A high-passage rabbit kidney RK-13 cell line  used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b. Pestivirus in the family Flaviviridae, which also includes border disease virus and classical swine fever virus  and Geneious 7.0.6 software .Bovine viral diarrhea virus (BVDV) is a nonenveloped, positive-sense, single-stranded RNA virus with a genome size of approximately 12.3 to 12.5 kb. The virus is a member of the genus r virus 36. In addr virus 3–10. It w virus 3––15. Manyvirus 3––. In thisvirus 3––. The seq- strain [GenBank accession no. JX419397.1]; 12,064 nt, 99.7% identity) (The complete genome of ncpBVDV contaminating the HP-RK-13 [KY] cell line (ncpBVDV HP-RK-13 [KY] strain) is composed of 12,271 nucleotides (nt) and contains a 5′ untranslated region (UTR) (386 nt), a single open reading frame (ORF) , and a 3′ UTR 188 nt). The single ORF encodes a 3,898-amino acid polyprotein, which is predicted to be cleaved into 12 proteins. The ncpBVDV HP-RK13 [KY] strain had 85.2% to 99.7% identity with 11 strains of BVDV-1b and 68.6% to 70.9% identity with eight strains of BVDV-2 at the whole-genome level. Interestingly, the ncpBVDV HP-RK-13 [KY] strain is very closely related to the previously described ncpBVDV present in RK-13 cells reported from Japan (RK13/E8 nt. TheKT355592.The complete genomic sequence of ncpBVDV KY-HP-RK-13 strain has been submitted to GenBank under accession no.


Cotton leaf curl Kokhran virus (CLCuKoV-Bur), and a recombinant form of CLCuMB have consistently been identified in cotton across the major cotton growing areas of Pakistan. Unusually a bipartite isolate of the begomovirus Tomato leaf curl virus was identified in CLCuD-affected cotton recently. In the study described here we isolated the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) from CLCuD-affected cotton. To assess the frequency and geographic occurrence of ToLCNDV in cotton, CLCuD-symptomatic cotton plants were collected from across the Punjab and Sindh provinces between 2013 and 2015. Analysis of the plants by diagnostic PCR showed the presence of CLCuKoV-Bur in all 31 plants examined and ToLCNDV in 20 of the samples. Additionally, a quantitative real-time PCR analysis of the levels of the two viruses in co-infected plants suggests that coinfection of ToLCNDV with the CLCuKoV-Bur/CLCuMB complex leads to an increase in the levels of CLCuMB, which encodes the major pathogenicity (symptom) determinant of the complex. The significance of these results are discussed.Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production on the Indian subcontinent, and is caused by monopartite begomoviruses accompanied by a specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Since the breakdown of resistance against CLCuD in 2001/2002, only one virus, the “Burewala” strain of  Cotton is an important commodity and the export of cotton products is crucial for the economies of India and, especially, Pakistan. The cultivation of cotton across Pakistan and northwestern India is severely affected by cotton leaf curl disease (CLCuD) , 2. The Begomovirus are whitefly (Bemisia tabaci)-transmitted single-stranded (ss)DNA viruses that belong to family Geminiviridae. Begomoviruses occur in all the warmer parts of the World and infect only dicotyledonous plants [ori) for virion-sense viral DNA replication. The sequence specific interaction between Rep and cognate iterons also ensures that the Rep of one virus will not initiate replication of the genome of a second virus. The DNA-A and DNA-B components of bipartite begomoviruses share a sequence, known as the common region (CR) that usually spans most of the intergenic region. The CR acts to maintain the integrity of the split genome ensuring that the DNA-A-encoded Rep can initiate replication of the virion strands for both components [Viruses of the genus s plants . In the s plants , 6. The s plants . DNA-B cmponents .The majority of monopartite begomoviruses are associated with additional small ssDNA molecules known as betasatellites and alphasatellites . BetasatMastrevirus, family Geminiviridae) [The alphasatellites  are notviridae) .Cotton leaf curl Multan virus (CLCuMuV), Cotton leaf curl Alabad virus (CLCuAV), Cotton leaf curl Kokhran virus (CLCuKoV) and Papaya leaf curl virus (PaLCuV) [Cotton leaf curl Burewala virus). CLCuKoV-Bu is a recombinant virus with some sequences derived from CLCuMuV [Bur) with some sequence derived from another betasatellite [CLCuD in Pakistan and northwestern India during the 1990s was shown to be associated with at least four monopartite begomoviruses including (PaLCuV) , 21. Of (PaLCuV) , 21. Aft CLCuMuV . Unusual CLCuMuV , 23 and atellite .Tomato leaf curl New Delhi virus (ToLCNDV) is an unusual begomovirus. It is one of very few bipartite begomoviruses in the OW and has been reported from a large number of different plants, including members of the Solanaceae, Cucurbitaceae and Malvaceae [Pepper leaf curl Bangladesh virus[Tomato leaf curl Palampur virus[Bhendi yellow vein mosaic virus [Tomato leaf curl virus, a begomovirus for which some isolates are monopartite [alvaceae –28. The esh virus, Tomato pur virus, Bhendi opartite .Recently we have identified an isolate of ToLCV with a DNA-B in CLCuD-affected cotton plants originating from the Punjab; the first time a bipartite begomovirus was identified in cotton on the Indian subcontinent . The stuLeaf samples from cotton plants with symptoms typical of CLCuD, consisting of leaf curling, vein thickening, vein yellowing, enations and stunted growth , and froThe National Institute for Biotechnology and Genetic Engineering (NIBGE) is a public sector institute, the employees of which areauthorized to visit farmer’s fields and collect plant samples. However, before going to any private field, verbal permission was sought from the owner of the field. The field studies did not involve endangered or protected species.GATATCATCATTTCAACGCCCGCATCGAA)/ToLCNDV_A2_Reverse (GATATCTGCTGGTCGCTTCGCCATAGTTC), ToLCNDV_A3_Forward (GAGCTCGTGCAGTTGTCCCCATTGCCCGCGTCAC)/ToLCNDV_A4_Reverse(GAGCTCCATAGGGGCTGTCGAAGTTG) for DNA-A and ToLCNDV_B1_Forward (AAGCTTCTGCTCGAACATGGACGGAAATGAC)/ToLCNDV_B2_Reverse (AAGCTTAGCCAGTTGAGGAATAGATGCATG), ToLCNDV_B3_Forward (GGTACCCGTAACGATCTTGAACTATGTCCC)/ToLCNDV_B4_Reverse (GGTACCCTATCTGGCTATAGGTCCGAACG) for DNA-B. The degenerate primers BegomoF and BegomoR were used to PCR amplify the genomes of monopartite and/or DNA-A components of bipartite begomoviruses [Rolling circle amplification (RCA) using phi 29 DNA polymerase  was used to amplify all circular DNA molecules in 31cotton samples . The DNAoviruses . Betasatoviruses , 37. Theoviruses  were usePCR reactions for cloning used RCA product as the template. Amplification products of ~2.8 kb, for virus, DNA-A or DNA-B, and ~1.4 kb for alphasatellite and betasatellite, were cloned into a T/A cloning vector . From each isolate 3–5 clones were sequenced. Sequences were determined by dideoxy nucleotide chain termination sequencing on an Applied Biosystems 3730XL DNA sequencer and were assembled and manipulated using the Lasergene package of sequence analysis software . The MUSCLE option of the Sequence Demarcation Tool  was usedReal-time PCR reactions consisted of a total volume of 25 μL with 12.5 μL of SYBER Green Super Mix , 0.25 μL of each primer (0.1 μM each), 2.5 μL of DNA (25 ng), and 9.5 μL water. The cycling conditions were an initial 94°C for 10 min, followed by 40 cycles of 30 seconds (s) at 94°C, 30 s at 57°C, 30 s at 72°C, followed by melt curve analyses. Reactions were performed in a 96 well microtitre plate format using an iQ5 thermal cycler . The 18S ribosomal RNA gene was used as a reference gene to normalize DNA levels in samples. Each sample was run in triplicate.CCTTTAATCATGACTGGCTT)/DNA_A_qPCR_Reverse (CATTTCCATCCGAACATTC) for begomovirus genome or DNA-A component, DNA_B_qPCR_Forward (GCCCATGATTCGTTCGGAC)/DNA_B_qPCR_Reverse (CACGTGGTACTGGAATATCGCA) for DNA-B and Betasatellite_qPCR_Forward (GATTTGACTTATATTGGGCCAATTTAAT)/Betasatellite_qPCR_Reverse (GATACTATCCACAAAGTCACCATCGCTAAT) for betasatellites.Standard curves for absolute quantification were obtained from five sets of tenfold serial dilutions (starting from 20 ng/μL) of a plasmid containing the cloned full-length CLCuMB  ToLCNDVClones of ~ 2.8 kb and ~1.4kb were obtained from 10 cotton plants with CLCuD symptoms Tables  and 4. TA closer analysis indicated that 6 of these clones have a truncated TrAP gene, with a potential coding capacity of 35 amino acids, whereas one clone (SAZ34) encoded a putatively full-length TrAP predicted to be of 136 amino acids . MutatioAnalysis of the sequence of clone SAZ34 showed it to have 94–96% sequence identity with the sequences of ToLCNDV component DNA-A available in the databases. A phylogenetic analysis showed the sequence to have low branch lengths to the DNA-A components of previously characterized ToLCNDV isolates , panel EThe remaining seven ~2.8kb clones had an arrangement of genes typical of the DNA-B component of bipartite begomoviruses, consisting of one gene encoded in each orientation . These sA total of 23 ~1.4kb clones were obtained from eight CLCuD-affected cotton plants. Analysis of the sequences showed them to encode either one large (~950bp) gene in the virion-sense, typical of alphasatellites, or a small (~350bp) gene in the complementary-sense, typical of betasatellites . The 9 pBur) rather than the “Multan” or “Shadadpur” strains  sequences available in the databases. In a phylogenetic analysis the new sequences group with short branch lengths with the sequences of earlier reported CLCuMB isolates , panel B strains , panel Gbreaking .The incidence and area over which coinfection of cotton with ToLCNDV and the CLCuKoV-Bur/CLCuMB complex was investigated by diagnostic PCR with primers specific for ToLCNDVDNA-B and CLCuKoV-Bur on DNA samples extracted from CLCuD symptomatic cotton originating from across the Punjab and northern Sindh provinces of Pakistan .. Of theThe results of a quantitative PCR analysis of the levels of virus (the primers used amplify both ToLCNDVDNA-A and CLCuKoV-Bur), ToLCNDVDNA-B and CLCuMB in CLCuD symptomatic cotton plants infected with only the CLCuKoV-Bur/CLCuMB complex or co-infected with CLCuKoV-Bur/CLCuMB and ToLCNDV are shown in Bur. Recently CLCuKoV-Bur and CLCuMBMul have been shown experimentally to be able to cause CLCuD in cotton [Begomovirus disease complexes are evolving rapidly by recombination, component capture and mutation to expand their host range and overcome sources of resistance. The resistance breaking begomovirus-betasatellite complex causing CLCuD evolved by recombination and mutation, 23, 45.n cotton . HoweverIn many of the cotton plants examined here identified the alphasatellite CLCuMA was identified. This indicates that, as was the case before resistance breaking, the virus causing CLCuD is associated with an alphasatellite. The study of Amrao et al.  which fiChickpea chlorotic dwarf virus [Okra enation leaf curl virus [The most surprising finding of the study presented here was the presence of the bipartite begomovirus ToLCNDV in cotton affected by CLCuD. A number of other geminiviruses have been identified in cotton including the mastrevirus rf virus , ToLCV [rf virus  and Okrarl virus . HoweverBur complex and ToLCNDV which leads to an increase in the amount of CLCuMBBur present in coinfected plants. Betasatellites encode a dominant symptom determinant [The quantitative PCR analysis suggests that in cotton there is a synergistic interaction between CLCuKoV-Bur/CLCuMBerminant , 51 and erminant . Any incBur complex and ToLCNDV is unclear. The DNA-A component of ToLCNDV, in the absence of the DNA-B, has been shown to be able to support the replication of CLCuMB in cotton and, at least transiently, induce typical CLCuD symptoms [Nicotiana benthamiana showed the presence of CLCuMB to enhance the viral DNA levels but the presence of DNA-B depressed CLCuMB levels [The nature of a possible synergistic interaction between the CLCuKoV-Bur/CLCuMBsymptoms . A studyB levels . NeverthB levels .CLCuD is a major constraint to cotton production in Pakistan and India. At this time there are no commercially available cotton varieties with resistance to the disease. The appearance of a form of the virus-complex causing the disease with potentially enhanced pathogenicity is thus not good news. Further studies will be needed to monitor the situation and see whether the coinfection persists and precisely what the effects are on the yield of cotton. Additionally, any efforts towards developing resistance to the disease, either by conventional or non-conventional means, would be wise to take into account the possibility of a more complex situation becoming important in cotton in the future.


Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)2Cys6 protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active Gαi protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by Gαi. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed.Proteins containing Zn(II) Wherease whiter . This efe whiter . In addie whiter . Therefoenotypes . This copcz1 plays a role in the vegetative growth of P. roqueforti, positively regulating this process.Transformants M9 and M11 showed a reduced rate of growth in all the media tested . For exapcz1 in P. roqueforti resulted in a drastic reduction in conidia formation  involves a network of regulators, which lead to a central conidiation pathway, composed of the genes brlA, abaA and wetA encoding transcription factors that control sporulation [pcz1 silencing, we quantified the expression of these genes by qRT-PCR , abaA transcripts , and wetA transcripts , suggesting that Pcz1 regulates positively the expression of conidiation-specific genes.The current model of genetic control of conidiation , transformants M9 and M11 showed enhanced conidial germination compared with the wild-type strain. Approximately 12.7% of conidia from transformants were able to germinate after 48 h in minimal Czapek medium lacking a carbon source, whereas at the same time, 3.8% of conidia from the P. roqueforti wild type strain had germinated  or other industrial filamentous fungi , the functional characterization of genes from P. roqueforti using genetics strategies is still in its infancy. Thus, this work is one of the few examples of functional characterization of a gene in this fungus.pcz1 strongly represses conidiation , which are in turn negatively regulated by the repressor protein SfgA [A. nidulans, overexpression of sfgA resulted in reduced levels of mRNA of brlA, suggesting that SfgA inhibits the expression of this gene [brlA expression. In addition to sfgA, three other C6 genes (oefC from A. nidulans and MoCOD1 and MoCOD2 from Magnaporthe oryzae) are involved in the control of conidiation [Upstream of the conidiation central pathway, there are a group of activators of BrlA  that stimulates the intrinsic GTPase activity of a Gαi subunit. Thus, SfgA helps in the promotion of vegetative growth through Gαi signaling in Aspergillus [pcz1 represses vegetative growth in P. roqueforti  in P. roqueforti and other fungi, such as P. chrysogenum, Fusarium sporotrichioides and F. fujikuroi [Aspergillus, Gαi signaling has a negative effect on the apical growth rate. Because the Gαi subunit downregulates pcz1 in P. roqueforti  through the cAMP-independent pathway [pcz1 resulted in 80–94% less conidiation than in the parental strain  or subtracted cDNA from P. roqueforti pga7 (B). Those clones harboring putative differentially expressed cDNAs in the wild-type strain should hybridize only to A. Numbers in red are nomenclature of the clones. Clone 321 was differentially expressed in the wild-type strain (highlighted in the red box) and contains pcz1 cDNA. C+: Positive control for hybridization.The dot blot assay was carried out as described in Klagges et al. . Briefly(TIF)Click here for additional data file.S2 Fig2Cys6 DNA binding domain is shown. Asterisks indicate fully conserved residues. The six key conserved cysteines are shown in blue. Sequence belonging to Pcz1 is bolded and underlined. At the left, the name of each organism and the Genbank accession number for each sequence is indicated. The names of some fungi are abbreviated: M. anisopliae: Metarhizium anisopliae; S. chlorohalonata: Stachybotrys chlorohalonata; P. destructans: Pseudogymnoascus destructans; S. sclerotiorum: Sclerotinia sclerotiorum; M. thermophila: Myceliophthora thermophila; L. maculans: Leptosphaeria maculans; C. apollinis: Coniosporium apollinis; B. compniacensis: Baudoinia compniacensis; C. psammophila: Cladophialophora psammophila; T. stipitatus: Talaromyces stipitatus; P. brasiliensis: Paracoccidioides brasiliensis; C. posadasii: Coccidioides posadasii; T. interdigitale: Trichophyton interdigitale; T. verrucosum: Trichophyton verrucosum; C. yegresii: Cladophialophora yegresii; C. carrionii: Cladophialophora carrionii; P. fijiensis: Pseudocercospora fijiensis.Alignment was performed with Clustal Omega using default parameters. Full sequences were aligned, but only the region spanning the Zn(II)(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Artificial insemination is widely used in many cattle breeding programs. Semen samples of breeding bulls are collected and closely examined immediately after collection at artificial insemination centers. Only ejaculates without anomalous findings are retained for artificial insemination. Although morphological aberrations of the spermatozoa are a frequent reason for discarding ejaculates, the genetic determinants underlying poor semen quality are scarcely understood.ARMC3) that was compatible with the supposed recessive mode of inheritance. The deletion is expected to alter the reading frame and to induce premature translation termination (p.A451fs26). The mutated protein is shortened by 401 amino acids (46 %) and lacks domains that are likely essential for normal protein function.A tail stump sperm defect was observed in three bulls of the Swedish Red cattle breed. The spermatozoa of affected bulls were immotile because of severely disorganized tails indicating disturbed spermatogenesis. We genotyped three affected bulls and 18 unaffected male half-sibs at 46,035 SNPs and performed homozygosity mapping to map the fertility disorder to an 8.42 Mb interval on bovine chromosome 13. The analysis of whole-genome re-sequencing data of an affected bull and 300 unaffected animals from eleven cattle breeds other than Swedish Red revealed a 1 bp deletion  in the eleventh exon of the armadillo repeat containing 3-encoding gene  contains supplementary material, which is available to authorized users. Artificial insemination (AI) is widely used instead of natural mating in many cattle breeding populations. Ejaculates of breeding bulls are collected once or twice a week and closely examined immediately after semen collection at highly specialized AI centers. Only ejaculates without apparent abnormalities are retained for AI. Up to 20 % of all collected ejaculates are rejected because they do not comply with current standards for AI . Diagnosin vivo. Morphological aberrations of the sperm tail compromise sperm motility and impair fertilization. Such aberrations are collectively referred to as multiple morphological abnormalities of the flagella . Diagno, humans –5, pigs , humans , 7 and m, humans –10. HoweBulls with MMAF have been observed in Holstein-Friesian, Ayrshire and Indobrasil cattle –15. The ARMC3 that most likely causes the sperm tail disorder in Swedish Red cattle.Here we present the phenotypic manifestation and the genetic analysis of a recessively inherited tail stump sperm defect in the Swedish Red cattle breed. The application of homozygosity mapping facilitated the mapping of the fertility disorder to a short segment on bovine chromosome 13. The analysis of comprehensive whole-genome sequence data revealed a frameshift mutation in Three young bulls 11 months) of the Swedish Red cattle breed born in 2008, 2009 and 2012, were reported from an AI center because they produced ejaculates with immotile spermatozoa during a semen collection period of 5 months. Examination of the bulls’ fresh ejaculates revealed a reduced sperm concentration (~140 million spermatozoa per ml) despite normal ejaculate volume (~4 ml). The sperm count was only 10–20 % of the average sperm count of control bulls. All spermatozoa were immotile because of multiple flagellar abnormalities such as rudimentary , short length and absent tails. A proximal droplet surrounded most rudimentary tails Fig. . The pro1 months The analysis of the pedigree records of three affected bulls revealed a common ancestor born in 1987) in their paternal and maternal path  in three affected bulls. Only two genomic regions were consistently homozygous in all affected animals: a 1.13 Mb segment on BTA22  and an 8.42 Mb segment on BTA13  Fig. . The segTo pinpoint the mutation causing the tail stump sperm defect, the whole genome of an affected bull was sequenced to an average read depth of 9.29. In addition, we exploited data of 300 previously sequenced animals from eleven cattle breeds other than Swedish Red for the identification of the mutation. Deleterious recessive mutations are assumed to have occurred after breed formation and are thus likely to be breed-specific. Thus we assumed that the causal mutation should not segregate among the sequenced control animals. Multi-sample variant calling in the 8.42 Mb region of extended homozygosity on BTA13 yielded genotypes at 81,925 single nucleotide and short insertion and deletion polymorphisms . In addition, 11,505 structural variants were detected in the genome-wide sequence data of the affected bull and 226 control animals with genome coverage of at least eight-fold.ARMC3, Chr13: 24,301,425 bp, ss1815612719, Fig. Seventy-seven variants were compatible with recessive inheritance that is homozygous for the reference allele in 300 control animals and homozygous for the alternate allele in the affected bull. Bioinformatic analysis revealed that 76 variants were located in non-coding regions of the genome and one variant resided in the coding region of the armadillo repeat containing 3-encoding gene . Sanger sequencing confirmed homozygosity for the deletion variant in two bulls with the tail stump sperm defect. The 1 bp deletion is expected to alter the reading frame and to change the amino acid sequence from position 451 onwards resulting in a premature translation termination at position 476 (p.A451fs26). The mutated protein should be shortened by 401 amino acids (46 %). Bioinformatic analysis revealed that the protein sequence of bovine ARMC3 contains ten armadillo/beta-catenin-like (ARM) repeats  that segregated with the tail stump sperm defect. Forty-one variants in non-protein-coding regions were also associated with the disorder. However, we consider the frameshift in ARMC3 as the most likely causal mutation because it is predicted to result in a protein that lacks 401 amino acids. The function of the truncated ARMC3 protein may be severely compromised, since it lacks domains that are likely required for normal protein function [The analysis of pedigree records indicated that the sperm tail disorder is inherited in an autosomal recessive fashion. Sequence variants underlying recessive traits are traditionally identified by comparing allele counts of dense molecular markers in affected and unaffected individuals . The lie.g., . The taie.g., . Three ae.g., –26. Compin vivo. Apart from immotile spermatozoa, the bulls were healthy. The morphological aberrations of the spermatozoa are similar to those observed in the Ayrshire cattle breed [ARMC3 occurred in a common ancestor of the two breeds and that it might also be associated with the sperm tail disorder in Ayrshire cattle. However, the genetic underpinnings of apparently similar phenotypes may be completely different across breeds , 31in viARMC3 with morphological abnormalities of the sperm flagellum. However, deleterious mutations in other genes encoding armadillo repeat-containing proteins have already been shown to compromise sperm motility [ARMC3 were immotile because of severe flagellar abnormalities. A previous study demonstrated that dysfunction of ARMC4, a paralog to ARMC3, impairs physiological function of the cilia and sperm flagella in humans [Gudu, a gene highly homologous to ARMC4, is essential for an undisturbed spermatogenesis in Drosophila melanogaster [ARMC3. Such findings suggest a crucial role of ARMC3 for physiological spermatogenesis.To our knowledge, our study reveals for the first time an association of a mutation in motility , 32. In n humans . Proper nogaster . Our invSPEF2 [in vivo both in natural service and AI. The phenotypic manifestations of the two defects differ only slightly. Spermatozoa of animals being homozygous for the ARMC3 frameshift mutation mostly lack the midpiece with mitochondria, which is, however, commonly present in spermatozoa of animals homozygous for the SPEF2 mutation [The morphological aberrations of the spermatozoa observed in our study are similar to those observed in Yorkshire boars with a loss of function mutation in SPEF2 . Both demutation .ARMC3 that most likely causes a sterilizing tail stump sperm defect in Swedish Red cattle. Our findings suggest that impaired function of ARMC3 compromises spermatogenesis and thereby results in severely disorganized sperm tails, which prevents successful fertilization in vivo. Compared to mutations that manifest in idiopathic male sub- or infertility [e.g., genotyping assays on customized genotyping arrays.The combination of high-density genotype and whole-genome re-sequencing data revealed a recessively inherited frameshift mutation in bovine ertility , spermati.e., AI center) of the bulls. None of the authors of the present study was involved in the decision to slaughter the bulls. Testicles of an affected bull were collected after slaughter. Consent from the owner of the bulls was obtained to use the semen and tissue samples for this study. No ethical approval was required for this study.All animals were housed at an approved commercial AI center in Örnsro, Sweden. Semen samples were collected by employees of the AI center as part of their regular breeding and reproductive measures in cattle industry. Bulls with the tail stump sperm defect were slaughtered because their semen was not suitable for artificial insemination. The decision to slaughter the bulls was made solely by the owner . A drop of semen  and from a wet mount formol-saline sample using a phase contrast microscope with 1000× magnification, respectively. Moreover, sperm head morphology was assessed in dry smears stained with carbol fuchsin according to Williams  and LageP > 0.0001)), 46,035 SNPs were retained for further analyses. Beagle genetic analysis software [Twenty-one bulls  of the Swedish Red cattle breed were genotyped using the Illumina BovineSNP50 Bead chip . The chromosomal position of the SNPs corresponded to the UMD3.1 assembly of the bovine genome . Mitochosoftware  was usedhomozyg-function implemented in the whole genome association analysis toolset PLINK [Segments of extended homozygosity were identified in three affected bulls using the et PLINK , 40. Duefastq-files were generated with the CASAVA bcl2fastq conversion software . The alignment of the reads to the University of Maryland reference sequence . The sample was sequenced on an Illumina HiSeq2500 system using TruSeq SBS v3 chemistry  and the 2x100 bp paired-end read module. The  was per Aligner . The resrd-tools .n = 12), Nordic Finncattle (n = 6), Fleckvieh (n = 153), Original Simmental (n = 15), Holstein-Friesian (n = 31), Brown Swiss (n = 50), Murnau-Werdenfelser (n = 2), Ayrshire (n = 2), Red-Holstein (n = 21), Original Braunvieh (n = 8)) other than Swedish Red [mpileup function of SAMtools [Pindel software package [Variant Effect Predictor tool [Single nucleotide and short insertion and deletion polymorphisms were genotyped in the affected bull together with 300 previously sequenced animals from eleven cattle breeds  and the lW2 tool  was usedrch Tool .http://www.ebi.ac.uk/ena) under accession number PRJEB12739.The data supporting the results of this article are included within the article and its additional files. Whole-genome sequencing data of a bull with the tail stump sperm defect were deposited in the European Nucleotide Archive (


Once again the world is forced to face an emerging disease threat; this time arising from a mosquito-borne agent that has spread by stealth around the globe.Aedes africanus mosquitoes,1 giving an early indication that Zika virus is essentially a sylvatic infection transmitted from one animal host to another. Humans were long regarded as being the only incidental hosts. Thus, from its discovery until 2007 Zika virus remained an obscure mosquito-borne virus thought to be restricted to a narrow equatorial belt across Africa and Asia. Only 14 or so sporadic laboratory-confirmed cases had been reported until Zika virus spread across the Indian Ocean to South East Asia and Polynesia, with a large epidemic being reported on Yap Island in the Federated States of Micronesia:2 nearly 75% of the population were infected. The first indications of the neurological involvement came from the data collected in Polynesia describing Guillain–Barré syndrome and other neurological complications.4 Of greater concern, however, is the upsurge in incidence of microencephaly among the new-borns in Brazil over the past year that public health officials believe is linked to Zika virus infection during pregnancy. Although the circumstantial evidence is at first sight convincing, rigorous case-control studies and wider epidemiological studies are needed to confirm this causal linkage.Zika virus was first discovered in 1947 in the Zika Forest of Uganda during an intensive search for yellow fever virus funded by the British Government and the Rockefeller Foundation. The virus was isolated from both a sentinel rhesus monkey and independently from a pool of 5 Where serological studies have been undertaken, it is clear that Zika virus infection has been substantially under-reported. Phylogenetic data from outbreaks in Africa, Asia and Polynesia strongly indicate Zika virus is evolving within human populations: three main genetic lineages have already been identified, one likely originating in Malaysia and being responsible for the 2007 outbreak in Micronesia.6 However, the divergence in nucleotide sequence below 12% should allow for the use of Zika virus-specific primers for routine PCR and allow distinction to be made from other flavivirus infections.Initial observations from South America suggest that only one in four adult cases are symptomatic. One issue that needs resolving urgently is an assessment as to the extent of infection in all endemic areas. Such studies are made more difficult, however, as early clinical signs can easily be mistaken for dengue or chikungunya. Human cases present with fever, headache, myalgia and rash, symptoms common to many tropical diseases. Retrospective seroepidemiology studies using virus-specific antibodies must be a priority. As with many emerging diseases, the extent to which a given virus has entered the population affected by an epidemic is often underestimated. For example, there is clear evidence of Ebola virus activity in West Africa prior to the explosive outbreak of 2014.Aedes aegypti being the principle vector for human-to-human transmission. Worryingly the virus also can be transmitted by Aedes albopictus the ‘tiger' mosquito, the female of the species feeding silently but aggressively on humans at all times of the day and night. This species is replacing A. aegypti in many urban areas and has a much longer life of up to eight weeks. More importantly, adaptation to A. albopictus may lead to the emergence of Zika virus strains with a heightened pathogenicity for humans, as has been seen for Chikungunya virus, where a single amino acid change in the E envelope glycoprotein increased virus growth in A. albopictus.7 We know glycosylation patterns of the Zika E protein are influenced by passage history and this may be a focus of future studies for clinical isolates.As is the case with yellow fever, Zika virus in Africa is most likely maintained in a sylvatic transmission cycle within populations of non-human primates, with humans being incidental hosts. In regions where non-human primates are not available to act as primary reservoirs of infection, humans have become the primary amplification hosts, with the urban mosquito, 8 How this will translate into treatment and control strategies in the short term remains to be seen, however, work is needed in developing an animal model of teratogenic infection. Unravelling the complex interactions between the mother's infected tissues and foetal development will be an arduous task. It may well be that other co-factors also are important in pregnancy and detailed analyses of risk factors will take time.Little is known regarding the pathogenesis of Zika virus infection. There must be a substantial viraemia following the incubation period to allow the ease of human–mosquito–human transmission that is so evident in current and past outbreaks. Whether or not these levels are the same among asymptomatic cases needs to be established before mathematical models of transmission will prove useful. There is some evidence of sexual transmission, not surprising if the viraemia is high, although the risk of travellers indulging in unprotected sex needs to be better defined. High and prolonged virus excretion in urine for at least 10 days after onset presents a further risk for containment. Early laboratory studies show that the virus grows well in cultured skin fibroblasts, and we can expect rapid progress at the molecular level.9 But even if a vaccine becomes available, the largely unpredictable nature of Zika virus emergence and the task of immunising large numbers of people living in endemic zones would mean that mass vaccination would not be cost-effective, especially as presently adult infections are relatively minor or asymptomatic. Perhaps more efficient prevention might be obtained using any Zika vaccine in women of child-bearing age. Caution will be needed, however, to ensure no harmful teratogenic effects result from immunopathogical reactions to Zika virus proteins.It has been widely commented that a Zika vaccine could be developed rapidly, no doubt encouraged by the rapid deployment of experimental vaccines during the recent West African Ebola virus epidemic. But there were already a number of candidate Ebola virus vaccines under development and thus such optimism that a Zika vaccine could be developed within a year are misplaced. It should be remembered that the development of a vaccine against the four serotypes of the serologically related dengue virus has been fraught with difficulties. More encouragingly perhaps, existing vaccine development platforms, for example, chimeric vaccines using yellow fever 17D vaccine as a ‘backbone', have been successfully used for the development of a vaccine against the flavivirus West Nile that entered North America in 1999.A. aegypti is the most urgent need, all the more so given the ease in which other flaviviruses can spread into indigenous mosquito species and thus promulgate virus spread. Present day public resistance to the use of insecticides needs to be overcome plus more effort given to educating residents in affected areas to cover even the smallest pools of stagnant water. An excellent example of how such control can be implemented is the state of Singapore that pursues an aggressive policy of both inspection and public awareness campaigns. Lessons are also to be had from history: in 1900 the seminal work of Walter Reed and colleagues led to a drastic reduction in the incidence of yellow fever in Cuba once A. aegypti had been confirmed as the principal vector. The Panama Canal was only completed once mosquito eradication significantly reduced the risk of disease. As is so often the case, however, a political will to implement effective strategies is imperative.For the present, the control of That Zika virus has essentially followed the expansion of chikungunya around the world should alert us all to other potential risks, such as the potential expansion of other African viruses, for example, the alphavirus O'nyong nyong. Zika virus also brings into the spotlight once more the matter of surveillance. Much of what we know today as to mosquito and tick-borne infections is thanks to extensive surveillance studies undertaken by a small cohort of scientists in the 1950s and 1960s working mainly in the tropics with either government funds or philanthropic support. Sadly, these efforts have largely ceased or reduced into programmes of hypothesis-driven research into known diseases. Surveillance studies are neither fashionable nor popular with those seeking scientific advancement. Yet were countries with the resources prepared to invest in applying modern technologies to better understanding the ecology of diseases and the balance between human advancement and manipulation of the world around us, we would be better prepared when the unexpected emerges.


Background: costs incurred at the end of life are a main contributor to healthcare expenditure. Urban–rural inequalities in health outcomes have been demonstrated. Issues around geographical patterning of the association between time-to-death and expenditure remain under-researched. It is unknown whether differences in outcomes translate into differences in costs at the end of life.Methods: we used a large representative sample of the Scottish population obtained from death records linked to acute inpatient care episodes. We performed retrospective analyses of costs and recorded the most frequent reasons for the last hospital admission. Using a two-part model, we estimated the probability of healthcare utilisation and costs for those patients who incurred positive costs.Results: effects of geography on costs were similar across diagnoses. We did not observe a clear gradient for costs, which were lower in other urban areas compared with large urban areas. Patients from remote and very remote areas incurred higher costs than patients from large, urban areas. The main driver of increased costs was increased length of stay.Conclusions: our results provide evidence of additional costs associated with remote locations. If length of stay and costs are to be reduced, alternative care provision is required in rural areas. Lower costs in other urban areas compared with large urban areas may be due to urban centres incurring higher costs through case-mix and clinical practice. If inequalities are driven by hospital admission, for an end of life scenario, care delivered closer to home or home-based care seems intuitively attractive and potentially cost-saving. Previous research has shown that costs incurred at the end of life, in particular for inpatient care, are a main contributor to total healthcare expenditure (HCE) –8. IncreEnsuring appropriate and equitable care is provided across all geographic areas within a country is important but can be difficult to achieve in practice. Several studies have described differences in mortality dependent on where people lived –13. RuraThere is additional evidence on local variation in spending on HC services for older people with higher spending per capita reported in Orkney and Shetland compared with all other mainland areas . What isScotland is well suited to descriptive work looking at the association between health service utilisation and geographic location, with ∼21% of the population living in rural or remote areas. It has been shown that geographical variation in mortality is higher in Scotland than in the rest of the UK . Health The aim of this study was to investigate whether geographic inequalities in mortality also translate into differences in costs incurred for the last admission to hospital prior to death.We used a longitudinal dataset comprising a 5% sample of all Scottish decedents derived from national mortality data  and randomly drawn by the Information and Services Division (ISD) Scotland. ISD linked these mortality data to inpatient hospital records . Data on death, preceding hospital admissions and associated costs were available from 1997 to 2012. The costing method that we used employed Healthcare Resource Groups (HRGs) as the basis on which we assigned unit costs to a Continuous Inpatient Stay (CIS) . These aWe identified the 10 most frequent reasons for the last admission to hospital prior to death for various geographical areas. The clinical data of interest were not the recorded cause of death, but the reason for admission to hospital as these better defines the costs incurred for that episode. We used the primary diagnostic code  to identify the reasons for admission. These codes refer to the main medical condition that was managed during the hospitalisation . To bestAlthough this classification does not directly measure driving time or distance to the nearest hospital, it gives a very good estimation of driving time to the nearest bigger settlement, and this can be used as a proxy for distance to major hospital site and overall accessibility of HC services. For our analysis, we ordered the 8-fold urban–rural categorisation in terms of driving time to the next larger settlements.A = age at death categories (reference category: <45 years); S = sex ; TTD = remaining time to death in quarters (reference category: 12th quarter before death); U = urban/rural indicator (reference category: large urban area); D = Scottish Index of Multiple Deprivation (SIMD) deciles (reference category: most deprived decile (1)); Y = time period of admission; iu = robust standard errors for patient i at time t.We based our analyses on hospital admissions over the last 3 years of life, identifying the last admission prior to death within this time period. We measured TTD in quarters, with each individual patient contributing at least 12 observations. If there was no hospitalisation in a particular quarter, these were assigned zero costs. To obtain unbiased estimates of costs, these observations were included in the analysis and a two-part model was employed, estimating the probability of hospital utilisation in its first part  conditional on a set of explanatory variables X (Equation 1). Costs were then estimated in the second part conditional on positive utilisation.From the second part of the model, costs were estimated for those patients who incurred positive costs; i.e. greater than zero and expected costs were obtained for each decedent. The same explanatory variables were used in the second modelling part (Equation 2).xβ representing the linear predictor for HCE. We estimated costs by fitting a generalised linear model (GLM) clustered on patient identifier with a Gamma distribution and a log link. The distributional family takes into account the heavily skewed nature of HCE data and clustering accounts for multiple hospital admissions for the same patient. In a third step, both modelling parts were multiplied, and cost estimates were recorded for each of the geographical regions (Equation 3). Separate models were estimated for each disease classification.with We calculated the total cost per disease by multiplying individual-level estimates for each area with their respective sample sizes and aggregated over areas. We then calculated the proportional share of costs for each area.P value of <0.05 was considered to indicate a statistically significant difference.All analyses were undertaken using Stata/SE12.1 software . A Age and Ageing onlineSupplementary data, Table S1, available in ). The group not accessing hospital prior to death  were more likely to be younger, male and lived in rural areas.Hospital admission records and related costs (1997–2012) were available for 39,366 patients with a total number of related observations of 618,333 over the last 3 years of life . Costs are shown as estimated costs from the two-part model with associated P values, sample size and proportional share of overall costs.After adjusting for age, sex, year of admission, social deprivation (Scottish Index of Multiple Deprivation SIMD) and remaining TTD, the overall effects of urban–rural area classification on costs for the last admission to hospital were similar across diagnoses (P < 0.001). The proportional share of total costs is broadly similar across diseases, and large urban areas contribute most to overall costs.Costs in very remote, rural areas were significantly higher than costs in large, urban areas Figure . We founAge and Ageing onlineSupplementary data, Figure S3, available in ). We found that particularly for stroke, infections of the lung and disorders of the urinary system, LOS was markedly longer in very remote areas than in more accessible, urban areas.A post hoc investigation of the main driver of increased costs in more remote areas revealed that LOS in hospital was longer in more remote, rural areas than in urban areas . A.L. is funded by the Medical Research Council and the Chief Scientist Office of the Scottish Government Health and Social Care Directorate (MC_UU_12017/13).This work was supported through a post-doctoral fellowship awarded by the


Phlebotomus. Infections are usually asymptomatic but the virus is known to cause aseptic meningitis and/or meningo-encephalitis in the Mediterranean countries. Dogs are good sentinels for detection of viral circulation and are more easily accessible than wild animals.Toscana virus (TOSV) is an arbovirus belonging to the Bunyaviridae, a family of negative-stranded, enveloped RNA viruses. The virus can be transmitted to humans through the bite of an infected female sand fly of the genus In 2013 and 2014, we collected sera from 231 adult dogs living in 26 counties in two departments in Corsica, a French island in the Mediterranean. The virus microneutralization-based seroprevalence assay revealed a seropositivity of 3.9 % dogs on the eastern coast of Corsica.Our study confirms the circulation of TOSV in Corsica. Accordingly, in geographical areas where dogs possess TOSV neutralizing antibodies, direct and indirect TOSV diagnosis should be implemented in patients presenting with febrile illnesses and central nervous system infections such as meningitis and encephalitis. Phlebotomus. The infection has previously been reported in countries located on the northern shores of the Mediterranean Sea , as well as in the east (Cyprus and Turkey)  and in Corse-du-Sud . Among the 231 dogs sampled, 147 were male (63.6 %), and 84 were female (36.4 %). The average age of dogs was four year-old (3–12 year-old) and most were wearing collars to reduce the likelihood of arthropod infestation. The dogs came from a range of sources including hunters’ dogs, shepherds’ dogs, military working dogs and some pet dogs. They all appeared to be in good health at the time of sampling and were examined with the assistance of their owners. Blood samples were centrifuged within 24 h of collection and the sera were subsequently frozen at -20 °C until being processed in the laboratory.In 2013 and 2014, we collected sera from the radial veins of 231 adult dogs. The dogs were living in 26 communes in the two departments of Corsica  was adapted from the previously described protocol . The MN n = 5) and 1/40 (n = 4), which was expected when a virus challenge dose of 1000TCID50 was used, thus rendering the assay more stringent compared with the commonly used 100TCID50; previous studies have shown a good correlation between both protocols [Leishmania infantum [The virus microneutralization-based assay is the most discriminative serological test adapted to differentiate the affinity of antibodies against different closely related viruses . Of the rotocols . The resrotocols . In Bizerotocols . In Algerotocols . In continfantum . In Graninfantum . Howeverinfantum . This isinfantum  and whicP. mascitii, P. perniciosus and Sergentomyia minuta on the whole island at altitudes lower than 800 m, with the latter two sand fly species being largely dominant. Phlebotomus mascitii has been described only in the north-eastern region. Phlebotomus papatasi, P. ariasi and P. perfiliewi were never reported [The seroprevalence of TOSV found in dogs in this study was fairly low. Historical records describe reported . Thus, treported . Our resreported , suggestOur study indicates that dogs are promising sentinels for exposure to TOSV transmission by sand flies in the Mediterranean region. However, the potential of dogs as reservoirs of Toscana virus is yet unknown. Our data extend the knowledge of the distribution of this virus in Corsica, indicating higher prevalence of Toscana virus along the eastern coasts of Corsica. In geographic areas where dogs possess TOSV neutralizing antibodies, direct and indirect TOSV diagnosis should be implemented in patients presenting with febrile illnesses and central nervous system infections such as meningitis and encephalitis.ELISA, enzyme-linked immunosorbent assay; MN, microneutralization; TCID50, tissue culture infective dose producing pathological change in 50 % of the cell culture inoculated; TOSV, Toscana virus


The safety of a multi-span waler beam subjected simultaneously to a distributed load and deflections at its supports can be secured by limiting the maximum stress of the beam to a specific value to prevent the beam from reaching a limit state for failure or collapse. Despite the fact that the vast majority of accidents on construction sites occur at waler beams in retaining wall systems, no safety monitoring model that can consider deflections at the supports of the beam is available. In this paper, a maximum stress estimation model for a waler beam based on average strains measured from vibrating wire strain gauges (VWSGs), the most frequently used sensors in construction field, is presented. The model is derived by defining the relationship between the maximum stress and the average strains measured from VWSGs. In addition to the maximum stress, support reactions, deflections at supports, and the magnitudes of distributed loads for the beam structure can be identified by the estimation model using the average strains. Using simulation tests on two multi-span beams, the performance of the model is evaluated by estimating maximum stress, deflections at supports, support reactions, and the magnitudes of distributed loads. Safety monitoring of multi-span beam structures under distributed loads has been an issue of interest since structural health monitoring (SHM) has been introduced ,5,6,7,8.To estimate the maximum strain or stress of multi-span beams, mathematical models based on measured average strains have been developed and applied to monitoring the safety of beam structures . In the However, for the case of beam structures subjected to deflections at the supports, the mathematical models of multi-span beam without consideration of the effect of deflections at supports on the magnitude of the maximum stress cannot be used in the assessment of the safety of beam structures. A typical example of a beam structure subjected to deflections at its supports is the waler beam in an anchored retaining wall system shown in As shown in Therefore, in this paper, an analytical model to estimate the maximum stress for a multi-span beam with deflections of supports under distributed loads is presented. For the estimation, the relationship between the maximum stress and the average strain of each span is derived and the average strain is measured at the middle of each span. In addition to estimation of the maximum stress, using average strains from VWSGs, estimation models for deflections at supports, support reactions, and the magnitudes of distributed loads are presented. For the safety monitoring of multi-span beam structures in retaining wall systems, the model is applied to estimation of the maximum stress and distribution of moments along the length of a beam.n + 1 ground anchors in a retaining wall system is modeled a linear elastic n-span continuous beam with n + 1 supports. ith span, respectively. ith supports, respectively. It is assumed that the deflection at each support has a different value.In ith span along the axis of the beam can be written as:For a linear elastic multi-span beam as shown in the beam . Using t sensors ,17. In a sensors ,23,24.ith span of the waler beam are set to ith span of the waler beam, As shown in ith span, Then, the relationship between the bending moment at the n + 1 supports, n − 1 three moment equations can be formulated using Equation (A1) in M}, {w} and {δ} are the bending moment vector of dimension n + 1 supports, the vector of dimension C′] is a matrix of dimensions ith support can also be found by multiplying the stiffness ik by the deflection δi of the support. Then, the reaction vector in Equation (5) can be expressed by substituting the moment vector {M} in Equation (4) into Equation (5).K] is the stiffness matrix for the n + 1 supports in w} and {δ} is found by:Based on the three moment theorem, the relationship among the bending moments, R} and distributed lateral loads {w}, the bending moment of each span of the beam Using the support reactions {ith span can be evaluated by numerical integration over the gauge length of By considering the gauge length of a VWSG in Equation (3), the relationship in Equation (8) for the Then, by substituting n-span waler beam can be determined from the average strains measured by VWSGs.From Equation (11), deflections at supports of the waler beam The setup for simulation of the four-span test beam is shown in 1 = ε4 = 344 με and ε2 = ε3 = 457 με. For the given values for average strains obtained from the numerical analysis with the properties of the model in To test the conceptual model, it is assumed that the vibrating wire strain gauges with the gauge length of 15 cm are installed to measure the average strains in The deformed shape based on estimated support deflections, support reactions, and applied loads of the test beam is shown in In 2 and ε3, may be limited to the value of 780 με to prevent the waler beam from reaching the allowable stress of 160 MPa for SS400 grade steel beam used in the construction site.As a practical application of the analytical model presented in the paper, the measured values of the average strain can be limited to a specific value to prevent the waler beam from reaching one of limit states for failure or collapse of waler beams. For the safety monitoring of the four-span waler beam, the average strains at the second and third spans, εA schematic diagram for the eight-span test beam is shown in w1 = w2 = w7 = w8 = 100 kN/m, w3 = w4 = 200 kN/m, and w5 = w6 = 300 kN/m. As input for the estimation model in Equation (12), the average strains from VWSGs are listed in The waler beam was designed to carry three different magnitudes of distributed design loads of The deformed shape based on estimated support deflections, support reactions, and applied loads of the test beam are shown in In this paper, an analytical model for estimation of the maximum stress of a multi-span beam with deflections of its supports is presented. The analytical model is derived by defining the relationship between the maximum stress and the average strain of each span of the multi-span beam. Based on the use of the average strains obtained from VWSGs as the input to the analytical model, the analytical model allows estimation of deflections at supports, support reactions, and the magnitudes of distributed loads. Using tests on two multi-span beams, the performance of the model is evaluated by estimating maximum stresses, support deflections, support reactions, and the magnitudes of distributed loads. From the simulation test, it can be concluded that safety monitoring of multi-span waler beams is made possible by comparing the given allowable stress of the beam with the estimated maximum stress with consideration of any deflections at the supports. Through periodic estimation of the maximum stress by the proposed method, the safety of retaining walls at construction sites will be improved.


Influenza A virus (IAV) in wild bird reservoir hosts is characterized by the perpetuation in a plethora of subtype and genotype constellations. Multiyear monitoring studies carried out during the last two decades worldwide have provided a large body of knowledge regarding the ecology of IAV in wild birds. Nevertheless, other issues of avian IAV evolution have not been fully elucidated, such as the complexity and dynamics of genetic interactions between the co-circulating IAV genomes taking place at a local-scale level or the phenomenon of frozen evolution. We investigated the IAV diversity in a mallard population residing in a single pond in the Czech Republic. Despite the relative small number of samples collected, remarkable heterogeneity was revealed with four different IAV subtype combinations, H6N2, H6N9, H11N2, and H11N9, and six genomic constellations in co-circulation. Moreover, the H6, H11, and N2 segments belonged to two distinguishable sub-lineages. A reconstruction of the pattern of genetic reassortment revealed direct parent-progeny relationships between the H6N2, H11N9 and H6N9 viruses. Interestingly the IAV, with the H6N9 subtype, was re-detected a year later in a genetically unchanged form in the close proximity of the original sampling locality. The almost absolute nucleotide sequence identity of all the respective genomic segments between the two H6N9 viruses indicates frozen evolution as a result of prolonged conservation in the environment. The persistence of the H6N9 IAV in various abiotic and biotic environmental components was also discussed. Orthomyxoviridae with a genome composed of eight distinct negative-sense RNA segments. The segmented genome and the lack of proofreading activity of the virus RNA polymerase provide a basis for extreme genetic diversity.Influenza A virus (IAV) is a member of the genus Monitoring studies carried out during the last two decades provided a large body of knowledge regarding the ecology of IAV in wild birds. Multiyear studies utilizing data from various bird populations sampled in different areas worldwide allowed us to identify the reservoir bird species and revealed the main ecological characteristics like the prevalence, subtype diversity, seasonality, environmental persistence, transmission routes, geographic distribution, intercontinental exchange, etc. Nevertheless, there are still aspects of IAV evolution and ecology in wild birds which have remained to be elucidated. For example, contrary to the IAV prevalence and subtype variety reported in the monitoring studies, only a few projects were focused on revealing the subtype diversity, genomic complexity, and dynamics of genetic interactions between the viruses occurring at the local-scale level, i.e. in the wild bird population residing in a single locality or in a single pond Another sparsely reported and not fully understood issue is the phenomenon of “frozen evolution” of the influenza virus in nature. The hypothesis of frozen evolution or frozen replication (both terms are used in the literature) is used to explain occasional evidence of anachronistic influenza virus genomes or genomic segments So, the frozen evolution is a hypothesis explaining significantly lover nucleotide mutation rate than expected th century. The ponds are often interconnected with streams into cascades resulting to a dense network of pond and lake systems through the landscape the role of which in the ecology and perpetuation of the IAV has not been fully investigated yet.The objective of our study was to help to elucidate these aspects of IAV evolution by investigating the genetic diversity and evolutionary relationships between the viruses detected one year apart from two neighboring localities. In 2009, unusually high IAV subtype diversity was observed in a sample pool from a mallard flock inhabiting a pond in the South Bohemian Region in the Czech Republic. A year after, the viruses collected in the vicinity of the first locality exhibited exceptionally high sequence similarity to that of the previously identified IAV strains. South Bohemia is known for its countless ponds and lakes which have been established since the 12We analyzed the subtype diversity of AIV in each of the study localities. Further, the eight genomic segments of the co-circulating virus strains were examined to determine the nucleic and amino acid sequence diversity, genotype constellations, and patterns of genetic reassortment. Finally, we focused on the estimation of the evolutionary relationships between the viruses from the two localities.The phylogenetic analysis and genotyping revealed remarkable genomic variability, with the identification of direct parent-progeny relationships between the newly emerging IAV genotypes. In addition, mutual comparison of IAV from the two localities indicated between-year frozen evolution.Cloacal and tracheal swabs  were collected post mortem from hunting harvested mallards (Anas platyrhynchos) during the National avian IAV surveillance in the Czech Republic in 2009 and 2010. As the National surveillance program no specific permissions were required to access the sampling localities and for sampling activities. The sample collection was coordinated with the Regional Veterinary Administration of the Czech Republic and the given hunting organizations. Our study did not involve endangered or protected species. All the specimens were collected post mortem after the hunting harvest. The GPS coordinates of the sampling localities were provided in The swabs were re-suspended in PBS buffer and the suspensions were then divided into aliquots and used either for molecular detection or virus isolation. Total nucleic acid was extracted using the MagNA Pure Compact and MagNA Pure LC extractors (Roche), employing the Total Nucleic Acid Extraction Kit (Roche) with an input volume of 200 or 400 µl and an elution volume of 50 µl. The extracts were screened for IAV by the RT-qPCR method of Nagy et al. Virus isolation was performed according to the methodology in reference nd passage of the co-infected sample P/18K (hemagglutination test titer of 128) positive for both the H6 and H11 in RT-PCR was serially diluted in a range of 10−1 to 10−4 in distilled water. Then, 100 µl of each dilution series were mixed with 100 µl of H6 or H11 antibodies and incubated for 30 min at room temperature. Subsequently, 200 µl of allantoic fluid-antibody mixture for each dilution was inoculated into the allantoic sac of two specific pathogen free (SPF) embryonated chicken eggs and incubated at 37°C. After embryo death, the allantoic fluid was recovered and tested by the hemagglutination test Allantoic fluid derived from the 2Partial or whole genome amplification  was performed with various combinations of the previously described primers The sequences were assembled and edited and the sequence quality was evaluated by the SeqScape software (Life Technologies). BLAST analysis The results of phylogenetic analyses were summarized by employing the digital genotyping approach The sequences were deposited in GenBank with the accession numbers listed in Cloacal and tracheal swabs from mallards (Anas platyrhynchos) inhabiting a pond in the South Bohemian Region  locatedClostridium botulinum and are released to the respective ponds. They are kept outdoors until the hunting harvesting takes place in autumn. Next year the entire re- and de-population cycle repeats. The hunting organizations were not in mutual contact and the ducklings for the two ponds were bought independently from different suppliers. The bank of pond P was lined with wooden huts attracting wild mallards to nest  to four  and three (NS); . In addiThe nucleic acid sequence alignment of the regions used for phylogeny estimation showed diversity with peaks in a range from 5.1 to 7.5% between the segments, namely: PB2 , PB1 , PA , NP , MP . Regarding the segments encoding for the surface antigens, the major differences were identified within the H6 , N2  and H11  sequences. The N9 amplicons were identical at the nucleotide sequence level .The high nucleotide variation contrasted with the high identity at the amino acid sequence level. The differences spanned below 6 residues within the investigated regions regardless of the segment considered. The only exception was the NS segment with 18 amino acid differences in the NS1 and 21 between the NS2 protein fragments, respectively, which corresponds to the known dual allelic structure in avian viruses.Contrary to the high genetic diversity within locality P, the phylogenetic analysis of the H6N9 strains  did not reveal any discrete sequence clustering and the two representative genomes were 100% identical at the nucleotide sequence level .Finally, we established the relationships between the IAV strains from localities P and H both in terms of phylogenetic analysis and sequence identity. The results of phylogenetic analyses revealed that the PB2, PB1, and H6 segments of the H/H6N9 strains were closely related to those of the P/H6N2 sub-lineage 2 viruses while the PA, NP, N9, MP, and NS segments clustered within the P/H11N9 sub-lineage 2. This was further supported by almost 100% nucleotide sequence identity between the H/H6N9 and corresponding P/H6N2 and P/H11N9 segments .The summarization of the results of phylogenetic analysis within the segment identity matrix (SIM) revealed at least five distinct IAV genotypes which co-circulated in locality P : H6N2 geFinally, the mutual comparison of the SIM columns derived from locality P and H IAV genomes clearly showed that the H/H6N9 genome was assembled from the P/H6N2 genotype 2 and P/H11N9 viruses at a 3∶5 ratio . Indeed,The results of phylogenetic analysis and digital genotyping indicated that the H/H6N9 strains detected in 2010 were apparently direct progenies of the previously co-circulating P/H6N2 genotype 2 and P/H11N9 viruses.The close genetic relationships uncovered between H6N9 viruses from localities P and H drew our attention back to the H6/H11 co-infected cloacal swab specimen P/18K and to the IAV strains retrieved by virus isolation on ECE from that specimen.First of all, we focused on the allantoic fluid and estimated the IAV subtype composition by the H6, H11, N2, and N9 specific RT-PCR tests. Surprisingly, the results revealed positivity for all segments but N2, which indicated that the allantoic fluid of P/18K evidently represented a mixed population of the H6N9 and H11N9 subtypes. Subsequently, these subtypes were successfully separated using H6 and H11 subtype-specific monoclonal antibodies and confirmed by RT-PCR tests and sequencing.de facto 100% similarity at the nucleotide sequence level . This sufficiently proved that, contrary to the previous observations of Lindsay and colleagues in vitro reassortment and ruled out artificial generation of the P/H6N9 subtype during the virus isolation efforts. So, the co-infected specimen evidently contained both the H6N9 and H11N9 viruses.The analysis of the co-infected specimen from locality P showed the presence of two HA subtypes, H6 and H11, and a single NA subtype, N9. This constellation was observed both in the screened allantoic fluid and the original swab material. We used specific H6 and H11 antibodies to separate these two subtypes. A similar strategy was applied previously to investigate IAV co-infections in wild ducks Two conclusions can be drawn regarding the emergence of the H6N9 virus: i) the H6N9 subtype was evidently present, if not originated, in locality P, ii) the H6N9 virus persisted in the same area as was suggested by its re-detection roughly one year apart in the nearby locality H.Genotyping of the antibody separated P/H6N9 virus showed that it was a 3∶5 reassortant of the P/H6N2 genotype 2 and P/H11N9 viruses with almost 100% identity of the respective genome segments at the nucleotide sequence level. At first sight, it indicates that the P/H6N9 virus represents a possible progeny of the P/H6N2 genotype 2 and P/H11N9 viruses. However, considering the close co-circulation of these viruses, it was not possible to determine which one is the parent and which one is the progeny. Furthermore, it is not clear whether the P/H6N9 virus emerged within the co-infected mallards or originated from elsewhere and subsequently co-infected the same bird along with the related  P/H11N9 strain. Nevertheless, the genotype constellations favor the suggested parent-progeny scenario.Despite a roughly one year interval between the P/H6N9 and H/H6N9 detection, both of the viruses retained identical subtype and genotype constellation. Unexpectedly, the entire genomes exhibited almost absolute nucleotide sequence identity. Considering the rapid evolutionary dynamics of avian IAV So, which mechanism would account for the exceptionally high sequence conservation of the H6N9 virus? To address this question, we performed epizootological investigations which included visiting the sample collection sites as well as communication with the hunting association representatives and field veterinarians who assisted in specimen collection. The investigation excluded any mutual contacts, cooperation, trade or involvement of other man-associated routes to allow artificial transmission, one-year preservation, and re-appearance of the H6N9 virus. Therefore, it is reasonable to hypothesize that the observed conservation of the H6N9 virus resulted from its environmental persistence and frozen evolution. We suppose that some of the H6N9 infected birds in locality P might have been frightened by hunters and escaped to nearby pond H where they disseminated the virus into the environment. Then, the H6N9 strain persisted in the environment and infected the new and immunologically naive mallard flock re-populating pond H next-year.The frozen evolution is long considered as one of the mechanisms of influenza virus perpetuation in nature It has been thought that IAV do not prevail in the form of latent infection in birds. In addition, pond H underwent annual re and de-population cycles. Hence, the genetic conservation of the H6N9 virus suggests some kind of environmental persistence. This raised another important question: On which environmental matrix could the virus persisted? The H6N9 virus had to survive through winter, spring, and especially summer, which is relatively hot in the temperate zone, to re-appear during the autumn. Although recent data suggests that unfavorable environmental conditions during summer do not prevent circulation of avian IAV in the environment Nevertheless, a mechanism has been characterized to date which can fit our assumptions. It has been demonstrated that feathers covered with preen oil could efficiently capture and concentrate the avian IAV from water Although our conclusions probably raise more questions than they answer the results of the presented study suggest that the IAV subtype and genotype diversity between the IAV at the local-scale level can be admirably complex. Evaluation of the most intimate genetic links between such viruses can reveal rarely observed phenomena like direct parent-progeny relationships between the co-circulating strains or frozen evolution. Further and more detailed studies are required to fully elucidate whether between year persistence and frozen evolution observed here is an isolated unique event or represents a more regular but yet unrecognized phenomenon in the evolution of the influenza virus in aquatic birds.File S1Figure S1. Locality P. The samples in 2009 were collected from mallards inhabiting the pond called Putim . The green flag on the map S1c represents the position of the S1a view. Figure S1b shows wooden huts distributed along the shore. The map was generated by the www.mapy.cz. Figure S2: Locality H. The samples in 2010 were collected from mallards resided the pond called Kahoun which is situated near the Hajany village . The green flag on the map S2b represents the position of the panoramic view S2a. The map was generated by the www.mapy.cz. Table S3: Nucleic acid sequence identity matrices of the locality P and H avian influenza virus segments. The matrices were constructed using the BioEdit program on the basis of nucleotides 1251–2288 (1038) of PB2, 1465–2289 (825) of PB1, 783–1401 (649) of PA, 816–1724 (909) of H6, 679–1274 (596) of H11, 748–1544 (797) of NP, 568–889 (322) of N2, 1050–1431 (382) of N9, 203–1006 (804) of MP, and 540–870 (331) of NS. The tables were highlighted with a segment specific color which is corresponding to (PDF)Click here for additional data file.


Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms. Animals relocate objects as needed by active motion; even in human bodies, liposomes make use of active transport to move chemicals with molecular motors in the cytoskeleton13457810111213141834510111213141516In this article, we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. Neither the entropy of the bead distribution nor the interaction between beads and the floor surface govern the dynamics. Rather, this surprising bead-transport phenomenon is produced by small, non-thermal fluctuations in the local number density of beads and its autocatalytic growth, both of which are generated by nonlinear droplet dynamics in a strongly non-equilibrium state. This may provide an avenue for designing active transport systems in the laboratory. Technologies based on active transport may produce highly sophisticated micro/nano systems wherein chemical and physical substances migrate as in living organisms.18TACl)2021We attempted to produce an active transport system with loading and unloading using a simple oil/water system. An oil droplet containing iodine and iodide anion(s) spontaneously moves on a glass surface in water containing a cationic surfactant, trimethyloctadecylammonium chloride  moving on a circle. This circle was divided based on its azimuth into twelve sections by Δthe ring .Pin and Pout depend on the course width. Thus, experiments to measure of these probabilities were carried out. We placed an oil droplet in the concentric ring and changed the course width. Five glass beads were distributed randomly on the course. We counted the events where the droplet passed through a section occupied by the bead(s). The number of events is Ntotal. In some cases, the droplet took up the bead(s). The number of uptake events was denoted as Nuptake. Pin  as a function of the course width. Pin increases with a decrease in the width because the collision probability between the droplet and the bead(s) is larger in a narrower course.Pout at a section with the residence time T is given by 1−exp(−T/τout) . A longer τout value leads to smaller Pout. Pout also depends on the residence time T of the moving droplet in each section: in wider positions, a droplet can move along the radial direction in addition to the circumferential one. This degree of freedom in the droplet’s motion increased residence time in the wider sections. Using the course with wr = 2.0, the residence time when the droplet resided transiently in each section was measured. The average residence time T is shown in Pout was shown in Pout increases with an increase in the width. This characteristic results from the width-dependency of T, while τout is irrespective of the width.out) see . Pout isPin and Pout shown in Calculations were performed with the correlation curves for Pin should be a decreasing function of the aggregate size n. Thus, the probability should be denoted as Pin(n), where Pin(1) is equal to the Pin shown in Pout should be an increasing function of the aggregate size. Here, the probability should be denoted as Pout(n), where Pout(0) is equal to the Pout shown in When two or more beads were contained in a droplet, they tended to form an aggregate owing to the presence of oil. Once the aggregate had been released, the droplet rarely took up the aggregated beads . This mePin(n) and Pout(n); other examples are shown in n-dependency in Pin(n) and Pout(n) does affect the quantitative aspect of the results. However, most important is that both Pin(n) and Pout(n) provide autocatalytic growth of the fluctuation of the number of beads: the growth of the cluster is accelerated with an increase in cluster size.We found an abiotic active transport system with loading and unloading. The glass beads are transported to the widest place by a self-moving droplet that carries the beads. This phenomenon can be explained by autocatalytic accumulation seeded by random release. This scenario may be developed to design the transport systems with a semblance of life and their applications to future technologies, such as active transport in microfluidic devices.Trimethyloctadecylammoniumchloride (purity >98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. Nitrobenzene (purity >98.0%), potassium iodide (purity >99.9%), iodine (purity >99.8%), and agar of reagent grade were provided by Wako Chemicals Inc. All chemicals were used without further purification. Glass beads of 2 mm were provided by AS ONE Corporation. All glass beads and Petri dishes were treated by vacuum plasma so as to remove surface contamination (FEMTO Science CUTE-MP(MP/R)). After treatment, the glass surfaces were wetted with KOH aqueous solution (1 M) and rinsed with deionized water.D (<9 cm) was cut out using a cookie cutter. Next, a ring-shaped gel was obtained, with inner and outer diameter equal to D (cm) and 9 cm, respectively. A cylindrical gel of diameter D was also obtained. From the cylindrical gel, another cylindrical gel with diameter d (<D) was cut out by a cookie cutter. Next, two pieces of gel, the ring-shaped gel and the cylindrical gel with the diameter d, were obtained. These gels were soaked in an aqueous solution of C18TAC (3 mM). The aqueous solution was refrigerated for 24 hrs. After treatment, the gels were removed from the refrigerator and washed in deionized water. These gels were put in a Petri dish of 9-cm diameter so as to form the desired layout. The central gel shown in d) and the outer (D) diameters were 3.4 cm and 6.4 cm, respectively. Thus, the widest width Lmax was related to wr by Lmax = {(D–d) wr} / (1 + wr). The Lmax of wr = 2.0 was 2.0 cm; this was approximately two times larger than the average diameter of an oil droplet (approximately 0.8–1 cm). The aqueous solution containing trimethyloctadecylammonium chloride (3 mM C18TAC) was poured into the ring-shaped course so that the depth of the C18TAC-containing aqueous solution was around 20 mm: that is, a large enough depth compared to the droplet’s diameter. The glass beads of 2-mm diameter were placed randomly in the ring course filled with the aqueous solution. The number of beads was usually ten. This was nearly the maximum number of the glass beads, because too many beads restricted the free motion of the oil droplet. After setting, a nitrobenzene droplet that contained 50 mM I2 and saturated KI was placed in the ring. The droplet volume was 400 μL, and its diameter was approximately 8–10 mm. The experimental result was recorded by a CCD camera (Keyence Corporation VW-6000/5000), and the movie was analyzed by the software MovieRuler (Photron limited).An aqueous solution containing 4% agar was poured into a Petri dish 9 cm in diameter. The initial temperature of the solution was approximately 80 °C and it was allowed to cool to room temperature. Special care was taken not to introduce bubbles to the gel. After gelation, a cylindrical gel mass of diameter How to cite this article: Goto, Y. et al. An Abiotic Glass-Bead Collector Exhibiting Active Transport. Sci. Rep.5, 14348; doi: 10.1038/srep14348 (2015).


Flavivirus. WNV is preserved in the environment through cyclic transmission, with mosquitoes, particularly Culex species, serving as a vector, birds as an amplifying host and humans and other mammals as dead-end hosts. To date, no studies have been carried out to determine the prevalence of the WNV antibody in Malaysia. The aim of this study was to screen for the seroprevalence of the WNV in Malaysia’s Orang Asli population.West Nile virus (WNV) infection is an emerging zoonotic disease caused by an RNA virus of the genus Serum samples of 742 Orang Asli were collected in seven states in peninsular Malaysia. The samples were assessed to determine the seroprevalence of WNV immunoglobulin (Ig)G with the WNV IgG enzyme-linked immunosorbent assay (ELISA) method. For each individual, we documented the demographic factors. Anti-dengue and anti-tick-borne encephalitis virus IgG ELISA were also performed to rule out a cross reaction. All statistical analyses were performed using the GraphPad Prism 6 ; p values of less than 0.05 were considered significant.The serosurvey included 298 men (40.16%) and 444 women (59.84%) of Malaysia’s Orang Asli. Anti-WNV IgG was found in 9 of the 742 samples (1.21%). The seroprevalence was 0.67% (2 of 298) in men and 1.58% (7 of 444) in women. The presence of anti-WNV IgG was found not to be associated with gender but, however, did correlate with age. The peak seroprevalence was found to be 2.06% (2 of 97) in individuals between 30 to 42 years of age.Culex mosquitoes as the main vector and a high degree of biodiversity, including migratory birds that serve as a reservoir to the virus.No previous studies have examined the seroprevalence of the WNV antibody in the human population in Malaysia, and no clinical reports of infections have been made. Screening for the WNV seroprevalence is very significant because of many risk factors contribute to the presence of WNV in Malaysia, such as the abundance of  The WNV is a member of the virus family Flaviviridae, which belongs to the Japanese encephalitis virus (JEV) serogroup of flaviviruses and is closely associated with other human pathogens such as dengue virus (DENV), yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV) . The flaPhylogenetic lineage studies show that approximately 1000 years ago, WNV emerged as a distinctive virus and had developed into two distinct lineages . LineageIn 1957, in an outbreak amongst aged patients in Israel, the virus was recognized as a cause of severe human meningitis or encephalitis  . Since tThe presentation of clinical illness in humans ranges from asymptomatic infection to viral syndrome to neurologic disease . In the Culex genus, in particular Culex pipiens that serve as a vector, birds that serve as intensifying hosts and humans or others mammals that serve as dead-end hosts [WNV is a mosquito-borne flavivirus transmitted to humans by mosquitoes, primarily the nd hosts -21. Zoonnd hosts . Studiesnd hosts . In promnd hosts . Host send hosts -25. Manynd hosts . Humans nd hosts . A case nd hosts .At present, no studies have been carried out in Malaysia regarding the seroprevalence of the WNV antibody in the human population, and no clinical reports of infections have been made. This could be due to undiagnosed or misdiagnosed subclinical infections or because the WNV has not yet reached Malaysia. The objective of this study was to screen for the prevalence of antibodies against WNV, particularly immunoglobulin (Ig)G, among Malaysia’s Orang Asli, the native people, who make up less than 1% of the Malaysian population. Most of them reside at the frontier of the jungle and rural areas (61%) and in the center of the jungle (37%), and only a small number of them are found in or close to an urban area (1%). Because dengue fever is endemic in Malaysia, a comparison with the anti-dengue IgG antibody result is also discussed to rule out cross reactivity.The study acknowledged distinctive consent from the Department of Orang Asli Development (JAKOA) and attained approval from the Ethics Committee of the University Malaya Medical Centre (UMMC) (MEC Ref.824.11). Contribution in this study was virtuously voluntary. The volunteers were given a briefing on the project and were allowed adequate time for consideration. Written informed consent was acquired. An additional agreement form was completed by the parents or guardian of volunteers below legal age (less than 18 years old). The samples were prepared with rigid obscurity. All volunteers had completed a given questionnaire to identify symptoms they had experienced and their lifestyle. All volunteers gave written consent for the use of blood samples after the samples were made anonymous.All blood samples were collected between September 2012 and February 2013 from 742 Orang Asli from seven states in peninsular Malaysia  with the assistance of a proficient medical assistant. Most of the Orang Asli were forest inhabitants. They were interviewed and given a standard questionnaire regarding demographic features, medical history and disclosure of potential risk.TMELISA  according to the manufacturer’s instructions [WNV IgG testing was done by WNV IgG capture DxSelectructions ,27,28. TCategorical data were assessed in two-way contingency table analyses using Fisher’s exact test. The correlation of age and reactivity of WNV infection were determined by means of the Spearman nonparametric correlation. All statistical analyses were performed using GraphPad Prism 6 , and p values of less than 0.05 were considered significant.An overview of the characteristics of the 742 serum samples are shown in Figure P = 0.3270) groups (Table P = 1.000). A summary of serum prevalence by age and gender is presented in Table P = 0.3270). The seroprevalence was 0.67% (2 of 298) in male subjects and 1.58% (7 of 444) in female subjects  .In general, anti-WNV IgG was discovered in 9 of 742 samples 1.21%). There was no significant difference in the seroprevalence between the male  and female . Although it is not considered endemic, a prolonged study should be done. In 2011, a study conducted among captive birds to determine the presence of the WNV antibody was carried out in four selected areas in Selangor and had showed 4.41% seropositivity rate . As for nfection , disseminfection .Aedes aegypti and Culex pipiens are the main vector for DENV and WNV, respectively, which can be found abundantly in rural areas but not in the forest [We discovered that the cross-reactivity in IgG antibodies of WNV and DENV (0.13%) was lower than with TBEV (11.59%) . The lowe forest ,38. ThisDue to some limitation in this study, neutralisation tests were not executed for the confirmation of positive WNV antibodies by ELISA tests. According to the previous study, patients with positive WNV infection by ELISA were also confirmed to be positive by neutralisation tests . The WNVBecause there was doubt about cross-reactivity with other viruses, the IgG ELISA test was performed on two other flaviviruses, TBEV and dengue virus. However, only 86 of 742 samples (11.59%) exhibited cross-reactivity for anti-TBEV IgG and only 1 of 742 samples (0.13%) for anti-dengue IgG, whereas 14 of the 742 samples (1.89%) were found to have cross-reactivity for all three types of viruses.Another study showed that the seropositivity of WNV did not correlate with age and gender , neverthAlthough WNV is not endemic, our study demonstrates that the presence of WNV in several states in peninsular Malaysia could be an early warning of transmission of the virus. Transmission of the virus will be very difficult to prevent and control because the public health infrastructure is insufficient to cope with vector-borne and zoonotic diseases . In the Culex mosquitoes as the main vector and a high degree of biodiversity, including migratory birds that serve as a reservoir to the virus.No previous studies have examined the seroprevalence of the WNV antibody in the human population in Malaysia, and no clinical reports of infections have been made. Screening for the WNV seroprevalence is very significant because many risk factors contribute to the presence of WNV in Malaysia, such as the abundance of