to the distribution given in https://github.com/StanDeSiecle/Ihaventdonethisyet.In order to quantify the extent to which point mutations create new stop codons, we simulated To determine the frequency of mutations leading to stop codons in the data generated from next generation sequencing of p1 viruses, each codon in the 68U201 genome was read sequentially. Using the substitution data from the p1 NGS data, we generated alternative codons if a substitution of any base was found to have occurred at any locus within that codon. We then recorded whether that substitution resulted in a stop codon. The proportion of stop codons to other mutations was then calculated for each virus and replicate.S1 Fig(TIFF)Click here for additional data file.S2 FigThe table shows the significance as determined by two-way ANOVA p<0.0001. Individual changes are found in the table. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 FigA). At 10^6 pfu/ml infection where the bodies were statistically different from the wild-type by two way ANOVA at P<0.0001, all fidelity clones showed differences from the wild-type by Dunnetts multiple comparisons test. B). At 10^5 pfu/ml infection where the bodies were statistically different by ANOVA p = 0.0077. However, by Dunnett’s multiple comparison test only the G14R showed significant difference from the wild-type at p<0.01.(TIFF)Click here for additional data file.S5 FigThe relationship of titer to clone for A, B & C infections with low dose for G7R, 3x and 4x RdRp mutants, respectively. D, E & F, infections with high dose for G7R, 3x and 4x RdRp mutants, respectively. All titers were calculated from using standard curves run concurrently with the samples. G. The correlation between number of clones and titer for each of the samples for the G7R, 3x and 4x samples.(TIFF)Click here for additional data file.S6 FigMice were infected with an equal mixture of 8 clones and perfused to remove infected blood before tissues were sampled.(TIFF)Click here for additional data file.S1 TableThe genome was sub-divided into 100 bins and the first column corresponds to the bin centers for that bin. This table was used to produce (DOCX)Click here for additional data file.


Manis javanica) as well as the molecular epidemiology of dominant pathogenic viruses between Malayan Pangolin and other hosts. A total of 62,508 de novo assembled contigs were constructed, and a BLAST search revealed 3600 ones (≥300 nt) were related to viral sequences, of which 68 contigs had a high level of sequence similarity to known viruses, while dominant viruses were the Sendai virus and Coronavirus. This is the first report on the viral diversity of pangolins, expanding our understanding of the virome in endangered species, and providing insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into other mammals.Pangolins are endangered animals in urgent need of protection. Identifying and cataloguing the viruses carried by pangolins is a logical approach to evaluate the range of potential pathogens and help with conservation. This study provides insight into viral communities of Malayan Pangolins ( Manis javanica), a representative mammal species of the order Pholidota, is one of the only eight pangolin species worldwide. Four of them are from Asia , whereas another four from Africa  [The Malayan pangolin (minckii) . Unlike minckii) . Becauseminckii) , implicaminckii) ,7,8,9,10Viruses are infectious agents that replicate only inside living cells and have the ability to infect a variety of hosts . There hThe Guangdong Wildlife Rescue Center received 21 live Malayan pangolins from the Anti-smuggling Customs Bureau on 24 March 2019; most individuals, including adults and subadults, were in poor health, and their bodies were covered with skin eruptions. All these Malayan pangolins were rescued by the Guangdong Wildlife Rescue Center, however, 16 died after extensive rescue efforts. Most of the dead pangolins had a swollen lung which contained a frothy liquid, as well as the symptom of pulmonary fibrosis, and in the minority of the dead ones, we observed hepatomegaly and splenomegaly. We collected 21 organ samples of lung, lymph, and spleen with obvious symptoms from 11 dead Malayan pangolins to uncover the virus diversity and molecular epidemiology of potential etiologies of viruses based on a viral metagenomic study. This study will be beneficial to pangolin disease research and subsequent rescue operation.The study design was approved by the ethics committee for animal experiments at the Guangdong Institute of Applied Biological Resources  and followed basic principles outlined by this committee.In our study, organ samples of lung, lymph and spleen were collected from dead Malayan pangolins at the Guangdong Wildlife Rescue Center. Preparation of viral-like particles followed a previous published paper . Total nAs raw sequencing reads always include some low-quality data, it is necessary to perform processing to improve the accuracy of reads for follow-up analyses. To this end, we used SOAPnuke version 1.5.6  to removM. javanica (NCBI Project ID: PRJNA256023) utilizing BWA version 0.7.17 [To avoid the confusion cause by ribosomes and host sequences, all clean reads that passed quality control were mapped to the ribosomal database (silva) and the host reference genome of n 0.7.17 ; only thClean reads without ribosomes and host sequences were mapped to an in-house virus reference data separate from the GenBank non-redundant nucleotide (NT) database to primarily identify virus reads. According to the NCBI taxonomy database annotation information, reads were classified into different virus families. To improve the accuracy, we removed the alignment results with a coverage below 5 reads.de novo assembled using MEGAHIT version 1.0 [−5. Contigs with significant BLASTx hits were confirmed as virus sequences.Clean reads were sion 1.0 . BWA version 1.0  was usedsion 1.0  was usedhttps://www.viprbrc.org/brc/home.spg?decorator=vipr). Virus sequences from Malayan pangolins and other hosts were aligned using MAFFT version 7.427 [http://tree.bio.ed.ac.uk/software/figtree/).Whole genome sequences of virus strains, the same species as the dominant viruses in Malayan pangolins, from different hosts were downloaded from ViPR database . In total, 233,587 reads were best matched with viral proteins available in the NCBI NR database . The number of viral-associated reads in each sample varied from 2856 to 78,052 . In the ce reads .de novo assembly using MEGAHIT version 1.0 [Paramyxoviridae, Flaviviridae and Caudovirales . After sequence alignment conducted by MAFFT version 7.427 , the best substitution model analyzed by iqtree was GTR+F+I. Then phylogenetic analysis revealed the closest relationship between the 13,232 bp length contig from Malayan pangolin and Sendai virus strains isolated from humans (AB005795.1), but distant from strains isolated from the mouse . For several of these pangolin samples, larger contigs were produced, and the length ranged from 503 to 2330 bp  isolated from different hosts were downloaded from the ViPR database (https://www.viprbrc.org/brc/home.spg?decorator=vipr). Together with 16 contigs confirmed as Coronavirus in this study, all the sequences were aligned utilizing MAFFT version 7.427 . The best substitution model analyzed by iqtree was GTR+F+R7, and the phylogenetic analysis therefore showed multiple relationships between Coronavirus contigs and the four Coronavirus genera , which strongly suggests the possibility that the Sendai virus is transmitted between pangolins and humans. Sendai virus is a member of the paramyxovirus subfamily eristics . Sendai eristics , North Aeristics  and Euroeristics , and is eristics ,47, withBesides the Sendai virus, Coronaviruses were also detected as potential pathogens of Malayan pangolins. The phylogeny of Coronavirus sequences assembled and strains from four Coronavirus genera demonstrated complex genetic relationships and high species diversity of the Coronavirus in Malayan pangolins. Coronaviruses can cause a variety of severe diseases including gastroenteritis and respiratory tract diseases, and have been identified in mice, rats, chickens, turkeys, swine, dogs, cats, rabbits, horses, cattle and humans ,49. SomeWe found high viral diversity of dead Malayan pangolins, and the Sendai virus and Coronavirus may be the dominant pathogens responsible for their death. The Sendai virus showed a close relationship between the Malayan pangolin and the strain isolated from humans, whereas Coronavirus sequences showed a high species diversity. Further investigations are required to compare the incidence of these viruses in healthy and diseased pangolin individuals in order to better elucidate their pathogenic role. To date, this is the first metagenomic study of virus diversity in pangolins in China. This study expands our understanding of the viral diversity in endangered species and the capability of directly or indirectly crossing over into other mammals.