Glossina pallidipes salivary gland hypertrophy virus  is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies . GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host’s mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene).G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes.We show that in response to hemocoelic injections of GpSGHV into Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.The upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in The online version of this article (10.1186/s12866-018-1298-1) contains supplementary material, which is available to authorized users. Glossina pallidipes salivary gland hypertrophy virus , a large double-stranded DNA (dsDNA) virus pathogenic specifically to Glossina spp., [G. pallidipes, although the majority of cultured and wild tsetse fly species are asymptomatically infected by GpSGHV (low virus titers), some unknown factors can trigger symptomatic infections (high virus titers) [G. pallidipes, SGH epizootics reduce fly survival and productivity, and have caused the collapse of three colonies; two in the Insect Pest Control Laboratory (IPCL) in Seibersdorf, Austria (in 1987 and 2001), and one in the mass rearing facility in Kality, Ethiopia (in 2012) [G. pallidipes from the Southern Rift Valley of Ethiopia [Tsetse flies (Diptera: Glossinidae) are naturally infected by the na spp., , 2. In G titers) . This, i titers) , 5. In tin 2012) . These Gia in 198 and 20012 viral genome copies/fly) released by asymptomatic flies via saliva during feeding [G. pallidipes potentially represents a homeostatic equilibrium between the host’s immune system and the viral escape strategies. Consequently, the viral infection is kept under control (asymptomatic state), but is not completely eliminated from the fly.Although it is uncertain how the virus is maintained within tsetse populations in nature and in laboratory colonies, three hypothetical scenarios may account for the maintenance of asymptomatic GpSGHV infection state. The first is a persistent infection whereby the virus remains in specific host cells with low-level production of virions, but without causing substantial cell damage . The sec feeding . In addi feeding  may reflDrosophila melanogaster, DCR1 is involved in both siRNA and miRNA pathways, while DCR2 is only involved in the siRNA pathway. Additionally, Drosophila AGO1 and AGO2 enzymes may participate in both the siRNA and miRNA pathways [Amongst the possible host mechanisms that keep GpSGHV under control is the insect’s RNA interference (RNAi) machinery, which regulates both host and viral gene expression by use of small RNAs that bind to their complementary messenger RNA (mRNA) targets , 13. Thipathways .DCR2 enhanced the susceptibility of Helicoverpa armigera to infection by H. armigera single nucleopolyhedrovirus (HearNPV) [Drosophila during infection by Flock House virus (FHV), Drosophila C virus (DCV), and Sindbis virus (SINV) [The siRNA-mediated RNAi pathway is a potent antiviral immune pathway in insects , 26 and HearNPV) . Similars (SINV) . In addis (SINV) . To estas (SINV) –34. In ts (SINV) .G. pallidipes and whether this would downregulate the development of SGH and instead induce a covert infection state (persistent or latent). To accomplish this, we performed comparative analyses of the expression of AGO, DCR and Drosha between artificially (intra-hemocoelic) virus injected and uninfected (PBS injected) individuals, and between asymptomatic and symptomatic infected flies (with overt SGH symptoms). It should be noted that artificial injection of the virus does not result into overt SGH in the same  generations but in the progeny flies [G. pallidipes. The data obtained in this study offer a rationale for similar studies on other Hytrosaviridae family members and may open novel strategies to manage SGHVs in insect production facilities.In the current study, we evaluated whether GpSGHV infection induces an RNAi response in ny flies . We compG. pallidipes, G. m. morsitans, G. f. fuscipes, G. p. palpalis, G. austeni and G. brevipalpis resulted in the identification of AGO 1, 2 and 3 in all these species  and the PIWI domain (executioner of the RNase activity)  Fig. . These rDrosophila DCR1 protein in G. pallidipes and G. brevipalpis, but the other Glossina species included in this study contained a DCR1 protein homolog  as reported in Drosophila [Drosha was present in all six tsetse species investigated  protein homologs, which were organized as in their orthologs in D. melanogaster , DCR2, and Drosha in flies injected with the virus suspensions compared to the PBS-injected control flies. Additionally, we correlated the expression levels of these RNAi-related genes to the level of virus replication by quantifying the expression levels of GpSGHV odv-e66, a conserved and late viral gene. Compared to the control (PBS-injected) fly group, the expression of odv-e66 increased significantly with time   and AGO2  but not AGO3 , of which the AGO2 (involved in siRNA pathway) was the most upregulated  in response to the virus injection  in the virus-injected flies compared to the levels observed in the PBS-injected flies  Fig. , implyinion Fig. . Howeveries Fig. . This suDCR and AGO family genes in the virus-injected G. pallidipes flies described above with the expression levels in symptomatic (flies with overt SGH symptoms and high virus titers) and asymptomatically infected individuals (flies with low virus titers)  , AGO2  and Drosha  were significantly downregulated in symptomatic G. pallidipes flies as compared to asymptomatically infected flies , or AGO3  expression between the asymptomatic and symptomatic infected flies  Fig. . We founies Fig. . There wies Fig. .Fig. 4CoAGO2 knockdown on GpSGHV infection. The AGO2 gene, which is involved in the siRNA pathway, was chosen for knockdown largely because its expression levels were significantly modulated in both the virus-injected flies   , DNApol , SGHV038  and SGHV091  compared to the controls  Fig.  and tset01) Fig. , respectG. pallidipes by quantifying the expression of both the host  and four viral genes. The finding that AGO and DCR proteins in Glossina species contain the functional domains or motifs known to mediate the RNAi response strongly indicate that the RNAi machinery is functional in tsetse, presumably in a similar version as reported in Drosophila [Drosophila.In the current study, we have investigated the potential involvement of the RNAi machinery during GpSGHV infections in osophila . It is kosophila  discoverosophila . MoreoveDCR2 in G. pallidipes and G. brevipalpis, and only DCR1 in G. p. palpalis and G. austeni suggest that in these species only one of the respective proteins is involved in the siRNA and miRNA pathways. In contrast, genes for both DCR1 and DCR2 proteins were found in G. m. morsitans and G. f. fuscipes. Given that in Drosophila DCR1 and DCR2 are involved in the miRNA and siRNA pathways, respectively [Glossina species as well. Alternatively, they might be involved in both pathways, as suggested for the other Glossina species as there were no differences in DCR1 or DCR2 protein sequences of species with one or both DCR proteins, which could be linked to the RNAi pathways. The DCR proteins can be involved in both siRNA and miRNA pathways [Glossina species [The presence of only ectively , it is ppathways  or be inpathways . Taken t species , 41.G. pallidipes, and the reactivation from viral persistence/latency to overt symptomatic infection that is associated with overt SGH symptoms [AGO2 in GpSGHV-injected flies suggests that the virus infection induces the host’s siRNA-mediated response, presumably to inhibit the virus infection. Notably DCR2, which might be involved in both siRNA and miRNA pathways in G. pallidipes, was similarly upregulated during virus infection. The upregulated expression of both AGO2 and DCR2 post virus injection in G. pallidipes, which are key components in the dsRNA-mediated gene silencing in several insects, suggests a functional RNAi-mediated innate immunity response in Glossina species. However, more work is required to elucidate the precise details of this pathway in Glossina as well as the involvement of the RNAi machinery in other members of the Hytrosaviridae family.Currently, the mechanism(s) permitting the covert (asymptomatic) GpSGHV infection in symptoms , are pooAGO2 and DCR2 in virus-injected flies, the comparative analysis of the expression of the two genes between asymptomatic and symptomatic infections showed a significant downregulation of the expression of AGO2, but insignificant upregulation of DCR2 in the flies exhibiting diagnostic SGH symptoms. The high expression of siRNA pathway genes in the virus-injected flies suggests a tight control of the virus via the RNAi response during asymptomatic GpSGHV infections. However, during symptomatic infections as evidenced by increased virus titers, the siRNA pathway may be compromised (as supported by the low expression of AGO2) enabling the virus to escape the RNAi-mediated innate immunity, thereby increasing virus titers and in turn causing the detectable SGH symptoms. A similar outcome has been documented in the case of the African malaria mosquito, Anopheles gambiae, where dsRNA-mediated silencing of AGO2, which functions in conjunction with DCR2 in this mosquito, resulted in increased O’nyong-nyong virus (ONNV) viral loads [DCR2 was not considered a suitable candidate for the knockdown assays in this study. Therefore, we assessed the potential involvement of dsRNA-mediated gene silencing (siRNA pathway) in G. pallidipes by knockdown of AGO2.In contrast to the above-mentioned increased levels of al loads . It shouG. m. morsitans flies could persistently silence expression of tsetse EP, a gene that is demonstrated to protect the fly against establishment of trypanosome infections in the midgut [AGO2 in G. pallidipes did not alter the transcript levels of the selected GpSGHV genes implying that AGO2 knockdown had no effect on the GpSGHV infection. These findings are in contrast to previous results; for instance in Drosophila melanogaster in which flies that were deficient in the DCR2 protein showed increased susceptibility to infection by members of three different RNA virus families; i.e. FHV (Nodaviridae), DCV (Dicistroviridae), and SINV (Togaviridae) [DCR2 and AGO2) or the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway, are not only more sensitive to infection by several viruses, but also harbor higher viral titers than their wild-type counterparts. The JAK-STAT pathway is also a conserved insect innate immune antiviral response [D. melanogaster defective for the AGO2 were found to be hypersensitive to infections by DCV, an infection which also supported a 1000-fold increased production of progeny virus [Several examples of the function of RNAi in insects have been clearly demonstrated, including for species from the orders Diptera, Dictyoptera, Isoptera, Hymenoptera and Orthoptera , 44. In e midgut . In the viridae) . In generesponse , 38, 47.G. pallidipes did not cause a significant impact on the fly immunocompetence. Notably, in addition to the RNAi pathway investigated in this study, the JAK-STAT, immune deficiency (imd) and Toll immune pathways are also involved in elimination of viruses [G. pallidipes flies in our study alternative antiviral pathways in these flies were able to control the virus infections.The results obtained from the current study showed that reduction of RNAi efficiency in  viruses –50. The Glossina spp. We detected RNAi key genes in all the analyzed Glossina species, which may indicate a functional antiviral role of RNAi machinery in tsetse flies. In G. pallidipes, the model Glossina species in this study, the siRNA pathway genes AGO2 and DCR2 were upregulated during virus infection, which confirmed the involvement of the RNAi response in the flies’ defense against GpSGHV. We also noted that in G. pallidipes, the siRNA pathway was compromised during symptomatic infection as evidenced by the low expression of AGO2. However, although knockdown of AGO2 in G. pallidipes did not have an impact on virus infection, it would be worthwhile to further investigate the long-term effects of the gene knockdowns on GpSGHV transmission and the expression of SGH symptoms in F1 progeny produced by the parents with the knockdown. The F1 generation is of interest since induction of SGH symptoms does not occur in the parental generation of flies that are intra-hemocoelically injected with the virus but are observed in the subsequent F1 generation produced by injected mothers [Hytrosaviridae family members and may open novel strategies to control SGHVs in insect production facilities.Given the high diversity of pathogens, their elimination by host organisms is challenging and therefore many organisms, including insects, employ multiple mechanisms to deal with them. The RNAi machinery, particularly the siRNA pathway, plays a central role in insects by specifically recognizing and eliminating invading pathogens and other invasive elements such as transposons. This study has elucidated important milestones in the infection of GpSGHV in  mothers . It may  mothers . The nexG. pallidipes flies used in this study were obtained from the colony maintained at the Joint FAO/IAEA IPCL, Seibersdorf, Austria. G pallidipes was selected for this study because, unlike other species, it shows both asymptomatic and symptomatic virus infections [G. pallidipes flies were maintained in controlled insectaria with 70–80% relative humidity, 24 ± 1 °C temperature and 12 h photo-phase. The flies were fed for 10–15 min, 3 times per week on defibrinated bovine blood using an in vitro membrane feeding system [G. pallidipes male flies; viral titers in the gland homogenates (in PBS) were estimated by qPCR as described previously [6 virus genome copies per μl.The fections . The expg system . The vireviously . The expG. pallidipes genome contains the key RNAi pathway genes, AGO, DCR and Drosha sequences were retrieved from the VectorBase database [G. morsitans morsitans [− 2). To determine the conservation of RNAi in Glossina species, sequences of these core genes for G. fuscipes fuscipes, G. palpalis palpalis, G. austeni and G. brevipalpis were similarly retrieved from VectorBase. The functional domain architecture of the retrieved AGO, DCR and Drosha sequences was analyzed using the ScanProsite tool [D. melanogaster for which RNAi mechanisms and pathways have been demonstrated. Multiple alignments of protein-coding loci of the identified gene sequences were performed in BioEdit [To determine whether the database  using asorsitans   were injected with either the virus inoculum as described above, or phosphate buffered saline (PBS) as control. Following the injections, four females and four males were sampled within 1 h post injection and at 7, 14 and 21 days post injection (dpi). Total RNA was extracted from individual whole bodies of the sampled flies using Trizol reagent  according to the manufacturer’s instructions. Contaminating DNA was removed from the extracted RNA by treating the samples with DNase 1 , after which the concentration of the RNA was quantified using a Nanodrop ND-1000 spectrophotometer . Complementary DNAs (cDNAs) were synthesised using the SuperScript® III Reverse Transcriptase kit  following the manufacturer’s instructions. The iQ SYBR green supermix  was used for RT-qPCR analysis. The viral infection was assessed by quantifying the expression of GpSGHV odv-e66, a conserved, late viral gene , followed by expression analysis of AGO, DCR and Drosha transcripts, using the PCR cycling conditions: 95 °C for 3 min, followed by forty cycles of 95 °C for 10 s, 60 °C for 1 min, then 95 °C for 1 min and 55 °C for 1 min, using the primers shown in Table β-tubulin was used to normalize gene expression.To investigate the impact of GpSGHV on the core RNAi genes in AGO, DCR and Drosha between asymptomatic and symptomatic infected flies, 10-day old F1 progeny flies produced by virus injected mothers were screened under a stereo microscope for the occurence of diagnostic SGH symptoms. Total RNA was extracted from whole bodies of 8 asymptomatic and 8 symptomatic infected flies . The viral infection was estimated by quantification of the GpSGHV odv-e66 transcripts, followed by expression analysis of the three genes as described above. These expression analyses were replicated three times .To determine the differential expression of AGO2 mRNA for the synthesis of dsRNA constructs were determined by siRNA design software (default setting) [− 2) search of VectorBase. Following identification of the siRNAs, primers for dsRNAs synthesis were designed to flank the most effective siRNAs based on the above-described steps and a T7 promoter sequence added on each primer  , which uAGO2, total DNA was isolated from G. pallidipes using the Qiagen DNeasy Blood and Tissue kit . The extracted DNA was subsequently used to produce T7 tailed PCR amplicons of AGO2 using primers designed to contain 5′-T7 promotor sequences .To generate dsRNA to knockdown  tsetse, , 46 was AGO2 knockdown on GpSGHV infection, teneral G. pallidipes flies were divided into three groups each consisting of 40 females and 40 males and offered one blood meal. After 48 h, two of three groups were injected with 4 μl of either AGO-2 or tsetse EP dsRNAs (2.5 μg/μl dsRNA in RNase free water)  AGO2 and tsetse EP gene transcripts using the qPCR primer sets listed in Table AGO2 knockdown on GpSGHV infection was assessed by RT-qPCR quantification of mRNA transcripts of the selected conserved GpSGHV genes; the per os infectivity odv-e66 (SGHV005) gene, DNApol (SGHV079) gene involved in DNA replication, a tegument gene (SGHV038) and capsid gene (SGHV091) [odv-e66 gene transcripts and the total virus copy numbers has been previously reported [AGO2 knockdown on virus replication.To assess the effect of SGHV091) , 60. Notene SGHV08 and capλ-1)/λ in other cases. T-tests were used for the comparison of RT-qPCR data.All quantitative RT-qPCR results were representative of at least three independent biological experiments, each with three technical replicates. Statistical differences in the expression of the above described host and viral genes between the different treatments and the controls were performed with RStudio v1.0.143   and the degrees of freedom (D.F) in column 8. (DOCX 23 kb)Regression parameters applied in the statistical analysis. For each regression line (column 2) found in the figures (column 1), the confidence interval of lambda values (column 3) obtained based on the Box-Cox routine to determine the method of transformation (column 4) of the data, the intercept values (column 5), the t value (column 6) the Additional file 2:AGO2 dsRNA design and off-target prediction. A). AGO2 mRNA sequence; the primers flanking the knockdown sequence are highlighted in cyan, predicted siRNAs in grey and qPCR primers in yellow. (B) Shows a graphical view of effective siRNAs candidates. (TIF 9175 kb)Result of Additional file 3:tsetse EP dsRNA design and off-target prediction. A). Tsetse EP mRNA sequence; the primers flanking knockdown sequence are highlighted in cyan, predicted siRNAs in grey and qPCR primers in yellow. (B) Shows a graphical view of effective siRNAs candidates. (TIF 8491 kb)Result of Additional file 4:AGO2 on virus infection in G. pallidipes. Teneral G. pallidipes flies were collected and offered one blood meal. 48 h later the flies were divided onto three groups and injected with 4 μl AGO2 dsRNAs, tsetse EP dsRNAs or RNase-free water. Five days later, each of the 3 groups was divided into two and each injected with either 2 μl of the virus suspension or with PBS. Three females and 3 males were sampled from each of the described treatments at 1 h post injection, and at 7, 14 and 21 days post injection to determine the effect of AGO2 knockdown on virus infection. (TIF 1030 kb)Step by step procedure to determine the effect of Knock-down of


The viruses they discovered were long tailed giant viruses named Tupanviruses. The genomes of Tupanviruses are 1.4–1.5 Mb of DNA, harboring the most complete set of genes for the translation apparatus among known viruses with up to 70 tRNAs and 20 aminoacyl-tRNA synthetases, implying the markedly reduced dependence of Tupanvirus replication on the host molecular machinery. Tupanviruses were consistently shown to infect a broad range of protist species including Acanthamoeba spp., Vermamoeba vermiformis, Dyctiostelium discoideum, and Willartia magna. This wide range of hosts is atypical among known giant viruses. Another interesting finding was that when infections by Tupanviruses were tested against the bacteria-feeder Tetrahymena, Tupanviruses failed to replicate in Tetrahymena; however, their virions exerted cytotoxic effects against this non-host protist. Therefore, Abrahão et al. performed in vitro experiments that simulated multiple virus-cell interactions, in which each of the Tupanviruses and Mimiviruses was cultured with their host (Acanthamoeba) and predator (Tetrahymena), the latter of which feed on giant viruses. The Tupanvirus population was maintained in the co-culture experiment because their virions diminished the activity of the predator (Tetrahymena) and successfully replicated in the host (Acanthamoeba). In contrast, Mimivirus particles, which are not toxic to Tetrahymena, were rapidly eaten by Tetrahymena, and, thus, did not successfully replicate. This finding indicates an ecological strategy not reported for viruses whereby their “seeds  are used as a weapon to control the population of their predators; however, this has not yet been validated in natural microbial communities.Viruses express phenotypes to alter their surrounding environments, thereby increasing their fitness. These surrounding environments include host metabolism and behavior. A recent study by Abrahão et al.  suggester “seeds ” . It integrates into the genome of the marine protist C. roenbergensis and resides in the host genome as a provirophage. Genes of the mavirus provirophage are transcriptionally silent, but are activated when the host protist encounters the giant CroV virus, the predator of the protist. Activation of the provirophage leads to mavirus virion production, the unsuccessful replication of CroV, and lysis of the protist. Disseminated infectious mavirus virions then suppress CroV replication and, thus, enhance the survival of the host protist population. Therefore, the mavirus provirophage induces the altruistic behavior of its host, in which host cells choose death in order to protect their sisters. In this manner, mavirus increases the chances of replicating itself (through the activation by CroV infection and subsequent co-infection with CroV to the protist host) and maintaining the host population (by suppressing the propagation of giant CroV viruses).Besides this outstanding discovery, a series of studies described the remarkable survival strategies of viruses that control viral host behavior and metabolism . VirophaLymantria dispar nucleopolyhedrovirus; LdMNPV) that causes tree-top disease in infected lepidopteran larvae  of infected larvae.Viral strategies to manipulate host behaviors are widely recognized in insect-virus systems , 47. A nn larvae , 36. Inf  infecting a fungal endophyte (Curvularia protuberate), which is associated with a plant (the panic grass Dichanthelium lanuginosum). In this tripartite virus-fungus-plant symbiotic system, the RNA virus confers thermal tolerance on the plant, which grows on geothermal soil in Yellowstone National Park .e.g., psbA encoding the photosystem II core reaction center protein D1) are known to be expressed during infection and contribute to maintaining the photosynthetic activity of cyanobacterial cells during the lytic infection cycle of viruses   .31 viral particles on Earth . The estimated abundance of virocells among living cell populations in nature eloquently explains the ecophysiological impact of unseen viral phenotypes, such as the significant regulation of microbial food webs and biogeochemical cycles (The above described phenotypes (or “extended phenotypes ”) of viron Earth , and preon Earth , 40, 41.on Earth . These fon Earth , which rl cycles , 17, 28.et al. . It currently remains unclear whether each of these viral families has the large genetic diversity of “Megaviridae”; however, notable diversity has been recognized in an RNA virus family  may exc” family ” is curr” family , 43, 44.s family , althougy (e.g., , 45, 46


Primula have shown that the controlling self-incompatibility (S)-locus is not allelic, but is instead a small hemizygous region of only a few genes in the thrum genotype. How these genes alter the development of floral morphology and the specificity of self-incompatibility is still not completely clear. In order to start to identify genes regulated by the S-locus and elucidate the large-scale biological processes affected, we used RNA-seq data from floral buds of heteromorphic P. vulgaris pin  and thrum  morphs at early and late developmental time points. Differential expression between the two morphs was assessed at both time points and Gene Ontology term analyses of these gene sets were conducted. Our findings suggest that the S-locus regulates a large number of genes outside its physical bounds and likely sets up a cascade of expression changes. Additionally, we found evidence to suggest that there may be a timing difference in pollen development between the morphs, with pin pollen development proceeding earlier than thrum pollen development. This finding provides insight into how morphological differences in pollen between the morphs may be established, but intriguingly, could also be related to the self-incompatibility phenotype.Recent findings on the molecular basis of heteromorphic self-incompatibility in  Primula vulgaris possesses a classical HSI breeding system that combines distyly with physiological self-incompatibility and has historically been of considerable interest to geneticists and evolutionary biologists  is one of many breeding systems that have evolved in the angiosperms to prevent inbreeding and promote out-crossing . Primulaiewed in ,3). P. vlization ,5. Theselization ,7.Primula identified a single diallelic genetic locus (the S-locus) controlling the phenomenon. The short-styled thrum morph was considered heterozygous (Ss) for the S-locus, while the long-styled pin morph was considered homozygous recessive (ss) [S-locus super-gene complex, and classical genetic analyses of these homostyles interpreted as indicating the presence of at least three genetic sub-components of the locus, termed G , P (pollen size and incompatibility phenotype), and A  [Classical genetic analyses of HSI in ive (ss) . Rare inive (ss) ,10,11. TPrimula [S-locus is a hemizygous insertion found only in the thrum morph genome, and contains just five genes. As one of the S-locus genes (PvGLO2) has homology to MADS-box transcription factors, and another is likely involved in hormone regulation (CYP734A50), it is almost certain that many genes outside of the S-locus are under its control. Previous studies have reported transcriptomes of Primula species, but were focused on identifying morph or species-specific transcripts [Primula. Examination of these downstream processes, controlled by the S-locus components, which ultimately lead to the phenotypes seen in HSI, is a necessary aspect for understanding the evolution of this breeding system in Primula.Several major breakthroughs have recently revolutionized our understanding of the genetic control of HSI in Primula ,13,14. Inscripts ,16. No pP. vulgaris pin and thrum flower buds at early and mature developmental stages, using floral bud size as a metric for overall flower development. These reads were mapped to our previously reported transcriptome [P. vulgaris. Most significantly, a potential difference in the timing of pollen development between floral morphs was identified. Here, we utilize RNA-seq data from criptome  and usedThe sequence data analyzed here were originally generated and assembled into a reference transcriptome in . BrieflyPreviously unreported RNA-seq metrics with significance to this study are as follows: a total of 425.75 Mbp of sequence from 2,584,552 reads were generated. After quality filtering and adapter trimming, a total of 343.61 Mbp from 2,413,892 reads remained . These r−10 in the National Center for Biotechnology Information (NCBI) Non-Redundant Protein (nr) database. The remaining transcripts (~10%) are most likely either unique to Primula vulgaris, or too small to generate a significant match using BLASTx. Out of the transcripts with significant BLASTx scores, 13,767 (92.9%) were assigned putative Gene Ontology terms.BLAST2GO software  was usedp-value < 0.001 to be significantly differentially expressed. As the thrum morph genome should be the same as the pin, except for the hemizygous insertion of the S-locus, we considered any difference in expression between floral morphs to be resultant of the action of the S-locus. Therefore, transcripts more highly expressed in thrum flowers compared to pin flowers were considered S-locus up-regulated, while those with lower expression in thrum were considered S-locus down-regulated. Using these criteria, we identified a total of 540 transcripts as DE early in development and 3101 transcripts as DE at the later stage . We considered transcripts with greater than or equal to three-fold difference in normalized expression and a Benjamini–Hochberg false discovery rate (FDR)-corrected er stage . To asseS-locus up-regulated, and 311 transcripts as S-locus down-regulated. A full table of these DE transcripts can be found in S-locus gene PvGLO2 in the up-regulated set, but we did not find evidence of expression for the other S-locus genes. In 3–4 mm thrum flower buds, 229 transcripts were identified as S-locus up-regulated transcripts (nearly 20% in 3–4 mm buds) were annotated with gene expression related GO terms. At deeper ontology levels, we found more specific terms, such as negative regulation of gene expression and post-transcriptional gene silencing.S-locus down-regulated transcripts were found to have a very different composition of over-represented GO terms. The largest class (~20%) was annotated with GO terms involved in oxidation-reduction processes. Likely related to these, a large number of transcripts involved in lipid and hormone metabolic processes were also present in this group. Upon closer examination at deeper ontology levels, several terms related to brassinosteroid metabolic processes were identified. One of the DE transcripts annotated with these terms was a homolog of the Arabidopsis gene ROTUNDIFOLIA3, a cytochrome P450 involved in brassinosteroid biosynthesis [S-locus down-regulated transcripts, such as gametophyte development, pollen development, pollen wall assembly, pollen exine formation, pollen sperm cell differentiation, and male gamete generation. A full list of over-represented GO terms can be found in Supplementary Tables S5 and S6Early t class ~% was annynthesis . There wS-locus and 1612 down-regulated  proteins. One of these had been previously identified as differentially expressed in Primula by suppressive subtractive hybridization [At the 10+ mm stage, 1489 transcripts were found to be up-regulated by the dization . These tS-locus up-regulated transcripts in 10+ mm buds revealed a sizable portion (~11%) annotated with carbohydrate metabolic process related terms. Likely associated with these were several over-represented terms related to cell growth, such as external encapsulating structure organization, wax biosynthesis process, cell wall organization, and cell wall modification. In contrast to the early developmental stage, sexual reproduction terms, such as sexual reproduction, cellular process involved in reproduction, gamete generation, pollen germination, and male gamete generation, were all over-represented in this up-regulated set. Finally, unlike the 3–4 mm S-locus up-regulated transcript set, over-representation of gene expression related terms was not seen in the genes up-regulated by the S-locus at this later stage. Over-represented Biological Process GO terms in levels 4–6 are shown in S-locus down-regulated set, we found the largest percentage of transcripts was annotated with terms related to biosynthetic processes. Unfortunately, it is not readily clear which specific process might be covered by this rather broad term. Cellular communication terms, including cell communication, signal transduction, and cell–cell signaling, were also found to be over-represented. The only developmental process that was over-represented in the down-regulated transcript set was embryo development. Upon examination of deeper ontology terms, we found several related to gene regulation, including both gene silencing and positive regulation of gene expression  than early in development (540). To interpret this result, we considered several factors. First, the 3–4 mm early flower stage is the first point at which developmental differences become noticeable between the two morphs ,18. Secove genes ,13,14. Wgene set . FurtherRALF-like transcript was identified as DE between pin and thrum flowers [RALF-like transcripts almost exclusively expressed in the thrum morph. Critically, these RALF-like genes are not found in the S-locus, indicating that they are instead up-regulated by the effects of the S-locus at the later developmental stage. RALF proteins are a family of small, secreted, cysteine rich peptides implicated in numerous developmental processes, particularly cell elongation and expansion . In floectively ,25,26. Iin roots . Tantaliin roots . These oS-locus genes previously identified [PvGLO2 was the only one with detectable expression within our dataset. While a detailed time course expression analysis of S-locus genes has not yet been carried out, there are two possible explanations for the lack of detection. One possibility is that they are not expressed at these time points. The second is that they are expressed at very low levels below the sensitivity of our methods. The latter possibility is supported by previously reported expression data, in which PvGLO2 was expressed at a dramatically higher level than the other four S-locus genes, albeit at an unreported time point [PvGLO2 spans a broad period of time, from early to late floral development. As PvGLO2 shows homology to MADS-box transcription factors and CYP734A50 is likely involved in brassinosteroid regulation, these genes represent strong candidates for setting up the large-scale shift in gene expression observed between the early and late time points. Out of the five entified ,13,14, PS-locus gene CYP734A50 is through degradation of brassinosteroids (BR), causing a reduction in cell expansion in developing pistil cells, and leading to the short style found in the thrum morph [Arabidopsis ROTUNDIFOLIA3, a cytochrome P450 thought to convert typhasterol to castasterone [S-locus may have co-evolved to down-regulate expression of these genes, reinforcing the short style phenotype. Alternatively, BR may be acting in other organs besides the style. There is a precedent for such a role in relation to breeding systems. It has recently been reported that higher than optimal BR limits petal-cell proliferation in self-compatible Capsella rubella, leading to a smaller petal size than in the outcrossing species C. grandiflora [The proposed mechanism of action for the um morph . In viewasterone ,21. Castasterone . Our finndiflora . These pS-locus, while later in development, they were up-regulated. This is suggestive of pollen developmental program differences between the pin and thrum morphs when compared with the other floral organs, with pollen development being delayed in thrum. This finding could underlie some of the differences observed between pin and thrum pollen, with pin producing roughly twice as many pollen grains, but those grains being only half the size (volume) of those of thrum pollen [S-locus components and structure now known, it appears likely that the mechanisms of self-incompatibility differ between the morphs. Indeed, this has previously been suggested because of the very different sites of SI action that are observed between the two morphs [S-locus gene initiating the observed differences in pollen related gene expression is PvGLO2. First, PvGLO2 has homology to GLOBOSA type B-functional MADS-box transcription factors, and hence is likely to directly impact transcription of other genes. Second, our preliminary investigations have found PvGLO2 to be primarily expressed in the anthers and petals . Finally, transposon insertion into PvGLO2 has been correlated with the development of short homostyly [PvGLO2 in the control of anther height. While we find this highly likely in this analysis, as the transcriptomes were from whole buds, we are unable to identify specific downstream pathways though which PvGLO2 might control anther height. Future RNA-seq, using specific tissues or comparing thrum and short homostyle flower buds or stamen, will be necessary to address this.Intriguingly, we found that pollen development related genes were differentially expressed at both early and late stages. Early in floral development, we found they were down-regulated by the m pollen ,31. Furto morphs ,33,34. To morphs . For sevomostyly . This laP. vulgaris plants were grown under greenhouse conditions. Floral buds  were collected from both mating types at two developmental time points (3–4 mm and 10+ mm bud sizes), flash frozen, and stored at −80 °C. For each sample, 4 g of floral buds from six independent plants were used for RNA extraction and subjected to mRNA purification prior to library construction. Sequencing libraries for each mRNA sample were constructed from 200 ng of polyA mRNA using the Rapid RNA Library Kit , and sequenced with a Roche 454 FLX titanium . To increase sequencing depth in the young bud samples, mRNA from 3–4 mm thrum and 3–4 mm pin samples was also used to generate Ion Torrent sequencing libraries using the Ion Total RNA Seq V.2 kit , and sequenced with an Ion Torrent PGM. All library construction and sequencing was performed by the Washington State University Genomics Core Laboratory. Sequencing reads were trimmed to remove adapters, quality filtered using default parameters, and then used for de novo transcriptome assembly. Assembly was performed using the SeqMan NGen assembler , with default parameters .For more details, see . Briefly−10 and a “max hits” of 15. Gene Ontology term annotation of the transcripts was then performed using default settings.Annotation of the reference transcriptome was carried out using the BLAST2GO software package . Using Bp-value < 0.001 were considered significantly differentially expressed. All differential expression, the differences between thrum and pin, was considered to be a result of the actions of the S-locus. Putative functions of DE transcripts were considered based on GO terms annotated to them. For each up- and down-regulated set of transcripts, BLAST2GO was used to identify Biological Function GO terms that were over-represented when compared with the transcriptome as a whole. A p-value < 0.05 filter was used to identify over-represented GO terms. Transcript abundance calculations and differential expression analysis were performed using CLC Genomics Workbench (CLCBio). Read mapping was performed using default parameters and transcript abundance was normalized as Reads Per Kilobase per Million. Two DE analyses were performed; one comparing expression between 3–4 mm pin and thrum flower buds, and a second comparing expression of 10+ mm pin and thrum flower buds. Sequences with three-fold or greater difference in expression and a Benjamini–Hochberg false discovery rate (FDR)-corrected Sequence read data is available at the NCBI, BioProject Accession number PRJNA294594. Specific gene sequences discussed in this manuscript have been deposited in GenBank.Primula S-locus [S-locus lead to large-scale transcriptional differences between the morphs throughout floral development. Furthermore, analysis of differentially expressed transcripts forms a basis for proposing hypotheses regarding the mechanisms behind HSI. These include the potential for additional roles for brassinosteroid signaling beyond controlling style length, and that differential expression of pollen development genes may contribute with the observed differences in pollen number, morphology, and potentially self-incompatibility. The results of this preliminary study suggest that detailed examination of Primula pollen development in both morphs is warranted, as well as transcriptomic investigation of homostyle flowers to examine how each S-locus component contributes to the observed transcriptomic differences.Our findings here, coupled with the new findings about the nature and composition of the  S-locus ,13,14, s


Bovine parainfluenza virus type 3 (BPIV3) is one of the most important known viral respiratory pathogens of both young and adult cattle. It is also named “heat stress in transport”, causing morbidity and mass death. New variants of BPIV3 have been detected or isolated in China since 2008. Here, we isolate one BPIV3 strain (named BPIV3 BJ) in Madin-Darby bovine kidney (MDBK) cells from nasal samples collected in China. Phylogenetic analysis showed that our isolate is related to BPIV3 of the genotype A. The comparison of BPIV3-BJ and the reference Chinese isolate NM09 showed that these strains are highly divergent. We found many differences in the amino acid composition in the nucleocapsid (NP) protein among these genotype A strains. Since the NP protein has been implicated in immunization studies, our BPIV3 isolate will be useful for the development of immune assays and vaccine studies. The diversity of BPIV3 lineages that we found in China indicated ongoing evolution for immune escape. Our study highlights the importance of genetic surveillance for determining the effect of BPIV3 variability on pathogen evolution and population-scale immunity. Bovine respiratory disease complex (BRDC), a multi-factorial disease, is an economically important health problem of cattle worldwide. The disease is commonly referred to as “Shipping fever” and causes an increase in morbidity mortality rates . The mulBovine parainfluenza virus type 3 (BPIV3) was one of the most important viruses associated with BRDC in cattle . It was Up to now, based on phylogenetic analysis, BPIV3 has been divided into three genotypes: Genotype A, genotype B, and genotype C ,14,15,16Here, we describe the cell culture isolation and genomic sequencing of a BPIV3 genotype A strain isolated from cattle in China. Although BPIV3 is endemic in cattle, little is known about the pathogenesis of this virus and information regarding antigenic variation owing to the genetic variability is rare . The phyTen nasal swabs from cattle with a slight cough and nasal discharge in an auction market were collected from Shandong Province, China, in 2010. Nasal swabs were placed in virus collection tubes . RNA extraction was conducted following the manufacturer’s instructions accompanying the Viral RNA Rapid Extraction Kit . Potential infectious pathogens of BRDC, including BPIV3, BVDV, BHV-1, and BRSV, were detected by PCR. Nasal swabs were placed in virus collection tubes . RNA extraction was conducted following the manufacturer’s instructions accompanying the Viral RNA Rapid Extraction Kit . Potential infectious pathogens of BRDC, including BPIV3, BVDV, BHV-1, and BRSV, were detected by PCR. The conditions of PCRs for the detection of BVDV, BPIV3, and BRSV were as follows: Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, and 35 cycles for this stage; extension at 72 °C for 10 min; and storing at 4 °C for forever. The conditions of PCRs for the detection of BHV-1 were as follows: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 50 s, annealing at 60 °C for 40s, extension at 72 °C for 30 s, and 35 cycles for this stage; extension at 72 °C for 10 min; and storing at 4 °C for forever. Nasal samples, detected as BPIV3-positive, were used for virus isolation on Madin-Darby bovine kidney (MDBK) cells. Primers for PCR detection are shown in ® CCL-22™) were cultured at 37 °C with 5% CO2, in DMEM  supplemented with 8% horse serum. Virus isolation and determination of the median of tissue culture infective dose were performed on MDBK cells. Virus isolation was performed as follows: The three nasal swabs, positive for BPIV3, were filtrated through a 0.22 μm filter  and inoculated to a monolayer culture of MDBK cells cultured in Dulbecco’s modified eagle medium  supplemented with 8% horse serum . MDBK cells were maintained at 37 °C in an atmosphere of 5% CO2. The cytopathic effect (CPE) was examined daily. The storage solution was exposed to a ten-fold dilution and filtered with a 0.22 μm filter. Then, the filtrate was inoculated into MDBK cells for 1 h. Finally, the culture medium was replaced with DMEM containing 2% horse serum. MDBK cells inoculated with filtrate were cultured in an incubator continually for 72 h. The propagation of the virus was performed three times.Bovine kidney cells  were frozen and thawed three times before inoculating MDBK cells. MDBK cells inoculated with the virus produced typical cytopathic effects characterized by rounding, shrinkage, and cracking off . The sucThe whole genome of BPIV3-BJ was assembled using SeqMan. The genomic annotation was performed according to the blastx search. The complete genomic length of BPIV3-BJ was 15,480 bp with 36.1% GC content. The percentage of nucleotide sequence similarities was assessed with these BPIV3 strains for which complete genomic sequences were available in GenBank. BPIV3-BJ showed a low nucleotide similarity to BPIV3 strains located in genotype B (83%–83.6%) and genotype C (81.7%–82.1%). It shared a higher nucleotide identity with isolates located in genotype A. In total, 99% of nucleotide identity was observed between BPIV3-BJ and 910N (Genbank number: D84095). The virus isolated in this study showed a lower nucleotide identity with Chinese BPIV3 strains. Phylogenetic analysis showed that BPIV3-BJ  protein among some strains are summarized in In summary, we have described the isolation and genomic sequencing of BPIV3 genotype A strains isolated from cattle in China. Our isolate is highly divergent from the previously described isolate from China, NM09. The BPIV3 genotype A infection has been infecting livestock animals in China and causing enormous economic loss to the cattle industry. For this reason, the isolation and genomic characterization of our divergent BPIV3 genotype A strain will help the development of molecular diagnostic tools and vaccine studies.


Muscle flexibility is a main component of health-related fitness and one of the basic components of fitness for the performance in some sports. Sport and health professionals require the flexibility profile of soccer to define quantitative aims in the training of flexibility. The aim of this study was to identify age-related differences in lower extremity flexibility in youth soccer players.2) completed this study. Measures of eleven passive hip (hip extension (HE), hip adduction with hip flexed 90°(HAD-HF90°), hip flexion with knee flexed (HF-KF) and extended (HF-KE), hip abduction with hip neutral (HAB) and hip flexed 90°(HAB-HF90°), hip external (HER) and internal (HIR) rotation), knee (knee flexion (KF)) and ankle dorsiflexion (ankle dorsiflexion with knee flexed (ADF-KF) and extended (ADF-KE)) ranges of motion (ROM) were taken. Descriptive statistics were calculated for hip, knee and ankle ROM measured separately by leg (dominant and non-dominant) and age-group . The data was analysed using a one-way analysis of variance (ANOVA) to examine the interaction of 11 ROM in the different players’ age-group.Seventy-two young male soccer players , HAB , HIR , HER  and HAB-HF90° ROM, showing a progressive and irregular decrease in these ROM until the U19 team.Generally, U10 and/or U12 soccer players obtain the highest mean value in almost all ROM evaluated . Nonetheless, significant differences between the players’ age-groups are just found in HAD-HF90°(The findings of this study reinforce the necessity of prescribing exercises aimed at improving HAD-HF90°  ROM in U16, HAB ROM in U14, HIR ROM in U16 and U19, HER ROM in U12 and U19, and HAB-HF90°  ROM in U16 and U19 players within everyday soccer training routines. Soccer is by far the world’s most popular sport . AccordiSoccer requires players to perform many repeated high intensity movements such as sudden acceleration and deceleration, lots of changes of direction, jumping and landing tasks, as well as many situations in which players are involved in tackling to keep or obtain possession of the ball . OptimalMuscle flexibility is a main component of health-related fitness, and one of the basic components of the performance in some sports ; in soccSeventy-two young soccer players completed this study. The participants were recruited from five different teams  of a youth soccer academy . All theThe exclusion criterion was history of orthopaedic problems to the knee, thigh, hip, or lower back in the last 3 months due to the fact that residual symptoms could have an impact in the habitual players’ movement competency and/or lower extremity ROM profile. The study was conducted at the end of the preseason phase of the year 2016. The time frame of the study was selected to make sure that the players recruited to each team were definitive and stable within the testing period.Before any participation, experimental procedures and potential risks were fully explained to both parents and children in verbal and written form, and written informed consent was obtained. The experimental procedures used in this study were in accordance with the Declaration of Helsinki and were approved by the Ethics and Scientific Committee of the University of Murcia (Spain) (ID: 1551/2017).The passive hip extension (HE), hip adduction with hip flexed 90° (HAD-HF90°), hip flexion with knee flexed (HF-KF) and extended (HF-KE), hip abduction with hip neutral (HAB) and hip flexed 90° (HAB-HF90°), hip external (HER) and internal (HIR) rotation, knee flexion (KF), ankle dorsiflexion with knee flexed (ADF-KF) and extended (ADF-KE) ROM of the dominant and non-dominant leg were assessed following the methodology previously described  Fig. 1)Fig. 1).95) were calculated from the results of subsequent measurements. Results of pre-and-post-measurements showed a high reliability coefficient in all the tests . The MDC95 for each flexibility measure ranged from 3.7° to 6.9° .These tests were selected because they have been considered appropriate by American Medical Organizations, One week before the start of the study, all the soccer players completed a familiarization session with the purpose of getting to know the correct technical execution of the exploratory tests by means of the practical realization of each one of them. The dominant leg was defined as the participant’s preferred kicking leg. All tests were carried out by the same two sport scientists (one conducted the tests and the other ensured proper testing position of the participants throughout the assessment manoeuvre) under stable environmental conditions. The sport scientists were blinded to the purpose of the study and to the test results from previous testing sessions.Prior to the testing session, all participants performed the dynamic warm-up designed by After the warm-up, soccer players were instructed to perform, in a randomised order, two maximal trials of each ROM test for each leg, and the mean score for each test was used in the analysis. Soccer players were examined wearing sports clothes and without shoes. A 30s rest was given between trials, legs and tests.® Stainless) and “lumbosant” -lumbar support- to standardize the lumbar curvature  was used with an extendable telescopic rod , a metalurvature .® Stainless) was used.Before each assessment session, the inclinometer was calibrated to 0° with either the vertical or horizontal axis. The angle between the longitudinal axis of the mobilized segment was recorded (following its bisector) with the vertical or the horizontal . RegardiOne or both of the following criteria determined the endpoint for each test: (a) an examiner palpable or appreciated some compensation movement that increased the ROM onset of pelvic rotation, and/or (b) the soccer player feeling a strong but tolerable stretch, slightly before the occurrence of pain .p > .05). Descriptive statistics including means and standard deviations were calculated for hip, knee and ankle ROM measurements, separately by leg (dominant and non-dominant). Dependent sample t-tests were carried out to assess differences between the values of the dominant and non-dominant sides. Also, data was analysed using a one-way analysis of variance (ANOVA) to examine the interaction of 11 ROM at different youth teams. Post hoc comparisons were made using the Bonferroni test for pair wise comparisons. Analysis was completed using SPSS version 20 . The effect sizes (ES) of each variable were tested using eta squared (η2) between groups , HAB , HIR , HER  and HAB-HF90°  ROM, showing a progressive and irregular decrease in these ROM until the U19 team. On the contrary, the greatest mean value in the HF-KE  and HF-KF  ROM is observed in U19 players, but these differences are not statistically significant.Statistical analysis reported no differences between dominant and non-dominant sides for each ROM value, so the mean scores were used for comparing age-groups. p = .035) in HAD-HF90°, U12 (p < .001) and U19 (p = .001) players in HER, and U14 (p = .003), U16 (p < .001) and U19 (p < .001) players in HAB-HF90°. U12 players obtained the highest ROM in HAB and HIR; the Bonferroni post hoc comparisons showed statistical differences with U14 (p = .001) and U16 (p = .04) in HAB, and U16 (p = .001) and U19 (p = .003) in HIR. The Bonferroni post hoc test also indicated significant differences between U12 players and U14 (p = .019), U16 (p = .002) and U19 (p = .001) soccer players in HAB-HF90° ROM. Finally, the Bonferroni post hoc test showed significant differences in U14 vs. U10 (p = .011) and U14 vs. U19 (p = .021) in HAB, and U16 vs. U12 (p = .005) and U16 vs. U19 (p = 0.01) soccer players in HER , but not all (HF-KE and HF-KF) of the ROM assessed in comparison with their counterpart older players.Flexibility seems to decline with age . Johns &On the contrary, older players have shown better values of ROM in HF-KF and, mainly, in HF-KE. The HF-KE test assesses the flexibility of hamstring muscles. Some previous studies have also shown similar increases in older soccer players’ hamstring flexibility in comparison with their younger counterparts using the sit-and-reach test . During Appropriate body status requires a minimum of flexibility to respond to the demands of sport practise. Although there is no strong scientific evidence, flexibility appears to be also necessary for sport performance in youth. In U16 soccer players, greater flexibility of the lower back and upper thigh might discriminate players with high skill levels ; in younHowever, it is unclear what level of flexibility is optimum to improve performance and to prevent injuries in soccer. Optimal values may vary between muscle groups and different sports ; while aOne of the principal limitations of this research was the sample size used in each group ; the limited number of players in each of the youth academy teams hinders the athletes’ involvement. Other limitation could be that neither index of maturity nor player position was assessed within the study. Future studies should analyse possible differences in flexibility in regard to players’ maturational status and tactical position.U10 and U12 soccer players display highest values in five of the ROM assessed . On the contrary, older players (U19) showed better ROM in HF-KE and HF-KF tests in comparison with their younger peers. The ROM reductions in internal and external hip rotation and hip adduction and abduction movements throughout the studied ages could explain an adaptive response of the muscles involved in playing soccer. The ROM restriction could predispose players to abnormal movement patterns and increase the risk of future injuries in the joints involved. Thus, the findings of this study reinforce the necessity of prescribing exercises aimed at improving HAD-HF90° ROM in U16, HAB ROM in U14, HIR ROM in U16 and U19, HER ROM in U12 and U19, and HAB-HF90° ROM in U16 and U19 players within everyday soccer training routines.10.7717/peerj.6236/supp-1Supplemental Information 1Raw dataClick here for additional data file.


Begmovirus, family Geminiviridae) causing cassava mosaic disease in Asia, poses serious threats to cassava cultivation in Asia. However, the transmission of SLCMV in the areas into which it has recently been introduced remain largely unexplored. Here we have compared the transmission efficiencies of SLCMV by three widely distributed whitefly species in Asia, and found that only Asia II 1 whiteflies were able to transmit this virus efficiently. The transmission efficiencies of SLCMV by different whitefly species were found to correlate positively with quantity of virus in whitefly whole body. Further, the viral transmission efficiency was found to be associated with varied ability of virus movement within different species of whiteflies. These findings provide detailed information regarding whitefly transmission of SLCMV, which will help to understand the spread of SLCMV in the field, and facilitate the prediction of virus epidemics.In recent years, Sri Lankan cassava mosaic virus (SLCMV), a begomovirus (genus  Thanks to its inherent tolerance to abiotic stresses such as drought and infertile soils, cassava is now being widely grown in tropical Africa, Asia and Latin America, making it one of the most important crops in the world  and Sri Lankan cassava mosaic virus (SLCMV) were characterized in Asia . As for Due to the fact that cassava plants are normally vegetatively propagated, inter-regional spread of CMBs entails the transport of infected cuttings . For exaBemisia tabaci, a species complex consisting of more than 36 genetically distinct but morphologically indistinguishable cryptic species , tobacco (Nicotiana tabacum L. cv. NC89) and cassava (Manihot esculenta cv. HLS11 and SC8) were used. All cotton and tobacco plants were grown in a greenhouses under natural lighting supplemented with artificial lighting at controlled temperatures of 25 ± 3 °C, 14 L: 10 D. For insects, three whitefly cryptic species, of which two are invasive worldwide including MED and MEAM1, one is indigenous species in Asia, namely Asia II 1, were used. These three whitefly species were chosen as they exhibit abundant distribution in regions where SLCMV occurred, including Vietnam, Cambodia and South China (COI) GenBank accession codes are GQ371165 (MED), KM821540 (MEAM1) and DQ309077 (Asia II 1). Whiteflies of all three species were maintained on cotton plants in separate insect-proof cages in artificial climate chambers at 26 ± 1 °C, 14 h light/10 h darkness and 60–80% relative humidity. The purity of each whitefly culture was monitored every three generations using the mtCOI PCR-RFLP technique and sequencing as described before . Then equal amount (OD) of agrobacteria containing pBINPLUS-1.9A and pBINPLUS-1.9B were mixed. Agro-inoculation was performed with 1 mL syringe when tobacco plants reached 3–4 true leaf stage. Approximately one month later, infection of tobacco plants was examined by inspection of symptoms  were collected and released onto each test plants to feed for 96 h. Three replicates, each containing 10 plants were conducted for each whitefly species. When cassava plants were used as test plants, groups of 30 whiteflies (Asia II 1 only) were collected and released onto each plant to feed for 120 h. Two test plants were used for each of the two cassava varieties used. Leaf-clip cages were used to enclose the whiteflies on the test plants . Then wh2.4actin as a reference gene (For quantification of SLCMV DNA in whitefly whole body after various virus access periods (AAPs), whitefly adults were collected in groups of 15 and lysed in lysis buffer  followed by 1.5 h incubation at 65 °C and 10 min at 100 °C to obtain the template for the subsequent virus quantification. Sample preparation of whitefly honeydew after whiteflies have been feeding on infected plants for 48 h and 96 h were conducted as described before . For orgnce gene .2.5For PCR detection of SLCMV in whitefly whole body, whiteflies were collected individually after various AAPs. For organs, midguts were dissected and collected individually, and haemolymph from one whitefly was collected as one sample. For primary salivary glands, a pair of them was dissected from the same whitefly and analyzed as one sample. All the samples were then subjected to DNA extraction using lysis buffer as mentioned above and PCR with primers SLCMV-A-PCR-F and SLCMV-A-PCR-R .2.6Immunofluorescence was performed as per the protocol described by 2.7-△Ct as normalized to whitefly actin. For the comparison of transmission efficiency and quantity of virus, normal distribution tests were performed prior to analysis, and then Kruskal-Wallis test was used for analysis of significance. All data were presented as the mean ± standard errors of mean (mean ± SEM). The differences were considered significant when P < 0.05. All statistical analyses in the present study were undertaken using SPSS 20.0 Statistics and EXCEL.For the quantification of virus in whitefly whole body and organs, all real time data were calculated using 233.1B. tabaci complex, namely Asia II 1, MEAM1 and MED were compared. The average transmission efficiencies were 87.2% for Asia II 1, 3.3% for MEAM1 and 16.7% for MED as indicated by symptom . As shown in 3.42 = 26.495, 24.879, 14.873 for midgut, haemolymph and primary salivary gland, P < 0.05 in all cases; After a 96 h AAP, whitefly midguts, haemolymph and primary salivary glands samples were prepared and subjected to SLCMV quantification. In all three organs, the copy number of virus differed significantly among three whitefly species . For midguts, while SLCMV signals were detected in the midguts of Asia II 1 and MED whiteflies after 48 and 96 h AAPs, respectively, no viral signal was detected in the midguts of MEAM1 whiteflies even after a 168 h AAP; and in the midguts of Asia II 1 and MED whiteflies, viral signals, mostly found in the filter chamber, became stronger as AAP increased; notably, stronger viral signals were found in midguts from Asia II 1 than those from MEAM1 after whiteflies were given 96 h and 168 h AAPs . A simil4Arabidopsis thaliana plants  when only Asia II 1 and MEAM1 are considered, suggesting something in common in those three viruses, probably in their coat proteins considering the function of coat proteins . For begTaken together, here we show that indigenous Asia II 1 whiteflies were able to readily transmit SLCMV and invasive MEAM1 and MED whiteflies can only transmit this virus with very low efficiency. Further analysis revealed that the differential transmission might be due to the differential capacity of SLCMV to be retained by different whiteflies and to transport across the midgut of different species of whiteflies. To the best of our knowledge, this study is the first to explore the detailed whitefly transmission profile of an Asian CMBs. Our findings identified Asia II 1 whiteflies, but not MEAM1 or MED whiteflies as efficient vectors for SLCMV, which will help to evaluate the potential threat of SLCMV to cassava production in many regions and to facilitate the prediction of virus epidemics.Yao Chi: Conceptualization, Methodology, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing.Li-Long Pan: Conceptualization, Methodology, Formal analysis, Data Curation, Writing - Original Draft, Writing - Review & Editing.Sophie Bouvaine: Conceptualization, Validation, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.Yun-Yun Fan: Investigation.Yin-Quan Liu: Resources, Supervision, Project administration, Funding acquisition.Shu-Sheng Liu: Conceptualization, Resources, Supervision, Project administration, Funding acquisition.Susan Seal: Conceptualization, Validation, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.Xiao-Wei Wang: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition.


Visualization of algal viruses has been paramount to their study and understanding. The direct observation of the morphological dynamics of infection is a highly desired capability and the focus of instrument development across a variety of microscopy technologies. However, the high temporal (ms) and spatial resolution (nm) required, combined with the need to operate in physiologically relevant conditions presents a significant challenge. Here we present a short history of virus structure study and its relation to algal viruses and highlight current work, concentrating on electron microscopy and atomic force microscopy, towards the direct observation of individual algae–virus interactions. Finally, we make predictions towards future algal virus study direction with particular focus on the exciting opportunities offered by modern high-speed atomic force microscopy methods and instrumentation. Considering the sheer diversity and abundance of algal viruses ,2, and tVirus structure knowledge took a leap forward in the year 1956. Crick and Watson suggested the hypothesis that ‘rod’ and ‘spherical’ viruses could be built from regularly packed identical sub-units. The basis for this idea was the high degree of order seen in X-ray photographs of crystallized plant viruses . These tPost-1956, research included the probable icosahedral structure of Tipula iridescent virus using EM with double shadowing , direct The main technique for ultrastructural studies in this early era was electron microscopy (EM) having been invented in 1935 . This de2 . Using only basic electron micrograph and X-ray diffraction data the pair were able to generate a theory on virus structure using geometric rules, later proven correct by direct visualization. EM and cryo-EM data has been iteratively improving since its inception and use in icosahedral viral analysis, increasing resolution for PBCV-1  methods for virus samples  for an ir PBCV-1  three-foEM techniques are not only useful for intact virus structural study but can also be used in investigation of viral dynamics and the analysis of individual components. For example, when using TEM and its various derivatives such as scanning transmission electron microscopy tomography, immuno-electron microscopy and whole-cell focused ion beam-scanning electron microscopy ,25. ThesAnother form of microscopy and virus structure analysis tool is atomic force microscopy (AFM). Invented in 1986 by IBM scientists , the AFMAlthough a cryo-atomic force microscope was developed in 1996 , this teFor the next twelve years the California group published extensively their research into AFM study of viruses (reviewed in ), with tIt is of note that a 2002 study by the California structural group performed a similar viral infection experiment to the IBM scientists from 10 years previous , however−1  sho shoSacchEmiliania huxleyi in natural and modified forms. Also, discovery of specific membrane areas involved in algal viral adhesion, and fusion entry and exit should be possible.To measure these algal host-virus interactions, there are currently two main approaches that could be pursued. One is taking advantage of the speed of HS-AFM to truly visualize and gather data on dynamic infection processes. To achieve this, taking advantage in improvements in live cell imaging techniques and using a HS-AFM with the resolution and frame rate necessary to observe fast biological processes is key. The second avenue is an aspect of AFM not thus far mentioned. Atomic force microscopes can be operated in a point-measurement mode that enables them to collect force-distance curves at different locations on the nanoscale surface. These curves can then be processed to obtain data about the material properties of the sample surface. With these methods it may be feasible to quantify lipid domains/sphingolipid raft size structure and characteristics with conceivable inference of sphingolipid production rates in examples such as Having the sensitivity to measure piconewton interaction forces enables AFM to study the interaction forces between single biomolecules  and to pThere are other imaging modes available for AFM  that havC. variabilis cells and AFM of osmotic shock treated virus was used to examine single virions and their emerging DNA with associated proteins (AFM, in its basic form is fundamentally a surface imaging technique similar to rudimentary SEM. SEM in truth does have an interaction volume, giving information about electronic and elemental characteristics. AFM can provide physical and mechanical characteristics of samples such as friction and stiffness. However, in the context of this section SEM and AFM are considered as surface imaging techniques. The idea that AFM in this respect provides insight into the internal features of various biological samples seems counter-intuitive considering the mechanism of data acquisition. Indeed, few studies have explored this idea. With current technology, techniques including Cryo-ET, confocal and fluorescence based microscopy arguably produce better results with less effort. However, some intriguing progress has been made into using AFM to examine the structures hidden below the surface of viruses. As eluded to above, in the case of virus structural study, the disruption of the capsid shell and in some cases external lipid membrane either by accident or on purpose, often exposes the features not immediately available by AFM or SEM as seen in proteins b. FurtheThe focus of algal virus study has followed the same paths as generally pioneering studies into medically relevant, human pathogenic viruses. This being the initial interest into the unseen structure of these nanoscale biological particles, how their structure relates to and follows geometric rules, the structure of the individual building blocks and evenually insight into infection dynamics and strategies. The principles outlined early in this review of theory and orthogonal instumentation and methodology have been key in every stage of the improving understanding. Theory suggests hypotheses to later be proven by improved instrumentation, and the improved instumentation providing data from which more detailed theory can be hypothesised. Key to this process, and outlined in part in this review, is the constant iteratively refining methodology for analysing these host-viral systems on such a small scale. We have covered the basic history of some of the methods available for use in experimental studies at this time, with a particular focus on different forms of microscopy and especially atomic force microscopy. Atomic force microscopy, we believe, has potentially been overlooked as purely a structural tool, but has showed recent promise as a method for dynamic observations. We have confidence that with reducing costs and increased availablity and when applied in parallel/correlatively with other virology approaches; these techniques can be effectively functional in the study of algal viruses to determine replication mechanisms and to collect evidence for existing theory. Hopefully, as is usually the case, once methods are established they become far easier to perform and more widely available, leading to a far wider range of algal viruses being studied in greater detail when compared with the limited test systems being used in recent histroy and current work.What is the limit of resolution when using these techniques? For EM, we are currently at near atomic resolution  and for


Henipavirus, causes severe disease and stuttering chains of transmission in humans and is considered a potential pandemic threat. In May 2018, an outbreak of Nipah virus began in Kerala, > 1800 km from the sites of previous outbreaks in eastern India in 2001 and 2007. Twenty-three people were infected and 21 people died (16 deaths and 18 cases were laboratory confirmed). Initial surveillance focused on insectivorous bats (Megaderma spasma), whereas follow-up surveys within Kerala found evidence of Nipah virus in fruit bats (Pteropus medius). P. medius is the confirmed host in Bangladesh and is now a confirmed host in India. However, other bat species may also serve as reservoir hosts of henipaviruses. To inform surveillance of Nipah virus in bats, we reviewed and analyzed the published records of Nipah virus surveillance globally. We applied a trait-based machine learning approach to a subset of species that occur in Asia, Australia, and Oceana. In addition to seven species in Kerala that were previously identified as Nipah virus seropositive, we identified at least four bat species that, on the basis of trait similarity with known Nipah virus-seropositive species, have a relatively high likelihood of exposure to Nipah or Nipah-like viruses in India. These machine-learning approaches provide the first step in the sequence of studies required to assess the risk of Nipah virus spillover in India. Nipah virus surveillance not only within Kerala but also elsewhere in India would benefit from a research pipeline that included surveys of known and predicted reservoirs for serological evidence of past infection with Nipah virus (or cross reacting henipaviruses). Serosurveys should then be followed by longitudinal spatial and temporal studies to detect shedding and isolate virus from species with evidence of infection. Ecological studies will then be required to understand the dynamics governing prevalence and shedding in bats and the contacts that could pose a risk to public health.The 2018 outbreak of Nipah virus in Kerala, India, highlights the need for global surveillance of henipaviruses in bats, which are the reservoir hosts for this and other viruses. Nipah virus, an emerging paramyxovirus in the genus  Nipah virus is an emerging zoonotic virus that spills over from bats to humans causing severe disease and chains of transmission in humans. In May 2018, in Kerala, India, Nipah virus infected 23 people, killing 21. We reviewed and analyzed published records of Nipah virus surveillance in bats and identified eleven species that occur in India and have had evidence of henipavirus infection or exposure. However, almost all of these bat species were sampled outside of India. Using a trait-based machine learning approach, we identified at least four additional Indian bat species that are likely to have been exposed to Nipah virus or cross-reacting henipaviruses. We suggest surveillance of these species as well as studies on the ecological dynamics of Nipah virus and epidemiology of spillover transmission to humans. This work will help prioritize a research agenda for responding to the recent outbreak of Nipah virus in Kerala, India and elsewhere. Paramyxoviridae that infect humans directly or via domestic animals that function as bridging hosts , 39rotl To make predictions of bat species that may carry Nipah virus in India and the surrounding region, we trained a generalized boosted regression model on data that characterized 48 traits of 523 extant bat species with geographic ranges in Asia, Australia, and Oceana. By learning the intrinsic features of species that have previously been found to have evidence of Nipah virus- infection , the objective is to identify additional bat species whose trait profiles suggest a high probability of being Nipah virus-positive. In addition, by examining those traits that are most predictive of Nipah virus-positive species, we may also glean ecological insights about why some bats are found to be Nipah virus-positive compared to others in this region. While examination of these suites of shared traits can be insightful, it is important to note that these methods are designed for pattern recognition rather than to identify mechanisms; however, in some cases, mechanisms may be suggested ).We acquired range maps from the International Union for Conservation of Nature (IUCN) . We obtaWe conducted a second generalized boosted regression analysis to diagnose whether greater data availability for better-studied species leads to trait profiles that describe well studied bat species rather than species where evidence of Nipah virus infection has been reported. In this model, we used the number of citations in Web of Science for each species’ scientific name as a proxy for study effort at the time this study was conducted. As before, models were trained on 80% of the full data set and were comprised of 30,000 trees specifying a Poisson error distribution and built with 10-fold cross-validation to prevent overfitting. Hyperparameter values and outputs for generalized boosted regression models can be found in Cynopterus brachyotis, C. sphinx, Eonycteris spelaea, Rousettus leschenaultii, and P. medius [formerly P. giganteus]) and two non-Pteropodidae  ) , 41. Serenaultia , 57 werein India , 41 .Nipah virus has a wide host breadth in both reservoir bat species and recipient animal species. Therefore, identifying the reservoir in a new location can be challenging. We used a systematic literature search to collate data from previous studies of Nipah virus in bats. We then prioritized surveillance of bats in Kerala, and more generally in India, on the basis of these data. We applied a trait-based generalized boosted regression that identified species with traits similar to those associated with serological or virological evidence of Nipah virus. Nipah virus was detected by PCR in only one species occurring in India, Our work provides a list of species to guide early surveillance and should not be taken as a definitive list of reservoirs. A series of further studies are required to triangulate on the reservoir hosts that pose a risk to humans. A major reason these studies do not identify definitive reservoirs is because almost all previous Nipah virus studies relied on serology, but serological assays often lack specificity; detection of Nipah virus may represent cross-reactions to closely related viruses . For exaIn addition to suggesting potential reservoir species, the associative traits that predict reservoir capacity inform the ecology of potential bat reservoirs, which may guide epidemiological studies of Nipah virus infection. However, the utility of these traits as predictors of reservoir capacity should be interpreted as associative rather than causal. Some of the traits in the generalized boosted regression , the maximum latitudinal extent of each species geographic range, the richness of mammal species found within a species’ geographic range, and the trophic level of each species see . In geneP. medius, as well as the distributions of other potential reservoirs proposed here. Studies in wildlife and humans must cover this broad geography to assess future risk in India. Moreover, the last comprehensive and systematic taxonomic study on the bats in India was conducted more than a century ago. There are several cryptic species or species with unresolved taxonomic status in India, and it is possible that species with Nipah virus detections outside of India may have been misidentified. Therefore, our conclusions may change after detailed and systematic taxonomic studies are done on Indian bats.A current constraint on progress towards understanding the epidemiology of Nipah virus in India is the dearth of virologic and taxonomic studies on bats in India. The majority of studies used for these analyses were conducted outside of India and no studies, to our knowledge, investigated Nipah virus in Kerala prior to this outbreak. India encompasses many different bioregions. The outbreak in Kerala shows that the ecological niche for Nipah virus is very wide and could include the entire distribution of Once serological evidence of Nipah virus is detected in potential reservoir hosts, longitudinal spatial and temporal surveillance of these hosts will be necessary. Detection of virus at a single point in time and space conveys limited information and could represent a spillover event from another species. To confirm reservoirs status of a species, virus must be consistently found within that species . MoreoveIdentifying reservoir hosts and then characterizing the diversity of their viruses and their virus shedding patterns are critical steps in understanding spillover. However, the transmission of Nipah virus from bats to humans requires alignment of a number of other ecological and epidemiological factors , includiIn addition to sampling bat reservoir hosts, sampling plans should consider that henipaviruses could be maintained in domestic recipient hosts. These hosts, with closer and more frequent contact with humans, can become bridge hosts for human infections . For exaProjecting the risk of Nipah virus outbreaks in humans requires identification of the reservoir hosts and the dynamics of Nipah virus within those hosts. Our predictions inform initial sampling that can be followed by a sequence of studies that investigate the bat species highlighted here. The machine learning approaches presented here can be the first step in a research pipeline to eventually understand the mechanisms underpinning epidemiologically important cross-species contacts.https://caryinstitute.figshare.com/s/8f79fff6795132cabc1aData is available at S1 TableAll variables included in the generalized boosted regression analyses, together with the coverage of each variable across species, and how each variable is defined according to source references. Variable names are consistent with those reported in original citations.(PDF)Click here for additional data file.S2 TableResults of the first analysis (columns B and C) treat Nipah virus positivity as a binary response variable. Results of the second analysis (columns D and E) treat study effort as a Poisson distributed response variable .(PDF)Click here for additional data file.S3 TableA list of bat species in descending order NiV positivity as predicted by a generalized boosted regression model on species-level traits (pNiV+), and a binary column (NiV+) indicating seropositivity from all previously published work.(PDF)Click here for additional data file.S1 FigThe frequency histogram of trait value distribution across all bat species (blue bars) is overlaid with a black line indicating model sensitivity to trait values for classification accuracy.(PDF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.S2 ChecklistTable of included papers.(PDF)Click here for additional data file.S1 Diagram(PDF)Click here for additional data file.


Mycoplasma pneumoniae, and Chlamydia.Respiratory viruses, such as influenza viruses, initially infect the upper airways but can manifest as severe lower respiratory tract infections in high‐risk patients with significant morbidity and mortality. For syndromic diagnosis, several multiplex nucleic acid amplification tests have been developed for clinics, of which SureX 13 Respiratory Pathogen Multiplex Kit (ResP) can simultaneously detect 13 pathogens directly from airway secretion specimens. The organisms identified are influenza virus A, influenza virus A pdmH1N1 (2009), influenza virus A H3N2, influenza virus B, adenovirus, boca virus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, This study provides performance evaluation data of this assay by comparing with pathogen‐specific PCRs from oropharyngeal swab samples.Ten pathogens were detected in this assay, of which rhinovirus, adenovirus, and influenza virus A pdmH1N1 (2009) were the most common. The overall agreement between the ResP and the comparator tests was 93.8%. The ResP demonstrated 86.5% agreement for positive results and 97.8% agreement for negative results.The ResP assay demonstrated a highly concordant performance comparing with pathogen‐specific PCRs for detection of respiratory pathogens in oropharyngeal swabs from outpatients and could aid in the diagnosis of respiratory infections in a variety of clinical scenarios. The symptoms caused by these pathogens are largely similar, thus definitive diagnosis requires effective laboratory testing. By using multiplex assay targeting these pathogens, early diagnosis can be made in a timely manner. Consequential antimicrobial or antiviral therapy may thus be administrated promptly and appropriately.Acute respiratory infections (ARIs) are common and contribute significantly to morbidity and mortality. They are the leading causes of outpatient visits and hospitalizations in all age groups, especially for children under 5 years of age.The routine clinical laboratory testing for respiratory viruses is largely conducted by direct fluorescent‐antibody assays and rapid antigen tests in China. Given the poor sensitivity and complicated manual operation, these methods have been gradually replaced by nucleic acid amplification tests (NAATs), which are more sensitive and more specific. However, majority of the NAAT kits are based on real‐time polymerase chain reaction (PCR), which can only detect one or two pathogens of ARIs within a single tube, thus are not syndromic testing.SureX 13 Respiratory Pathogen Multiplex Kit (ResP) is a syndromic multiplex molecular test for simultaneous detection of 13 pathogens in a single tube. The aim of this study was to evaluate the application of the ResP for detection of respiratory pathogens in outpatients with flu‐like manifestations.22.1The inclusion criteria for this study were as follows: (a) patients admitted to hospitals between Feb. 2017 and Aug. 2018; (b) oropharyngeal swabs were collected from hospitals and Centers for Disease Control in Guangzhou; (c) patients had the following flu‐like manifestations: (a) fever (>38°C); (b) cough or sore throat. After sampling, specimens were kept in 4°C and transferred to the laboratory for testing within one week.2.2g for 20 minutes, and the supernatant was aspirated. About 50 µL of RNA was extracted from 140 µL supernatant using the QIAamp Viral RNA extraction kit , according to the manufacture's instruction and was stored at −80°C.The specimen was shaken vigorously for 5 minutes in phosphate‐buffered saline solution, centrifuged at 9.6 2.3Influenza virus nucleic acid detection was performed by Influenza A/B Influenza Virus Nucleic Acid Detection Kit . Positive samples were further tested for influenza virus A pdmH1N1 (2009) and seasonal influenza virus H3N2 using a separate kit . Both tests were carried out on ABI Quant Studio 7 System (Thermo Fisher Scientific) according to the instructions. A typical S amplification curve and Cq value ≤35.0 were determined positive.2.4Mycoplasma pneumoniae (MP), and respiratory syncytial virus (RSV), using corresponding NAAT kits from Daan Gene. All tests were carried out on ABI Quant Studio 7 System (Thermo Fisher Scientific) according to the instructions. A typical S amplification curve and Cq value ≤38.0 were determined positive.For influenza virus‐negative samples, more PCR tests were performed to detect the following pathogens: adenovirus (ADV), bocavirus (BOV), human rhinovirus (HRV), parainfluenza virus (PIV), human metapneumovirus (HMPV), 2.5chlamydia, human metapneumovirus, Mycoplasma pneumoniae, coronavirus, and respiratory syncytial virus. The PCR product was subjected to capillary electrophoresis using GenomeLab™ GeXP Genetic Analysis System (Beckman Coulter) according to the instructions. Each pathogen, if detectable, produced a distinctive fragment size after PCR amplification. The results of fragment analysis were used to determine the outcomes of testing. In brief, if the peak height of a targeted fragment size is lower than the lower peak of the signal standard, the targeted pathogen is determined negative; if the peak height of a targeted fragment size is higher than the higher peak of the signal standard, the targeted pathogen is determined positive; if the peak height of a targeted fragment size is between the higher and the lower peaks of the signal standard, the targeted pathogen is determined uncertain and the test should be repeated.The nucleic acid was subjected to multiplex amplification for all specimens using SureX 13 Respiratory Pathogen Multiplex Detection Kit  on ABI GeneAmp PCR System 9700 (Thermo Fisher Scientific). The 13 respiratory pathogens were as following: influenza A virus, influenza A virus H1N1 (2009), seasonal H3N2 influenza virus, influenza B virus, adenovirus, boca virus, rhinovirus, parainfluenza virus, 2.6P value was calculated by CHITEST, and P < .05 indicates statistical significance.The results were analyzed using EXCEL2007. The Cohen's kappa statistics were calculated to measure the agreement between pathogen‐specific PCRs and multiplex PCR results .3A total of 420 oropharyngeal swabs were enrolled from 10 hospitals and 10 CDCs in Guangzhou from 2017 to 2018. Samples were collected from a wide range of ages, with the average age of 27.2 (Table A pathogen‐positive result was determined when the pathogen‐specific fragment(s) was positive, as shown in Figure Chlamydia. In six of the ten detected pathogens, the Cohen's kappa values were over 0.8 with P value <.01 Our study showed almost perfect kappa statistics for the ResP on rhinovirus, adenovirus, influenza virus A pdmH1N1(2009), respiratory syncytial virus, and influenza virus B, suggesting that the performance of ResP on these viruses was as effective as pathogen‐specific PCRs. On human metapneumovirus, the kappa statistics were lower than 0.8, presumably due to the small number of positive cases. Overall, this assay demonstrated 86.5% PPV and 97.8% NPV. This work suggested that the performance of ResP was sufficient enough be used for respiratory pathogen identification in outpatients with flu‐like manifestations.Mycoplasma pneumoniae, boca virus, influenza virus A H3N2, coronavirus, and Chlamydia. Further investigation is needed to evaluate the performance of ResP on these pathogens.The major limitation of this study is the small number of human metapneumovirus, parainfluenza virus, In conclusion, the performance of ResP showed a high‐degree agreement with pathogen‐specific PCRs in oropharyngeal swabs from outpatients. The implementation of ResP may facilitate the diagnosis of respiratory infections in a variety of clinical scenarios.The study was supported by the grants from the National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX10103011), the Project for Key Medicine Discipline Construction of Guangzhou Municipality (2017‐2019‐07), Science and Technology Program of Guangdong Health Department (A2017489), Science and Technology Program of Guangzhou Health Department (20181A011052).


Herpes simplex virus 2, or human herpesvirus 2, is a ubiquitous human pathogen that causes genital ulcerations and establishes latency in sacral root ganglia. We fully sequenced and manually curated the viral genome sequence of herpes simplex virus 2, strain 333 using Pacific Biosciences and Illumina sequencing technologies. Herpes simplex virus 2, or human herpesvirus 2, is a ubiquitous human pathogen that causes genital ulcerations and establishes latency in sacral root ganglia. We fully sequenced and manually curated the viral genome sequence of herpes simplex virus 2, strain 333 using Pacific Biosciences and Illumina sequencing technologies. Alphaherpesvirinae subfamily within the Herpesviridae virus family is a highly prevalent human pathogen which infects through the genital mucosa, remains latent in dorsal-sacral root ganglia, and produces a lifelong infection. The worldwide prevalence rates are close to 20 to 25%, depending on the country and economic status (KP192856) was described as having 153,333 bp, based on alignment of Illumina reads against the HG52 reference genome (Herpes simplex virus 2 (HSV-2) is commonly found as the causative agent of genital tract infections. This member of the c status , 2. HSV-c status , 4. A pa2 culture dishes of Vero cells at 0.1 multiplicity of infection (MOI). After 2 h, the viral inoculum was removed, and the cells were overlaid with 20 ml fresh Dulbecco’s modified Eagle’s medium complete (DMEMc) with 2% fetal bovine serum (FBS). Then, 48 h postinfection, cells and supernatant were collected and concentrated with low-speed centrifugation. Viral particles were purified in a 36% sucrose cushion with ultracentrifugation at 20,000 × g over 1 h. Then, the eluted pellet was treated with DNase I to remove cellular DNA. Viral genomic DNA was purified by a proteinase K-SDS treatment followed by phenol-chloroform extraction.A single viral clone was passaged 3 times in cell culture with plaque isolation. Then, that purified clone was amplified by infecting 5 subconfluent P150-cmde novo assembled with HGAP v.3 (A 20-kb PacBio library was prepared using the BluePippin size selection protocol (7-kb cutoff). This library was sequenced on a PacBio RS II instrument using P6-C4 chemistry with a 360-min movie time, in one single-molecule real-time (SMRT) cell, at the Norwegian Sequencing Centre, University of Oslo (Norway), and we obtained 80,266 reads. Reads longer than 12 kb were HGAP v.3 , which gUL32 and UL36 genes were truncated relative to strain HG52 due to the presence of 2 single-nucleotide deletions and 1 single-nucleotide deletion, respectively. Using Illumina read mapping, those indels contained in the annotated coding DNA sequence (CDS) were corrected manually in the PacBio contig. The final assembly , was 828 bp longer than the strain HG52 reference sequence (GenBank accession no. NC001798) and showed small differences in both ends but shared 99% genome identity. Despite the use of long reads for the assembly, the “a” sequences at both ends were not fully resolved, and only the central one was complete. Although repeated regions are considered equal, small differences were observed between them.The HG52 reference strain was used to annotate strain 333 using BLAST alignments followed by manual curation. LS480640.The viral genome sequence of HSV-2 strain 333 was deposited in ENA/GenBank under the accession no.