RNA viruses associated with honeybees represent a potential threat to other insect pollinators, but the extent of this threat is poorly understood.Declining populations of bee pollinators are a cause of concern, with major repercussions for biodiversity loss and food security. EID) transmission between managed and wild pollinator species across a wide range of RNA viruses.This study aims to attain a detailed understanding of the current and ongoing risk of emerging infectious disease (RNA viruses in co‐occurring managed honeybee (Apis mellifera) and wild bumblebee (Bombus spp.) populations. We then construct models that compare virus prevalence between wild and managed pollinators.Within a structured large‐scale national survey across 26 independent sites, we quantify the prevalence and pathogen loads of multiple RNA viruses associated with honeybees are widespread in sympatric wild bumblebee populations. Virus prevalence in honeybees is a significant predictor of virus prevalence in bumblebees, but we remain cautious in speculating over the principle direction of pathogen transmission. We demonstrate species‐specific differences in prevalence, indicating significant variation in disease susceptibility or tolerance. Pathogen loads within individual bumblebees may be high and in the case of at least one RNA virus, prevalence is higher in wild bumblebees than in managed honeybee populations.Multiple RNA viruses between managed and wild bee pollinators, pointing to an interconnected network of potential disease pressures within and among pollinator species. In the context of the biodiversity crisis, our study emphasizes the importance of targeting a wide range of pathogens and defining host associations when considering potential drivers of population decline.Our findings indicate widespread transmission of  The ongoing biodiversity crisis threatens human health and global food security , with the exotic ectoparasitic mite, Varroa destructor, meriting particular attention. The mite has risen to prominence due to its ability to act as a vector of several RNA viruses that previously persisted relatively benignly in honeybee colonies, most notably deformed wing virus (DWV), but also viruses belonging to the acute bee paralysis virus (ABPV) complex  understand the contemporary landscape prevalence of common RNA viruses thought to be associated with honeybees, (ii) quantify and compare the individual infection levels of RNA viruses in bee foragers, (iii) assess the extent to which RNA virus spillover is occurring between honeybees and bumblebees (in either direction). We show that multiple RNA viruses are prevalent in wild bee populations and present evidence for recent and widespread circulation of viral diseases between Britain's primary managed and wild bee pollinators.et al.  to collect 20 A. mellifera and 20 Bombus spp. individuals was recorded as an estimate of abundance. Honeybees and up to four species of bumblebees from each site were then screened for the presence and quantity of a range of viruses. Honeybee or bumblebee abdomens were bisected longitudinally, one‐half of which was submerged in RLT buffer and disrupted in a Tissue lyser II  at 30 Hz for 2 min followed by 20 Hz for 2 min prior to RNA isolation. Total RNA was extracted manually using the RNeasy mini kit  following manufacturer's instructions.Field sampling methodology and RNA extraction follows Fürst et al. . Briefly® kit . We used probes designed for the positive strand of the following six composite positive‐sense single‐stranded RNA virus targets ; (iii) acute bee paralysis virus, Israeli acute paralysis virus and Kashmir bee virus (ABPV/IAPV/KBV); (iv) slow bee paralysis virus (SBPV); (v) sac brood virus (SBV); and (vi) chronic bee paralysis virus (CBPV) and, as a housekeeping (control) gene, β‐actin. Notable viruses such as the Lake Sinai viruses (LSV 1 and 2) have been recorded in North America  using the RT‐MLPACt) of 35 was set for β‐actin, above which samples were not included in quantitative analysis. Given the previous positive detection of virus by MLPA, an upper threshold for virus quantification by qRT‐PCR was not applied. We used specific primers for the following viruses: BQCV; DWV; VDV‐1; ABPV; IAPV; KBV; SBPV; SBV . Following PCR, DNA was denatured for 1 min at 95 °C and cooled to 55 °C for 1 min. A melting profile was generated from 55 to 95 °C (0·5 °C per second increments). Quantification was calculated using duplicate DNA standard curves of purified flanking PCR products  or plasmids , with efficiencies of 98·4% (DWV), 99·9% (VDV‐1), 96·2% (BQCV), 101·3% (ABPV) and 93·1% (SBPV), and correlation coefficients (R2) from 0·995 to 0·999.For each MLPA positive virus target, samples were analysed by qRT‐PCR to identify the specific virus (in the case of DWV/KV/VDV‐1 and ABPV/IAPV/KBV) and to estimate individual viral load. This allowed us to differentiate between VDV‐1 and DWV/KV and between ABPV, IAPV and KBV. KV and DWV are very closely related and were not differentiated by qRT‐PCR. Total cDNA was synthesized using M‐MLV Revertase  following manufacturer's instructions, using 500 ng of sample RNA. For absolute quantification, duplicate qRT‐PCR was performed for each sample with a Bio‐Rad C1000, using SYBRgreen Sensimix  in the following program: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 30 s at 57 °C and 30 s at 72 °C (read). Duplicate β‐actin reactions were also amplified for all samples as an internal reference marker. A negative control containing RNA‐free HPLC water and a virus‐positive sample were included as controls in each reaction run. To account for potential variation in sample quality, an upper cycle threshold  and cloned directly using the pGEM T Easy Vector system  following manufacturer's instructions. Plasmid DNA was isolated using a Spin Miniprep Kit . Up to five clones per sample were sequenced in forward and reverse orientation , and aligned by eye to genome references of BQCV (NC_003784), ABPV (NC_002548) and SBPV (NC_014137). DWV and VDV‐1 sequences have been analysed previously (Fürst r v 3.0.2 (R Core Team 2 test), using a Bonferroni correction  for comparisons between species. So that differences among species of different samples sizes could be meaningfully compared, we estimated true prevalence and 95% confidence intervals using the r library ‘epir’ v0.9‐54, with sensitivity and specificity both set at 95%. Disease prevalence was mapped to sites using the ‘mapplots’ package v1.4, or estimated using Gaussian kernel estimators using the package ‘prevr’ as described previously . V. destructor mites have caused both an increase in viral load and prevalence of several RNA viruses in western honeybee populations, including DWV . Site G was removed prior to statistical analysis as no A. mellifera foragers were collected at this site. A. mellifera and Bombus spp. abundance were log‐transformed, and all quantitative predictors were standardized to a mean of zero and standard deviation of one prior to analysis. Models were simplified by backward stepwise selection based on AIC (‘drop1’ function). We used variance inflation factors (VIF) to check for colinearity among our explanatory variables, applying a cut‐off value of 3. Variables with a high VIF were removed one at a time until all VIF values were below 3  and marginal  values are shown.To explore possible drivers of RNA virus prevalence in managed and wild bees, we performed generalized linear mixed models (GLMM) with binomial error structure using the package ‘A. mellifera and Bombus spp. We modelled Bombus prevalence as dependent on A. mellifera virus prevalence, A. mellifera abundance, Bombus abundance, latitude, longitude and landcover type. As before, abundance was log‐transformed and quantitative predictors were standardized. As before, we also conducted a GLM with A. mellifera virus prevalence as the response variable. Model selection was performed using an automatic approach  to explore the overall relationship of virus prevalence between A. mellifera (n = 237); 100% B. hortorum (n = 30); 100% B. jonellus (n = 1); 90% B. lapidarius (n = 169); 93% B. lucorum (n = 89); 90% B. pascuorum (n = 55); 100% B. monticola (n = 7); 100% B. pratorum (n = 3); and 64% B. terrestris (n = 92). B. terrestris β‐actin was identified to contain a ligation‐site sequence mismatch, and the proportion of samples passing quality control was lower for this species. In future cross‐species comparisons, MLPA probes should be designed for a wider range of housekeeping genes from which uniform markers across bee species can be selected.Of 792 sampled bees, the following passed β‐actin quality control : 92% A. mellifera and Bombus spp., we detected VDV‐1 and DWV/KV from the DWV/VDV‐1/KV complex, but only ABPV from the ABPV/IAPV/KBV complex. DWV complex strains are closely related at proteolytic sites  could be attributable to qRT‐PCR primer mismatches preventing amplification). Analysis of nucleotide sequences further confirmed the sequence identity of BQCV, ABPV and SBPV in infected A. mellifera and Bombus spp. foragers . Unrooted trees for SBPV and BQCV sequences are given in Fig. S2 (Supporting information) . SBPV clones from B. pascuorum and A. mellifera are similar or identical, whereas B. terrestris is represented by two diverging haplotypes. For BQCV, clones from each species were more clearly separated, but this is unsurprising given that each individual host bee originated from a different site. Interestingly, B. terrestris was again represented by two distinct haplotypes.For both Both MLPA and qRT‐PCR assays did not specifically amplify the negative strand of RNA viruses, and as such, they did not test for actively replicating virus directly. Nevertheless, our methods provide a reliable indicator as to the presence and potential severity of viral infections in bee foragers by employing a multiplexed presence/absence screen followed by quantification.A. mellifera and 23% (95% CI: 19–27%) in Bombus spp. . Most viruses occurred singly, with co‐occurrence of two and three viruses being detected in, respectively, 7% (95% CI: 4–12%) and 1% (95% CI: 0–3%) of A. mellifera individuals, and 3% (95% CI: 2–5%) and 0·2% (95% CI: 0–1%) of Bombus spp. individuals . The proportion of coinfected individuals did not depart from null expectations . The most prevalent virus was DWV in honeybees  and ABPV in bumblebees  in A. mellifera and Bombus spp.  and DWV  were more prevalent in A. mellifera, whereas ABPV was more prevalent in Bombus spp. . Although SBPV and SBV were more prevalent in Bombus spp. and A. mellifera, respectively, differences between host genera were not statistically significant . In a comparison of virus prevalence among the five commonest host species , we found that DWV and SBPV were significantly more prevalent in A. mellifera and B. hortorum, respectively  and wild (Bombus spp.) bees were linked. In GLMMs of individual viruses, we found that prevalence of BQCV , ABPV  and DWV  in A. mellifera had a positive effect on BQCV, ABPV and DWV prevalence in Bombus spp., respectively . In a GLM of combined RNA viruses , we found that disease prevalence in A. mellifera also had an overall positive effect on disease prevalence in Bombus spp. . Sites harbouring highest overall RNA virus prevalence were concentrated in SE England. On the other hand, those harbouring lowest RNA virus prevalence were located in remote western regions, including two V. destructor mite‐free islands  that contained the lowest overall disease prevalence across all sites , with putatively low‐level infections (104–106 virus particles) predominating in bees from both genera. For DWV, viral loads in A. mellifera foragers were greater than in Bombus spp. , consistent with the presence of high‐level infections in A. mellifera (1010–1011 virus particles) vs. low‐level infections in Bombus spp. (104–106 virus particles). We detected a wide range of ABPV and SBPV virus loads in Bombus spp. (104–1011 ABPV and 105–1011 SBPV particles), but sample sizes were inadequate in A. mellifera (ABPV and SBPV: n = 2 each) to be able to compare distributions between pollinator genera. SBV was not detected in any of the samples by qRT‐PCR .We quantified BQCV, DWV, ABPV, SBPV and SBV from the positively detected ers Fig. . For BQCIn a comprehensive field survey of managed and wild bee pollinators, we found that common RNA viruses previously associated with honeybees are widespread in bumblebee populations, and that viruses vary substantially in terms of individual pathogen load and population‐level prevalence. Significantly, we demonstrate a positive association in disease prevalence between managed and wild bees, indicating that disease spillover may be an important general aspect of RNA virus epidemiology in bee pollinators.RNA viruses are prevalent in wild bee populations and occur broadly in the landscape. More than one in every five bumblebee foragers sampled contained at least one of the RNA viruses that were screened for. We note that the total impact of RNA viruses on wild bees is likely to be higher than suggested from our prevalence data, as severely affected individuals may be less likely to fly and forage.et al. et al. et al. et al. V. destructor mites in vectoring this virus. Surprisingly, we find that ABPV and SBPV are more prevalent in bumblebee than honeybee foragers , and that bumblebees harbour a wide range of pathogen loads, including a substantial proportion of putatively high infections .Of the targeted viruses, we found that BQCV, DWV, ABPV and SBPV occurred commonly in bumblebee foragers. Previous studies have indicated that wild bees could harbour RNA viruses typically associated with honeybees , suggesting that differences in host susceptibility or quality may exist . We also cannot exclude the possibility that one or more unknown species from the wider invertebrate community is the primary disease reservoir. Equally, the main reservoir may be a low‐prevalence host, whereupon entering a second host the virus spreads rapidly, resulting in an epidemic and higher observed prevalence. For example, it is plausible that increased ABPV prevalence in bumblebees is due indirectly to the increased exposure to infectious virus particles emerging from V. destructor‐infested honeybee colonies that contain higher than normal loads of ABPV  to harbour RNA viruses, often at putatively low levels. In combination with other stressors, such pervasive disease pressures could have a general and profound impact on the long‐term health of bee populations. Recent studies have reported interactions between sublethal doses of neonicotinoid pesticides and pathogens, with significant impacts on virus replication and host immunity  of RNA viruses in A. mellifera and Bombus spp.Click here for additional data file.Fig. S4. Linear regressions of raw A. mellifera and Bombus spp. RNA virus prevalence data.Click here for additional data file.


Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins  with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other . However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.Viruses belonging to the species  Orbivirus, family Reoviridae. They have icosahedral and non-enveloped virus-particles, containing a genome composed of ten linear pieces of dsRNA. The orbiviruses currently include 22 virus species  that have been recognised by the International Committee for the Taxonomy of Viruses (ICTV), as well as 14 unclassified isolates  Culicoides [biting midges] and sand flies) and collectively have a wide host-range that includes domestic and wild ruminants, equines, marsupials, sloths, bats, birds and humans The orbiviruses are classified within the genus Macropus fuliginosis), red kangaroos (Macropus rufa), Eastern grey kangaroos (Macropus giganteus) and euros or wallaroo (Macropus robustus). Many thousands of kangaroos were affected in the states of New South Wales, South Australia and Victoria, suffering from non-suppurative choroiditis, retinal degeneration with inflammation and secondary degeneration of the optic nerve Eubenangee virus were also isolated from wallabies affected by ‘Tammar Wallaby Sudden Death Syndrome’ (TSDS) Orbiviruses, including Wallal virus (WALV), Warrego virus (WARV) and Eubenangee virus (EUBV) have previously been isolated from marsupials, and there is serological evidence for infections by Wongorr virus (WGRV) Wallal virus contains three distinct serotypes/strains: Wallal virus (WALV), Wallal K virus (WALKV) and Mudjinabarry virus (MUDV) Culicoides collected in Charleville, Queensland, Australia Culicoides marksi collected in the Alligator Rivers area of the Northern Territory of Australia The species Warrego virus contains three distinct serotypes/strains: Warrego virus (WARV), Warrego K virus (WARKV) and Mitchell River virus (MRV). WARV virus was first isolated in 1970 from adult Culicoides collected in Charleville, Queensland, Australia Culicoides spp. trapped in the Mitchell River region in Australia The species Eubenangee virus contains four distinct viruses: Eubenangee virus (EUBV), Tilligerry virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) Culicoides spp. and antibodies to these viruses have been detected in cattle and marsupials Orbivirus species The species Culicoides borne orbivirus (CBO) include Bluetongue virus (BTV) (the type species of the genus Orbivirus), African horse sickness virus (AHSV) and Epizootic haemorrhagic disease virus (EHDV), which are thought to include the most economically important orbiviruses. Complete genomes have been sequenced for representative isolates of BTV, EHDV, AHSV as well as some other CBO species including Palyam virus (PALV) and Equine encephalosis virus (EEV) (the members of which infect domesticated livestock) Culicoides, The different species of Recent advances in RT-PCR and high throughput DNA sequencing methods have generated full-genome sequence data for multiple orbivirus isolates, helping to clarify the genetic relatedness and taxonomic status within and between different orbivirus species, including BTV, AHSV and EHDV Orbivirus species, we report full genome nucleotide sequences for WALV, MUDV and WARV, and present a comparison to isolates from other Orbivirus species as an aid to help identify novel orbivirus isolates. The full-genome sequences of WALV and MUDV indicate that they represent different serotypes within the Wallal virus species and that WARV represents a distinct species within the genus Orbivirus.As part of a programme to generate representative data for all The viruses used in this study  were obtained from the Orbivirus Reference Collection (ORC) at The Pirbright Institute. These viruses were originally taken from naturally infected animals by qualified veterinarians, as part of normal diagnostic testing procedures in the respective countries. MUDV was propagated in BHK-21 cells , while WALV and WARV were grown in BSR cells (a clone of BHK) g for 10 min at 4°C. Single stranded RNA (ssRNA) was removed by 2 M LiCl precipitation at 4°C overnight, followed by centrifugation at 10,000 xg for 5 min. An equal volume of isopropanol containing 750 mM ammonium acetate was added to the supernatant, then mixed and the viral dsRNA was allowed to precipitate for a minimum of 2 hours at −20°C. The dsRNA was pelleted by centrifugation at 10,000 xg for 10 min, washed with 70% ethanol, air dried and suspended in nuclease free water (NFW). The RNA was either used immediately or stored at −20°C.Intact genomic dsRNA was extracted from WALV, MUDV and WARV infected cell cultures using a guanidinium isothiocyanate extraction procedure as described by Attoui et al The genome segments of WALV, MUDV and WARV were reverse-transcribed using a ‘full-length amplification of cDNA' (FLAC) technique described by Maan et al Amplified cDNAs were purified and cloned. MUDV genes were cloned into the Strataclone blunt-end PCR cloning vector ‘pSC-B-amp/kan’ supplied in the StrataClone Blunt PCR cloning kit (Stratagene). WALV (only Seg-2) and WARV amplicons were cloned into the ‘pCR-Blunt’ vector supplied with the Zero Blunt PCR Cloning Kit (Invitrogen). Recombinant plasmid-vectors containing MUDV inserts were transformed into Solopack competent cells (Agilent Technologies), while WALV and WARV plasmids were transformed into One Shot TOP10 competent cells supplied with the respective cloning kits. Clones containing relevant inserts were identified by touch PCR using M13 universal primers. Plasmids were extracted from the clones identified using the QIAprep Spin MiniPrep Kit (Qiagen). The plasmids and PCR products were sequenced using an automated ABI 3730 DNA sequencer (Applied Biosystems). Some of the MUDV sequencing primers detected other segments of WALV and using the sequences generated, running primers were designed to obtain full sequence for the remaining nine segments of WALV.http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). Multiple alignments of consensus sequences were performed using ClustalX (Version 2.0) http://www.ebi.ac.uk/Tools/msa/clustalo/) and MAFFT http://www.cbs.dtu.dk/services/RevTrans/) for further nucleotide analysis. The best fit amino acid (aa) and nucleotide (nt) models for Maximum likelihood (ML) analysis were determined using ProtTest 3.0 and jModeltest respectively ‘Raw’ ABI sequence data were assembled into ‘contigs’ using the SeqManII sequence analysis package (DNAstar version 5.0). The ORFs of WALV, MUDV and WARV were identified and translated into aa sequences for further analysis using EditSeq (DNAstar version 5.0). The putative function of each protein was identified by BlastX comparisons to homologous orbivirus (e.g. BTV) proteins in GenBank (WALV (isolate - AUS1978/09), MUDV (isolate AUS1982/03) and WARV (isolate AUS1969/01) all induced severe cytopathic effects (CPE) in BHK and BSR (a clone of BHK cells) cell monolayers at 48–72 hours post infection. When analysed by 1% agarose gel electrophoresis (AGE), the genomic dsRNA of these viruses migrated as distinct groups, consisting of 3 large segments, 3 medium size segments, 3 small segments, and 1 very small segment (giving a 3-3-3-1 pattern) . This paFull length sequences of Seg-1 to Seg-10 of WALV (AUS1978/09), MUDV AUS1982/03) and WARV (AUS1969/01) have been submitted to GenBank with accession numbers KJ495745 to KJ495754; KJ495765 to KJ495774 and KJ495755 to KJ495764 respectively. These data were compared to other orbiviruses (accession numbers for the data used are given in Table S1 in /03 and WU/AA………..AC/AA/GCU/A/CUAC-3′). WARV shares six fully conserved nucleotides at the 5′ ends and four at the 3′ ends (+ve: 5′-GUAAAA………..AC/AA/U/CUAC-3′). Two terminal nucleotides at the 5′ end and three nucleotides at the 3′ end (5′-GU……UAC-3′) of all three viruses are characteristic of the genus Orbivirus, and the first and last two nucleotides represent inverted complements (http://www.reoviridae.org/dsRNA_virus_proteins/CPV-RNA-Termin.htm).The different genome segments of WALV and MUDV have the same conserved termini, sharing five fully conserved nucleotides at their 5′ and 3′ends  (36.66% in PHSV to 45.86% in EEV) but less than tick-borne orbiviruses (TBOs) (51.93% in St Croix River virus (SCRV) to 57.29% in Great Island virus (GIV)) Like other orbiviruses, most genome segments of WALV, MUDV and WARV have shorter 5' than 3' terminal non-coding regions (NCRs). However, Seg-8  of WALV and MUDV, and Seg-5  of WARV have longer 5′ NCRs . ExceptiOrbivirus species), encoded by a downstream ORF (+2 frame) on Seg-9  was identified in BTV and several other orbiviruses The coding assignments of the genome segments of WALV and MUDV  are simiAUGG), except for the first ORFs of Seg-9 and Seg-10, which have ‘weak’ (UAGAUGU) or ‘moderate’ (GUCAUGU) contexts respectively, potentially allowing read-through of the upstream initiation codon, enhancing expression of NS4 and NS3a respectively. Similarly, most ORF's of the WARV genome-segments have a strong (RNNAUGG) to moderate Kozak sequence [Seg-4 (AUCAUGC) and Seg-5 (UCUAUGA)], except Seg-9 (UGGAUGU) and Seg-10 (GUCAUGC), both of which have a weak ‘context’.Most of the WALV and MUDV ORF's have a ‘strong’ Kozak sequence (RNNAAGAUGG) encoding an 87 aa protein, and in WARV has a moderate Kozak sequence (GGGAUGC) encoding 78 aa protein. In all three viruses WALV, MUDV and WARV, the second initiation site (for NS3a) in Seg-10 has the strong Kozak context (AA/CGAUGG).Weak or moderate Kozak sequences have previously been observed in several of the genome segments of other orbiviruses, although they still appear to be translated effectively The conserved nature of the orbivirus RNA dependent RNA polymerase (Pol) (encoded by Seg-1) has previously been used to help classify orbivirus isolates at the genus and species level based on sequence comparisons and identity levels Orbivirus species. In comparisons to other orbiviruses they show a maximum of 70.96%/66.97% aa/nt identity with EUBV and a minimum of 36.08%/43.55% with SCRV (Orbivirus species within the CBOs.Seg-1/VP1(Pol) of WALV and MUDV share 99.16/93.57 aa/nt identity, confirming that they belong to the same ith SCRV . VP1 is the most abundant of the orbivirus structural proteins and is a strongly immuno-dominant serogroup/species-specific antigen  is the mCulicoides transmission. An unrooted NJ phylogenetic tree constructed for OC1 of different orbiviruses (Orbivirus species.Orbivirus outer capsid protein (OC1) determines virus serotype and is highly variable, both in sequence identity levels and in size. VP2(OC1) of the CBOs is encoded by Seg-2 (VP2), by Seg-3 (VP3) of the MBOs and by Seg-4 (VP4) of the TBOs iviruses  identifiiviruses , 3 and 4Orbivirus species were initially recognised as distinct ‘serogroups’, sharing common antigens detectable by complement-fixation (CF), agar gel immuno-diffusion (AGID), fluorescent antibody (FA) tests, or enzyme-linked immune-sorbent assays (ELISA) Orbivirus species The majority of the existing The advent of more rapid and reliable sequencing methods for dsRNA virus genomes has generated data for reference strains of the individual virus species, that can be easily disseminated for phylogenetic comparisons Orbivirus species. Consequently, these proteins and their comparisons have been used as ‘markers’ for identification and classification of existing and novel orbivirus isolates Bluetongue virus and Epizootic haemorrhagic disease virus . The genes encoding these proteins have also served as targets for development of orbivirus genus-specific and species-specific RT-PCR assays that can be used for diagnosis and virus discovery The orbivirus sub-core-shell ‘T2’, polymerase ‘Pol’ and outer-core ‘T13’ proteins are all highly conserved, with >83%, >73% and >73% aa identity respectively within a single Nucleotide variation of up to 31.6%  has been observed in the serotype controlling VP2(OC1) gene between different BTV isolates within a single serotype, with a maximum of 71.5% nt identity between different types providing useful ‘bench marks’ for typing of isolates in other orbivirus species co-speciation’ with their arthropod vectors Phylogenetic analysis of the three most conserved VP1, T2 and T13 proteins/genes and the highly variable OC1 protein of the orbiviruses, that are presented here show three distinct ‘clusters’ that correspond to the arthropod vectors used by each virus , 3 and 4Orbivirus species cluster more closely together, while members of distinct species branch separately. The trees constructed for orbivirus Pol, T2 and T13 genes are almost entirely congruent, also showing similarities to the OC1 tree, although with higher levels of identity within each virus species. This suggests that functional interactions between the different viral proteins/RNAs may restrict the exchange of genome segments (reassortment) between virus species.Phylogenetic trees for the different genome segments/proteins of WALV, MUDV and WARV confirm that members of the same Orbivirus species - Wallal virus. The data presented here, showing that WALV and MUDV share >99%/90% aa/nt identities in their conserved Pol, T2 and T13 proteins support this conclusion , the mechanisms involved in virus movement and emergence, and their impact on disease in vertebrate populations. The generation of full-genome sequences for reference strains of Orbivirus species recognised by ICTV. These data will provide a basis for development of relevant diagnostic assays, and support molecular epidemiology/evolutionary studies, to promote our understanding of orbivirus disease in marsupials and other host groups.Full genome references sequences are now available for fourteen of the twenty two File S1Table S1 and Figure S1. Table S1. Nucleotide accession numbers for sequences used in phylogenetic analysis. Figure S1. Unrooted Neighbour-joining phylogenetic tree comparing orbivirus T2 proteins.(DOCX)Click here for additional data file.


Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str]  demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to  Tick-borne encephalitis virus (TBEV) is recognised as a re-emerging human pathogen with an estimated 16,000 cases recorded annually over 30 European and Asian countries  viruses was not affected by multiple freeze-thawing cycles. Incubation at 37°C for 48 hours significantly reduced the titres of all viruses and this was also the case after multiple freeze-thawing cycles followed by incubation at 37°C for 48 hours. The infectivity of all viruses was stable when incubated at 40°C for 40 minutes, followed by 37°C for 40 minutes. However, incubation at 42°C for 48 hours caused a significant decrease of infectivity across all viruses and indeed, Hypr[Vs str] was totally inactivated under these conditions.A single cycle of freezing and thawing had no significant affect on the virus titres. However, six sequential cycles of freezing and thawing caused a remarkable decrease in infectivity of Hypr[Vs str] by approximately 1 logIn conclusion, under increased stress conditions, the Hypr[Vs str] chimaera was more labile than the other viruses possibly indicating reduced virion integrity. However, all the viruses were sufficiently stable when subjected to relatively mild physical conditions corresponding to the experimental procedures. Thus, virion lability is unlikely to have been responsible for the observed decrease of replication efficiency of Hypr[Vs str] refer to .The relative contribution of different structural and non-structural regions of the TBEV to NVT was measured for chimaeric viruses following tick co-feeding. Briefly, following co-feeding between two TBEV infected females and 15 uninfected nymphs, the female ticks and nymphs were individually tested for the presence of the virus. NVT rate was evaluated as percentage of nymphs positive for TBEV . Highly The most remarkable result was the increased NVT rate of chimaeric virus Vs[Hypr str] (~ 85%) which was even higher than that of HyprIC virus (~ 65%) p = 0.002). Surprisingly, no transmission was detected for Hypr[Vs str] at all . Thus, N. Surpris10 pfu/ml for VsIC and 4.3 log10 pfu/ml for HyprIC, p = 0.001) (10 pfu/ml) in adult ticks was recorded for chimaera Vs[Hypr str] which also showed the highest overall NVT rate. This supports the concept that the structural genes of TBEV largely determine the differences in TBEV replication efficiency, as observed between Vs and Hypr TBEV in adult ticks. Correspondingly, engineering Hypr E and prM-E genes into the Vs genome also caused an increase in Vs[Hypr E] and Vs[Hypr prM-E] titres in adult ticks when compared with the control VsIC virus. The increment was very small (p > 0.05) but was in concordance with increased NVT rates of Vs[Hypr E] and Vs[Hypr prM-E] when compared with VsIC  . Furtherith VsIC .10 pfu/ml compared to 4.3 log10 pfu/ml for HyprIC .Substitution of the entire VsIC 5`UTR-C-prM-E gene cluster into the Hypr genome (Hypr[Vs str]) significantly (p = 0.0002) down-regulated the TBEV replication in adult ticks by ~ 2 orders of magnitude, compared with control HyprIC virus . The repr HyprIC . Introdu10 pfu/ml, with the exception of Hypr[Vs str], which exhibited reduced reproduction in adult ticks by ~2 log10 pfu/ml compared with control HyprIC virus (with the longer 3'UTR). Control HyprIC and chimaeric Vs[Hypr str] viruses generated the highest infectious titres in I. ricinus adult ticks whilst Hypr[Vs str] virus generated the lowest titres. The other chimaeric viruses exhibited a range of intermediate virus titres, with insignificant differences in repeated experiments . Nevertheless, these small yet reproducible differences could have an impact on overall virus biological characteristics , which again is in concordance with results in adult ticks.Levels of TBEV infectivity in nymphs reflect virus accumulation and reproduction during tree days of co-feeding with infected female ticks . With thfor VsIC . The hig10 pfu/ml and was not significantly different from HyprIC (p > 0.05).The VsIC-based viruses, with substituted Hypr E and prM-E genes, exhibited increased infectivity in comparison to control VsIC virus—although for Vs [Hypr prM-E], the differences were not statistically significant (p > 0.05). In correlation with this, the HyprIC based chimaeras with E and prM-E of VsIC virus had lower infectivities in comparison with HyprIC but for Hypr [Vs prM-E] this was also not statistically significant. Similarly, as with the NVT data, no infectious Hypr[Vs str] virus was recovered from nymphs following co-feeding with infected adults . The infvice versa for the Hypr genes as they were progressively introduced into the Vs-based chimaeras, though the statistical support for these differences was not always significant  at the end of the co-feeding experiments . DiffereTo evaluate further whether or not the observed changes in NVT rate were caused by modifications of the chimaeric viral genomes rather then titre, we applied three independent methods of regression and correlation analysis. Firstly, analysis of correlations between the NVT, titres of TBEV in salivary glands of females and in nymphs of different chimaeric viruses; secondly, regression analysis of correlations between TBEV genome fragment replacements with NVT rate and thirdly, regression analysis of correlation between NVT rate and amino acid similarity of the structural proteins of studied viruses  between 2 = 1). Under this model, the differences in NVT rate of chimaeric Vs and Hypr viruses resemble sinusoidal curves which are in direct opposition. Thus, maximal NVT rate values of Vs-based chimaeric viruses correlate with minimal values of NVT rate for Hypr-based chimaeric viruses and vice versa . Then we plotted the amino acid distances against the NVT rate of corresponding viruses and performed the linear regression analysis. The NVT rate had a strong positive correlation with genetic distance from VsIC estimated as amino acid substitutions per site . HoI ricinus and I persulcatus respectively) revealed previously unidentified adaptive characteristics to competent tick species. Comparative analysis of a panel of chimaeric viruses supported this conclusion, revealing that in spite of the ability of divergent TBEV subtype to replicate in I. ricinus, the virus titre in female salivary glands is significantly higher for EU-TBEV and that this correlates with the rate of NVT. At the molecular level the efficiency of NVT appeared to be primarily determined by the structural gene cluster 5'UTR-C-prM-E, with a strong indication of an important contribution by the 5'UTR—C genome region. Indeed, the addition of the heterologous 5'UTR-C region to [prM-E] chimaeras was an essential requirement to increase the NVT rate of Vs[Hypr str], to values comparable with those of the control wild type HyprIC virus and in the directly opposite case, i.e. Hypr[Vs str], transmission was completely abrogated. The prM functions as a chaperone for authentic folding of the E protein; it is present in immature (intracellular) virions and is separated into its individual components, ie pr and M protein by furin-mediated cleavage during virion egress from infected cells. Consequently, mature extracellular virions contain only the M protein anchored in the cell-derived virion membrane together with the E protein ) were generally more stable than those containing the 5`UTR-C from Vs in a backbone of HyprIC (Hypr[Vs str]). Thus, the replication dynamics in PS cells, physical stability of virions and infectivity in salivary glands of tick females for Vs[Hypr str] were comparable to controls or better. However, the reciprocal chimaera Hypr[Vs str] exhibited delayed replication dynamics in cell culture, lower thermal stability of virions and significantly decreased production of infectious particles in tick salivary glands Figs , 5 and 6In spite of the fact that virus cytopatogenicity for PS cells was primarily determined by the non-structural part of the genome, the pathogenicity profile in mice depended primarely on structural genes. Most observed differences in mice reflect the impact of the genetic modifications within the TBEV genomes rather than that of virus challenge dose. The titres of chimaeras HyprIC short, Vs IC, Vs[Hypr prM-E] and Hypr[Vs E] in tick salivary glands fall within a range of 3–3.5 log10 pfu/ml and therefore differences in their pathogenicity profiles are probably caused by genetic alterations in the virus genome rather than by variability of the infecting virus dose delivered by the ticks. The significantly attenuated properties of Hypr[Vs str] in mice could be attributable to the low infectious dose (2.5 log10 pfu/ml) in ticks. However, attenuated properties of this virus were also observed in PS cells where the other viruses did not show these differences.in vitro cell culture methods. All the TBEV strains tested exhibited similar replication kinetics in cell culture but exhibited remarkable differences in their relative ability to replicate in adult ticks and nymphs. These differences ultimately determined the efficiency of NVT during tick co-feeding, a fundamental requirement for virus dissemination and survival in the natural environment. When combined with previous studies of TBEV transmission between ticks, these studies are beginning to define the underlying molecular processes that have evolved to ensure maximum virus fitness for the differing molecular machinery of distinct tick species.In conclusion, the modelling of TBEV transmission in conditions that closely resemble those in nature has revealed results previously inaccessible using I. ricinus ticks were bred in the Institute of Zoology, Slovak Academy of Science, Bratislava  an an47] anrotocols . The infrotocols  and HyprE. coli, we previously developed the approach of sub-cloning single virus cDNA molecules into two overlapping plasmids that could be ligated in vitro to re-create a full-length cDNA, which acts as a template for SP6-transcription to produce synthetic full-length genomic RNA [To overcome the issue of TBEV IC cDNA genome instability in omic RNA . A shortomic RNA , 36, 49.Hypr virus genome cDNA was amplified in 2 sets of long high-fidelity PCR, one representing the structural region between nucleotides 1–2461 and the second the non-structural region between nucleotides 3154–10835. These PCR fragments were cloned as two plasmids, pDGHypr1-2450 and pATHypr3154-10835. A silent BsuI site was introduced into pDGVs660-3216-BsuI at the boundary between the E and NS1 proteins  or non-structural (through NS1-NS5-3UTR) pregion of the TBEV genome were digested with overlapping enzymes and ligated eviously , 49. Eacing mice , 49 or ting mice  and by R10 pfu/ml.Aliquots of virus diluted in serum-free RPMI 1640 medium (250 μl) were added to PS cell monolayers in 24-well plates. After virus adsorption for 1 h at 37°C the inocula were removed and cells were overlaid with RPMI 1640 medium supplemented with 2% FCS and 1% SeaPlaque Agarose . After incubation at 37°C for 5 days monolayers were fixed with 4% formaldehyde and stained with 0.05% crystal violet. Plaques were counted and virus titre was expressed as log2 atmosphere for 96 hours. The plates were fixed with 4% formaldehyde, stained with 0.05% crystal violet (CV), washed, air dried and photographed. The stain from monolayers was extracted with methanol and viable cells were quantified by measurement of optical density of extracts at wavelength 590 nm. Previously, it has been shown, that optical density of crystal violet extract is linearly related to the number of stained cells [Confluent monolayers of PS cells in 24-well plates were infected at least in four replicates at an estimated MOI = 1 pfu/cell. Infected cells were overlaid with 1 ml of support media and incubated at 37°C with a 5% COed cells . TherefoPorcine kidney PS cells were grown overnight in 50 ml flasks to confluence and then inoculated with 0.2 ml of TBEV suspension at MOI = 1 pfu/cell. After 1 h of adsorption at 37°C, the inocula were removed; cells were washed 5 times with serum-free RPMI 1640 medium and overlaid with 5 ml of support medium containing 2% FCS. The supernatant medium from infected cells was collected at 0, 4, 8, 12, 16, 20, 24 and 72 hpi and frozen at –80°C prior to further analysis. The experiments were performed in triplicate. The titres of infectious virus at different time points were determined by plaque assay.One millilitre of cell culture supernatant medium (CCSM) from PS cells infected with TBEV was clarified by centrifugation at 12,000 g for 10 min. Each CCSM was aliquoted (100 μl) in sterile tubes and subjected to one of the following procedures 1) frozen at -80°C ; 2) freeze-thawed once and then frozen at -80°C; 3) freeze-thawed 6 times and then frozen at -80°C; 4) incubated at 37°C for 40°C and then at 40°C for another 40 min and then frozen at -80°C; 5) incubated at 37°C for 48 hours and then frozen at -80°C and 6) incubated at 42°C for 48 hours and then frozen at -80°C. Virus infectivity was then determined by plaque titration. Each experiment was performed in triplicate.I. ricinus female ticks were infected with 500 pfu of TBEV chimaeric or wild-type strain Hypr or Vs virus by inoculation into the coxal plate of the second pair of legs. Infected ticks were incubated at room temperature (24 ± 4°C) and 85–90% RH in a desiccator for 14 days. Two infected adult female ticks were allowed to feed on each of 4–8 Balb/C mice for three days simultaneously with 15 uninfected I. ricinus nymphs that were attached in close proximity to the feeding donor females. Surviving nymphs and salivary glands of donor females were used to determine the titres of infectious virus using plaque assays. The NVT rate was estimated as the proportion of nymphs that became infected. In total, 114 individual transmission experiments were taken into analysis that provided at least four independent replicates for every virus. Transmission experiments for the control viruses HyprIC long 3`UTR and VsIC H216 were also replicated in triplicate.Experiments on TBEV co-feeding transmission were essentially performed as described previously , 16, 29.Host mice after tick co-feeding were monitored during 21 days post tick bite and day of onset of symptoms of encephalitis was documented to evaluate morbidity. Mice that survived 21 days post tick bite were euthanized and haemagglutination inhibition assays were performed to detect anti-TBEV antibodies and confirm the infection. At least 8 mice were monitored for each virus and their survival probabilities and morbidity rates were calculated. The usage of animals in the experiments was approved by the State Veterinary and Food Administration of the Slovak Republic  and by the Committee for biomedical ethics of FSPSI “Scientific Centre of Family Health and Human Reproduction Problems” . All possible efforts were taken to ameliorate animal suffering, including minimization of the number of animals used for the study, anesthesia, and euthanasia of moribund animals at the outbreak of clinical symptoms. The animals were checked twice a day in the morning and late afternoon. The general fitness of mice—based on body weight, food consumption, mobility and motion coordination were used to assess animal health, body condition, and well-being. The humane endpoint symptoms included rough fur as indicator of fever, loss of balance, and\or back limb paralysis. Consequently, the mice were euthanized the same day they showed the symptoms. No animals died without anaesthesia. Animals were euthanized by cervical dislocation under deep anaesthesia induced using carbon dioxide.TBEV NVT rates and infectivity are presented as mean values. Confidence intervals of 95% were estimated for each mean value and outlying variables were excluded using quartile method according to . BetweenS1 FigSchematic representation of intermediate plasmids constructed to recover recombinant TBEV strains . The pol(TIF)Click here for additional data file.S1 TablePlasmids representing structural and non-structural proteins of the TBEV regions  were use(DOC)Click here for additional data file.


Nowadays natural products such as pure compounds and plant extract scan provide unlimited opportunities for new antiviral drugs. Newcastle disease virus (NDV) is one of the most important viral diseases in poultry industry. Vaccination could provide protection against NDV outbreaks, but it is not sufficient because infections by NDVs have remained frequent around the world.Achillea millefolium and Thymus vulgaris antiviral activity against Newcastle disease virus (NDV).The current research aimed to study The antiviral activity of the plants was measured by the reduction assay of viral titer, and explained by inhibition percentage (IP).1.75, which indicated the ability of the extracts to reduce the viral potency by more than 56 folds.Inhibition percentage was determined as 10 Both plants were found effective against Newcastle disease virus. Pseudo-fowl pest or Newcastle disease (ND), a devastating disease of poultry seen in chickens and turkeys, caused by Newcastle disease virus (NDV). The signs of disease are high mortality, hemorrhagic intestinal lesions, severe respiratory distress, decrease of egg production, and nervous disorders . The NDVVaccination programs can provide protection against NDV outbreaks, but they are not sufficient because infections by NDVs have remained frequent around the world in the recent years . There iAchillea millefolium (yarrow) is a powerful medicinal plant which is widely distributed and has been used by different cultures for thousands of years , a Mediterranean aromatic plant, has been frequently used for medicinal purposes. T. vulgaris has been used in traditional medicine for several of its properties such as antiseptic, antimicrobial, antifungal, antioxidative, and antiviral activities , with centrifugation at 3000 r/min for 15 minutes, and then the supernatant was collected. This process was repeated three times. Solvents were then removed by evaporation  was used for A field strain of NDV was obtained from the veterinary medicine department of the University of Tehran. This virus was isolated from fowl disease. Stock suspensions of the virus were prepared in the following manner. The received virus was inoculated to 9-day embryonated eggs. After 4 days the allantoic fluid was harvested and HA test was applied to confirm the virus .The antiviral activity of plant extracts was assayed in the following manner. The stock allantoic fluid suspension of the virus was diluted in concentrations of 10-1, 10-2, 10-3, 10-4 and 10-5 in sterilized phosphate buffer saline. Next, 500μLof the plant extracts were mixed separately with 500μLof the diluted concentrations of virus and the mixture was incubated for one hour at 4°C to allow reaction occurrence . Then 20Triplicate tests were applied for each virus concentration with and without the extract . Next, tA. millefolium and T. vulgaris were 10 and 50 μg/mL concentrations, respectively. At higher concentrations, chickens were dead, but at lower concentrations they were born alive and healthy. Therefore, for the antiviral assay of A. millefolium and T. vulgaris, 10 and 50 μg/mL concentrations were selected, respectively. The results of the overall in ovo screening are summarized in in ovo against NDV in allantoic cavity and their gross antiviral activity was evaluated in terms of haemagglutination inhibition. Egg toxicity assay was performed using embryonated eggs to determine the maximum non-toxic concentration (MNTC). The obtained MNTCof The extracts were considered active if they reduced EID50 of the virus in the case of NDV, and EID50 of the virus and the virus/extract was measured by the Reed and Muench method. Antiviral activities were measured in terms of the virus titer difference between treated and untreated infected control eggs and inhibition percentage (IP). The EID50s of the virus stock suspension used in these series of experiments and extract/virus suspension related to both plants were 10-4.25/mL and 10-2.5/mL EID50, respectively. Finally, antiviral activity was determined as 10 1.75, which indicated the ability of the extracts to reduce the viral potency by more than 56folds. Both extracts were found effective.In ovo injection method was designed because studying the plants in vitro and in vivo is time consuming and expensive.. In this method, the plants were first screened for antiviral activity in ovo and then the best plants were selected for the next step. Nowadays natural products such as pure compounds and also plant extract scan provide unlimited opportunities for new antiviral drugs . InfectiThe amount of active constituents of the plants depend on the geographical distribution, season of collection, and climatic and ecological condition of the collection site . StudiesT. vulgaris (thyme) has essential oils which contain mixtures of different volatile and lipophilic substances, such as sesquiterpenes, monoterpenes, and phenylpropanoids. These substances have been proposed to be part of the preformed defense system of higher plants (T. vulgaris was found effective against HSV-1/HSV-2. This plant, with the help of essential oils, interferes with the virus envelope, and also masks viral components which are necessary for attachment, penetration, or entry into host cells (r plants . Reportsst cells . A. millefolium show the presence of essential oils, tannins, flavonoids, sesquiterpene lactones, alkamides, inulin and ascorbic acid in the plant. The essential oil of A. millefolium possesses antioxidant and antimicrobial properties. This plant also reduces virus titer in vivo(in vitro and in vivo.Chemical analyses of r in vivo. The NDV


P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics.A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus  were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions ( Since March 2013, A novel H7N9 influenza virus first crossed the species barrier and infected humans in East China, causing disease with unusually high morbidity and mortality. It is a reassortant virus which consists of the gene fragments from earlier H7N9, H7N3 and H9N2 viruses . The patIn recent years, many new discoveries have been made concerning H7N9 infection. Sakai et al  demonstrAll procedures performed in studies were in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki declaration and were approved by the Medical Ethics Committee of Shenzhen Center for Disease Control and Prevention (SZCDC), Shenzhen, P. R. China.A549 cell line, H7N9 and H1N1pdm09 influenza virus were obtained from Shenzhen Center for Disease Control and Prevention (SZCDC). All the reagents for DIGE were purchased from GE Healthcare . DL-Dithiothreitol (DTT), Iodoacetamide (IAA), CHAPS were purchased from Sigma–Aldrich . The antibodies against CAPZA1, OAT, PCBP1, EIF5A, PAFAH1B2 were purchased from Abcam . The antibody against β-action was purchased from Santa Cruz . The secondary antibodies of goat anti-mouse and goat anti-rabbit were purchased from Thermo Fisher . The restriction enzyme (BamH I and Not I) were purchased from NEB(USA).Lipofectamine2000 and pcDNA3.1/Neo(+) were purchased from Invitrogen .2. To ensure most of the cells were able to grow adhering to the wall at 72hpi, a multiplicity of infection (MOI) of 0.001 was used , which is essential for EIF5A function . The proPlatelet-activating factor acetylhydrolaze (PAFAH) can inactivate the potent pro-inflammatory phospholipid platelet-activating factor (PAF) by removing the acetyl group at its sn-2 position. This family of enzymes is comprised of one secreted (plasma) and at least four intracellular isozymes  ,46. IsofOur study showed platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2)to be significantly down-regulated at 72h in H7N9-infected A549 cells. The level of PAF would be up-regulated, which was related to the later clinical symptoms of patients infected with H7N9 virus, such as pneumonia, ARDS, encephalopathy and so on. Moreover, the increase of PAF induces large amounts of platelets to aggregation which can produce a wide range of micro thrombus in the host. It maybe the reason that the patients infected with H7N9 virus exhibit thrombocytopenia and coagulopathy ,9,11.We utilized proteomic approaches to identify differential expressions of cellular proteins in A549 cells infected with H7N9 and H1N1pdm09 influenza virus at 24, 48, 72hpi. It suggests that these differentially expressed proteins are involved in the pathogenic mechanisms of H7N9 infection at the cellular level. A study by Simon et al  infected


Infectious bursal disease virus is a relevant avian pathogen that affects poultry production. Here, we report the full-length coding sequence of the Uruguayan strain dIBDV/UY/2014/2202, isolated from a commercial broiler flock. The strain belongs to the distinct IBDV lineage that is widely distributed in South America. Avibirnavirus within the family Birnaviridae. This nonenveloped icosahedral virus has a double-stranded RNA genome consisting of two segments named A and B  belongs to the genus  A and B .IBDV is a highly contagious avian pathogen affecting commercial poultry production worldwide . All patThe IBDV strain was collected in 2014 from a 27-day-old commercial broiler flock suffering from respiratory problems and increased mortality. Viral RNA was isolated from bursae tissue using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using random hexamers (Thermo Scientific). The IBDV was diagnosed as a non-very virulent virus by real-time PCR . The comThe coding region of segment A  contains two open reading frames that encode VP5 (145 aa) and a VP2-VP4-VP3 polyprotein ; segment B encodes VP1 (879 aa), the viral RNA polymerase.The phylogenetic analysis of the VP2 hypervariable region indicates that the virus belongs to the dIBDV lineage. This was confirmed by the presence of a unique and conserved molecular signature  that is a diagnostic character for the classification of dIBDVs . SegmentComparative analysis of the complete segment A shows higher nucleotide similarity (96%) with classical strains . In the case of VP1 (segment B), the highest similarity (96.4%) was found with strains that are not very virulent .The comparison of the VP2 hypervariable region of dIBDV/UY/2014/2202 with most vaccine strains commonly used in South America showed an amino acid similarity ranging from 88.9 to 92.1%; D78 (Nobilis D78) and Winterfield 2512 (Cevac IBD L) were the most similar strains.A comprehensive study of more genomic sequences of these dIBDVs is needed to understand the virus’s epidemiology and to contribute to the effective control of IBDV infection.KT336459 (segment A) and KT336458 (segment B).The full-length coding sequence of strain dIBDV/UY/2014/2202 has been deposited in GenBank under the accession numbers


Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease and for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development. Since the wild-type EBOV is avirulent in rodents, the adaptation series of passages in these animals are required for the virulence/lethality to emerge in these models. Here, we provide an overview of our several adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups. Several animal models for Ebola virus infection have been established in rodents and nonhuman primates (NHPs). The NHPs, including rhesus and cynomolgus macaques, are best suited for pathogenesis, treatment, and vaccine studies, since only they can be lethally infected by nonadapted EBOV strains with the resulting pathology closely resembling the human EBOV disease . HoweverIn primates, the pathogenesis of EBOV infection is associated with the viral replication in several major cell targets accompanied with immune dysregulation and coagulopathies. Viral reproduction in primary targets, the mononuclear phagocytes of spleen and lymph nodes, is followed by a massive replication in the liver, mostly, in macrophages and hepatocytes, and the virus spread to the other organs and tissues . A bystander lymphocyte apoptosis by an unknown mechanism is proposed to be the cause of severe lymphopenia occurring in EBOV infection. Inhibition of IFN-mediated response mediated by viral proteins VP24 and VP35 blocks the innate antiviral defense. Vascular damage either occurring directly, due to lytic virus reproduction in the endothelial cells, or induced indirectly by the effects of proinflammatory cytokines on the vascular wall is an important factor of pathogenesis. The mechanisms of coagulation dysfunctions, such as disseminated intravascular coagulation (DIC) and hemorrhages, as well as thrombopenia occurring in primate EBOV infection, are still to be investigated in more detail [10 PFU/g in different tissues including spleen, liver, adrenals, lungs, kidneys, and pancreas) on days 1–9 of the infection, and the peak viremia in blood is reached on day 7 with ~105 PFU/mL [In guinea pigs, the lethal EBOV variants are established through the sequential passages 4–8 times) of an originally wild-type virus, in which, first, incomplete and, further, complete lethality in the groups of inoculated animals are acquired –8 times . The guiWhile resembling the course of EBOV infection in primates in many aspects, the EBOV disease in rodents has some important differences. Fever and maculopapular rash, which are the typical signs of infection in primates, are both not present in mice infected with mouse-adapted Ebola virus (MA-EBOV) . In guinHere, we present a comprehensive overview of the virologic, pathomorphologic, and hematologic data obtained in the EBOV adaptation experiments in guinea pigs (including four passage series performed in our laboratory) and discuss the value of those findings obtained in the rodent models for the general understanding of pathology of EBOV infection.Serial passage of different viruses in animals or cell cultures can result in the selection of mutants with different reproduction characteristics and either higher or lower virulence, as compared to the original wild-type virus , 14. AduThe original EBOV, strain Mayinga, stock of the first adaptation series 8mc) was passaged 24 times in Vero E6 cell line prior to the guinea pig adaptation experiments , while tmc was pa10 PFU/mL to 5.5 ± 0.7 log10 PFU/mL (as determined by plaque forming assay in Vero cells). The first lethal outcomes were detected at passage 3, and the lethality and infectivity of the adapting virus isolate were gradually increasing until passage 8. In guinea pigs inoculated with the virus isolates of late passage 8, the fever occurred at days 4–7 and represented a double-peaked pattern with the second peak at days 11–13. Fever episodes were associated with the maximum levels of tissue virus titers. The adapted virus strain was designated EBOV-8mc, and several clones were derived from it and proved to have similar features as the noncloned EBOV-8mc virus itself [At first passage of 8mc adaptation series , a group10 PFU/mL at passage 1 and did not change throughout the passage series. The resulting virus strain was designated K-5 [As mentioned above, the original EBOV stock of K-5 series was passaged twice in NHPs and further 15 times in L68 cell culture prior to its adaptation to guinea pig, and its infectivity and virulence were evolving in the passage series. The biological titer of the virus harvest in PFU decreased from the first to third passage and then began to increase, reaching its plateau by passage 12 and further remaining stable. However, the virus infectivity in guinea pigs was unchanged throughout the L68 passages. Following the cell culture passages, the virus cloning was performed, and the biological titer (in PFU) of the intermediate virus stock  and the three obtained clones were shown to be equal. When adaptation to guinea pigs was started, clone 2 turned out to cause no febrile reaction, and virus isolation from blood samples of inoculated animals failed. However, clone 1 caused a rise in the rectal temperature in guinea pigs up to 40.5–41.8°C after the first passage, affecting up to 100% of animals. Beginning from the fourth passage, lethal outcomes were recorded. After the fifth passage, the virus became highly lethal for guinea pigs. The virus presence in the blood was recorded at a titer of 3.5 logated K-5 .10 PFU/mL) could only be detected in the serum after the virus concentration by centrifugation, while no virus could be isolated from the whole blood. The tissue virus titers were detectable at 1.6 log10 PFU/mL (in liver) and 3.22 log10 PFU/mL . The first lethal outcomes were detected at passage 2 (on day 10), concomitant with the increase of serum and tissue virus titers to 4.2 log10 PFU/mL. Interestingly, the second peak of fever was not observed in the surviving animals. At passage 3, the adapting EBOV became fully lethal to the guinea pigs with all animals dying between days 5 and 7, and the serum and tissue virus titers stabilized at the levels of 3.0–4.0 log10 PFU/mL. The GPA-EBOV strain obtained in this series was used for a comprehensive analysis of pathomorphologic, hematologic, immunologic, and coagulation patterns of EBOV disease in guinea pigs.In the third passage series, GLA , the aniIn the last series, GPA-P7 , the pre11 PFU/mL at later passages. Of note, the EBOV titers in the liver even at the first passage reached 3.5 × 109 PFU/mL on day 11. This mouse-adapted virus variant was named D-5 [Our several attempts were made to adapt two original Mayinga strain EBOV stocks (of adaptation series 8mc and K-5) to adult ICR mice, using the same approach as the adaptation series to guinea pigs. All attempts to adapt the K-5 virus stock failed; however, using high doses of the first EBOV stock, we obtained a paradoxical variant of EBO. This passage series in adult ICR mice caused no lethal cases but resulted in the increasing virus titer in the liver reaching 5.55 × 10All adaptation series and the designations of all original and derivative viral stocks are provided in The guinea pig-adapted EBOV strains, 8mc, K-5, GLA, and GPA-P7, as well as two other adapted variants obtained by the other research groups , 7, thus de novo and some rare mutations can be amplified and fixed in the adapted virus stocks in the process of adaptation, comparison of genetic changes in adapted lethal and preadaptation nonlethal EBOV genomes and matching of those to the virulence of different EBOV variants can yield invaluable information on the genetic determinants of virulence/lethality.The information on the genetic markers of EBOV virulence is important for the understanding of EBOV infection pathogenesis, as well as for the development of vaccines and antivirals. Since some new mutations (as compared to the wild-type virus stocks) can occurIn our first adaptation series, 8mc, a detailed analysis of nucleotide sequences of the EBOV genomes of the original and guinea pig-adapted virus stocks revealed several changes in genome regions encoding for the five viral proteins , 16. TheContrarily to the above mutations, at least some of the three nucleotide changes in the coding region of vp24 gene, which led to amino acid substitutions Met→Ile(71), Leu→Pro(147), and Thr→Ile(187), may be linked to the adaptation process, since the gel mobility of 8mc-EBOV VP24 is lower than its wild-type counterpart, suggesting a major structural rearrangement of this protein and, probably, the crucial role for VP24 in the adaptation process.A conservative amino acid substitution Thr→Ala(820) in L gene, similarly to Phe→Leu(648) substitution in NP gene, does not seem to be responsible for virus virulence. However, since both are found in all five EBOV-8mc clones, they may have a supportive role in the increase in virulence .A comparative nucleotide sequence analysis of the virus variants of the second adaptation series (K-5) reveals two single nucleotide substitutions in vp24 ORF in the adapted virus strain K-5 , which lead to the amino acid substitutions Asp→Gly165) and His→Tyr(186) . Since t5 and HisTo further evaluate the contribution of genetic differences present in guinea pig-adapted EBOV variants in the virus virulence, two different approaches were employed. The first one was another adaptation series started with a cloned EBOV stock (pre-GPA), in which the genetic events occurring were determined at every passage and compared to the pathogenic reactions in guinea pigs reflecting the emerging EBOV virulence . The othIn the former study, the GPA-P7 adapted strain genome was found to differ from the genome of the pre-GPA clone by 17 nucleotide changes. No genetic differences were found in the terminal untranslated regions of the GPA-P7 genome (3′-leader and 5′-trailer). The nucleotide sequence of the GPA-P7 NP gene contained 12 nucleotide substitutions, 8 of those being synonymous and 4 leading to amino acid changes in the encoded NP protein (Leu→Pro(544), Asn→Ser(566), Ser→Pro(598), and Asn→Asp(663)). Three substitutions leading to amino acid changes were detected in the nucleotide sequence of the GPA-P7 VP24 gene (Met→Ile(71), Leu→Pro(147), and Arg→Leu(154)). The L gene (encoding the RNA-dependent RNA polymerase) of GPA-P7 contained two nucleotide substitutions, one synonymous and one nonsynonymous, the latter resulting in amino acid change ) [Correlation of the mutations in GPA-P7 with the time of their occurrence (passage number) and the emerging virus pathogenicity in guinea pigs allows one to evaluate their roles in the virulence. Of 17 nucleotide substitutions that distinguish between GPA-P7 and the pre-GPA clone, 12 occurred as early as the first passage (GPA-P1 variant). These 12 substitutions comprise nine synonymous (eight in NP gene and one in L gene) and three nonsynonymous substitutions resulting in 3 amino acid changes in NP gene (Leu→Pro(544), Ser→Pro598), and Asn→Asp(663)). Those mutations were considered not related to the emerging EBOV virulence, since they occurred before the acquisition of lethality. Two other mutations emerged at passage 3, leading to the amino acid changes Asn→Ser(566) in NP protein and Leu→Pro(147) in VP24 protein. Of note, the first lethal outcome in this series was recorded at passage 3, although no lethal outcomes among the infected animals were recorded at passage 4. Those two loci remained heterogenous until the substitutions were fixed in the genome of the GPA-P5 variant (at passage 5) representing increased virulence for guinea pigs and causing the disease with 40% lethality . At passage 6, a mutation in the VP24 gene sequence, G10557A, occurred resulting in amino acid change Met→Ile(71). This passage was accompanied by a further increase in EBOV virulence for guinea pigs, appearing as an 80% lethal disease . At passage 7, a nucleotide substitution G10805U, resulting in an amino acid change Arg→Leu(154), was fixed in the VP24 gene sequence, and a heterogeneous site ) appeared in the L gene sequence. Emergence of these mutations correlated with a further increase in EBOV pathogenicity for guinea pigs, which manifested as 100% lethality (between days 8 and 12). Thus, only the three amino acid changes in VP24 protein , one change in NP protein (position 566), and one change in L-polymerase (position 236) may have an essential contribution to the gradually emerging virulence of EBOV in the course of GPA-P7 series , and Asn.In the latter study , recombiOf interest, a similar major contribution of VP24 mutations in the EBOV adaptation to mice has been observed, with the requirement for the supportive mutations in NP . The rolA summary of all genetic determinants revealed in the guinea pig adaptation series of EBOV is provided in Ebola virus infection in primates is associated with diffuse parenchymal necrosis in the liver without marked inflammatory changes . Unlike In our study, we were able to observe the dynamics of pathomorphologic changes in wild-type EBOV infection in guinea pigs , which wThe gradual emergence of EBOV virulence to guinea pigs in a series of passages is reflected in the changes of pathomorphological pattern of the infection. At early passages (1–3), only quantitative differences in the numbers of activated and infected liver macrophages were observed. The pathomorphological pattern of infection changes at passage 4, with greater numbers of liver macrophages involved, as well as the virus reproduction first detected in hepatocytes. However, only the formation of nucleocapsids, not the proper virus budding, occurs in hepatocytes at this stage, making it unclear whether the infection in these cells is productive. In general, the liver lesions are more severe than at earlier passages, and the signs of spleen and kidney involvement become evident. All observed morphological changes gradually become more prominent at passages 5-6, with liver focal lesions becoming widespread and number of infected cells and virions increasing. With the acquisition of full lethality of the adapted virus to guinea pigs (passage 8), the hepatic tissue becomes filled with infected hepatocytes and macrophages with intracellular nucleocapsids, as well as numerous virions. Some cells contain structurally abnormal viroplasm and nucleocapsids. Areas of liver necrosis containing lysed hepatocytes, macrophages, and virions become all-pervading. Other organs and tissues show greater severity of lesions, as well .In GPA-EBOV-infected guinea pigs, we, similarly, observed the necrotic hepatocytes  and viruDay-by-day dynamics of pathomorphologic changes occurring in the GPA-infected guinea pigs illustrated by Connolly and colleagues  is in a  in vitro by the recombinant virus obtained by reverse genetics and carrying the adaptive mutations in VP24 identical to those occurring in the 8mc adaptation series show characteristic inclusion bodies containing viral nucleocapsids. Contrarily, no typical viral nucleocapsids but only massive protein inclusions were found in the cells infected by the wild-type virus, which correlated with the lack of infectious virus release from those cells. This data suggested an efficient block of wild-type EBOV infection already in the primary target cells, probably, at the stage of VP24-mediated nucleocapsid assembly/budding [An independent morphological study of the wild-type and guinea pig-adapted EBOV infection reveals an inability of the nonadapted virus to reproduce in its primary target, the macrophages . Periton/budding .Thus, the morphologic patterns of wild-type and adapted EBOV infection in guinea pigs are suggestive of the inability of the virus to establish productive infection in macrophages and, particularly, in hepatocytes. Failure to massively produce infectious wild-type EBO virions may result in the host defense successfully blocking further virus spread by locking the infection in granuloma-like inflammation foci with a subsequent virus clearance. The adaptive mutations in the EBOV genome can restore the ability of a virus to reproduce in its major targets, macrophages and hepatocytes, thus, facilitating systemic virus spread in the infected animals that results in high virulence. On the other hand, this mechanism of adaptation would present itself as the increase of blood and liver virus titers in the adapted virus-infected animals, as compared to those inoculated with the wild-type virus, which is not the case for two adaptation series, K-5 and GPA-P7. There should be, therefore, an alternative mechanism of virulence increase during adaptation, which is relevant to those series. A morphological study of K-5 and GPA-P7 virus infections, which has not been done as yet, and its comparison with the morphological patterns of infection described earlier would, thus, be of interest. in vivo despite the progression of lymphoid necrosis, and they are resistant to infections by different EBOV strains in vitro [Wild-type EBOV infection causes serious hematologic aberrations in its natural hosts including leukocytosis due to marked neutrophilia and, frequently, lymphopenia in terminally ill primates. Thrombocytopenia develops in acutely ill human patients and experimentally infected primates experiencing hemorrhagic syndrome. Unlike that, both neutrophilia and lymphopenia can be observed in EBOV-infected guinea pigs as early as day 2 p/i and are progressing over the course of the disease. According to some reports, the scale of those hematological shifts in individual infected animals can be as great as a >3.5-fold increase in the number of neutrophils and >3.0-fold drop in the number of lymphocytes. This finding appears intriguing, since lymphocytes are not infected by EBOVin vitro . The bysin vitro .It appears, though, that the leukocyte counts should be taken with care, since a wide-range variation of different leukocyte types between the animals and their experimental groups is natural to guinea pigs . TherefIn one of our adaptation series, GLA, we specifically aimed to study the evolution of different blood indices in the course of adaptation . In thisThe percentage of young thrombocytes demonstrated a 3-fold increase at passage 2 and an overall 12-fold increase by passage 6. The total thrombocyte count remained stable until it fell abruptly (2-fold) at passage 6. The occurrence of young thrombocytes at early passages, before the fully lethal EBOV has been selected, indicates the important shifts in the thrombocytic hemostasis, which resulted in a progressive loss of platelets observed in the next passages.An interesting finding was the change of erythrocyte morphology in the EBOV adaptation. The percentage of normal red blood cells was progressively decreasing at every passage concomitant with the occurrence of echinocytes and erythrocytes with inclusions, which suggests the exit of immature erythrocytes from the bone marrow, apparently, to compensate for the deficiency of red blood cells in the organism. The occurrence of echinocytes in the peripheral blood has been noticed in the acute viral hepatitis and some other viral and inflammatory diseases ; howeverThe shifts of the peripheral blood counts were accompanied by the changes in bone marrow composition, firstly, by the increase in the number of blast cells as early as passage 2, reaching its peak of 65 pro mille by passage 5 but further dropping. This may indicate compensatory activation of bone marrow function with subsequent exhaustion. Megakaryocyte counts started growing at passage 2 and reached their peak at passage 5, clearly reflecting the upward dynamics of young thrombocytes.Finally, the phagocytic function of neutrophils was progressively decreasing from passage 2, although the number of neutrophils was variable at different passages. Contrarily, the phagocytic function of peritoneal macrophages was remaining stable for 6 passages and then dropping abruptly , 23.Although many of the hematologic indices were fluctuating in the passage series or changing at later passages , some were evidently concomitant with the increasing pathogenicity or even appearing prior to the acquiring of full lethality by the adapted virus. Those include the increase of the percentage of young thrombocytes along with the growing number of blast cells and megakaryocytes and the decrease of phagocytic activity of neutrophils, all commencing as early as passage 2, alongside the significant growth of blood and tissue virus titer and the occurrence of the first lethal cases in the inoculated animals. It is, thus, tempting to hypothesize that the evolving wild-type EBOV acquires new characteristics triggering those early responses in hematologic parameters . Those kα production by peripheral blood macrophages infected in vitro by another filovirus, Marburg virus, were proposed to be the major pathogenetic factor of Marburg virus infection due to its ability to induce hemorrhagic shock [A rather intriguing immunologic finding was the lack of correlation between the blood and peritoneal TNF levels and the pathogenicity of EBOV infection in experimental animals. The guinea pigs infected with wild-type Mayinga or lethal adapted 8mc EBOV demonstrated similar, rather low, levels of TNF activity in blood and no TNF activity in peritoneal exudates . Contraric shock . Thus, oSince coagulopathies, such as disseminated vascular coagulation (DIC) or massive hemorrhages, are the hallmark of EBOV disease in primates  and consThe dynamics of complement activity in wild-type EBOV-infected guinea pigs and in the intact or EBOV-preimmunized animals infected with a lethal adapted EBOV strain demonstrates striking differences, which can be important for the understanding of EBOV-induced coagulopathies. In wild-type EBOV-infected animals, the complement activity shows a double-peak profile with a 2-day lag, the first, higher peak at days 4–7 and second, lower peak at days 9-10. Contrarily, in animals infected with the adapted lethal 8mc strain, the complement activity demonstrates a rapid peak at 24 h p/i followed by an abrupt decline on day 2, return to the baseline by days 4-5, and further drop to the background levels by days 9–11, followed by the animal death. Finally, the guinea pigs preimmunized with formalin-inactivated EBOV and infected by a lethal 8mc strain showed even a more steep rise of complement activity on day 1 and a more abrupt fall on day 2 p/i with a further decline to near-zero values by day 6, also followed by the animal death by day 8 .The rate of complement activity growth in the lethally infected guinea pigs suggests the involvement of an alternative pathway of complement activation. In preimmunized animals, the classic activation can additionally occur, due to the presence of anti-Ebola immunoglobulins, which may be the possible cause of higher complement activities than in nonimmunized animals. Alternatively, slower kinetics of complement activation in guinea pigs infected with nonlethal strain indicates classic activation with the two peaks corresponding to IgM (before day 7) and IgG production (days 7–14). Rapid complement activation by alternative pathway appears to be a unique ability of the 8mc lethal EBOV strain to immediately hyperactivate complement production with subsequent fast and complete exhaustion of its synthesis . ImportaInterestingly, the pattern of serum concentrations of circulating immune complexes (CICs) is closely resembling the kinetics of complement activity both in intact and in preimmunized lethally infected animals, with a typical rapid rise in the first day and an immediate decline to near-zero values by days 8-9 or 5-6 in intact or preimmunized animals, respectively, followed by the animal death. Slow CIC and complement kinetics are also very similar in the nonlethal EBOV infections of guinea pigs. The significance of circulating immune complexes (CICs) in the pathogenesis of Ebola disease still remains to be studied .Since no coagulation abnormalities are occurring in the mouse-adapted EBOV model, which is a general feature of mouse models for acute viral infections , the guiSince the first isolation of wild-type EBOV strains, the problems with the animal model for the study of biology and therapy/prevention of this infection became evident. Resistance of the most convenient rodent model animals  to EBOV infection prompted the researchers to use adaptation of wild-type EBOV strains to the small animal models. Practically, the EBOV adaptation to guinea pigs was the easier and the first to be achieved. The lethality of the guinea pig-adapted EBOV variant to the nonhuman primates remained unchanged . In the We and others were able to demonstrate the mutations in EBOV VP24 protein to be the most important determinants of the virus virulence and lethality , 16, 18.The need to reproduce the consistency of processes occurring in the course of adaptation and establish the details of adaptation process required the repeated adaptation series starting from the virus stocks with different passage histories including the cloned EBOV stocks.The differences in the dynamics of hemolytic complement activity found in the infections of guinea pigs by the wild-type and adapted EBOV strains allowed us to be able to predict the outcome of EBOV infection in the first hours p/i .Also, our studies indicate strong suppression of neutrophilic phagocytosis in the infections of guinea pigs with GPA-EBOV strain, while only its mild activation on day 5 of wild-type EBOV infection was observed. This suggested that the reproduction of EBOV in macrophages, on the one side, with concomitant suppression of neutrophils, on the other, results in the disturbance of neutrophil/macrophage interaction as a driving force of a lethal EBOV disease.Another interesting discovery was the presence of young thrombocytes in GPA-EBOV-infected animals. The phenomenon, which was lacking in the wild-type EBOV-infected animals, together with the increase of megakaryocyte counts in the bone marrow, can be the evidence of the activation of megakaryocytic hematopoiesis and the exit of nonmature platelets into the circulation.α levels in the animals infected with a nonlethal wild-type than in those infected with lethal adapted EBOV, thus, demonstrating lack of TNF involvement in GPA-EBOV pathogenesis.Previously, the elevated levels of proinflammatory cytokines have been reported to occur in the course of EBOV disease and contribute to the infection lethality in humans and NHPs, due to their role in the development of EBOV coagulopathies. However, in guinea pigs we, paradoxically, found higher (while comparable) TNF-


The H3N8 virus and the H3N2 virus are the main subtypes of canine influenza virus (CIV). H3N8 CIV mainly circulates in America, and H3N2 CIV mainly circulates in Asia. However, there was an outbreak of the Asian H3N2 virus in the United States (US) in 2015. Thus, it is important to evaluate the presence of subtype H3N8 virus in dogs in China. From May 2015 to November 2015, 600 sera from pet dogs were collected from Guangzhou, Shanghai, Beijing and Shenzhen for hemagglutination inhibition (HI) assays and microneutralization (MN) assays. Fifty-two (8.66%) of the 600 sera were positive for the subtype H3N2 virus, which matched the previous reports. Five (0.83%) of 600 sera were positive for the subtype H3N8 virus (H3N8 EIV or H3N8 AIV or H3N8 CIV), which is the first report of subtype H3N8 virus infection among dogs in China and remind us to play more attention to this subtype virus. Therefore, further serological and virological surveillance of influenza virus infection among dogs in China is imperative. Under most circumstances, there are species barriers that hamper interspecies transmission of influenza viruses. However, evolution can help viruses surmount species barriers to sustain transmission in a new host species . RecentlThe research history of CIV is relatively short because dogs were long regarded as unsusceptible to influenza viruses. This perception did not change until H3N8 CIV was first identified in the US from what was known as an equine-origin H3N8 influenza virus in January 2004 , and thiFrom May 2015 to November 2015, sera from 600 pet dogs (150 specimens per city) were collected for serology from animal hospitals in Guangzhou, Shanghai, Beijing and Shenzhen and were preserved at -80°C for future testing. The dogs’ characteristics were recorded by the research staff. The samples were tested for EIV-H3N8: A/equine/Heilongjiang/SS1/2013 (H3N8); AIV-H3N8: A/avian/Guangdong/J/2012 (H3N8); CIV-H3N2: A/canine/Guangdong/01/2014(H3N2). Negative control serum was collected from an influenza-negative dog whose serum did not contain antibody against H3N2, H3N8, H9N2, or H1N1 as indicated by HI tests. Positive control sera were prepared from immune rabbits using inactivated viruses. These viruses and control sera were obtained from the Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, the College of Veterinary Medicine, South China Agricultural University.Denka Seiken 340016 (370013)) and absorbed with erythrocytes to remove nonspecific inhibitors before the tests. The sera were further diluted to a 1:10 dilution. The samples were two-fold serially diluted in 96-well V bottom microtiter plates, and 4 hemagglutination units (HAU) of the virus were added to each well. The sera and virus mixtures were incubated at room temperature for 30 min. Then, 1% red blood was added to all wells. The plates were incubated at room temperature and read after 30 min. The serum titer was expressed as the reciprocal of the highest dilution of serum at which hemagglutination was inhibited. All assays were conducted twice with triplicate wells each time, and the final titer was only accepted when both replicates yielded matching results.We used a WHO-recommended HI assay . Briefly50/50 μl were mixed with the diluted sera. After incubation for an hour, 1.5×104 MDCK cells were added to each well. After incubation for 18–22 hours, the monolayers of MDCK cells were washed with PBS and fixed in cold 80% acetone for 10 minutes. Finally, the viral nucleoprotein (NP) was detected by enzyme-linked immunosorbent assay . An HI titer ≥ 20 and an MN titer ≥ 80 is considered positive evidence of previous H3N8 virus infection. Additionally, the specimens with an HI titer ≥ 20 against H3N8 virus were further evaluated for human H3N2 influenza virus (HuIV) antibody using an enzyme-linked immunosorbent assay  according to the manufacturer’s instructions.Sera from dogs with an HI titer ≥ 20 were confirmed with MN recommended by the WHO . BrieflyAfter the outbreak of H3N2 CIV in Chicago, US, in April 2015, we sought to evaluate whether subtype H3N8 virus infection had occurred among dogs in China. Thus, from May 2015 to November 2015, we collected sera from 600 pet dogs in Guangzhou, Shanghai, Beijing, and Shenzhen. In total, using a cutoff of HI ≥1:20, 52 (8.66%) of the 600 sera were positive for canine H3N2 virus, 8 (1.33%) of the 600 sera were positive for equine H3N8 virus, and 4 (0.67%) of the 600 sera were positive for avian H3N8 virus . FurtherSince CIV was first identified in the US, the H3N8 CIV has mainly been circulating in America, and H3N2 CIV has mainly been circulating in Asia , 6. HoweH3N8 EIV was first reported to surmount species barriers to cause sustained transmission among dogs in the US in 2004 , 20. In Here, we first reported the seroepidemiological evidence of subtype H3N8 virus infection among dogs in China. Further and continuous surveillance needs to be performed for this subtype virus among dogs in China. Currently, dogs are infected with different subtypes of influenza viruses, such as H3N8 , H1N1 2, H3N1 1, H9N2 2, H5N2 2 and H5N1In this study, there are two potential limitations should be kept in mind. First, although no cross-reactivity between subtype H3N8 virus and H3N2 CIV or H3N2 HuIV, the strong cross-reactivity between H3N8 EIV and H3N8 AIV can not be eliminated. Nonetheless, the subtype H3N8 virus infects in dogs is been verified in this study. Second, no H3N8 CIV that is known as an equine-origin was used in this study to testify the results, but the origin H3N8 EIV was used as a substitute.This study protocol was reviewed and approved by the Institutional Review Board of South China Agricultural University.