Together, these results further our understanding of honey bee antiviral defense, particularly the role of a non-sequence specific dsRNA-mediated antiviral pathway.Recent high annual losses of honey bee colonies are associated with many factors, including RNA virus infections. Honey bee antiviral responses include RNA interference and immune pathway activation, but their relative roles in antiviral defense are not well understood. To better characterize the mechanism(s) of honey bee antiviral defense, bees were infected with a model virus in the presence or absence of dsRNA, a virus associated molecular pattern. Regardless of sequence specificity, dsRNA reduced virus abundance. We utilized next generation sequencing to examine transcriptional responses triggered by virus and dsRNA at three time-points post-infection. Hundreds of genes exhibited differential expression in response to co-treatment of dsRNA and virus. Virus-infected bees had greater expression of genes involved in RNAi, Toll, Imd, and JAK-STAT pathways, but the majority of differentially expressed genes are not well characterized. To confirm the virus limiting role of two genes, including the well-characterized gene,  Apis mellifera) and other insects are important pollinators of plants in both natural and agricultural landscapes. Insect pollination services are valued worldwide at $175 billion annually1, and in the United States honey bee pollination is valued at $14.6 billion annually2. Commercially managed honey bee colonies, which are the primary pollinators of numerous agricultural crops, have experienced high annual mortality in the U.S.  and parts of Europe5. Multiple abiotic and biotic factors, including pathogens, contribute to colony losses7. Pathogen incidence and abundance have been positively correlated with Colony Collapse Disorder (CCD)-affected colonies in the U.S.8 and colony losses in different regions of North America, South America, and Europe17. Honey bees are eusocial insects that live in colonies comprising approximately 40,000 sterile female workers, hundreds of male bees, and a single reproductive queen bee. Honey bees are often infected with pathogens including viruses, fungi, bacteria, and trypanosomatids, and they are typically parasitized by the Varroa destructor mite  per bee21, thus they likely affect bee physiology and health.The largest group of honey bee infecting pathogens are positive sense single-stranded viruses, including several Dicistroviruses , Iflaviruses , as well as taxonomically unclassified viruses -mediated pathway is one of the primary insect antiviral defense mechanisms37. Correspondingly, several studies have shown that administration of virus-specific dsRNA or siRNA reduced viral load in honey bees40. Furthermore, CCD-affected colonies had higher amounts of virus-specific 22 nt siRNAs as compared to non-CCD-affected colonies37 and early field studies suggested that honey bees fed IAPV-specific dsRNA had increased honey production and larger colony size41.Like other insects, honey bee antiviral responses include autophagy, apoptosis, eicosanoid biosynthesis, endocytosis, melanization, the JAK/STAT , Toll, NF-κB (Nuclear Factor κB), JNK , and MAPK (Mitogen-Activated Protein Kinases) pathways, and RNA interference (RNAi)40, non-sequence-specific dsRNA (ns-dsRNA) has also been shown to reduce virus abundance and affect gene expression in honey bees and bumble bees45. The more prominent role of ns-dsRNA mediated reduction in virus abundance in eusocial hymenopteran insects , as compared to solitary insects  that do not exhibit this response, may reflect an evolutionary adaptation to limit virus transmission within colonies using general non-virus specific antiviral responses. In mammals, dsRNA serves as a virus-associated molecular pattern (VAMP) that is recognized by pathogen recognition receptors (PRRs), such as Toll-like receptor 3 (TLR3), Protein kinase R (PKR), Retinoic acid-inducible gene 1 (RIG-I), and Melanoma differentiation-associated gene 5 (MDA-5), and results in induction of the antiviral interferon response46. Similarly, Dicer, which is the endoribonuclease involved in RNAi, also serves as a dsRNA sensor that induces expression of antiviral defense genes  in fruit flies, mosquitoes, and bumble bees49. However, the role of specific genes in honey bee antiviral defense, particularly nonspecific dsRNA-mediated antiviral responses, are not well characterized.While experimental introduction of virus-specific dsRNA reduced honey bee virus infections, likely via RNAi22. To further investigate the mechanisms of dsRNA triggered antiviral defense and the dynamics of virus infection and corresponding immune responses in honey bees, we performed a time series experiment  that included transcriptional profiling of individual virus-infected and dsRNA-treated bees. We determined that honey bee gene expression varied with the progression of virus infection and included genes involved in endocytosis, development, transcriptional regulation, RNAi, and the Toll, Imd, JAK-STAT, and JNK pathways. Interestingly, bees that exhibited decreased virus abundance in the context of dsRNA treatment exhibited increased expression of two RNA helicases, one JNK pathway member, and several genes involved in dsRNA transport. Furthermore, we performed in vivo studies that confirmed the importance of the genes dicer and cyclin-dependent serine/threonine kinase (MF116383), which exhibited increased expression in virus-infected bees, in honey bee antiviral defense. Together these results further our understanding of honey bee antiviral defense mechanisms and the effects of dsRNA on honey bee gene expression and may lead to the development of strategies that limit virus infection in honey bees. Development and increased use of siRNAs and dsRNAs to reduce pathogens and pests  in crops that are frequently visited by pollinators51 also underscores the need to further examine the effects of these molecules on bee health.Previously, we determined that treating honey bees with either virus sequence-specific-dsRNA (sp-dsRNA), or non-sequence specific dsRNA (ns-dsRNA), decreased virus abundance at 72 hours post-infection (hpi)41, but nonspecific dsRNA-triggered antiviral response pathway(s) also play a role in honey bee antiviral defense22. To further investigate honey bee antiviral defense mechanisms, including RNAi and nonspecific dsRNA-mediated mechanisms22, we infected bees with a model virus in the presence or absence of dsRNA , nonspecific dsRNA corresponding to Drosophila C virus sequence , and luciferase sequence . In addition, co-administration of polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimic of dsRNA, or nucleoside triphosphates (NTPs), served as positive and negative controls, respectively; mock-infected controls were also performed. Bees from each experimental treatment group were collected at 6, 48, and 72 hpi, a time course that allowed for assessment of both early and late antiviral responses. Relative virus abundance was examined via fluorescence microscopy and quantified based on relative protein and RNA abundance using Western blot analyses and quantitative PCR (qPCR), respectively  viruses, which replicate via a dsRNA intermediate. RNAi mediated by virus-specific dsRNA is an important antiviral defense mechanism in honey beesely Figs  and S2.FHoney bees treated with sp-dsRNA or ns-dsRNA had reduced virus abundance. Fluorescent microscopy of bees that were infected with SINV-GFP in the absence or presence of dsRNA provided qualitative evidence that dsRNA treatment reduced SINV-GFP at 72 hpi  of individual virus-infected bees, bees infected with virus in the presence of sp-dsRNA or ns-dsRNA, dsRNA-treated bees in the absence of virus, and mock-infected bees at 6, 48, and 72 hpi Fig. . Forty-s53.Transcriptome analysis of virus-infected bees over the course of infection  determined that virus-infection altered the expression of hundreds of genes as compared to mock-infected bees Figs  and S5. cyclin-dependent serine/threonine kinase (MF116383), apid1, DNAn6-methyl adenine demethylase (loc412878), orb2-like, solute carrier organic anion transporter family member 3a1-like (sloc3a1), and titin-like carbonic anhydrase 1, venom acid phosphatase acph-1-like, and odorant binding protein 16, which also exhibited decreased expression in virus-infected bees  was also differentially expressed throughout all dsRNA-treated bees. In dsRNA-treated bees 6 and 48 hpi, several genes encoding antimicrobial peptides  exhibited increased expression , which plays a role in dsRNA uptake in D. melanogaster55 and may play an analogous role in honey bees. Together, our analyses indicate that dsRNA-treatment alters gene expression in honey bees, and that there are common and unique aspects between differential gene expression in virus-infected bees and virus-infection in the context of dsRNA.Venn diagram analysis of dsRNA-treated bees 6, 48, and 72 hours post-treatment identified 14 shared DEGs, three of which exhibited decreased expression: ees Fig.  and S8. ion Fig. , most ofion Fig. . In addicyclin-dependent kinase, orb2-like, titin-like, DNA n6-methyl adenine demethylase, slco31-like, hsp90, abaecin, ago2, dicer, igfn3-10, mfs-transporter, jra, fam102b) that exhibited increased expression in virus and/or dsRNA treated bees at 48 and/or 72 hpi via qPCR of sequenced bees 72 hpi  were confirmed to have increased expression in virus-infected and/or dsRNA-treated bees via qPCR  were also confirmed to have increased expression in biological replicate experiments that utilized pooled virus-infected honey bee samples (72 hpi) from two different colonies, likely with different genetic backgrounds  exhibited increased expression Fig. . Some ofsolute carrier organic anion transporter family member 3a1-like (sloc3a1) consistently exhibited increased expression in virus-infected bees   and peptidoglycan receptor s3 (pgrp-s3), and the toll-like receptor toll-10 exhibited increased expression in virus-infected bees  binding to the diaminopimelic-containing peptidoglycan of Gram-negative bacteria29. The Imd pathway is also important for fruit fly antiviral defense against some viruses, including Sindbis virus58.In the context of virus infection, extracellular receptors can serve in pathogen recognition and host defense or be co-opted by the virus to facilitate entry. The membrane localized se) Figs  and S7. ees Fig. . The JAKees Fig. . Similarigfn3-5, igfn3-10, and igfn3-11) exhibited increased expression in virus-infected bees . In mosquitoes, thioester proteins bind to invading bacteria which promotes phagocytosis of these pathogens66, but thioesters are also associated with improved defense against Dengue and West Nile viruses via unknown mechanisms67.Endocytosis, including phagocytosis, is an immune effector function carried out by hemocytesees Fig. . LamininD. melanogaster S2 cells, genes involved in receptor-mediated endocytosis are important for dsRNA uptake, including the genes scavenger receptor c (scr-c), fam102b, and sap-r55. Bees treated with virus, dsRNA, or both exhibited increased expression of scr-c, but bees treated with both virus and dsRNA exhibited the greatest increase  that has high sequence homology to an Eastern honey bee (Apis cerana) probable cyclin-dependent serine/threonine kinase (XM_017051141.1), exhibited the greatest increase in expression in virus-infected bees as compared to mock-infected controls  , including dsRNA, results in activation of cellular transduction cascades that activate particular immune responsese) Figs. , and S79cactus 1 and cactus 2, which suppress NF-κB signaling, and tube, an adaptor protein that promotes NF-κB signaling  does not necessarily indicate complete or continuous repression of the pathway 70. Likewise, we determined that pirk, which represses Imd pathway signaling72, exhibited increased expression in virus-infected bees , had greater expression in bees that were both virus-infected and treated with dsRNA at 6 and 48 hpi, and increased expression in all virus-infected groups at 72 hpi  exhibited increased expression in virus-infected bees  that have bactericidal activity against Gram-negative bacteria76. Virus-infected and/or dsRNA-treated bees also exhibited increased expression of abaecin and hymentoptaecin  simply indicate activation of these pathways79. The activation of Toll, Imd, and JNK signal transduction cascades likely stimulate transcription of hundreds of genes, including antiviral effectors that await further characterization.Several antimicrobial peptides, which are effector molecules of Toll, Imd, and JNK pathways, exhibited increased expression in virus-infected and dsRNA-treated bees. Importantly, one Figs –S856, 7581. Our transcriptional level analysis identified several genes encoding heat shock and accessory proteins that exhibited increased expression in virus-infected bees including hsp90, activator of hsp90, 60 kda hsp, 10 kda hsp, hsp83-like, and hsf5  as part of the RNAi response83, but Hsp90 can also be exploited by both insect and human viruses  in order to stabilize RNA replication85. Future studies aimed at better understanding the functions of heat shock proteins, particularly Hsp90, in virus-infected honey bees will be exciting since these proteins may either be antagonistic or beneficial to specific viruses.Heat shock proteins (Hsps) are involved in general stress responses and protein degradation and stabilization. In fruit flies, these ubiquitously expressed proteins are important for defense against some viruses39. Notably, there was greater expression of genes involved in RNAi , dicer, tudor-sn, hsc70-4, and tarbp2) in virus-infected bees , that after binding dsRNA, results in the increased activation of antiviral immune effectors 49. DWV-infected honey bees exhibited increased expression of Apis mellifera vago (loc503505), but differential vago expression was not observed in our data set; many host factors  may be involved, but perhaps vago expression is only increased in response to specific honey bee infecting viruses (Supplementary Table rna helicase ddx33 and rna helicase dhx52) was increased in virus-infected and dsRNA-treated honey bees , which activate the antiviral interferon responseees Fig. . RNA heldicer and a probable cyclin-dependent serine/threonine kinase (MF116383), we utilized RNAi-mediated gene knock down to reduce their expression and investigate the impact on virus abundance. We expected that reduced expression of these antiviral genes would result in increased virus abundance, as compared to the virus abundance in bees treated with ns-dsRNA. Virus-infected bees treated with cyclin-dependent kinase-specific dsRNA exhibited decreased expression by 48 hpi (40%) and 72 hpi (30%) compared to respective controls   , in limiting virus infection. Further investigation of this probable cyclin-dependent kinase and the proteins with which it interacts may lead to the discovery of novel honey bee antiviral pathways or aid in further characterization of known immune pathways.In order to further investigate the biological importance of two putative antiviral genes, e p < 0.0 Fig. 5)05) Fig. . Bees tr05) Fig. 91.This is the first study to examine individual honey bee antiviral responses to infection with controlled inoculum of a model virus at multiple time points. Other studies have examined honey bee responses to virus infection at the transcriptional level, but they vary by virus-challenge methodologies , purity and strain of virus inoculum, tissues examined, post-infection assay time, and bee developmental stage, which reduce commonalities in transcriptional results between studies24, SBV and DWV-infected bees25, adult honey bees naturally infected with IAPV9, and a synthesis of common DEGs that was recently generated from 19 gene expression data sets from Varroa destructor-parasitized and virus-infected bees92 essential for life-like, which encodes a protein in the small heat shock protein (Hsp 20) family, further supporting the role of heat shock proteins in honey bee antiviral defense  and symptomatic IAPV-fed bees 24. A few AMPs also exhibited differential expression in many of the DEG lists. For example, hymenoptaecin exhibited differential expression in SINV-GFP-infected bees, DWV and SBV co-infected bees, and in the virus and Varroa destructor DEG synthesis92  in honey bees, which may function as a form of social immunity in insect societies96.In spite of the methodological differences between this and other honey bee transcriptional level analyses, we identified common DEGs associated with virus infected honey bees. Using Venn diagram analysis, we compared our DEG lists to DEGs of symptomatic IAPV-fed beess92 Fig.  and S15.92 Fig. . Our stu11. In addition to other factors, several studies indicate that colony losses correlate with high pathogen incidence and abundance17, including infection by (+)ssRNA viruses56. The outcome of virus infection is influenced by many factors97, including virus strain98, agrochemical exposure97, nutritional status99, genetic diversity of the colony104, colony management, mite parasitism105, co-infections107, and immune responses at both the colony and individual levels. Honey bee antiviral responses include canonical immune pathways , RNAi, and nonspecific dsRNA-mediated immune pathways, though the relative roles of these pathways and the mechanistic details of honey bee antiviral immune responses are not thoroughly understood.Managed honey bee colonies in the US and parts of Europe have experienced high annual mortality levels Apis cerana gene (XM_017051141.1), in honey bee antiviral defense, future studies are required to further elucidate the honey bee antiviral defense network. Better understanding of honey bee antiviral defense mechanisms may aid in the development of strategies that reduce honey bee colony losses and furthers our knowledge of antiviral immune responses in insects, which may ultimately reveal evolutionary conserved pathways in other organisms.This is the first study to examine both individual and temporal honey bee transcriptional response to virus infection. Our results further indicate that honey bee antiviral defense includes canonical insect immune pathways, RNAi, and a nonspecific dsRNA-mediated antiviral defense mechanism. Transcriptional analysis of dsRNA-treated bees showed that dsRNA results in increased expression of genes involved in the JNK pathway, RNA helicases, and dsRNA transport, which parallels dsRNA transport and response mechanisms in fruit flies and mammals. While the results described herein implicate the role of numerous genes, several biological processes, and the involvement of Dicer and a probable cyclin-dependent kinase (MF116383), which had greatest sequence similarity with an 108.Frames of newly emerging bees were obtained from honey bee colonies maintained at Montana State University in Bozeman, MT, USA. Young (~24 hours post-emergence) female worker bees were utilized for experiments. The bees were housed in modified deli-containers at 32 °C and were provided water and bee candy56. We utilized a recombinant model virus, Sindbis virus expressing green fluorescent protein (SINV-GFP)109. There are several advantages to utilizing this virus including the ability to control the dose of virus inoculum, monitor the progression of virus infection using GFP, and the assurance that the honey bees were not previously infected with, nor exposed to, SINV-GFP. In addition, Sindbis virus does not encode a suppressor of RNAi (VSR)110. We and others have used SINV-GFP to investigate honey bee22, fruit fly109, and mosquito111 antiviral defense mechanisms, thus facilitating comparison of immune responses in both natural mosquito hosts and non-native hosts  that have not co-evolved with this virus. Honey bees were immobilized via incubation at 4 °C for 20 minutes and injected in the thorax with 3,750 plaque forming units (PFUs) of SINV-GFP22 diluted in 2 μl of 10 mM Tris buffer (pH 7.5) using a Harbo large capacity syringe equipped with disposable needles . The needles were prepared from borosilicate capillary tubes (0.8–1.10 × 100 mm) with a micropipette puller . To investigate the role of dsRNA in honey bee antiviral defense, SINV-GFP was inoculated with different types of dsRNA (1 μg each), including virus-specific dsRNA , nonspecific dsRNA matching Drosophila C virus sequence , or luciferase sequence  ; reactions were carried out at 37 °C overnight (8–10 hours). DNA was removed by incubating with RQ1 DNAse  for 15 minutes at 37 °C. ssRNA products were ethanol precipitated, suspended in 200 μl Rnase-free water, and annealed at 100 °C for 5 minutes and then slowly cooled to room temperature. dsRNA products were purified by phenol:chloroform extraction and ethanol precipitation. Quality was assessed by agarose gel electrophoresis and spectrophotometry. The dsRNA quantity based on gel band intensity was assessed using ImageJ113.dsRNA was generated by dicer (XM_016917734.1) and a novel transcript with 91% sequence identity with the A. cerana probable cyclin-dependent serine/threonine-protein kinase DDB_G0292550 (XM_017051141.1) was reduced by RNAi-mediated gene knockdown. In order to assess the effects of gene knockdown on virus abundance, bees were infected with SINV-GFP (using methods as above) and co-injected with 1 μg of either gene-specific or nonspecific (DCV-specific) dsRNA (control)  was added to individual bee thorax and abdomen homogenate and RNA was isolated following the manufacturer’s instructions. Prior to gene expression analysis by RNASeq or qPCR, RNA was further purified using Qiagen RNAeasy columns including on-column DNase t reatment (Qiagen) to remove DNA from samples. RNA was quantified using a spectrophotometer.Reverse transcription reactions (25 μl) were performed using 500 ng of total RNA and random hexamer primers (500 ng)  incubated with Maloney murine leukemia virus (M-MLV) reverse transcriptase  for 1 hour at 37 °C, according to the manufacturer’s instructions.114. All qPCR reactions were performed in triplicate using 2 μL of cDNA as template. Each 20 μl reaction was composed of cDNA template, 1X SYBR Green (Invitrogen), 1X Choice Taq Master Mix (Denville Scientific Inc.), 3 mM MgCl2, and forward and reverse primers (600 nM each). A CFX Connect Real Time instrument (BioRad) was utilized for qPCR, the thermo-profile for virus  and A. mellifera rpl8 analyses consisted of a single pre-incubation 95 °C (3 min), 40 cycles of 95 °C (5 s), 60 °C (20 s), 72 ºC (30 s), and a final elongation 72 ºC (4 min)) values and using the primer efficiency equation, (10(1/Slope)−1) × 100)  abundance in each sample target SINV-GFP qPCR amplicons were cloned into the pGEM-T (Promega) vector, as described in Flenniken and Andino 117. The ΔC(t) for each sample was calculated by subtracting the Am rpl8 C(t) from the SINV-GFP C(t). The honey bee gene encoding ribosomal protein 8, Am rpl8, was selected as an appropriate housekeeping gene for qPCR because it has been utilized in several other studies121 and analysis of the RNASeq data presented herein confirmed that rpl8 expression levels were similar in all sequenced libraries. The ΔΔC(t) was calculated by subtracting the average virus-infected ΔC(t) values from the ΔCt values for each treatment group. For host gene expression analyses and RNAseq validation, the percent gene expression for each gene of interest (GOI) was calculated using the following formula: 2−ΔΔC(t) × 100 = % gene expression, in which ΔC(t) = GOI C(t)- rpl8 C(t), and ΔΔC(t) = sample ΔC(t) – mock-infected control ΔC(t). Based on previous work44, we hypothesized that bees (n = 10) co-injected with dsRNA or poly(I:C) would have decreased relative virus abundance as compared to the virus-only treated group. To examine relative virus abundance between treatment groups  co-treated bees) that had equal variance and normal distribution we performed one-tailed Student’s t-tests. Analysis of honey bee host gene expression revealed unequal variance between treatments groups and thus Welch’s t-tests were used to identify statistical differences in host gene expression.The ΔΔC(t) method was used to calculate relative abundance of SINV-GFP in individual bees (n = 10) because it was most accurate; the ΔΔC(t) method ensures that results are not skewed by inadvertent differences in RNA reverse-transcription efficiencies and starting cDNA template abundance4–7 × 106 SINV-GFP copies) to no pre-existing infections for each treatment group and time point were selected for transcriptome sequencing for a total of 47 individual bees . Libraries were prepared and pooled by experimental time point and quantitated using an Illumina Library quantification kit (Kapa). Each pool was paired-end sequenced (2 × 100 nt) on a HiSeq. 2500 using a TruSeq SBS sequencing kit version 4, yielding ~12 million reads per sample, corresponding to at least 9.7 fold coverage  had q-value ≤ 0.05). Venn diagrams were generated using Vennt126. To further investigate the function of the DEGs, representative protein sequences (the longest sequence if there were splice variants) of every known honey bee gene were blasted against the D. melanogaster protein database via reciprocal BLAST+127 to identify fruit fly orthologs and homologs of the honey bee genes because there is a greater amount of gene ontology information for D. melanogaster genes compared to Apis mellifera genes. The honey bee genome encodes approximately 15,000 genes of which 13,592 genes are mapped and provided in the Amel4.5 genome annotation128. We annotated 8,944 genes (~66%) as homologs  to genes encoded by the fruit fly D. melanogaster genome, which encodes ~13,600 genes129. Biological processes (BP) functional enrichment analysis was performed with DAVID130. Gene ontology and biological processes (BP-FAT) enrichment analysis was performed with DAVID130.FastQC and fastx-toolkit were used to remove low quality reads (<Q30). Illumina adapters were trimmed with Trimmomatic24; SBV and DWV-infected bees25; adult honey bees naturally infected with IAPV9; and a common DEG list that was compiled from 19 gene expression data sets including Varroa destructor-parasitized and virus-infected bees92. We used NCBI Entrez Gene ID as a common identifier because DEG lists were generated using different technologies and versions of the Apis mellifera genome and transcriptome , which is longer than the originally annotated A. mellifera probable serine/threonine-protein kinase clkA .RNASeq analysis determined that reads aligning to LOC725387 were more abundant in virus-infected bees. To identify the gene or genes encoded by these differentially expressed reads, the consensus nucleotide sequence was used to query the NCBI Nucleotide collection (nr/nt) and A. cerana transcript annotated as a probable cyclin-dependent serine/threonine-protein kinase DDB_G0292550  as the top blastn result, which contained 95% of the submitted sequence and shared 91% identity ; additional top blastn hits included A. dorsata GATA zinc finger domain-containing protein 14-like. When the LOC725387 RNASeq consensus sequence was used to query the A. mellifera database, the top blastn result only covered 24% of the query sequence . To further characterize the LOC725387 transcript, we Sanger sequenced 5,027 nts (~2–3× coverage) and obtained the most 5′ end of this transcript from RNASeq data  , additional data is available from the corresponding author upon request, and sequence data may be accessed from the NCBI Sequence Read Archive (accession number SRP101337).Supplementary InformationSupplementary Tables


We detected influenza D virus in 18 nasal swab samples from cattle in Ireland that were clinically diagnosed with respiratory disease. Specimens were obtained from archived samples received for routine diagnosis during 2014–2016. Sequencing showed that viruses from Ireland clustered with virus sequences obtained in Europe within the D/swine/OK/1334/2011 clade. Orthomyxoviridae. This virus was originally detected in pigs in the United States , and a seroprevalance of 91% was found in persons working closely with cattle  <25 for further molecular characterization by using 3 primer sets (http://www.megasoftware.net/). We constructed phylogenetic trees by using the maximum-likelihood method in MEGA 5.01.We tested swab specimens by using real-time PCR for influenza D virus as described . We performed univariate statistical analysis by using Stata/SE14.1 . Herd factors investigated for a possible association with influenza D virus herd status were herd size, numbers of stillbirths, dairy cows in herd, beef cows in herd, inward movements from markets, inward movements to farm, and carcasses moved to knackeries.Herd information for 2015 was available for 84 herds of origin of these nasal swab specimens. Data were obtained from the Animal Identification and Movement System database of the Department of Agriculture, Food, and Marine Laboratory Sciences of Ireland .A total of 18/320 samples were positive for influenza D virus by PCR. Of the 18 positive samples, 13 were also positive by PCR for 1 or 2 other viral pathogens . Seven of the influenza D virus–positive specimens were from calves, 2 from weanlings, and 1 from a cow; other specimens were not described by animal age. Nine of the influenza D virus–positive samples had a CPhylogenetic analysis  Figure 1Herd information was available for 10 of the influenza D virus–positive herds and for 74 comparison herds . We found no associations between herd characteristics and influenza D virus status; this finding was determined by evaluating mean values with 95% CIs for infected herds and noninfected herds.This study confirms the emergence of influenza D virus in Ireland. Presence of the virus in nasal swab specimens submitted from routine respiratory disease cases supports the hypothesis that this virus plays a role in the bovine respiratory disease complex. Analysis of herds for infected cattle did not show any epidemiologic differences between influenza D virus infection and infection with other common respiratory viral pathogens. This finding is consistent with the hypothesis that influenza D virus might have limited effect by itself but can potentiate effects of other respiratory pathogens in causing respiratory disease (Detection of 2 virus lineages in Ireland clustering with viruses isolated in Europe within the D/swine/OK/1334/2011 clade raises the issue of how influenza D virus might spread internationally. Surveillance efforts could be targeted for data on trade of live cattle, which is extensive within Europe. Further research is planned to investigate the seroprevalence of influenza D virus in cattle in Ireland and to determine the effect of this virus in a cattle farming context in this country.Additional information on influenza D virus in cattle, Ireland.


Chickpea chlorotic dwarf virus (CpCDV), a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the “hard fruit syndrome” of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS) techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR), CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the “hard fruit syndrome” of watermelon. Citrullus lanatus) production in the world accounted to about 111 million tons in 2014 , with China being the top producer. This crop is largely cultivated in tropical and subtropical areas, and is also very popular in the Mediterranean countries, where it is one of the most common and popular summer fruit crops. Watermelon is widely cultivated in the Central and Southern parts of Tunisia, with a yearly production of 510,000 tons in 2014. A hard-fruit syndrome of watermelon (WHFS) has been reported in Tunisia since 1994, with up to 70% incidence in some areas; diseased plants produced unmarketable fruits exhibiting chlorotic mottling on the rind, white hard portions inside the flesh, and an altered taste. In recent years, the incidence has ranged between 10 and 40%, leading growers to switch to alternative crops. WHFS etiology is uncertain up to now, but two begomoviruses (Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus, (TYLCSV)) were recently found associated with this syndrome [Chickpea chlorotic dwarf virus,  [Cicer aretinum), where it was originally described [Vicia faba), lentil (Lens culinaris), bean (Phaseolus vulgaris) and wild legume species  in Sudan [Beta vulgaris in Iran [Carica papaya) in Burkina Faso [Solanaceae such as pepper (Capsicum annuum) in Oman [Lycopersicon esculentum) in Pakistan and Burkina Faso [Malvaceae as okra (Abelmoschus esculentus) in Pakistan [Gossypium hirsutum) in India [Cucurbita pepo) in Egypt [Cucumis sativus) in Pakistan [Watermelon (viridae) . Beside  in Iran , in papa in Oman , and tomin Egypt  and cucuPakistan . CpCDV, Pakistan . It is tTen watermelon fruits showing different degrees of hardness and discoloration of the flesh, with whitish inserts, deformation of fruits and seeds, and bad taste, were collected in several Tunisian areas  from indFor small RNA (sRNA) sequencing, total RNA was extracted from 10 symptomatic fruits using TRIzol Reagent , according to the manufacturer’s instructions. The RNA extracts were individually evaluated using NanoDrop spectrophotometer ND-1000  and pooled into one sample; three micrograms of the pooled RNA were sent to Human Genetic Foundation sequencing service  for librTotal RNA was also used for complementary DNA (cDNA) synthesis by High-Capacity cDNA Reverse Transcription Kit ; total nucleic acid extractions were performed from the same samples, by silica gel-mediated extraction .Raw data (Sequence Read Archive database acc. num. SRP119446) were checked for quality reads with FastQC software  and FastWatermelon mosaic virus (WMV) and to amalgaviruses  was used; for TYLCV and TYLCSV, DNA extracts from tomato plants experimentally-inoculated with TYLCV (Genbank Acc. No. DQ144621) and TYLCSV (Genbank Acc. No. X61153) agroclones [To confirm the presence of the viral sequences identified in the sRNA library, PCR and reverse transcription PCR (RT-PCR) assays were conducted separately on each of the 10 plants. Specific primers described in literature were used for CpCDV , TYLCV auses see . RNA wasthamiana ; for WMVroclones  were useNcoI/PstI from the full length CpCDV genome previously cloned with SpeI into pBluescriptKS+, followed by the insertion of a full-length genome in the remaining SpeI site. The obtained fragment, corresponding to a ca. 1.8mer of the viral genome, was recovered with HindIII/SacI restriction and transferred into the binary vector pBin19, linearized accordingly. The 1.8mer clone was transformed into Rhizobium radiobacter . Bacteria were grown in liquid YEB/kanamycin/rifampicin medium for 48 h at 28 °C, with shaking, pelleted and resuspended in sterile water. About 30–40 μL of the suspension were inoculated in the stems of watermelon seedlings at first true leaf stage or into the leaf axils of the model plants Nicotiana benthamiana, according to described protocols [For the infectivity assays, the agroinfectious clone of the CpCDV Tunisian isolate  [rotocols .Several watermelon cultivars for a total of 19 ‘Sugar Baby’, 3 ‘Crimson’, 32 ‘Bontà’, and 61 ‘Sentinel’ plants were tested for infection 28 days post agroinoculation (dpa) by tissue print or PCR. Tissue print positive reactions were always confirmed by dot blotting. Infected watermelon plants were maintained in confined environment in 20cm-diameter pots until fruit delivery. Healthy, non-inoculated watermelon plants were grown as controls in the same conditions. Fruits of about 10 cm in diameter were collected once ripening occurred, and samples from a mixture of flesh and rind were individually taken for DNA extraction.A. tumefaciens residual presence in the tissue. When PCR was performed for CpCDV detection in experimentally infected watermelon seedlings, primers amplifying a 1300 bp fragment of CpCDV genome (CpCDV-seq1 5′-GTTGCCACCTGCAACGATT-3′ and CpCDV-seq2 5′-CGACACATAAGGTTCAGGTTG-3′) were used. A digoxigenin-labelled probe was synthesized by PCR DIG probe Synthesis kit  according to manufacturer’s instructions, using the primer pair CpCDV-CP-F/R targeting the coat protein open reading frame (ORF) of the CpCDV Tunisian isolate [Leaf/stem prints (tissue print assay) or total nucleic acids extracts (dot blot assay), fixed on nylon membranes, were used to detect CpCDV. To visualize virus genomic forms, Southern blot assays were performed using total nucleic acids extracted from watermelon leaves and fruits with the TLES buffer-based method , according to . Analyse isolate .Watermelon mosaic virus   .CpCDV was represented by a total of nine contigs showing high identity (98%) with CpCDV isolate A Q2510 (GenBank accession No. KC172655) with a 100% coverage. About 12% of total reads mapped to the reference sequence of the CpCDV isolate A Q2510, indicating that overall, CpCDV was the most represented virus in the watermelon plants here considered. Examination of sRNA profiles revealed the presence of several hot spots along the viral genome, all located in coding regions a.WMV was represented by a total of 80 contigs, including a 10,051 nt-long contig that represented the entire viral genome and showed 97% identity with WMV isolate C05-270 (GenBank accession No. EU660585). The high number of contigs obtained for this virus highlights the high variability of reads, probably reflecting the occurrence of sequence variants in the viral populations present in our samples. About 1% of total reads aligned to the full genomic sequence of WMV isolate C05-270. Examination of sRNA profiles revealed a wide distribution of sRNA reads along the whole viral genome b.Blueberry latent virus and Rhododendron virus A , Ambrosia asymptomatic virus 2 , Cassava vein mosaic virus   .Amalgavirus genus suggests that a novel virus belonging to this taxonomic group is present in watermelon. This putative novel amalgavirus is provisionally named Watermelon amalgavirus 1 (WmAV1). The remaining contigs with amino acid identity to badnaviruses and a cavemoviruses (73% and 43% respectively) showed a reduced length (<250 nt), a low coverage (about 16%), and were therefore excluded from further analysis.The presence of contigs with an amino acid identity of around 55% with two viruses of the DNA and RNA extracted from each of the 10 watermelon fruits tested by sRNA NGS were assayed by PCR or RT-PCR, respectively, in order to validate the presence of CpCDV or WMV and the novel amalgavirus WmAV1 in individual samples. CpCDV was the only virus detectable in all samples tested, suggesting its key role in the development of the WHFS. Conversely, WMV was found in 4 out of 10 samples  and the putative amalgavirus WmAV1 in three out of 10 samples  . WmAV1 wWHFS has been recently linked to TYLCV and TYLCSV infection  and, forThe BLASTn analysis of the contig representing the full-length CpCDV genome (CONTIG494), showed maximum similarity with the CpCDV KX580024, obtained from watermelon collected in Tunisia in 2015, one year before the collection of the samples used in the present study. The two sequences are 2571 nt long and share a nucleotide identity of 99.1% (2549/2571 nt). The next-closest sequence is a CpCDV isolate from Syria (FR687959) , typed aCONTIG494 contains an unusual nonanucleotide TAATGTTAC in the stem-loop region, different from the canonical geminiviral nonanucleotide TAATATTAC. This non-canonical sequence is also present in CpCDV KX580024, and was confirmed by amplification and sequencing of additional watermelon samples collected in Tunisia in 2015 (2 samples) and 2016 (3 samples). However, all other sequences of CpCDV available in the GenBank repository (more than 200) have a TAATATTAC sequence. To verify whether the non-canonical sequence is correlated with the host plant species infected by CpCDV, we analyzed the two other CpCDV sequences isolated from cucurbits (KF692356 from squash and KT719388 from cucumber) and found that both contained the canonical geminiviral nonanucleotide.Chickpea chlorosis virus  Reference Sequence NC_014740), Chickpea chlorosis Australia virus (NC_022131.1), and Chickpea redleaf virus (NC_014739.1) [Dragonfly-associated mastrevirus (two records), and in one isolate of Maize streak virus.The non-canonical nonanucleotide is uncommon in geminiviruses, but can be found in some chickpea-infecting mastreviruses from Australia, e.g., 14739.1) ,30, as wN. benthamiana used as infection controls (222 out of 225 agroinoculated plants). A further experiment was performed, with the hypothesis that the virus concentration could be below the detection limit of the tissue print assay. In this new experiment PCR was used as detection tool, and 18 infected watermelons out of 32 were detected, resulting in a 56% infection rate. A possible explanation is that experimental watermelon infection may in some cases be below tissue print detection threshold, limiting the possibility of routine usage of this widely-used technique for mass diagnosis in these circumstances. Infected watermelon plants showed reduced growth and chlorosis, with smaller and deformed leaves compared to non inoculated plants.A CpCDV infectious agroclone was used to inoculate watermelon plants in controlled conditions using a tissue print assay to identify infected plants, to be grown for fruit delivery. The infection rate was about 7% (6 out of 83 tested plants), while it reached almost 99% with model plant Four CpCDV-infected watermelon plants a were keN. benthamiana. Conversely, no signal was observed in healthy plants.To evaluate the ability of CpCDV to replicate in the aerial parts and in fruits of experimentally inoculated watermelon plants, DNA samples were analyzed by Southern blotting using a CpCDV-specific probe. As it can be seen in This confirms that the CpCDV agroclone obtained from a symptomatic watermelon plant in Tunisia can efficiently replicate in watermelon plants and their fruits, that exhibit symptoms similar to those induced by WHFS.Over the past years, deep sequencing technologies have opened novel doors to reconstruct viral populations in a high-throughput and cost-effective manner. Up to now, an increasing number of studies have used NGS to either analyze known viruses by means of a reference-guided approach or discover novel viruses using a de novo-based strategy . In thisOrosius albicinctus (Distant) and Orosius orientalis (Matsumura), both originally found in Asia. O. orientalis was also reported in Tunisia [CpCDV, with its wide host range, the seriousness of symptoms induced and its expanding geographical distribution, is an emerging virus and has the potential to become a serious pest for several crops in tropical and Mediterranean countries and worldwide. The two known vectors of CpCDV are  Tunisia  and it iBeside the full-length sequence of the DNA virus CpCDV, the sRNA sequencing has allowed the assembly of the full-length sequence of the RNA virus WMV. WMV infection is widespread in Tunisia on all cucurbit species, at least since the late eighties ,34. The Amalgavirus genus: as reported for other viral species belonging to the same genus, its infection is apparently symptomless and transmitted through seeds. Amalgaviruses have small dsRNA genomes (about 3.4 kbp) and their virions have not yet been detected or identified; moreover, very little is known about their transmission, apart from seed involvement. The family Amalgaviridae is a recently recognized taxon, currently comprising four species of plant-infecting viruses  [The sRNA sequencing also allowed to identify a previously undescribed virus infecting watermelon, here provisionally named WmAV1, belonging to the virus M) ,36,37,38virus M) . It is nIn summary, this work confirmed that high throughput sequencing analysis of sRNAs is a powerful tool for the identification of both known and new viral sequences and for the definition of new etiological agents. The data obtained by this technique, supported by PCR validation and experimental infection in controlled conditions, allowed the causal link between CpCDV and WHFS to be highlighted for the first time.


On 1 February 2016, WHO declared the clusters of microcephaly and neurological disorders and their potential link with Zika virus outbreaks in Brazil and French Polynesia to be a Public Health Emergency of International Concern (PHIC). Since then, there are 75 countries and territories reporting continued mosquito-borne transmission of Zika virus, with 69 countries experiencing a first outbreak of Zika virus since 2015.[The current scientific evidence has now shown that infection with Zika virus in the prenatal period is associated with microcephaly and other serious brain abnormalities.  As of DeThere is currently a global scientific effort to gain a better understanding of the Zika virus, its vectors, modes of transmission, and the natural history of the disease. Key to this effort is having an accurate diagnosis of Zika virus infection; however, available tests are limited by several factors.On 18 November 2016, the Director-General of WHO declared the end of the PHEIC based on the recommendations made by the Emergency Committee on Zika and microcephaly convened under the International Health Regulations (IHR).WHO will continue its research and development (R&D) work on Zika under the WHO R&D Blueprint effort, which aims at reducing the time between declaration of a PHEIC and the availability of diagnostics tests, vaccines, antivirals, and other treatments that can save lives and avert a public health crisis. In order to address the limitations and gaps of current diagnostic tests, WHO led the development of consensus-driven target product profiles (TPPs) with United Nations Children’s Fund (UNICEF), Pan American Health Organization (PAHO), and other organizations. The target product profiles define the desired characteristics of Zika diagnostic tests and are aspirational in nature. Several consultation meetings were held with key stakeholders in the public health and scientific community. This was followed by a public consultation from 22 March to 11 April 2016. Feedback from the consultation was incorporated into the final TPPs, which were published on 13 April 2016. . Two TPP3 and 105 copies/mL. [Current diagnosis of Zika virus infection is challenging for a variety of reasons. Zika virus infection is generally considered a mild infection, which it still is in the majority of cases. Approximately 80% of Zika infections are asymptomatic, and 20% of Zika virus infections present clinically with symptoms of mild fever, maculopapular rash, arthralgia, myalgia, conjunctivitis, and headache.  These sypies/mL. –9 Based pies/mL.  Furthermpies/mL.  This fin6 copies/mL.[There are several small studies that suggest urine and saliva might be the preferred specimen type to diagnose active infection with Zika virus. ,13,14 Ziopies/mL.Cross-reactivity with other flaviviruses is a significant concern and complicates interpretation as well as development of new assays. ,9,15,16 It is currently unknown if there is a difference in the immune response of pregnant women with Zika virus infection. The effect of pregnancy on antibody titers to other viruses has been described previously.,21 In a 7 viral RNA copies per mg of tissue); in this report, Zika virus RNA was not detected in other tissue samples of the fetus. [Available data on diagnosis of prenatal and antenatal Zika virus infection are derived from testing tissues of fetal losses, full-term infants who died shortly after birth, or infants with microcephaly. Pathologic evidence for Zika virus in the setting of microcephaly has been described in a number of case reports and small case series, with detection of Zika virus in brain tissue, amniotic fluid, and cerebrospinal fluid. –27 Isolae fetus.  Zika vire fetus.  Adibi ete fetus. The majority of stakeholders felt that multiplexing capability allowing simultaneous detection of several pathogens in a single sample—particularly Chikungunya virus and Dengue virus—is highly advantageous. Aside from Dengue virus and Chikungungya virus, the choice of pathogens in a multiplexed diagnostic should consider clinically consistent presentations, actionable information, and the needs of epidemiological surveillance. Not all pathogens will be relevant to all settings; nonetheless, the need to differentiate Chikungunya virus, Dengue virus, and Zika virus for both clinical and epidemiologic purposes is of immediate importance and is likely to remain relevant in the future.Multiplexing using dual analytes in a single test   may alsoOperational characteristics  should have attributes common to other ideal tests intended for public health use in low and middle income countries. –33While discussion on testing policy is not a standard component of a TPP, and because the greatest impact of Zika virus infection appears to occur during pregnancy, it is worthwhile to highlight the uncertainty surrounding Zika virus in pregnant women, the need for highly accurate Zika virus tests, and the need for a diagnostic testing algorithm(s) that can be applied during routine antenatal care or counseling. In a cohort of pregnant women in Brazil, 40% of the women who tested positive for Zika virus infection using the best available diagnostic tests in 2015–2016 declined imaging studies. Fear, related to the possible identification of fetal abnormalities related to Zika virus infection, was one of the reasons cited.  In this Additional biologic, clinical, and social science research as well as consensus guidelines appropriate for different settings in which women receive antenatal care will be vital in establishing optimal testing strategies and algorithms for pregnant women.Commercial tests for Zika virus are becoming more available. There are at least seven nucleic acid detection lab-based tests (NAT), three serology tests that have received authorization for emergency use from regulatory agencies—Conformité Européene/in Vitro Diagnostic (CE/IVD), United States Food and Drug Administration Emergency Use Authorization (US FDA EUA), Agência Nacional de Vigilância Sanitária (ANVISA)—and several companies in various stages of product development.Following the declaration by the WHO Director-General of a PHEIC related to microcephaly cases potentially linked to Zika virus, the WHO Prequalification Team announced on 5 February 2016 that the Emergency Use Assessment and Listing (EUAL) procedure established during the 2014 Ebola virus disease outbreak was opened to candidate in vitro diagnostics (IVDs) intended for Zika virus diagnosis. The EUAL procedure was developed to expedite the availability of IVDs needed in public health emergency situations. It is intended to assist interested procurement agencies and Member States on the suitability for use of a specific IVD, based on a minimum set of available quality, safety, and performance data. The EUAL assessment includes a review of the product quality aspects, including manufacturing capability, an assessment of the studies supporting the performance of the test, and an independent performance evaluation complementing the manufacturer’s verification and validations studies.EUAL submission requirements for manufacturers were set following a meeting of experts, including regulators from the affected region and the US FDA. The goal was to identify a set of minimal requirements that are aligned internationally. To date, WHO has received 33 applications for EUAL assessment from 22 manufacturers with applications for a range of technologies, including rapid test format antibody tests, NAT, immunofluorescence, and antigen detection tests.A number of these applications have not met the WHO requirements for quality management systems, or have not undertaken sufficient verification and validation studies to comply with the EUAL requirements, and, as such, have withdrawn. Access to suitable specimens has been a major problem for manufacturers and has limited verification and validation studies. In addition, studies undertaken by manufacturers in Europe on infected travelers returning from the affected region may not provide sufficient challenge to the assay, as many of these patients have not previously been exposed to other flavivirus infections. This may limit the suitability of the assay in many of the Zika virus endemic countries. However, a number of the applications have evidence to indicate adequate performance and suitable manufacturing and are progressing through the assessment process. To date, two products—NAT assays—have passed the three-phase assessment process and were listed as eligible for WHO procurement.In addition to the various molecular tests being developed for commercial use, there are also in-house developed assays and kits used for research purposes.  These laFor Zika RNA, the biological standard for molecular tests had been characterized in an international study involving the majority of nucleic acid amplification technology (NAT)–based assays available. The compThe related work on WHO IS for serological assays detecting Zika antigen or anti-Zika antibodies is ongoing; unfortunately, the sourcing of suitable materials of human origin from Zika virus-affected regions is still an issue.There is an urgent need for new quality-assured diagnostics, particularly for decentralized use. WHO is facilitating the development, validation, and assessment of such diagnostics by issuing two consensus TPPs, by developing WHO reference preparations, and with its EUAL process.Lack of access to well-characterized clinical samples of Zika virus continues to be a challenge. After the recent Ebola outbreak, WHO has developed a framework for developing a specimen library that will allow equitable access for researchers and assay developers. However, this has not yet been realized for this current outbreak.While there has been some progress on the availability of Zika virus diagnostic tests, many challenges remain to meet the diagnostic needs of affected countries. Results of prospective studies will also be useful in designing the testing algorithm with the tests that will be available in the near term. There is a need to see beyond this current outbreak and prepare for the next one. WHO is also contributing to this effort through its R&D Blueprint.


The ability to learn allows organisms to take advantage of dynamic and ephemeral opportunities in their environment. Here we show that learning in belowground entomopathogenic nematodes has cascading multitrophic effects on their hosts, other nematodes, and nematophagous fungal predators. In addition to quantifying these effects, we show that social behavioral plasticity in these belowground parasitoids can amplify signaling by plant defense pathways and results in an almost doubling of insect herbivore infection by entomopathogenic nematodes. Cumulatively, these effects point to the critical role of plant signaling in regulating community structure while suggesting an equally important role for behavioral plasticity in shaping community dynamics. Organisms with high behavioral plasticity, the ability to modify behavior based on past experiences, can avail themselves of dynamic ephemeral opportunities for enhancing fitness that would not exist if genetic inheritance were solely responsible for adaption1. Primates, for example, benefit by taking advantage of resources made available from socially learned tool use3. Similarly, New Caledonian crows benefit from social transmission of tool designs that facilitate access to otherwise unavailable food resources4. While behavioral plasticity, especially social behavioral plasticity, has direct fitness benefits for the organism responding to ephemeral environmental cues, the ability of one individual or species to modify behavior based on past experiences may have cascading impacts on other trophic levels in a community.Social learning in higher organisms is thought to hold adaptive significance5. In contrast to a general predator-prey relationship where many predators affect multiple prey organisms, host-parasitoid relationships tend to be highly specific6. In a given system, the primary goal of a parasitoid is to find (often) a single specific host6. Likewise, hosts must (often) avoid a single specific parasitoid6. This unique dynamic sets the stage for an arms race where any ability to take advantage of ephemeral opportunities in a complex environment has immediate consequences for a tightly linked system.Evidence from parasitoids can be used to begin to elucidate the cascading trophic effects of social learning6. These images need not be merely visual; search images can also be auditory7 or olfactory. For example, olfactory cues are frequently used by insect and entomopathogenic nematode parasitoids to find their host9. While entomopathogenic nematodes, which infect and kill insect larvae belowground with the help of endosymbiotic bacteria, have differences with aboveground insect parasitoids11, their patterns of host infection and associated host mortality fit the functional definition of a parasitoid12. Indeed, there are similarities between the two. Aboveground, specific blends of herbivore induced plant volatiles recruit parasitic wasps13. Belowground, volatile compounds released by plants after induction of defense pathways recruit entomopathogenic nematodes. In maize, feeding belowground by larvae of the beetle Diabrotica virgifera virgifera induces release of (E)-β caryophyllene14 which recruits entomopathogenic nematodes. In certain citrus cultivars, induction of plant defense pathways through belowground herbivory by Diaprepes abbreviatus weevil larvae or foliar application of methyl salicylate induces belowground release of pregeijerene and d-limonene respectively18.For parasitoids, this advantage is often behavioral plasticity. Parasitoids rely on learned search images to better identify and locate their hosts19, extended multitrophic consequences of parasitoid learning have not been quantified. Here, we use a belowground model system involving plant volatiles, entomopathogenic nematodes (parasitoids of insect larvae), their hosts, and nematophagous fungi (predators of the nematode parasitoids) to understand the cascading consequences of parasitoid social behavioral plasticity throughout the system  was monitored in sand-filled bioassays. Entomopathogenic nematodes infect and kill insect larvae with the help of endosymbiotic bacteria and can be effective and efficient biological control agents22. Two of the entomopathogenic nematode species used (S. diaprepesi and H. indica) are commonly found infecting D. abbreviatus larvae in Florida citrus orchards23 where they inhabit a complex and dynamic belowground environment punctuated by the presence of transient plant volatile signals such as d-limonene, (E)-β caryophyllene, and pregeijerene18. Cohorts of these entomopathogenic nematode infective juveniles were exposed for 72 hours to either blank controls or one of either d-limonene, (E)-β caryophyllene, or pregeijerene. After exposure to the plant volatiles, nematode cohorts were then introduced to four-choice, sand-filled olfactometers and given the opportunity to infect D. abbreviatus larval hosts paired with one of the volatiles to which the cohorts had been exposed: either a blank control, d-limonene, (E)-β caryophyllene or pregeijerene. Host infection was then modeled using logistic regression to determine infection probabilities under different learning regimes.To quantify host effects of parasitoid behavioral plasticity, infection of D. abbreviatus larvae  on probability of host infection. With no exposure (i.e. exposure to blank control not containing plant volatiles), host infection probabilities were significantly greater  for larvae paired with the highly attractive herbivore induced plant volatile pregeijerene. Infection probabilities for hosts paired with pregeijerene were 15.5%  greater than blank controls, 20.2%  greater than d-limonene, and 12.5%  greater than (E)-β caryophyllene. Exposure to plant volatiles resulted in significant increases -β caryophyllene: 90.0% , P < 0.001; pregeijerene: 84.4% , P < 0.001) in host infection probability when the host was paired with that volatile. All nematode species behaved similarly; there was no significant effect of nematode species .Behavioral plasticity in response to exposure to plant volatiles affected infection of vae Fig. . Both trAnastrepha suspensa, mealworm Tenebrio molitor, and waxworm Galleria mellonella larvae was evaluated alongside infection of D. abbreviatus to cohorts of H. indica infective juveniles exposed to d-limonene or blank controls in four-arm, sand-filled olfactometers as above.Entomopathogenic nematodes may be somewhat unique in that they have the potential to infect a wide range of insect larvae that may not be considered traditional hosts. To investigate additional host effects of parasitoid behavioral plasticity, infection of Caribbean fruit fly D. abbreviatus  as in the host effects trials above, prior exposure to d-limonene also significantly increased infection probability of Caribbean fruit fly larvae  and waxworm larvae  over exposure to blank controls. Prior exposure to d-limonene marginally increased infection probability of mealworm larvae .Behavioral plasticity in response to exposure to d-limonene resulted in increased infections of all hosts Fig. . While pH. indica infective juveniles were exposed either to d-limonene or blank controls then introduced to four-choice bioassays which were either inoculated with the nematode-trapping fungus Arthrobotrys dactyloides24 or with blank controls. After passing through the nematophagous fungal (or a control) guantlet, infective juveniles had the opportunity to infect insect larvae paired with either d-limonene, (E)-β caryophyllene, pregeijerene, or a blank control.In addition to procuring host insects, entomopathogenic nematode parasitoids must contend with a variety of predators eager for a quick meal. To investigate effects of behavioral plasticity in entomopathogenic nematodes on their nematophagous fungal predators, cohorts of entomopathogenic nematode P = 0.0001) altered host infection probabilities reducing nonexposed nematode host infection probabilities by 88.1%  to essentially zero (lower 95% confidence level of 0.07%)  increasing host infection probabilities by 91.6%  to 18.1%  despite the nematophagous fungal gauntlet.The presence of nematophagous fungi significantly (7%) Fig. . Exposur25. Additionally, entomopathogenic nematodes can demonstrate social behavioral plasticity26. Cohorts of experienced entomopathogenic nematodes can influence the behavior of non-experienced cohorts through what is thought to be a follow the leader dynamic27. To investigate host effects of social behavioral plasticity in entomopathogenic nematodes, cohorts of 50 H. indica infective juveniles were exposed to either d-limonene or blank controls. Following exposure, those cohorts of 50 nematodes (the ‘leaders’) were then combined with cohorts of 2450 nonexposed infective juveniles (the ‘followers’) of either H. indica, S. diaprepesi, or S. riobrave. These combined groups were then introduced to four-arm, sand-filled olfactometers where they had the opportunity to recruit to D. abbreviatus larvae paired with either a blank control, d-limonene, (E)-β caryophyllene or pregeijerene. Fifty entomopathogenic nematodes  are often not enough to overcome host immune defenses25. Indeed, host infection probability from cohorts of 50 entomopathogenic nematodes is just 1.0% .Entomopathogenic nematodes spend much of their life in close proximity to other entomopathogenic nematodes and rely on group attack to overcome host immune systemsD. abbreviatus larvae paired with d-limonene significantly  increased by 97.2% . Joining cohorts of 50 H. indica infective juveniles that were exposed to d-limonene with 2450 nonexposed infective juveniles of either H. indica, S. riobrave, or S. diaprepesi further increased infection probability of D. abbreviatus paired with d-limonene by 13.5%   .Cohorts of 50 educated nematodes are able, however, to influence other entomopathogenic nematodes. When numbers of responding infective juveniles were increased to 2500, infection probabilities of 1%] Fig. . In factst) Fig. . AdditioIn examining cascading multitrophic effects, entomopathogenic nematode behavioral plasticity in response to d-limonene exposure directly increases host infection probability by 89.2% Fig. , Table 1C. elegans, for example, has a grand total of 302 neurons28) relative to the complex cerebral circuitry of such higher organisms, their demonstrated capacity for learning holds cascading implications for the rest of their belowground community.Increases in host infection probability following entomopathogenic nematode exposure to plant volatiles suggests that behavioral plasticity may hold adaptive significance in organisms far removed from primates and New Caledonian crows. While entomopathogenic nematodes have limited neuronal capacity , which can become rapidly unsuitable for colonization due to competition from other nematodes32. While this form of behavioral plasticity is not associative in the classical sense, the first contact entomopathogenic nematodes have after leaving the cadaver may be a sufficient signal to alter future recruitment behavior. Additionally, changes in host infection probability after exposure to pregeijerene suggest that such behavioral plasticity is not limited to mere recruitment behavior. Indeed, changes in numbers of nematodes recruiting after exposure to pregeijerene are minimal 26. The large increases in host infection probability after exposure to pregeijerene suggest that infection behavior, such as willingness to enter the host larva, may be plastic as well.First and foremost, behavioral plasticity in response to plant volatile exposure holds adaptive significance for those nematodes exposed to the volatiles themselves. The belowground environment is dynamic and punctuated by ephemeral signals released by plants, nematodes, and other organisms. Plant volatile responses to herbivory, for example, can peak at nine hours and disappear completely after twenty-fourSecond, behavioral plasticity of entomopathogenic nematode parasitoids in response to plant volatile exposure negatively impacts host species. Natural hosts show increases in infection probability which would likely translate to increased mortality in the field. Additionally, other hosts were also negatively affected by entomopathogenic nematode behavioral plasticity in our investigation. Increases in mortality of other hosts demonstrate the potential for appropriating parasitoid learning capabilities for broadly enhancing biological control of multiple insect pests. While using exposed – ‘trained’ - parasitoids for control of traditional hosts will likely increase efficacy of biological control, the same strategies could be employed to enhance control of and target non-traditional hosts.34. Behavioral plasticity of entomopathogenic nematodes allows them to overcome the nematophagous fungal gauntlet and infect their host insect larvae. Regulation of entomopathogenic nematode populations by nematophagous fungi benefits the insect host population while the ability of entomopathogenic nematodes to overcome such regulation through behavioral plasticity has negative consequences for both the host insect larvae and the nematophagous fungi which lose a food resource. The ability of entomopathogenic nematodes to overcome predation by nematophagous fungi after exposure to a plant volatile bears further investigation but could potentially be attributable to two mechanisms: (1) Learned responses to plant volatiles may motivate entomopathogenic nematodes to move faster, potentially making them harder to catch in fungal snares. (2) Conditioning of entomopathogenic nematodes to plant volatiles may alter the chemical ecology of nematode-fungal interactions. Increased response by entompathogenic nematodes to a given volatile after preexposure can be accompanied by a reduced response to other volatiles26. Training entomopathogenic nematodes to respond to a specific plant volatile may make them less likely to respond to other environmental stimuli, such as attractants released by nematophagous fungi36.Third, in addition to affecting host infection probability, behavioral plasticity in entomopathogenic nematodes affects a higher trophic level: nematophagous fungal predators. Nematophagous fungi are thought to play a critical role in regulating entomopathogenic nematode populations and affect biological control using entomopathogenic nematodes in the field37 as well as provide an explanation for observed aggregative movement in entomopathogenic38 and other parasitic nematodes39. While this aggregative movement holds benefits for nematodes overcoming host immune systems, the implications for inter-specific competition within hosts remains to be explored. Given the persistence of conserved communication in entomopathogenic nematodes, we may find that social behavioral plasticity persists due to other benefits for multiple species within a given host. Additionally, social behavioral plasticity in entomopathogenic nematodes resulting in group movement based on a leader-follower dynamic may provide a means of signal amplification in belowground ecosystems.Fourth, behavioral plasticity in entomopathogenic nematodes affects other nematodes. The ability of nematodes to follow each other, even members of different genera, suggests a remarkable level of conserved communication across diverse species and genera that holds broad implications for the belowground community. Such following behavior might augment the evolutionary utility of ‘sprinter’ individualsFrom a plant perspective, this signal amplification is advantageous. A signal that reaches large numbers of parasitoids is costly and difficult in a patchy belowground environment. Instead, plants can benefit from releasing a brief ephemeral signal that reaches a few parasitoids  which is then amplified by the social behavioral plasticity of belowground entomopathogenic nematode parasitoids. Ultimately, social behavioral plasticity of belowground parasitoids in response to exposure to plant volatiles results in a net 93.2% increase in probability of host infection Fig. . By expo40. Additionally, the cascade of multitrophic effects from social behavioral plasticity suggests an equally important role for behavioral plasticity in shaping community dynamics. Further understanding these effects, and their interactions with plant signaling, will enhance our ability to efficiently and efficaciously manage above and belowground natural systems.The adaptive significance of signal amplification by belowground behavioral plasticity highlights the importance of plants and their defense pathways  as key regulators of community structureDiaprepes abbreviatus weevil larvae was evaluated with cohorts of exposed and non-exposed entomopathogenic nematodes. Cohorts of 2500 Steinernema diaprepesi, Steinernema riobrave, Steinernema carpocapsae, or Heterorhabditis indica infective juveniles were placed into 5 ml water solutions. Nematode cohort solutions then received 5 μl of pentane (blank control) or 5 μl of 1 μg/μl of either pregeijerene, d-limonene, or (E)-β caryophyllene in pentane. Nematodes remained in respective exposure solutions for 72 hours. After 72 hours, nematode cohorts were washed with DI water and released into the central chamber of a four-arm, sand-filled glass olfactometer.To determine the effects of learning in entomopathogenic nematode parasitoids on their hosts, infection of 15 by closing off two of the six arms. Glass olfactometers were further modified by removing all mesh screens to allow entomopathogenic nematodes access to all parts of the olfactometer. Swingle citrumelo (Citrus paradisi Macf. x Poncirus trifoliata L. Raf.) rootstocks were placed in the distal portion of all four arms of the olfactometer along with a single fifth instar D. abbreviatus larva encaged in a stainless steel 1 cm2, 225 mesh screen cube. This mesh size allowed entomopathogenic nematodes to enter and infect larvae, but prevented larvae from feeding on citrus roots. Aliquots consisting of either 30 μl pentane (blank control) or 30 μl of 10 ng/μl solution of pregeijerene, d-limonene, or (E)-β caryophyllene in pentane were placed on filter paper and added to the root zone in each of the four arms. The olfactometer was then filled with washed autoclaved sand adjusted to 10% moisture by volume. Twenty-four hours after introduction of the entomopathogenic nematode infective juveniles, D. abbreviatus larvae were collected from each arm and placed on White traps41 to monitor for infection. Twenty replications of each exposure cohort-nematode species combination were conducted.Four-arm glass olfactometers were constructed from traditional six-arm glass olfactometers used in previous trialsD. abbreviatus, cohorts of exposed and non-exposed H. indica infective juveniles were presented to larvae of the weevil D. abbreviatus (host), the waxworm Galleria mellonella (used for rearing entomopathogenic nematodes), the beetle Tenebrio molitor, and the Caribbean fruit fly Anastrepha suspensa. As in the host effect trials, cohorts of 2500 Heterorhabditis indica infective juveniles were placed into 5 ml water solutions. Nematode cohort solutions then received 5 μl of pentane (blank control) or 5 μl of 1 μg/μl of d-limonene in pentane. Nematodes remained in respective exposure solutions for 72 hours. After 72 hours, nematode cohorts were washed with DI water and released into the central chamber of a four-arm, sand-filled glass olfactometer. Monitoring infection of larvae in olfactometers was conducted as described in Host Effects, with substitutions of alternative hosts for D. abbreviatus larvae. Twenty replications of each exposure cohort-nematode species combination were conducted.To determine the effects of learning in entomopathogenic nematodes on hosts other than H. indica infective juveniles were exposed to either a blank control or d-limonene for 72 hours as in Host Effects trials. After exposure, the cohorts were introduced into glass four-choice, sand-filled olfactometers whose central chambers were either inoculated 24 hours prior with the nematophagous fungus Arthrobotrys dactyloides or a blank control. Arrayed around the central chamber were insect (G. mellonella) larvae paired with either a blank control, d-limonene, (E)-β caryophyllene, or pregeijerene as described above. After allowing 24 hours for infection, insect larvae (G. mellonella in this case) were removed and monitored for infection. Twenty replications of each exposure cohort-fungal presence combination were conducted.To explore effects of nematophagous fungi on host infection by entomopathogenic nematodes, cohorts of H. indica infective juveniles were exposed to either a blank control or d-limonene for 72 hours as described in Host Effects above. After exposure, the cohorts of 50 exposed H. indica were combined with cohorts of 2450 nonexposed infective juveniles of either H. indica, S. riobrave, or S. diaprepesi and introduced to glass four-choice, sand-filled olfactometers as described above. The infective juveniles were given twenty-four hours in which to infect D. abbreviatus larvae paired with either a pentane blank control, d-limonene, (E)-β caryophyllene, or pregeijerene as described above. Host infection from these mixed cohorts was then contrasted with infection resulting from cohorts consisting of 2500 infective juveniles of either H. indica, S. riobrave, S. diaprepesi not exposed to any plant volatiles.To explore effects of social behavioral plasticity in entomopathogenic nematodes on host infection, cohorts of 50 H. indica infective juveniles exposed to d-limonene and 2450 infective juveniles not exposed to plant volatiles, or 2500 infective juveniles not exposed to d-limonene were compared using bioassay procedures described above for both H. indica and S. diaprepesi.To further explore abilities of small numbers of infective juveniles to affect larger populations, host infection by cohorts consisting of either 2500 infective juveniles exposed to d-limonene, mixed cohorts consisting of 50 Steinernema diaprepesi, Steinernema riobrave, Steinernema carpocapsae, or Heterorhabditis indica) were reared in late instar Galleria mellonella waxworm larvae42. After emergence from infected host carcasses, entomopathogenic nematode infective juveniles were collected on White traps, transferred to tissue culture flasks, and stored at 10 °C until use41. All infective juveniles used in experimental trials were less than two weeks old.All four species of entomopathogenic nematodes .A. dactyloides nematophagous fungi were originally isolated from citrus groves in Polk County, FL, USA then cultured at the Citrus Research and Education Center (CREC) in Lake Alfred, FL on quarter strength corn meal agar17. All fungi were sub-cultured monthly and maintained on agar within 9-cm diameter Petri dishes at 27.5 °C. Fungi were exposed to living S. diaprepesi 21 d prior to initiation of assays by spreading approximately 3,000 sterilized (0.2% streptomycin sulphate exposure for 24 h according to methods of ref. Citrus paradisi Macf. × Poncirus trifoliata L. Raf.) rootstocks were grown and maintained at the CREC in a greenhouse at 26 °C and 60–80% RH15.Swingle citrumelo  by entomopathogenic nematodes was modeled using logistic regression . Model selection was accomplished through examination of analysis of deviance, psuedo Overall net effects of behavioral plasticity on host infection probability were conservatively estimated by averaging means, lower 95% confidence levels, and upper 95% confidence intervals across trophic levels.readxl46 was used for Excel-R communication. dplyr47 and tidyr48 were used for data formatting, car49 for analysis of deviance, lsmeans50 for post-hoc comparisons and ggplot251 with scales52 for elegant graphics.Data were collected and collated in Microsoft Excel then analyzed using R version 3.3.1 in the RStudio development environment version 0.99.902. Additional packages were used to facilitate analysis.


Infection with Porcine Epidemic Diarrhea Virus (PEDV) causes vomiting, diarrhea, and dehydration in young pigs. The virus made its first appearance in the U.S. in 2013, where it caused substantial neonatal mortality and economic losses in the U.S. pork industry. Based on outbreak investigations, it is hypothesized that the virus could be transmitted through contaminated feed or contaminated feed surfaces. This potential risk created a demand for research on the inactivation kinetics of PEDV in different environments. Therefore, the objective of this study was to evaluate the survival of PEDV in 9 different feed ingredients when exposed to 60, 70, 80, and 90 °C, as well as the survival on four different surfaces .P > 0.05) in virus survival among the different feed matrices studied when thermally processed at 60 to 90 °C for 5, 10, 15, or 30 min. However, the time necessary to achieve a one log reduction in virus concentration was less (P < 0.05) when ingredients were exposed to temperatures from 70 °C (3.7 min), 80 °C (2.4 min), and 90 °C (2.3 min) compared with 60 °C (4.4 min). The maximum inactivation level (3.9 log) was achieved when heating all ingredients at 90 °C for 30 min. There were no differences in the amount of time necessary to cause a one log reduction in PEDV concentration among the different surfaces.Overall, there were no differences (The results of this study showed that PEDV survival among the 9 feed ingredients evaluated was not different when exposed to thermal treatments for up to 30 min. However, different combinations of temperature and time resulted in achieving a 3 to 4 log reduction of PEDV in all feed ingredients evaluated. Finally, PEDV survival was similar on galvanized steel, stainless steel, aluminum and plastic. Upon infection with Porcine Epidemic Diarrhea Virus (PEDV), pigs experience vomiting, diarrhea, and dehydration leading to high mortality in suckling pigs . The virRecent research on PEDV survival in feed ingredients has shown that it appears to survive longer in soybean meal (greater than 180 days) compared with other commonly used feed ingredients . The autIf a feed ingredient is contaminated when it enters the feed mill, it has been shown to contaminate the feed mill surfaces . PreviouThe objectives of this study were to measure the effect of thermal treatment on inactivation kinetics of PEDV in nine commonly used feed ingredients, and to determine the PEDV inactivation kinetics on various equipment and facility surfaces . We hypothesized that different chemical characteristics of the feed ingredients would affect PEDV survivability when subjected to different thermal treatments and PED virus inactivation would differ among material surfaces.2. The cells were examined daily for the appearance of cytopathic effects (CPE), usually appearing 4 to 5 days post-infection. After CPE was observed, the cells underwent 3 freeze-thaw cycles (−80 °C to 25 °C) and were then centrifuged at 2500×g for 15 min at 4 °C. After centrifugation, the supernatant was collected, aliquoted in 25 mL tubes, and stored at −80 °C until used.The NVSL strain of PEDV was grown in Vero-81 cells, which were grown in Dulbecco’s Modified Eagle Medium , 8% fetal bovine serum , 50 μg/mL gentamicin , 150 μg/mL neomycin sulfate , 1.5 μg/mL fungizone , and 455 μg/mL streptomycin . Before inoculation, the cells were washed 3 times with phosphate buffered Saline solution (pH 7.2). After inoculation, the cells were incubated at 37 °C allowing virus absorption using maintenance medium . After 1 h, new media were added to the flask and the cells were placed in an incubator at 37 °C under 5% COFeed ingredients  were obtained from the feed mill at the Southern Research and Outreach Center of the University of Minnesota . The sample of complete feed evaluated was a phase II starter diet that did not contain any animal by-products . All feed and feed ingredients were tested and confirmed negative for PEDV by real time RT-PCR. Samples were sent to Minnesota Valley Testing Laboratory  to analyze the nutrient composition of each ingredient (Table 4 TCID50/mL) was added to the samples. During this time, the samples were removed from the water bath for about 5 min to complete the inoculation procedure. The inoculated samples were then immediately placed back into the water bath for 0, 5, 10, 15, or 30 min.Five gram aliquots of each ingredient were weighed into plastic scintillation vials  and placed into sealed, airtight, and water proof containers. Preliminary experiments measured the temperature of the feed and feed ingredients after being placed in the water bath and determined that 1 h was required for feed to achieve the maximum temperature of the water bath. During the experiment, the containers were placed in a water bath at 60, 70, 80 and 90 °C for 1 h to reach water bath temperature. Once the samples reached the desired temperature, they were removed and 1 mL of PEDV  [To elute the surviving virus from the samples of feed and feed ingredients, an eluent solution, 3% beef extract  0.05 M glycine (Sigma), pH 7.2 was used. After various time points, this solution was added to the sample aliquot and mixed well. After light centrifugation to remove organic debris, the supernatant was collected. To determine the concentration of surviving virus, a titration was performed by preparing serial 10-fold dilutions of the supernatant in maintenance medium. These dilutions were inoculated into monolayers of Vero-81 cells grown in 96 microtiter well plates  at 100 μL/well using three wells per dilution. The inoculated cells were incubated at 37 °C under 5% COID50/mL) . The virA total of 4 surface materials were evaluated including stainless steel, aluminum, plastic, and galvanized steel. Stainless steel and aluminum sheets were purchased from Hardware Hank . For the plastic surface, 6-well plastic plates  were used, and galvanized steel  was obtained from Home Depot .The PEDV inoculation solution (40 μL) was applied to the center of a sample of stainless steel, aluminum, plastic, or galvanized steel. The virus was allowed to dry for 10 min, and the sheets and plates were then stored at room temperature (~25 °C) for up to 10 days. At 0, 1, 2, 5, and 10 days, surviving virus was eluted from the center of a surface using 400 μL of an elution buffer . To elute the virus, the elution buffer was applied to the surface and then removed off the surface with a pipette. At the time 0 elution point, 78% of the virus was recovered using this method. After elution, the sample was titrated in Vero-81 cells to determine virus concentration. This experiment was then repeated once more to provide a total of two experiments.Inactivation kinetics data on virus survival were analyzed using the Weibull model . The fitN is the surviving virus expressed as TCID50/mL, N0 is the initial virus titer at the start of the experiment, t is time (min), δ is the time of the first log reduction of virus concentration (min), and n is the shape parameter. The shape parameter (n) indicates the shape of the curve with a value n > 1 representing the formation of a shoulder-shaped curve and being convex, and n < 1 represented the formation of a tail-shaped curve and concave in shape, while n = 1 represented a linear function. The adjusted R2 value (Adj. R2) was used to evaluate how well the model fit the experimental data.In eq. P < 0.05. The experimental unit was a single vial.The delta values obtained from the Weibull model indicated the amount of time necessary to reduce the virus concentration by 1 log. The delta values were compared across treatments. Normality was assessed using the UNIVARIATE procedure of SAS. An ANOVA statistical analysis using the PROC-MIXED procedure of SAS was performed to determine statistically significant differences between feed ingredients and between temperature treatments. When evaluating virus survival between the four temperatures, feed ingredient was considered a random effect. Least squared means with a Tukey adjustment were used to determine differences among each treatment if P > 0.05) in survival of PEDV after the thermal treatment among the 9 feed materials evaluated (Table P < 0.05) than at 60 °C  were observed when the virus was exposed to 60 and 70 °C, but greater reductions (P < 0.05) in virus concentration were achieved at 80 and 90 °C. A reduction of 1.9 to 2.0 log was achieved at 60 °C for 15 min, or 70 °C for 10 min, and a 2.2 to 2.4 log reduction occurred after treatment at 60 °C for 30 min, 70 °C for 15 min, or 80 °C for 10 min. Greater than a 3 log reduction was observed when applying 80 °C for 30 min (3.4 log), or 90 °C for 15 min (3.3 log). The maximum log reduction (3.9 log) was achieved at 90 °C for 30 min.When comparing the log reduction achieved after 10, 15, or 30 min, no differences (2 TCID50/g) on each of the four surfaces after 10 days of incubation. When delta values were compared, there were no differences among all 4 surfaces ranging from 0.7 and 7.7 days . These results suggest that under the conditions evaluated in this study (high temperatures and long exposure times), rapid virus inactivation may occur independently of chemical composition of ingredients, and thus, similar processing conditions can be applied to all ingredients to achieve a similar reduction in virus concentration. Our results were unexpected because of the dramatic differences in the pH values of the feed matrices evaluated (3.49 to 8.40). Quist-Rybachuk et al. (2015) found that PEDV was more heat sensitive when the pH increased from 7.2 to 10.2 . The lacAlthough there were no differences in virus survival among the feed materials evaluated between 60 and 70 °C, greater virus inactivation was achieved at 80 and 90 °C. In order to optimize the thermal processing conditions (high temperature and short time) to inactivate PEDV, our data suggest that thermal treatment at 80 °C for 15 min was necessary for achieving a 3-log reduction. This extent of inactivation could also be achieved by thermal processing at 90 °C for 10 min or heating at 70 °C for 30 min. These findings and parameters are consistent with those reported by Hoffman and Wyler 1989), who found that PEDV was relatively stable at 50 °C, but at temperatures greater than 60 °C, the virus lost total infectivity within 30 min 89, who f.If a contaminated ingredient enters the feed mill, it has been demonstrated that this ingredient will contaminate feed mill surfaces and subsequent batches of feed . This reOne of the limitations for applying this combined knowledge into practice is the potential experimental methodology concern of adding 1 mL of media containing the virus to feed samples. The addition of liquid media necessarily increases the moisture content of the sample, and this may affect the virus survival. More research is necessary to compare the effect of moisture content and water activity on PEDV survival, and determine the extent that this factor plays in virus inactivation. In the surface experiment, however, the media was allowed to dry, eliminating this factor as a potential limitation. It is highly likely that the amount of virus excreted by an infected pig, and potentially transmitted via feed, would be much greater than the titer used in the present study. In a study that evaluated residual material in a suspected PEDV contaminated feed bin, CT values between 19.5 and 22.2 were determined . When usComplete feed, vitamin-trace mineral premix, and feed ingredients are potential biosecurity risk factors in the widespread of PEDV to pork production facilities around the world if they become contaminated. The results of this study indicate that there are no differences in virus survival among complete feed, premix, and ingredients with different chemical composition when thermally treated at temperatures greater than 70 °C, suggesting that similar processing conditions will be effective to inactivate PEDV across all types of feed materials. A maximum of 4-log reduction was achieved when applying 90 °C for 30 min. PEDV inactivation kinetics  did not differ among surfaces tested, which indicated that all surfaces have the same relative risk of PEDV transmission.