CpSlt2, an ortholog of the cell wall integrity (CWI) MAPK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica. The CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, and abnormal pigmentation. In addition, the CpSlt2-null mutant exhibited CWI-related phenotypic defects including hypersensitivity to cell wall-disturbing agents and other stresses. Electron microscopy revealed the presence of abnormal hyphae such as intrahyphal hyphae. In addition, virulence assays indicated that the CpSlt2 gene plays an important role in fungal pathogenesis. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. Although mycelial growth was partially recovered, the sectored progeny had dramatically impaired virulence, confirming the CpSlt2 gene has a role in pathogenicity. Compared to a previous mutant of the CpBck1 gene, a MAPKKK gene in CWI pathway, the CpSlt2-null mutant showed similar, although not identical, phenotypic changes and most phenotypic changes were less severe than those of the CpBck1-null mutant. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.We assessed the biological function of  Cryphonectria parasitica (Murrill) Barr that caused chestnut blight disease . Interestingly, single-stranded RNA Cryphonectria Hypovirus 1 (CHV1)-infected strains had characteristic symptoms of viral infection including decreased virulence3, sporulation, female fertility, pigmentation, laccase production, and oxalate accumulation6. In virus-infected strains, symptoms include changes in the host transcriptional profiles11. Since phenotypic changes in virus-infected strains are pleiotropic, a signal transduction pathway during viral symptom development was attributed as a coordinate and specific mechanism of virus-mediated fungal gene regulation. Thus, it has been suggested that in fungi, several signal transduction pathways are perturbed by hypovirus infection18.At the beginning of the 20th century in North America, the chestnut forests were devastated by 20 and are required for numerous processes related to growth, differentiation, and pathogenesis22. Five different MAPK pathways regulate mating, filamentous growth, osmotolerance, CWI, and spore wall assembly in Saccharomyces cerevisiae. Filamentous fungi are known to have three homologous signaling pathways that regulate the pheromone response, CWI, and the osmoregulation/stress response21.The mitogen-activated protein (MAP) kinases are a family of serine-threonine protein kinases, that comprise the signal transduction pathways of three functionally interlinked cascade kinases: MAPKKK (MAP kinase kinase kinase), MAPKK (MAP kinase kinase), and MAPK (MAP kinase). The MAPK signaling pathways are conserved across a wide variety of organisms from yeast to humansC. parasitica, several genes in MAPK pathways and their downstream effectors are regulated by hypovirus, or are involved in pathogenicity26. Among these pathways, the CWI pathway has aroused interest because Cpkk1, an ortholog of yeast Mkk2 (a CWI MAPKK) was found to be modulated by hypovirus and was important for virulence in the chestnut tree27. In addition, recent studies on a mutant of CpBck1, an ortholog of yeast Bck1 (a CWI MAPKKK), showed characteristic phenotypic changes related to CWI and virulence26. Interestingly, sporadic sectorization, which has not been observed in any other fungi, was observed in the CpBck1 mutant and sectored phenotypes were stably inherited in the progeny that were successively transferred from sectored mycelia. Thus, in this study, we analyzed the biological function of CpSlt2, an ortholog of yeast Slt2, to determine if only CpBck1, but not CpSlt2, is implicated in sectorization, and if the CWI pathway plays a key role in sectorization.In C. parasitica genome sequence (http://genome.jgi-psf.org/Crypa1/Crypa1.home.html) using yeast Slt2 gene identified CpSlt2, an ortholog of the yeast Slt2 gene, which showed high similarity to known fungal Slt2-like genes. The corresponding CpSlt2 gene was PCR amplified and the resulting 4,385-bp PCR amplicon was cloned and sequenced. Based on in silico sequence analysis, we performed RT-PCR to obtain a near full-length cDNA clone. The resulting 1,257-bp amplicon was cloned and sequenced. A sequence comparison with the corresponding genomic DNA revealed that the cloned gene consisted of five exons, with four intervening sequences of 89, 99, 86, and 125 bp. The deduced CpSlt2 protein product (CpSLT2) was shown to consist of 418 amino acids, with a predicted molecular size of 47.4 kDa and a pI of 5.47 (GenBank No. KY620036). The deduced amino acid sequence of CpSLT2 contained all conserved protein kinase subdomains and a canonical TEY sequence found in the phosphorylation site. The sequence environment around the start codon was in good agreement with Kozak’s consensus sequence, in that the −3 position was an A in CAAGATG. However, a typical poly(A) signal (AATAAA) was not found downstream of the stop codon.An analysis of the draft CpSlt2 gene showed that the cloned CpSlt2 gene exhibited high amino acid identities with other known fungal SLT2 homologs of Aspergillus nidulans (85% aa identity), A. oryzae (84%), Claviceps purpurea (84%), Colletotrichum lagenarium (91%), Fusarium graminearum (91%), Magnaporthe oryzae (91%), Mycosphaerella graminicola (83%), and S. cerevisiae (62%). In addition, the deduced amino acid sequence of the CpSlt2 gene harbored a Ser/Thr protein kinase domain found in proteins of the PKc-like superfamily between aa 23 and 314. This domain had high amino acid identities to that of A. nidulans (97% aa identity), A. oryzae (96%), C. purpurea (94%), C. lagenarium (97%), F. graminearum (98%), M. oryzae (98%), M. graminicola (93%), and S. cerevisiae (72%). A phylogenetic analysis of homologs from other signaling pathways, such as the pheromone responsive pathway (Fus3/Kss1) and the osmoregulation responsive pathway (Hog1), showed that the cloned CpSlt2 gene belonged to the Slt2-related group of MAPKs  gene-specific primers resulted in that two transformants showed a 2.6-kb PCR amplicon, consistent with the expected size of the replaced alleles of the CpSlt2 gene. Southern blot analysis confirmed the replacement of the wild-type allele with the replaced allele in these two transformants. As shown in Supplementary Fig. SacII-digested genomic DNA from the two putative CpSlt2-null mutants using a probe revealed a single hybridizing band at 3.7 kb, which differed from that of the wild type at 2.5 kb, consistent with the expected size of the replaced allele. In addition, the hybridizing band at 3.7 kb also hybridized to a probe from a 0.3-kb BamHI/SalI hygromycin B resistance (hph) gene fragment (data not shown), suggesting that a part of the corresponding 3′ proximal region of the CpSlt2 gene was replaced with the hph gene. Thus, PCR and Southern blot analysis showed that these two transformants (named TdSLT2-42 and −69 for 42th and 69th transformants resulting from deletion of SLT2) contained transforming vectors that had undergone double-crossover recombination at the CpSlt2 locus, resulting in the replacement of the wild-type allele with the appropriate part of the transforming vector without additional integration.We examined the biological function of the CpSlt2-null mutants exhibited considerably altered colony morphology , the phenotypic changes were markedly recovered compared to that of the CpBck1-null mutant  and the number of distinctive origins of sectorization per plate was reduced to one or two. When the CpSlt2-null mutants were cultured on minimal medium at a temperature of 20 °C instead of 25 °C, no differences in sectorization were observed (data not shown). Once sectored, the sectored progeny maintained their sector characteristics during successive subcultures and did not revert back to wild type or the parental CpSlt2-null mutant.In addition, as culture continued, the CpSlt2-null mutant and its sectored progeny in liquid culture were compared. The primary inoculum of 0.2 g of freshly harvested mycelial mat was homogenized in 50 ml sterile medium, and the resulting slurry was used to inoculate 150 ml liquid medium16. The parental CpSlt2-null mutant showed significantly reduced mycelial mass with abnormal, i.e., pale-greyish brown, pigmentation compared with that of the wild type. The sectored progenies of the CpSlt2-null mutant had a mycelial mass similar to that of the wild type, but their pigmentation was not fully recovered, indicating that the phenotypic changes of the mutants were not affected by the culture medium.The phenotypic changes of the CpSlt2-null mutants and a sectored progeny of a CpSlt2-null mutant (named TdSLT2–69-S1 for a sectored progeny of TdSLT2–69) underwent complementation via in trans transformation of corresponding mutants with a wild-type CpSlt2 gene. PCR amplification of the wild-type allele of CpSlt2 confirmed that all of the complemented transformants acquired a wild-type allele of CpSlt2. The in trans complemented strains (named TcSLT2-69 for a transformant resulting from complementation of SLT2) recovered the characteristics of wild type such as conidiation and pigmentation  showed that the CpSlt2-null mutants displayed characteristic proliferation of new intracellular hyphae, termed intrahyphal hyphae or cell-within-a-cell32. The sectored progeny of the CpSlt2-null mutants had normal elongated hyphae but a more compact mycelial distribution with numerous aggregations of individual hyphae, which can be attributed to geniculate rather than normal straight hyphae. However, no distinctive ultrastructural changes were observed in the sectored progeny.We conducted electron microscopy to examine ultrastructural changes in the ant Fig. . Scannin35, we examined CpSlt2-null mutant responses to various external stimuli.Since CWI increases multi-stress tolerance by fortifying the cell wall and repairing cell wall damage after stressCpSlt2-null mutant exhibited retarded growth, as expected. However, no discernable differences were found in the CpSlt2-null mutant in response to cold temperature at 20 °C. Specifically, there were no differences in the frequency of sectorization i.e., similar ratio of sectorization occurred at two different temperatures.We assessed temperature sensitivity at 20 and 25 °C. As shown in Supplementary Fig. CpSlt2-null mutant responses to the cell wall-perturbing agents Congo red (CR) and sodium dodecyl sulfate (SDS). These cell wall-perturbing agents are known to bind to cell wall components and thus interfere with cell wall composition. As shown in Fig. CpSlt2 mutant background, including the CpSlt2-null mutant and its sectored progeny, had more severe growth defects when cultured on plates containing cell wall-perturbing agents. This cell wall impairment, as evidenced by increased sensitivity to cell wall-perturbing agents, is consistent with the function of the cloned CpSlt2 gene as the MAPK component of the CWI signaling pathway.Next, we examined wild type and CpSlt2-null mutant. Interestingly, under high osmotic stress conditions (1.0 and 2.0 M sorbitol), a severe growth defect was observed in the CpSlt2-null mutant and its sectored progeny, suggesting that the CWI pathway may mediate multi-stress responses via crosstalk with other stress response signaling pathways such as the high osmotic glycerol (HOG) pathway37.We also analyzed responses to osmotic stress. As shown in Fig. CpSlt2-null mutant and its sectored progeny showed retarded growth in a dose-dependent manner up to 100 μM menadione. These results clearly indicate that the CpSlt2 gene plays an important role in ROS stress response  stress by growing the strains on PDAmb supplemented with appropriate concentrations of menadione. Compared with wild type, the CpSlt2-null mutant and its sectored progeny compared with the wild-type strain. The dark brown area was even smaller than that of the hypovirulent UEP1 strain. These results indicate that the production of phenoloxidase was greatly hampered by mutation of the CpSlt2 gene, regardless of the mycelial growth defects, suggesting that phenoloxidase activity may play a role in maintaining fungal CWI or, aside from CWI, this signaling pathway modulate phenoloxidase production resulting in new aspects of phenoloxidase-mediated biological function of the CWI pathway.We examined phenoloxidase activity using the Bavendamm reaction. As shown in Fig. CpSlt2-null mutant greatly decreased the colored area, we next examined whether the CpSlt2-null mutant and its sectored progeny could grow and produce normal stroma pustules on the chestnut tree. Almost no stroma pustules were observed, indicating that the CpSlt2 gene is very important for fungal growth and differentiation on its natural host .Sequence analysis of the cloned Mkk1 mutants were not included in this study, the phenotypic changes and responses to the majority of the tested conditions clearly showed that CpSlt2 and CpBck1 play consorted roles in CWI, fungal growth, morphogenesis, responses to external stimuli responses, host infection, and sectorization. However, CpSlt2-null and CpBck1-null mutants exhibited differences in apical hyphal growth units. Besides difference in the apical hyphal growth units, the CpSlt2-null mutant showed the same or less severe abnormalities, and less sensitivity to stress compared with the CpBck1-null mutant, indicating that the mutant did have differences. Therefore, it is possible that components even in the same CWI pathway may have its own, not-yet-identified, functions. In addition, crosstalk with other MAPK pathways was involved in the physiological response to a specific activating signal. Sequential activation of the HOG1 and CWI pathways has been identified in S. cerevisiae48. Considering the increased osmosensitivity of the CpSlt2-null mutant, it is conceivable that functional crosstalk between the CWI and HOG1 pathways occurs in this fungus. Therefore, crosstalk with other unknown pathways may further increase the complexity of component- and pathway-specific responses. Further studies on two similar, but not identical, mutants will allow us to define the pathway- and component-specific target genes, which will help us understand the global regulatory roles of the CWI pathway.Although C. parasitica strain EP155/2 (ATCC 38755) and its isogenic CHV1-713-infected strain UEP1 were maintained on a plate containing potato dextrose agar (PDA) supplemented with L-methionine (0.1 g/L) and biotin (1 mg/L) at 25 °C with constant low light49. The fungal culture conditions and methods for the preparation of the primary inoculum for the liquid cultures are previously described49. The harvested mycelia were stored at −70 °C and lyophilized for stability as described previously50.The wild-type C. parasitica was screened for the Slt2 homolog and PCR amplification was performed using the primers CpSlt2-gF1 (forward) 5′-CTAAACCAACTGTCGGATGC-3′ and CpSlt2-gR1 (reverse) 5′-CCAGCACAGGGTGTAAGT-3′. The resulting 6.8-kb PCR amplicon was cloned into a pGEM-T Easy vector  and sequenced using the dideoxynucleotide method with universal and synthetic oligonucleotide primers.The genome database of CpSlt2, PCR using reverse transcriptase (RT-PCR) was performed with primers CpSlt2-cF1 (forward) 5′-CAATTCAAGATGGCAGAGTTA-3′ and CpSlt2-cR1 (reverse) 5′-GTGAGACTCTATCTCCTCCCG-3′ at nucleotide (nt) positions −9 to 12 and 1,644 to 1,664 (relative to the start codon), respectively. The resulting 1.3-kb cDNA amplicon was cloned and sequenced.To obtain the cDNA clone of C. parasitica was extracted as described previously 52. The genomic DNA (20 μg) was digested with the restriction enzyme SacII, blotted onto nylon membrane, and hybridized with radioactive-labeled probes.Genomic DNA from CpSlt2 gene, and the hygromycin B phosphotransferase (hph) gene cassette. A 6.8-kb fragment containing full-length CpSlt2 was ligated into a pGEM-T Easy Vector, and the resulting plasmid was used as the template to obtain the 5′- and 3′- flanking regions of the CpSlt2 gene. The PCR amplicons of the 5′- and 3′-flanking regions were amplified with primers CpSlt2–5′FL-F1 (forward) 5′-TCTACCATGAGGCCGTTCGA-3′, CpSlt2-5′FL-R1 (reverse) 5′-TCCTTCAATATCATCTTCTGTCGACACCGAGGACAGGGTTCTGGA-3′, CpSlt2-3′FL-F1 (forward) 5′-GCATAAGGGAGAGCGTCGACTGCCGACGGGAGGAGATAGA-3′, and CpSlt2-3′FL-R1 (reverse) 5′-GCACGTTCATCCAAGCCCTG-3′ to incorporate the hph site for fusion PCR (underlined). The hph cassette was amplified with primers hph-F1 (forward) 5′-TCCAGAACCCTGTCCTCGGTGTCGACAGAAGATGATATTGAAGGA-3′ and hph-R1 (reverse) 5′-TCTATCTCCTCCCGTCGGCAGTCGACGCTCTCCCTTATGC-3′ to incorporate the 5′- and 3′-flanking regions of CpSlt2 for fusion PCR (underlined). Furthermore, three PCR amplicons were overlapped by fusion PCR with primers CpSlt2-5′FL-F1 and CpSlt2-3′FL-R1. The resulting 5.1-kb PCR amplicon was cloned into the pGEM-T Easy vector and sequenced. In the replacement vector pDSLT2, the hph cassette was inserted between −37 and 1,637 bp of the CpSlt2 gene relative to the start codon and was flanked by approximately 1.3 and 1.4 kb of the 5′ and 3′ sequences, respectively. The 5.1-kb NotI fragment of pDSLT2 was then used to transform the virus-free EP155/2 strain.The replacement vector pDSLT2, which was designed to favor double-crossover integration events, was constructed as follows: The pDSLT2 vector consists of three parts, the 5′- and 3′-flanking regions of the CpSlt2-null mutant was performed using the wild-type allele. The complementing vector pCSLT2 was constructed by inserting a NotI/XbaI-digested 5.5-kb fragment containing the full-length CpSlt2 gene into a NotI/XbaI-digested pSilent-Dual1G (pSD1G) vector containing the geneticin resistance cassette51. The resulting vector was then used to transform the CpSlt2-null mutant.Functional complementation of the C. parasitica were performed as described previously52. Transformants were selected from PDAmb plates supplemented with 150 μg/mL hygromycin B  or 150 μg/mL geneticin , passaged three to four times on selective medium, and single-spore isolated, as described previously53. PCR and Southern blot analysis were conducted with genomic DNA from the transformants to confirm the replacement and in trans complementation of the CpSlt2 gene.Protoplast preparation and transformation of CpSlt2-null mutant were compared with wild-type EP155/2 and its isogenic hypovirulent UEP1 strains. Phenotypic changes in growth rate, pigmentation, conidiation, and mating capability were measured using previously described methods50. Morphological characteristics were investigated on PDAmb medium with cell wall perturbing agents such as Congo red (CR), and sodium dodecyl sulfate (SDS)54. To examine the osmosensitivity, the CpSlt2-null mutant was transferred to PDAmb supplemented with four different concentrations  of sorbitol16. Responses to ROS stress were examined on menadione-containing PDAmb, which causes odidative stress55. A virulence test using a piece of chestnut tree bark was conducted according to Lee et al.56.The phenotypic and molecular characteristics of the C. parasitica hyphae. To quantify the changes in the branching pattern, ProRes® CapturePro software  was used to measure the straight length distance from an apical tip to a third-order branch of hyphae.Fungal mycelia were cultured for 5 d on PDAmb and viewed under differential interference contrast (DIC) microscopy  to analyze the blanching pattern of C. parasitica strains, fungal mycelia were cultured for 10 d on PDAmb and then analyzed by SEM and TEM. Briefly, mycelial fragments detached from the outermost region of fungal colonies were fixed with modified Karnovsky’s fixative in 0.05 M sodium cacodylate buffer supplemented with 2% glutaraldehyde and 2% paraformaldehyde at pH 7.257. After post-fixation with 1% osmium tetroxide in 0.05 M sodium cacodylate buffer at pH 7.2 and dehydration through a graded ethanol series of 30, 40, 50, 70, 80, 90, and 100% for 10 min each, the specimens were coated with gold using a Sputter Coater . The specimens were observed with a scanning electron microscope  at 20 kV31.To evaluate the CWI of hyphae of 58. For light microscopy, semi-thin (0.5- to 1-μm) sections were made with a diamond knife after staining with 1% toluidine blue O. Ultra-thin (70- to 80-nm) sections of embedded samples were cut with a diamond knife, stained with 2% uranyl acetate and Reynold’s lead citrate, and observed using a transmission electron microscope  at 100 kV.After dehydration of the mycelia, the fungal samples were embedded in EPON 812 resin following the standard procedures for specimen preparation for TEMp = 0.01.Colony diameters and canker areas were analyzed by ANOVA using SPSS software  and the significance of the effects of the strains and treatments was determined using the Tukey honest significant difference (HSD) test at Supplementary information


Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome.The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR.The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3′ UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato.Potyvirus genome-derived siRNAs and the potato transcripts.The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important The online version of this article (doi:10.1186/s12985-017-0803-8) contains supplementary material, which is available to authorized users. Potato virus Y (PVY) continues to be an economically important pathogen of potato (Solanum tuberosum L.), worldwide, that causes significant yield losses and is detrimental to quality of tubers [Potyviridae, and genus Potyvirus. It is a flexuous rod-shaped virus which exhibits a 9.7 kb + ss RNA genome [Potyiviridae ORF) encodes P3N-PIPO through RNA polymerase slippage mechanism in the P3-encoding region [f tubers –3. PVY bA genome . The geng region , 4–7.Potyvirus is characterized as one of the largest genera of plant-infecting viruses with more than 200 approved and tentative species of pathogenic viruses [The genus  viruses . The PVY viruses –11. Vari viruses , 13. Amo viruses . Potato  viruses .Plant-pathogenic virus infections usually lead to production of virus-derived small interfering RNAs (vsiRNAs) in infected plant cells, as a result of host RNA silencing-mediated defense mechanism –18. PlanThe RNA silencing process also comprises an amplification of signal strategy wherein host-derived RdRp are involved in generation of perfect dsRNA substrates for processing by DCL. This process leads to production of secondary siRNAs which further reinforces the activity of primary siRNAs –25. The With the increase in small RNAs datasets from deep sequencing studies, there is increased development of tools for prediction and identification of their prospective target genes in biological cells as way of functional characterization –31. The In this study, we aimed to identify the derivation of vsiRNAs from the genome of three biologically distinct strains of PVY  and to reveal their propensity for potato transcripts for post-transcriptional gene silencing. This deciphered the role of PVY-derived vsiRNAs as potential pathogenicity determinants in interfering with host potato physiological processes, stress responses and subsequent symptom development.The data for the study has not been obtained directly or indirectly from human or animal subjects nor has the study been conducted on human/animal subjects hence the research has been exempted from Washington State University Institutional Review Board’s (WSU-IRB) review.From our previous study , we obtaThe complete profile of vsiRNAs was categorized into ‘low’ (<50 reads), ‘high’ (> = 50 reads) and ‘very high expression’ (> = 1000 reads) based on the copy or count numbers. The vsiRNAs reads that were between 21 and 24 nt long and had at least 50 copies aligned to the respective PVY genomes were considered as query vsiRNAs for target transcript prediction analysis.S. tuberosum SolTub_3.0 cDNA sequences downloaded from Ensembl (ftp://ftp.ensemblgenomes.org/pub/plants/release-34/fasta/solanum_tuberosum/cdna/) [http://plantgrn.noble.org/psRNATarget/) [S. tuberosum SolTub_3.0 non-coding RNAs (ncRNAs) downloaded from Ensembl (ftp://ftp.ensemblgenomes.org/pub/plants/release-35/fasta/solanum_tuberosum/ncrna/) [http://www.cmbi.ru.nl/cdd/biovenn/) [The vsiRNAs profile was used as a query in psRobot small RNA target prediction algorithm utilizing the following default settings: target penalty score threshold – 2.5, five prime boundary of essential sequence – 2, three prime boundary of essential sequence – 17, maximal number of permitted gaps – 1, position after which with gaps permitted – 17 . Potentim/cdna/) . Lists oTarget/)  was alsoTarget/) . The psR/ncrna/)  using thiovenn/)  was usediovenn/) . CompariS. tuberosum annotation was calculated using the g:Profiler online tool (http://biit.cs.ut.ee/gprofiler/gconvert.cgi) [Solanum tuberosum, Hierarchical filtering - Best per parent group (strong).Gene ontology (GO) enrichment analysis of the target coding transcripts from the ert.cgi)  under thert.cgi) . Transcrhttp://solanaceae.plantbiology.msu.edu/pgsc_download.shtml) [S. tuberosum Group Phureja DM1–3 Genome Annotation v3.4 (based on v3 superscaffolds) [http://www.genome.jp/tools/kaas/) [Pathway mapping of the target coding transcripts was performed using the transcripts IDs as query and ‘PGSC_DM_v3.4_cds_nonredundant.fasta’ (d.shtml)  of S. tuaffolds)  as KEGG s/kaas/)  input fis/kaas/) . The parS. tuberosum cv. Russet Burbank. The transcripts were selected based on their predicted target score threshold  and abundance of vsiRNAs targeting them as predicted by both algorithms.Ten coding transcripts, representing various molecular functions, predicted by both algorithms and found to be commonly targeted by strain-specific vsiRNAs of PVY-N, PVY-NTN and PVY-O were selected for qRT-PCR validation in PVY-NTN infected S. tuberosum cv. Russet Burbank plants, grown under greenhouse conditions as described by Naveed et al. 2014 [Total RNA was extracted from leaves of PVY-NTN-inoculated al. 2014 , using Thttps://www.biosearchtech.com/support/tools/design-software/realtimedesign-software) [-∆∆Ct method was used to determine relative gene expression [Reverse transcription into cDNA was conducted in a 20 μl reaction of 4 μl of 5X iScript Reverse Transcription Supermix , 1 μg of RNA and made up to 20 μl with nuclease-free water. The following conditions were used: priming at 25 °C for 5 min, reverse transcription at 42 °C for 30 min and inactivation at 85 °C for 5 min. qRT-PCR was performed in 20 μl reaction containing 10 μl of 2X SsoAdvanced Universal SYBR Green Supermix , 2 μl of 50 ng cDNA, 0.7 μl (10 μM) of each primer and 6.6 μl of nuclease free water. The primer sequences used for the expression analysis of target transcripts  . Previouoftware) . The reaThe total clean 21-24 nt reads obtained from PVY-N library aligned to PVY-N genome were 3,504,410  whereas total clean reads for PVY-NTN and PVY-O libraries were 4,397,244 (41%) and 3,277,411 (28%), respectively  of PVY strains PVY-N, PVY-NTN and PVY-O, respectively. When unique reads were mapped to respective individual genes of PVY genomes, it revealed considerable differences in the population of small RNAs originating from various genomic positions of the PVY genome. More so, the vsiRNAs exhibited strand bias toward the sense strand of the virus genome over the antisense strand  transcripts as commonly targeted by vsiRNAs of PVY-O, PVY-N and PVY-NTN  could not be assigned to any pathway, however 5541 transcripts were mapped to at least one pathway in KEGG database. Similarly, for the vsiRNAs generated from PVY-NTN and PVY-O libraries, 7172 out of 60,513 and 4624 out of 41,254 targets were mapped to at least one pathway in KEGG database respectively.KEGG pathway enrichment of target coding transcripts of PVY-N library-derived vsiRNAs revealed top target pathways that belonged to plant hormone signal transduction with 316 hits followed by RNA transport (175), starch and sucrose metabolism (157), spliceosome (146) amongst others Fig. . The PVYThe general scheme for the vsiRNAs of all the strains entailed targeting plant hormone signaling pathway, genetic information processing pathways like RNA transport, ribosomes, spliceosomes followed by transcripts involved in plant-pathogen interaction Fig. . This suThe target coding transcripts Table  were anaThe 7073 coding transcripts, that were commonly targeted Fig. . In the Ten coding transcripts were selected for qRT-PCR validation based on their predicted target score threshold  and abundance of vsiRNAs targeting them as predicted by both algorithms. In psRobot predictions, the transcript PGSC0003DMT400075191 and transcript PGSC0003DMT400077373 Fig. . The psRP < 0.05, Student’s t-test). Transcript expression level of HSP-90 was reduced by log2(FC) = −6.07 and β-1,3 galactosyltransferase 2 was reduced by log2(FC) = −1.88  = −1.08, log2(FC) = −1.15 and log2(FC) = −1.09, respectively, while for the other transcripts it was log2(FC) < 1  and PGSC0003DMT400074517 - β-1,3 galactosyltransferase 2 showed significant change in expression level (The targeting of heat shock protein (HSP)-90 suggests its potential involvement in plant defense and stress responses . The dowThe predicted transcripts that showed no significant change in expression level could also suggest the involvement of other processes or factors in their gene regulation that may include, RNA-silencing suppressors coded for by the virus, abundance and complementarity of vsiRNAs, incorporation of vsiRNAs into the AGOs and their efficiency on mRNA degradation .S. tuberosum SolTub_3.0 cDNA sequences and S. tuberosum SolTub_3.0 non-coding RNAs) were employed effectively to identify genes that are potential targets for vsiRNA-induced silencing. It is vital to note that different small RNA target prediction algorithms predicted target transcripts at varying degrees, as such, intersections in outputs by different algorithms deliver precision and reduces false positives [We studied the role of highly frequent PVY-derived vsiRNAs of three biologically distinct strains of PVY  for their putative global effects on the host transcripts. The important resources of small RNA target prediction algorithms, psRobot and psRNATarget, to map vsiRNAs targets on the transcripts of potato and the potato genome annotations  are known to elicit varied biological responses and differential expression of their genome-derived vsiRNAs , 15. WitThe PVY-NTN strain is known to cause tuber necrosis in the potato tubers of susceptible plants and has been associated with manifestation of the potato tuber necrotic ringspot disease (PTNRD) , 52, 53.It must be stated that, while some target coding transcripts were exclusive to each strain of PVY, majority of the predicted transcripts were common amongst them Fig. . The samFunctional annotation indicated that the target potato transcripts encompassed broad functional properties. The ncRNAs MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5 were identified amongst top target candidates for vsiRNAs of PVY. The MIR821 has been shown to be involved in metabolic processes and stress responses –61. The Nicotiana benthamiana to Pepper mottle virus, a Potyvirus [Cucumber mosaic virus, a + ss RNA virus, in tomato [In our study, the HSP-90 was identified to be significantly downregulated upon PVY-NTN infection in potato  = −1.15  aquaporin transcripts are high in lines resistant to Tomato yellow leaf curl virus (TYLCV) as opposed to susceptible lines [The β-1,3 galactosyltransferase 2 was also significantly downregulated in PVY-NTN infected potato. Galactosyltransferases are proteins exhibiting membrane-spanning domains, and through glycosylation of glycoconjugates , are involved in a diversity of functions that include cell wall structure, cell–cell interactions and signaling and host–pathogen interactions –71. A β-.15 Fig.  in PVY-N.15 Fig. . Moreove.15 Fig. . It has le lines . This suSugarcane mosaic virus (SCMV)-derived siRNAs with maize mRNAs have been proven and it was revealed that host mRNA involved in ribosome biogenesis, other biotic and abiotic stress-related pathways were the targets [SolWD40-repeat) were identified for small RNAs derived from Pospiviroid infecting tomato and downregulation of host mRNAs was demonstrated [The findings presented here are in accordance with the earlier works on virus-derived siRNAs and off-target silencing of host transcripts , 78, 79. targets . SimilarPotyvirus genome-derived siRNAs and potato transcripts. It highlights the significance of deeper understanding of the role of vsiRNAs on viral replication, pathogenicity and host machinery. These clues on virus-host interactions can be applied in developing novel strategies for disease management.Evidence of vsiRNAs interfering with and silencing host plant genes has also been shown . In addiThe differential accumulation of PVY strain-specific vsiRNAs in potato cv. Russet Burbank was demonstrated. Most of the vsiRNAs populations were derived with high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN and P1, 3′ UTR and NIa for PVY-O genomic regions. The host genes targeted by the PVY-derived vsiRNAs were found to be involved in plant hormone signaling, genetic information processing and defense/ stress responses suggesting a counter-defense strategy by the virus to the host RNA-silencing machinery. The broad range of host genes targeted by PVY-derived vsiRNAs in infected potato suggests a diverse role for vsiRNAs. Future work could focus on the confirmation of the vast majority of the predicted transcripts, more so, on the transcripts exclusively targeted by the PVY strains.Additional file 1: Table S1.Primers used for qRT-PCR of target transcripts. (DOCX 13 kb)Additional file 2: Figure S1.Potato virus Y-NTN, Potato virus Y-N and Potato virus Y-O mapped to regions of respective Potato virus Y genome. Figure S2. Graphical representation of total virus-derived short-interfering RNAs originated from sense and antisense strands of Potato virus Y-NTN genome mapped to genomic regions of Potato virus Y-NTN. Figure S3. Graphical representation of total virus-derived short-interfering RNAs originated from sense and antisense strands of Potato virus Y-N genome mapped to genomic regions of Potato virus Y-N. Figure S4. Graphical representation of total virus-derived short-interfering RNAs originated from sense and antisense strands of Potato virus Y-O genome mapped to genomic regions of Potato virus Y-O. (DOCX 323 kb)Graphical representation of total virus-derived short-interfering RNAs of Additional file 3: Table S2.psRobot predicted PVY-N vsiRNAs target coding transcripts. (XLSX 992 kb)Additional file 4: Table S3.psRobot predicted PVY-NTN vsiRNAs target coding transcripts. (XLSX 1242 kb)Additional file 5: Table S4.psRobot predicted PVY-O vsiRNAs target coding transcripts. (XLSX 466 kb)Additional file 6: Table S5.vsiRNA abundance and scores to selected psRobot predicted coding transcripts. (DOCX 16 kb)


Surveillance and monitoring of viral pathogens circulating in humans and wildlife, together with the identification of emerging infectious diseases (EIDs), are critical for the prediction of future disease outbreaks and epidemics at an early stage. It is advisable to sample a broad range of vertebrates and invertebrates at different temporospatial levels on a regular basis to detect possible candidate viruses at their natural source. However, virus surveillance systems can be expensive, costly in terms of finances and resources and inadequate for sampling sufficient numbers of different host species over space and time. Recent publications have presented the concept of a new virus surveillance system, coining the terms “flying biological syringes”, “xenosurveillance” and “vector-enabled metagenomics”. According to these novel and promising surveillance approaches, viral metagenomics on engorged mosquitoes might reflect the viral diversity of numerous mammals, birds and humans, combined in the mosquitoes’ blood meal during feeding on the host. In this review article, we summarize the literature on vector-enabled metagenomics (VEM) techniques and its application in disease surveillance in humans. Furthermore, we highlight the combination of VEM and “invertebrate-derived DNA” (iDNA) analysis to identify the host DNA within the mosquito midgut. The recent epidemic of Ebola virus in Africa as well as the emergence of a hitherto unknown virus known as Middle East respiratory syndrome coronavirus (MERS-CoV), Bas-Congo virus in central Africa or of severe fever with thrombocytopenia syndrome virus (SFTSV) in China have repeatedly shown the global impact of emerging infectious diseases (EIDs) on economics and public health ,2,3,4. TParvoviridae, Flaviviridae, Togaviridae, Rhabdoviridae, Bunyaviridae, Reoviridae, Mesoniviridae, Tymoviridae and Birnaviridae [Togaviridae, Flaviviridae and Bunyaviridae, comprising highly pathogenic viruses such as Dengue virus, Chikungunya virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus and Rift Valley fever virus [To our knowledge, there are only few mosquito virome studies in the database as yet ,17,18,19aviridae ,17. In caviridae . The majer virus ,23.Culex tritaeniorhynchus, Anopheles sinensis, Armigeres subalbatus and Culex fatigans) from Hubei province in China [Anelloviridae, genus Iotatorquevirus) which is widely distributed in pigs [Most studies on mosquito viruses focus on mosquito-infecting viruses or mosquito-borne viruses, neglecting the presence of vertebrate viruses originating from the blood of the mosquitoes’ host that have accumulated in the mosquitoes’ intestine during blood feeding. Shi et al. give an example of such viruses in the metagenomic survey on viral abundance in mosquitoes  . The groin blood . Anellovin blood ,32. It iAedes albopictus mosquitoes as flying “biological syringes” to draw blood for virus titer determinations in small lab-housed vertebrates [Gallus gallus, house sparrows Passer domesticus; mammals: hamsters Mesocricetus auratus) were experimentally infected with West Nile virus (Flaviviridae) and Highlands J virus (Togaviridae). Blood samples were recovered from the mosquitoes’ blood meals after feeding on the mentioned lab animals and compared to blood drawn by syringe. Virus titers derived from these two methods were not significantly different. The technique has also been successfully used on small reptiles before, but was not compared to standard methods and viremia titers were not measured [If VEM and such approaches will be used to survey human and animal viruses circulating in a given area, it is of importance to understand how long viruses last in the mosquito’s blood meal. Following the introduction of VEM by Ng and others, several studies have shown that both DNA and RNA viruses are still detectable in the mosquito blood meal at least 24 h after ingestion. Kading et al. used tebrates . The labmeasured .Anopheles gambiae mosquitoes [Anopheles gambiae mosquitoes for up to 24 h post feeding. Furthermore, RNA virus recovery from mosquitoes that were allowed to feed artificially on a membrane feeding apparatus and from mosquitoes fed on hamsters with active replicating infections did not differ significantly.In a second feasibility study to use mosquito blood meals for virus detection with a novel approach called xenosurveillance, Grubaugh and others developed a laboratory model to explore variables influencing detection of human immunodeficiency virus 1, West Nile virus, Pirital virus, Lassa virus and Chikungunya virus in blood-fed squitoes . The patAnopheles gambiae mosquitoes were collected in villages in northern Liberia. Using NGS, the research group found human Epstein-Barr virus (EBV), a highly prevalent gamma-herpesvirus that infects B-lymphocytes [To assess the feasibility of xenosurveillance for the detection of human pathogens in real settings, indoor resting, blood-fed phocytes ,36. It iAnopheles stephensi mosquitoes were fed with Dengue virus-infected blood, and the efficiency of recovering viral RNA was analyzed after serial time points by qRT-PCR and NGS. Results showed that viral Dengue RNA in the blood meal is gradually degraded but still detectable 24 h after blood feeding.A third feasibility study for using mosquito blood meals as a tool for human and animal virus surveillance was conducted by Yang and others . The stuSince various blood-feeding animals may accumulate viruses of interest, VEM surveillance efforts should not be limited to mosquitoes exclusively. Here we summarize several groups of hematophagous arthropods and leeches that might be useful for surveillance purposes.Arachnida, subclass Acari) are a source of many highly pathogenic vector-borne diseases such as Crimean-Congo hemorrhagic fever virus (CCHFV) and tick-borne encephalitis virus (TBEV) [Rhipicephalus spp. ticks from Yunnan, China, revealed the presence of a virus belonging to the Anelloviridae that was closely related to torque teno canis virus, suggesting a vertebrate origin of the virus [Ticks (class s (TBEV) ,39,40. Ts (TBEV) ,42,43,44s (TBEV) . It has s (TBEV) ,47. The he virus ,13,30. Fhe virus . Howeverhe virus .Ceratopogonidae, Culicoides Latreille), small blood-sucking flies, have been found in almost all parts of the world in all environments [Biting midges  sampled near poultry farms have been used for the detection of H5N1 virus [Musca domestica) [Wild-caught carrion flies  ,58. Furtmestica) ,60. SincCimex lectularius and Cimex hemipterus, family Cimicidae) infestations are increasing worldwide also in industrialized countries [Common bed bug  are hematophagous insects which feed on mammals, including humans, and birds. They are known to transmit feline leukemia virus and myxoma virus ,66. To oHippoboscoidea, families Streblidae and Nycteribiidae) are specialized ectoparasititic insects only associated with bats [Bat flies  is able to take up to 15 mL of blood from its host [Leeches belong to the phylum Annelida. They can be found in freshwater, terrestrial and marine environments . They feits host . Upon feits host ,72,73. Iits host . Most spits host . TherefoPediculus humanus corporis) engorged in vitro with artificially infected human blood [Lice are wingless arthropods, which live as permanent ectoparasites of birds, mammals and humans. No zoonotic transmission of viruses from lice has been reported, but VEM has been shown successfully for human body lice  ,36. ThesHowever, VEM can also be applied to easily survey characterized viruses of known pathogenicity over space and time. Since the linkage of such identified viruses to the host is possible with iDNA approaches, the potential health risk might be identified if certain viruses increase remarkably in a given area or emerge in new areas or novel hosts.Climate and ecosystem changes, demographics and human behavior have contributed to the increased emergence and re-emergence of viral pathogens globally ,7,121. T


Although bats are natural reservoir hosts for numerous zoonotic viruses, little is known about the long-term dynamics of the host immune response following infection and how these viruses are maintained in nature. The Egyptian rousette bat (ERB) is a known reservoir host for Marburg virus (MARV). Following infection of ERBs with MARV, virus-specific IgG antibodies are induced but rapidly wane and by 3 months post-infection the bats are seronegative. To determine whether reinfection of ERBs plays a role in MARV maintenance, we challenge groups of ERBs that were “naturally” or experimentally infected with MARV 17–24 months prior. No bats in either group exhibit evidence of MARV replication or shedding and all bats develop virus-specific secondary immune responses. This study demonstrates that infection of ERBs with MARV induces long-term protective immunity against reinfection and indicates that other factors, such as host population dynamics, drive MARV maintenance in nature. Chiroptera) have been implicated as natural reservoir hosts for numerous zoonotic viruses including coronaviruses1, filoviruses2, lyssaviruses3 and paramyxoviruses5. Although much insight on the natural history of virus infection in bats has been gained from experimental infections11 and mathematical modeling15, with the exception of Nipah and rabies viruses17, relatively little is known about the long-term dynamics of the host immune response following primary virus infection. Of particular interest is whether or not long-term protective immunity is established following virus infection. This gap in knowledge can be attributed to: 1) the difficulties associated with obtaining serial biological samples from individual bats comprising large colonial, interconnected populations and 2) the uncertainty of whether short-term experimental models of virus infection in bats recapitulate natural bat-virus infection dynamics. Nonetheless, obtaining empirical data on the long-term dynamics of the bat immune response following virus infection is critical to understanding how bat-borne viruses are maintained in nature and identifying factors leading to virus spillover into humans.Bats  and Ravn virus (RAVV)) cause outbreaks of hemorrhagic disease in sub-Saharan Africa characterized by human-to-human transmission and high case fatality ratios18. The cave-roosting Egyptian rousette bat  has been identified as a natural reservoir host for the marburgviruses and a source of virus emergence in humans20. A longitudinal ecological study of marburgvirus infection in large ERB populations at Python Cave and Kitaka Mine, Uganda revealed an age-associated cyclical pattern of virus infection in which pups  are seemingly protected from virus infection through maternal antibodies until becoming independent, young juveniles at roughly 3 months of age 19. Acute virus infection levels increase in the juvenile population, peaking at 6 months of age , coincidental with the timing of the biannual birthing seasons. Juveniles enter the adult population at 7–8 months of age; a population that experiences year-round, consistent levels of virus infection . A susceptible-exposed-infectious-resistant (SEIR) mathematical model of marburgvirus transmission in a closed ERB population of 40,000 individuals with a twice-yearly birth pulse and a 21-day latent period predicted a prevalence of active infection (<2.0%) comparable to that observed in the Python Cave ecological study (2.5%), but predicted a seroprevalence (100.0%) remarkably higher than that which was observed in adults14. This discrepancy is likely due to waning marburgvirus antibody levels that may or may not be indicative of diminished protective immunity14.The marburgviruses 11, the oral mucosa from 5–19 DPI (91.7% of bats for a mean duration of 4.6 d)11, multiple tissues from 2–12 DPI10 and the spleen up to 28 DPI (66.7% of bats at this time point)8. MARV IgG antibodies peak by 28 DPI and then rapidly decline11, falling below the threshold of seropositivity by 3 months post infection (MPI)11. Short-term protective immunity against viral replication and shedding has been demonstrated in seropositive ERBs challenged with homologous virus 48 days following experimental inoculation with a moderately high dose of MARV9. However, whether natural MARV infection generates long-term immunity sufficient to fully protect seronegative ERBs from viral reinfection, replication and shedding remains unclear.Serological data gathered from experimental studies of MARV infection in captive ERBs11. In this study, we assess whether MARV infection confers long-term protective immunity against reinfection, replication and shedding by challenging groups of ERBs that had been experimentally or “naturally” infected 17–24 months prior during the previous transmission study with homologous virus. Following challenge, evidence of MARV replication in the blood and viral shedding from the oral mucosa is monitored for 14 days, MARV IgG antibody responses are monitored for 21 days and tissues obtained at necropsy at 21 days are tested for the presence of MARV RNA. Herein, we show that no bats in either group exhibit evidence of MARV replication or shedding. Further, all bats develop virus-specific secondary immune responses, demonstrating that infection of ERBs with MARV induces long-term, and likely lifelong, protective immunity against reinfection.We previously demonstrated horizontal MARV transmission between experimentally infected and naïve contact ERBsGroup 1 was comprised of 5 bats that were experimentally infected with MARV 24 months previously, group 2 was comprised of 5 bats that were “naturally” infected through contact with the experimentally infected bats 17–18 months previously and group 3 was comprised of 2 negative control bats  and 2   = 13.504, two-tailed P = 0.138) or the sexes over time  = 13.869, two-tailed P = 0.472). Group 3 bats tested uniformly MARV seronegative throughout the study.During the previous transmission study, MARV IgG (heavy and light chains) antibody levels for bats in groups 1 and 2 peaked and then rapidly declined falling below the threshold of seropositivity  ≥ 0.95) within 3 months11, the oral mucosa from 5–19 DPI (91.7% of bats for a mean duration of 4.6 d)11 and the spleen up to 28 DPI (66.7% of bats at this time point)8, and MARV IgG antibody levels are undetectable through 7 DPI, begin to rise at 9 DPI and peak between 14 and 28 DPI11. Prior to the initiation of this study, ERBs that were experimentally inoculated with MARV 24 months previously and those that were “naturally” infected through contact with infectious ERBs approximately 17–18 months previously were MARV seronegative11. Following subcutaneous challenge with a moderately high dose of MARV, virus was not detected in daily blood or oral swab specimens taken through 14 DPC and axillary lymph node, gonad, liver, salivary gland or spleen tissue taken at 21 DPC. A robust MARV IgG antibody response was observed at 7 DPC. The absence of MARV replication in the blood and spleen and virus shedding from the oral mucosa coupled with a rapidly attained, robust MARV IgG antibody response upon virus re-exposure is characteristic of a fully protective, secondary immune response.The virus infection and immune response dynamics following reinfection with MARV are in stark contrast to those observed following primary infection, where the virus can be detected in the blood from 1–16 DPI infection (100% of bats for a mean duration of 6.0 d)Eptesicus fuscus), which found that primary infection followed by a transient virus-specific neutralizing antibody response was not able to protect all bats from reinfection and mortality upon challenge 6 months later16. Evidence for Nipah virus recrudescence was reported in an adult female large flying fox (Pteropus vampyrus) who was seropositive at study entry, seronegative 1 month later, shed infectious virus in the urine ~10 months later and then returned to being seropositive within 2 weeks17. Other studies have also observed rapidly waning virus-specific IgG or neutralizing antibodies following experimental infection of bats with Nipah7, Hendra6 and Japanese encephalitis viruses21. Serological assays designed to detect virus-specific IgG or neutralizing antibodies in bats, particularly those with conservatively high seropositivity thresholds, likely underestimate the number of past infections as they are only able to detect those that have been infected recently (<3 months) or exposed to the virus more than once. This likely explains the discrepancy between observed and predicted seroprevalence in the SEIR model of marburgvirus transmission in ERBs14 and indicates that mathematical models of filovirus-natural host dynamics should continue to be parameterized using virus PCR prevalence data rather than seroprevalence data.Our data demonstrate that diminished MARV IgG antibody levels following primary infection are not associated with the loss of long-term protective immunity against virus reinfection, replication and shedding. This finding is in contrast to an experimental study on repeated rabies virus infection of big brown bats  to 0.53  for the black flying fox 22, 0.43 (juveniles) to 0.83 (adults) for the straw-colored fruit bat (Eidolon helvum)23 and 0.47  to 0.58  for the common fruit bat (Artibeus jamaicensis)24. An exponential life table created from ~16,000 captures of ~9,000 A. jamaicensis estimated the average lifespan of this bat in the wild to be 1.6 years24. Based on these estimates, it is likely that the average lifespan of wild ERBs does not exceed 24 months and that primary MARV infection provides lifelong protective immunity against reinfection.A previous study showed short-term protective immunity against MARV reinfection in seropositive ERBs challenged with homologous virus 48 days following experimental infection25, including bat-borne viruses. Reduced mortality of big brown bats (Eptesicus fuscus) during winter hibernation has been associated with rabies virus maintenance13 and the birthing and lactation stages of the seasonal reproductive cycle in little red flying foxes (Pteropus scapulatus) have been linked to a higher risk of Hendra virus infection26. Marburgvirus prevalence data collected during the longitudinal ecological investigation of ERB colonies at Python Cave and Kitaka Mine, Uganda revealed that distinct pulses of virus infection in newly susceptible six-month old ERBs temporally coincided the biannual birthing season19, which occurs just before the peak of the rainy season27. The SEIR model of marburgvirus infection14 found that the virus was only able to persist when biannual breeding was incorporated into the model. Mathematical modeling of infectious agent-host dynamics indicate that pathogen persistence increases gradually with population size28 and measles virus, perhaps the most comprehensively modeled virus-host system, was not predicted to persist in isolated populations of less than 250,000 individuals30. ERB colonies in tropical Africa where marburgviruses are found are large, often numbering over 100,000 individuals19, while ERB colonies in the Palearctic Region where marburgviruses have never been reported are considerably smaller, comprising no more than 1,500 individuals31. Accordingly, modeling predicted that marburgviruses were only able to persist in ERB populations > 20,000 individuals14. Extinction of infectious diseases in mobile populations is typically local as the agent can be reintroduced from spatially separated populations through the dispersal of infectious animals. Metapopulation dynamics have been shown to play a role in the persistence of measles virus in demographically heterogeneous human populations32, Hendra virus in flying foxes (Pteropus spp.) in Australia15 and Nipah virus in large flying foxes (P. vampyrus) and variable flying foxes (Pteropus hypomelanus) in Malaysia33. ERBs are patchily distributed throughout sub-Saharan Africa, with the limit of their geographic range extending north into southern Turkey and east into Pakistan34. Mark-recapture studies have documented movement patterns of ERBs ranging from local migration between roosts 35 km35 to 50 km19 apart to long-distance migration up to 500 km35. Infectious marburgvirus has been exchanged through the sub-Saharan metapopulation network, as evidenced through the detection of homologous marburgvirus sequences from bats and humans throughout sub-Saharan Africa20.The results of this study indicate that reinfection of bats with MARV as a consequence of diminished protective immunity does not significantly contribute to virus maintenance in natural ERB populations. A combination of other factors, such as seasonal variation in host and environmental factors, large population sizes and metapopulation dynamics, likely drive MARV maintenance and prevent virus extinction. Seasonal variations in precipitation and temperature, as well as host aggregation, births, deaths and immunity, have been shown to play a major role in the persistence of many wildlife diseasesMyotis spp. and Pipistrellus spp.36, pregnancy and lactation were significant risk factors for Hendra virus neutralizing antibodies in longitudinally-sampled spectacled flying foxes (Pteropus conspicillatus)37 and little red flying foxes (P. scapulatus)26, and nutritional stress was a significant risk factor for detectable Hendra virus neutralizing antibodies in longitudinally-sampled P. scapulatus26.Although valuable information on the long-term dynamics of the ERB immune response to MARV was obtained from this study, the data should be interpreted in light of its limitations. This study used a small number of 2-year old, laboratory-bred bats that were contained in a controlled environment throughout the study. If more bats had been included in this study, we may have observed individual variations in response to MARV challenge. Unlike their wild counterparts, these experimental bats were not subjected to stressors including pregnancy, lactation, poor nutrition, predation, high ectoparasite burdens and infection with other pathogens. It is possible that exposure to natural stressors such as these could compromise the host immune system, resulting in increased susceptibility to reinfection with marburgviruses in nature. Lactation has been significantly correlated with coronavirus detection in In conclusion, this study provides empirical evidence that “natural” infection of ERBs with MARV confers long-term protective immunity against virus reinfection, replication and shedding. This indicates that MARV maintenance in the ERB population is likely driven by other factors such as influxes of susceptible juveniles provided by the twice-yearly birth pulse, large population sizes and metapopulation dynamics. Further research is needed to determine the specific mechanisms involved in MARV clearance and protective immunity.2. Prior to use in this study, the virus was passaged twice in Vero-E6 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; sex: female, authenticated by ATCC) in maintenance media , 100 units/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies) and 2.50 µg/ml amphotericin B (Life Technologies)) at 37 °C in the presence of 5% CO2. The second passage stock virus (mycoplasma-free) was then titrated in the same cell line by tissue culture infectious dose 50 (TCID50) assay.The 371 bat strain of MARV  used in this study was isolated from pooled liver/spleen collected from an ERB in 2007 in southwestern Uganda8.The captive-born bats used in this study originated from the ERB breeding colony at the Centers for Disease Control and Prevention . This MARV-free colony was established from wild-caught ERBs imported from UgandaAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the CDC and performed according to the Guide for the Care and Use of Laboratory Animals . The CDC is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) fully accredited research facility.Procedures conducted with MARV or MARV-infected bats were performed at the CDC under biosafety level 4 (BSL-4) laboratory conditions in compliance with Select Agent Regulations . All bat cages were placed within bio-flow isolator units with HEPA-filtered inlet and exhaust air supplies .ad libitum.All bats were group-housed in a climate controlled BSL-4 animal area, with a 12 h day/12 h night cycle. Bats were provided daily with their body mass in fresh fruit supplemented with protein/vitamin powder  and received water The number of bats used in this study was based on their previous MARV infection status , as well as the amount of BSL-4 laboratory space required and the number of personnel needed to care for the bats over 24 months. The bat groups were sex-matched and no other methods of randomization were used throughout the study. Investigators were not blinded during the study.Rousettus aegyptiacus; 31 m of age; 6 males and 6 females; average weight of 164.0 g) were transferred from a previous 9-month long MARV transmission study that was initiated 24 months previously when the bats were 7 months of age (juveniles)11. The ERBs were divided into three groups  that were “naturally” infected through contact with the MARV-experimentally infected bats (group 1), as evidenced through the detection of MARV RNA in blood or oral swab specimens at 7 MPI and/or MARV IgG antibodies between 7 and 8 MPI. Group 3 was comprised of 2 negative control bats  that tested uniformly negative for MARV RNA and MARV IgG antibodies throughout the previous transmission study.A total of 12 healthy, adult ERBs  in the caudal abdominal region and bats in group 3 were inoculated in the same manner with 0.25 mL of sterile DMEM. Groups 1 and 2 were housed in separate cages maintained within a single bio-flow isolator unit, while group 3 was housed in a cage maintained in a separate bio-flow isolator unit.At 0 DPC, bats in groups 1 and 2 were subcutaneously inoculated under isoflurane anesthesia with 4 logBlood was taken daily from 0–14 DPC and at 21 DPC from the cephalic wing vein using a sterile lancet . Blood was tested for the presence of MARV RNA by Q-RT-PCR through 14 DPC and MARV IgG antibody responses were monitored weekly through 21 DPC. The oral mucosa was sampled daily through 14 DPC using two polyester-tipped applicators . One oral swab was tested for the presence of MARV RNA by Q-RT-PCR and the second swab was frozen in sterile media for attempted virus isolation of any MARV RNA positive swabs.At 21 DPC, the bats were euthanized by cardiac exsanguination under anesthesia followed by an overdose of isoflurane. At necropsy, the following tissues were collected: axillary lymph node, gonad, liver, salivary gland and spleen. One set of tissues was tested for the presence of MARV RNA by Q-RT-PCR and a second set were frozen for virus isolation attempts in the case that any tissues were MARV RNA positive.Nucleic acid was extracted on the MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies) from gamma-irradiated-Rift Valley fever virus (RVFV)-spiked (RNA extraction positive control) blood and oral swab specimens using the MagMAX Pathogen RNA/DNA Kit (Life Technologies) and from tissues using the MagMax Total RNA Isolation Kit (Life Technologies).56-FAM-ATC CTA AAC-ZEN-AGG CTT GTC TTC TCT GGG ACT T-3IABkFQ, probe 2: 56-FAM-ATC CTG AAT-ZEN-AAG CTC GTC TTC TCT GGG ACT T-3IABkFQ), large segment  and eukaryotic 18 S rRNA (Life Technologies) genes, respectively.Reverse-transcribed MARV RNA, RVFV RNA and eukaryotic18S rRNA, was detected on the ABI 7500 Real-Time PCR System  using the SuperScript III Platinum One-Step Q-RT-PCR Kit (Life Technologies) with amplification primers and reporter probes targeting the viral protein 40  and then incubated overnight at 4 °C. After washing the plates, a 1:100 dilution of gamma-irradiated bat whole blood was added to the first well and 4-fold serial dilutions were performed through 1:6,400. Following a 1 h incubation at 37 °C, the plates were washed and bound antibody was detected using a 1:2,000 dilution of goat anti-bat IgG . According to the manufacturer product datasheet, this antibody reacts specifically with IgG and with light chains common to other bat immunoglobulins, such as IgM. After incubation for 1 h at 37 °C, the plates were washed twice and the 2-Component ABTS Peroxidase System  was added. The substrate was incubated for 30 min at 37 °C prior to reading the plates on a microplate spectrophotometer at 410 nm. Adjusted sum optical density (OD) values were calculated by subtracting the ODs at each 4-fold dilution of wells coated with RESTV NP from their corresponding wells coated with MARV Angola NP. The average adjusted sum OD of duplicate runs was reported and the threshold for seropositivity was set at ≥ 0.95, as previously described11.ELISA plates were coated with 50 ng/well of purified recombinant MARV Angola nucleoprotein (NP) or Reston virus (RESTV) NP expressed in 2 = 12.479, d.f. = 5, two-tailed P = 0.030; 2: χ2 = 13.309, d.f. = 5, two-tailed P = 0.022), therefore degrees of freedom were corrected using Greenhouse-Geisser estimates of sphericity .Figure The authors declare that all data supporting the findings of this study are available within the article or from the corresponding author upon request.


ZIKA virus (ZIKA) has now become a global phenomenon. Since 2007, evidence of ZIKA transmission has been reported over 72 countries and territories. The transmission of ZIKA has made World Health Organization to categorize the situation under the ambit of a health emergency. This situation is serious because there appears to be a highly tangible link between infection during pregnancy and the occurrence of microcephaly and Guillain–Barré syndrome. In the context of this emergency situation, this review article intends to discuss the prevention and control strategies such as avoiding travel to infected area, being careful from mosquito bites, take precautions to reduce the risk of sexual transmission, and seek medical care for any acute illness with rash or fever. This review is an attempt to analyze the results of those campaigns, keeping in view the variables and constants that affect any such measures. Furthermore, this article will suggest proactive measures that can be employed to effectively combat the epidemic transmission of the ZIKA. Flavivirus. The virus is icosahedral, enveloped, non-segmented, single-stranded, 10 kb positive-sense RNA genome that closely relates to the Spondweni virus . The ZIKA infection symptoms usually appear in 3–12 days after the vector bite and end within 2–7 days after onset of symptoms  according to the CDC-issued guidance. The ZIKA RNA is detectable in serum after 7th days post-symptom onset. ZIKA RNA has been detected in serum in non-pregnant patients and pregnant patients after 62 days post-symptom onset and 53 days of after last known exposure in an asymptomatic pregnant women . ZIKA haCurrently, there is no specific vaccine for the preventive treatment of ZIKA infection. However, different groups of scientists are working world over to develop the ZIKA vaccine, and hopefully will be available in couple of years . For theAedes mosquito. Limiting the travel to infected areas also minimizes the risk of ZIKA. Fourth strategy could be the most helpful as well as it is an active strategy where the vector reservoirs are eliminated. In the case of ZIKA, proper drainage can reduce the stagnant water reservoirs to inhibit uncontrolled reproduction of mosquitoes. Adequate garbage management could also be used to hinder the vector proliferation. The fifth intervention that can be helpful is to avoid the recurrence of the disease where humans can act as the source of the virus for infection in non-human primates like monkeys. Avoiding mosquito bites to infected humans could be the aiding strategy for the prevention of spill over  recommended by WHO. Using the IVM model, overall sustainability, efficacy and cost-effectiveness of the strategy can be improved. The Integrated Management Strategy for the Prevention and Control of Dengue (IMS – Dengue) could be a basic model for ZIKA. Therefore, participation and collaboration of different organizations would play a great role in exploring IVM model. It has been reported that the VM Model . Some of• Monitoring of household and common areas to eliminate vector breeding sites, such as water reservoirs and waste drainage pipes.• Regular cleaning of garbage collection sites.• Development of physical, biological, and/or chemical methods to control mosquito breeding.• Use of appropriate insecticides as per WHO recommendations.• Fumigation of cargoes at ports and borders to prevent transport of larva by various means of transportation.In addition to the above control measure, one of the key control measures is to avoid contact with vector. Therefore, personal prevention is very important to avoid sickness. Patients suffering with ZIKA infection and their family members must be well-educated about the risk of transmission. Use of mosquito nets, wire-mesh doors and windows, skin protection, and use of repellents are some of the key prevention measures that need to be taken under consideration.Aedes species. In this subclass, authorities should be enabled to implement reliable water supply management, proper cleaning and maintenance of water storage systems, adequate solid waste management systems, and desirable alterations in human behaviors and residence systems. This includes proper cleaning of streets, maintenance and modifications of buildings/structures as well as housing units such as use of mosquito screens/nets on windows and mosquito proofing of water storages  genetic system  amino propionic acid ethyl-ester or icaridin can be used. There are no specific recommendations and restrictions regarding use of these mosquito repellents, unless there is any specific warning given on the label of the product .In 1947, Alexander Haddon and George Dick first identified ZIKA in monkey while studying yellow fever in the ZIKA forest of Uganda. Subsequently, the same virus was isolated from the Table 3). Travelers should be advised to carry necessary measures if traveling to ZIKA stricken regions. In addition, appreciate information about the symptoms of ZIKA infection and prevention should be handy at airports, bus terminals, railways stations, and so on. This information could be also printed on travel documents such as air tickets or webpages. It is very important that upon returning from ZIKA prone areas, travelers should contact their healthcare providers before returning home.Since there is a great risk of ZIKA infection due to travelers traveling from ZIKA infected regions to other non-infected regions, necessary precautions need to be taken. At present, there is no recommendation from the WHO as to travel bans to the affected countries. However, it is the government responsibility to educate the public while traveling to the areas known for epidemic spread (Aedes mosquitoes in South America. The virus entered and propagated in the country with conditions favorable for ZIKA like high population density, lack of immunity in targets and viral mutations. Global warming is a contributor in widespread of ZIKA as well (Aedes mosquito is on high alert, every day a new case emerges from other part of the world. ZIKA is progressing very fast and next few months are very crucial for stopping the attacks of Aedes mosquitos. High population directly affect the cases of ZIKA. So, if the virus outbreaks in those countries like India, China and USA, it would be impossible to control this virus. The current most workable suggestion to pregnant mothers is to avoid traveling to ZIKA affected areas. This is the best chance we have got till now, more research is necessary on this subject to find the cure for the disease. Cases of ZIKA has to be analyzed in detail to check the relationship of ZIKA and genetic mutation of Aedes to help understand why ZIKA is carried out in this type of mosquito (ZIKA with no doubt took the world as a storm because of  as well . On the mosquito .When ZIKA was identified, it caught us by surprise and no time could be leveraged to do anything in stopping this virus but there are major implications for researchers and doctors in studying ZIKA. It is still unidentified that how many more viruses of this type are present in our atmosphere . Due to IR wrote the initial draft of the paper, designed figures and SK updated and proofread the paper. VB and JL did the critical analysis of the data and Y-HP designed, analyzed and approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Host derived markers on virally infected cells or virions may provide targets for the generation of antiviral agents. Recently, we identified phosphatidylserine (PS) as a host marker of virions and virally-infected cells.Under normal physiological conditions, PS is maintained on the inner leaflet of the plasma membrane facing the cytosol. Following viral infection, activation or pre-apoptotic changes cause PS to become externalized. We have previously shown that bavituximab, a chimeric human-mouse antibody that binds PS complexed with β2-glycoprotein I (β2GP1), protected rodents against lethal Pichinde virus and cytomegalovirus infections.Here, we determined the antiviral activity of a fully human monoclonal antibody, PGN632, that directly binds to PS. Treatment with PGN632 protected 20% of guinea pigs with advanced infections of the hemorrhagic arenavirus, Pichinde, from death. Combining PGN632 with ribavirin improved the antiviral activity of both agents, such that the combination rescued 50% of animals from death.The major mechanisms of action of PGN632 appear to be opsonization of virus and antibody-dependent cellular cytotoxicity of virally-infected cells. PS-targeting agents may have utility in the treatment of viral diseases. Due H and Vκ chains linked to human IgG1 constant domains. It targets a lipid, phosphatidylserine (PS), which is normally intracellular -thymidine for eight hours . Stimulation index was calculated.Antigen-specific immune responses were analyzed using a on assay . Briefly2.14p values are reported. A value was considered significant if p ≤ 0.05.The significance between two means (histograms) was calculated by student’s unpaired t-test. Log-rank analysis for non-normally distributed data  was used to compare the survival of the animals exposed to different treatments, and pairwise comparisons of treatment means were made between groups to determine statistical significance. Chi-square, degrees of freedom, and 2.15Male Hartley guinea pigs , aged 4-6 weeks, and approximately 300-350g in weight, were used for all animal studies. The animals were maintained in a specific pathogen free (SPF) BSL3 facility, and fed a normal diet of pig chow and water (unrestricted access to both). Prior to each animal experiment, guinea pigs were randomized and grouped, weighed, and sub-cutaneously implanted with biometric transponder chips that recorded identification number and temperature . Infected and/or antibody-treated animals were monitored twice daily for the entire duration of the experiment. Animals were euthanatized when their body weight decreased by 20% of their total body weight, or when they were scored as ‘severe’ based upon appearance, clinical signs, or unprovoked behavior in accordance with UT Southwestern Institutional Animal Care and Use Committee guidelines.33.11). Binding was not increased in the presence of human β2GP1 (not shown). Binding was also not due to carryover of trace amounts of contaminating β2GP1, which has confounded other studies [1). PGN632 did not recognize any of the neutral phospholipids, sphingomyelin (SM), phosphatidylcholine (PC), or phosphatidylethanolamine (PE).PGN632 bound directly to PS-coated microplates. 50% maximal binding was observed with PGN632 at a concentration of 0.2 µg/ml (1.3nM) .Immunofluorescence microscopy showed that PGN632 bound specifically to cells infected with Pichinde virus. Uninfected Vero cells were not visibly stained. After infection, the cells developed the same punctate staining pattern that we have previously observed with bavituximab on virally infected cells . By 24-43.33a). To confirm that the PGN632 could bind to live infectious virions, bead depletion assays were performed in which magnetic beads coated with PGN632 were used to pull down infectious virus, and the number of infectious virus particles remaining in suspension were quantified by plaque assay. PGN632-coated beads removed 80% of the infectious virons, the same as for bavituximab-coated beads . Erbituximab-coated beads did not remove the virions, showing that the depletion caused by the PGN632- and bavituximab-coated beads was antigen specific. A further control compared the depletion with that of beads coated with polyclonal guinea pig IgG purified from guinea pigs that had been immunized with Pichinde virus. The polyclonal anti-Pichinde virus-coated beads removed the same amount of virus from the suspensions as did the PGN632-coated beads . Thus, PGN632 is capable of binding to PS on the external surface of the Pichinde virus envelope, at levels similar to those seen with bavituximab.PGN632 bound specifically to Pichinde virions adsorbed onto ELISA plates. The binding of the PGN632 was antigen-specific, since the erbituximab control antibody did not bind to the virions. PGN632 and bavituximab showed the same ability to bind to the immobilized virions .Uninfected guinea pigs treated with PGN632  gained body weight at the same rate as untreated animals. Their physical activity and appearance were indistinguishable from untreated animals. Because PGN632 binds to PS, it has the potential to be anti-coagulating through interference with coagulation factor binding to PS surfaces. We therefore measured PT and aPTT of blood drawn from uninfected guinea pigs 6 hours and 24 hours after treatment with PGN632 . Neither PT nor aPTT in PGN632-treated animals was prolonged relative to erbituximab-treated or bavituximab-treated animals.  of Pichinde virus, and treatment was begun when they showed signs of advanced infection . When administered as a single agent, PGN632 protected 20% of guinea pigs from dying from their infections, the same percentage as for bavituximab . While notable, the difference in survival between groups treated with anti-PS antibody and the PBS control animals was not significant (p = 0.1189). The guinea pigs started to regain body weight and gradually returned to having normal physical signs over the following 10-12 days. As expected, animals treated with erbituximab were not protected from challenge and did not survive past day 15.Guinea pigs were infected with 104b). This sub-optimal dose of ribavirin offered no protection by itself. However, when combined with PGN632, 50% of guinea pigs recovered from their infections and survived, as compared to 20% in the group treated with PGN632 alone . This difference trended towards, but did not reach, statistical significance (P=0.15). However, the difference in survival between the group treated with PGN632 and ribavirin, and the group treated with ribavirin alone was statistically-significant (p ≤ 0.0055). Thus, PGN632 appears to have additive anti-viral activity when combined with ribavirin.We next determined if PGN632 offered better protection when given in combination with ribavirin, the primary drug for treating Lassa virus infection. Animals with advanced disease (as above) were treated with PGN632 three times a week in combination with daily ribavirin  . In contrast, in the group of animals sacrificed on day 14, after 3 treatments, there was a 500-fold reduction in virus load in the blood, and a 10-fold reduction in virus load in the liver, lung, kidney, and heart relative to erbituximab-treated animals (P<0.0001). Bavituximab also reduced the virus load in blood and tissues, but to a somewhat lesser extent than did PGN632 . Virus load in the spleen was not reduced by either PGN632 or bavituximab treatment, probably because the spleen is a major site of clearance of opsonized virus from the blood.Guinea pigs were treated with PGN632 on day 4 after infection, when they showed signs of advanced disease, and were treated again on day 7. Animals were sacrificed ten days post-infection, and the virus load in blood and various tissues was determined. The virus load in the blood was reduced by 50-fold relative to the group of animals that received erbituximab. However, there were no significant reductions of viral load in the liver, lungs, kidneys, or hearts of animals treated with PGN632 as compared to erbituximab recipients and the virus load in the spleen was elevated. Bavituximab recipients also showed no significant reductions in virus load in these tissues . Bavituximab also reduced virus yield by 3-fold. Thus, neither PGN632 nor bavituximab are neutralizing antibodies.We examined the mechanisms by which PGN632 rescued animals dying from Pichinde virus infections. First, we determined whether PGN632 was able to induce direct neutralization of Pichinde virus. Vero cells were infected with Pichinde virus (MOI = 4) in the presence of PGN632, bavituximab or erbituximab. PGN632 reduced the yield of virus only slightly (3-fold) and not significantly  Thus, induction of humoral or cellular immunity does not appear to explain the survival of PGN632-treated animals.Next, virus-specific humoral immune responses to Pichinde virus were assessed in guinea pigs that had been infected with virus 14 days earlier, and then treated with PGN632, bavituximab or erbituximab as above. The day 14 time point was selected because this is the time at which differences in survival became apparent. There were no significant differences in levels of anti-Pichinde virus antibodies in the PGN632 or bavituximab-treated animals compared to erbituximab treated animals (results not shown). Similarly, splenocytes taken from guinea pigs that had been sacrificed 14 days after infection and treated with PGN632 or bavituximab showed no significant proliferative responses to Pichinde antigens compared with splenocytes from erbituximab-treated guinea pigs . Thus, opsonization of virus may contribute to the better survival of PGN632-treated guinea pigs.We determined whether antiviral activity could be due to opsonization of virus by PGN632 and subsequent clearance by the reticuloendothelial system. Guinea pigs were infected with Pichinde virus and treated i.p. with PGN632, bavituximab or erbituximab (6 mg/kg). Treatment with either PGN632 or bavituximab reduced the levels of circulating virus in the blood by 10 to 15-fold (P<0.01) relative to animals treated with erbituximab . PGN632 and bavituximab were equally effective at mediating ADCC  Thus, ADCC by innate immune cells likely contributed to the anti-viral action of PGN632.Lastly, we determined whether the antiviral activity of PGN632 could be due to ADCC of virus-infected cells. PGN632 and bavituximab efficiently mediated lysis of infected primary guinea pig fibroblasts by primary guinea pig macrophages 4in vitro [The use of antibodies as novel therapeutics against infectious diseases has gained traction in recent years as an effective way to combat invasive pathogens. Synagis  is a humanized monoclonal antibody that has been approved for the treatment of respiratory syncytial virus . Numerouin vitro . During 2+ that activate PS exporters while simultaneously inhibiting the major PS importers, the aminophospholipid translocases [We recently identified PS as a generic marker of virus-infected cells and enveloped virions . In uninslocases , 15. Altslocases . Importaslocases , cytomegslocases , 17, infslocases , HIV-1 [slocases , vaccinislocases , and HSVslocases . Envelopslocases . Virusesslocases .The PGN632 antibody has very similar phospholipid specificity to bavituximab, in that both antibodies recognize anionic phospholipids, including PS. Both anti-PS antibodies produced indistinguishable staining patterns on Pichinde virus-infected cells. PS is likely to be the major anionic phospholipid to become exposed on virus-infected cells since it is the most abundant, and it is the lipid whose asymmetry is most tightly regulated. However, it is possible that there is also exposure of the more-minor anionic phospholipids, including PI, PA and PG, which would also be detected by the antibodies. The major differences between PGN632 and bavituximab are: 1) PGN632 is human whereas bavituximab is chimeric and theoretically could induce antibodies against mouse determinants in patients and; 2) PGN632 binds to PS directly, without the need for the β2GP1 cofactor protein, which avoids possible depletion of β2GP1 in patients.6 pfu/ml generally survive [3-108 TCID50/ml [PGN632 treatment of guinea pigs with advanced Pichinde virus infections protected 20% of animals from death and reduced virus load in tissues. When combined with low-dose ribavirin, 50% of animals were protected, which would be expected for drugs with non-overlapping mechanisms of protection. We previously reported that 50% of guinea pigs could be protected from death by treatment with bavituximab alone, a higher percentage than has observed in the present studies. It is possible that these differences relate to the much higher virus inoculum used to infect the animals in the present study. It could also relate to the outbred nature of the guinea pig and genetic non-identity in the two studies. We chose to use the i.p. route for administration of the PGN632 antibody, as we had previously used this same route in our initial studies with bavituximab as an antiviral agent . It is p survive . Indeed,CID50/ml . In the CID50/ml . Combinain vitro. The mechanism was that PGN632 induced β-chemokine production which blocked the R5 coreceptor [Two mechanisms appear to explain the antiviral activity of PGN632: (1) opsonization of virus by the antibody; and (2) the induction of antibody dependent cellular cytotoxicity (ADCC) of virally-infected cells . PGN632 receptor . Viral Preceptor . Thus, iIn conclusion, using antibodies to target exposed PS on virus-infected cells and virions shows promise as an antiviral strategy. Because the antibodies act by different mechanisms, they should be used in combination with conventional antiviral agents. Other strategies for exploiting the exposure of PS on virus-infected cells and/or virus envelopes can be envisioned: antibodies, peptides or small molecules that target exposed anionic phospholipids might be used to deliver or direct agents that interfere with host cell or viral metabolism, or recruit immune effector cells.


Most scientists have a 2- to 3-minute prepared spiel about his or her research that can be given at a moment’s notice, upon request. We call this the “Elevator Speech” (or “Lift Speech” if you are British), and it is often delivered after you tell someone that you are a research scientist. While I’m prepared to explain my job, I am never prepared for the looks on individuals’ faces when I tell them, “I work on herpes,” which could be equated with the looks I might get if I asked them to borrow their toothbrush. I explain how pervasive herpesviruses are in the human population and how, on average, humans carry multiple herpesvirus infections with them for life. With looks of horror, the vehement denials begin. But this is when I remind them that chicken pox is caused by varicella-zoster virus, roseola is caused by human herpesvirus-6, that the college bout of mononucleosis they may have had was most likely caused by Epstein-Barr virus, each of which is a herpesvirus. Then I hit them with the bombshell: I tell them that I work on a virus they most likely have never heard of yet are most likely carrying in their bodies now and that, moreover, it is a significant pathogen and the cause of substantial medical burden. I work on human cytomegalovirus (HCMV), the largest human herpesvirus, with a genome size of a quarter million nucleotides encoding many viral proteins, of which we only understand the function of about two-thirds. While links have been made to certain brain tumors and atherosclerosis, if one has a healthy immune system, HCMV infections are usually inapparent. However, HCMV is highly problematic in individuals with undeveloped or compromised immune systems. With all the notoriety recently given to birth defects caused by Zika infections, these defects pale in comparison to HCMV. About 1% of United States births involved HCMV-infected babies, and 10% of those infants infected will show symptoms, making it the leading cause of congenital birth defects, including vision and hearing loss, learning disabilities, and microcephaly. Equally problematic are patients with weakened immune systems, including bone marrow transplant patients and solid organ recipients. Most individuals acquire HCMV as a toddler, after which it remains quiet and repressed by a person’s immune system, yet awaiting a chance to reawaken. In organ transplant patients, HCMV comes back with a vengeance and causes significant disease and organ rejection. HCMV is like that despicable kid in grammar school who you never really noticed before but who was the first one to kick you when you were down.I am often asked how I came to study HCMV. As a child, I never had intentions of pursuing a scientific career. Having grown up in the Bronx with my New York City detective father and Irish immigrant mother, I assumed that I would take the normal occupational path that most of my friends pursued—either become a criminal or join the New York Police Department (NYPD) or Fire Department New York (FDNY). My father explained to me that I would do far better in life if I went to college. His persuasiveness (and the threat of filicide) convinced me to apply. I opted for those on a national “Top 10” ranking of small liberal arts colleges and decided to go to the one with the highest ranking that accepted me. So, I arrived on campus at Grinnell College in Iowa, sight unseen, with the intent of just completing 4 years, then going back to take the NYC civil servant exam. It was there that a professor, Dr. Charles Sullivan, sold me on the beauty of biology and the analytical nature of the science. I am very mechanical by nature: I fix old wristwatches and build old BMW motorcycles for recreation. So perhaps it was natural that I was fascinated by biological research because I viewed it as dissecting a complex machine and figuring out how each part interacts with another. These characteristics are completely summed up in the field of virology. There is a Spanish term commonly used in Cuba called “rascuache,” used to describe individuals who can repair items with limited resources and those who can repurpose items to accomplish a different function. To me, viruses are the “Masters of Rascuache,” and I was drawn to study them. Of course, the path wasn’t direct by any means. In between my newly found deep respect for viruses and my serious pursuit of academic endeavors, there were countless speed bumps and diversions. I pursued many things following college as I was finding myself. I hitchhiked to and through Alaska with my best friend from the Bronx, worked as an NYC union carpenter walking girders and building skyscrapers, bartended, worked in parts/repair in a Texas Harley Davidson dealership, and played countless rugby games.However, the desire to study viruses never left me. I enrolled in a master’s program at Boston University to increase my admissibility into PhD programs. This proved to be the right move when I was admitted to the University of Iowa graduate school. There, I decided to join the laboratory of a very skilled virologist working on HCMV, Dr. Mark Stinski. Mark taught me that science should be fun, and if you don’t love what you are doing, then you are probably doing the wrong thing. After graduating, I left Mark’s lab to do a brief postdoc at Columbia University, and from there, I went to Princeton University to join the lab of Dr. Thomas Shenk. Tom’s lab was heaven to me. Beyond Tom being a very gracious mentor who truly helped shape the careers of each person in his lab, there were about 15 to 20 other highly skilled postdocs who were incredibly smart, always helpful, and quick to become lifelong friends. It was in this environment that my virology career truly blossomed. From Princeton, I became an assistant staff and then associate staff member in molecular genetics at the Cleveland Clinic, where my laboratory focused on the factors controlling HCMV latency. Following my promotion, I moved my laboratory to the Hepatitis B Foundation, where I run my academic laboratory, as well as serve as Head of Biology for a company founded by Tom Shenk making novel compounds to block viral infections.The major focus of my laboratory is to figure out how to keep HCMV “in check.” Everyone who is infected with HCMV  has the virus for life because HCMV can hide in stem cells within your bone marrow. An initial infection is resolved by one’s immune system, which continues to keep the virus in a repressed state. However, when one’s immune system becomes weakened, it can no longer effectively block the virus from waking back up, something we call reactivation. If one could block reactivation, one could effectively block the diseases caused by HCMV. My laboratory is investigating what factors and conditions are required for the virus to reactivate. We have found specific factors that keep the virus in a repressed state as well as factors that allow for efficient reactivation of the virus. We are now working on how to manipulate these conditions to create an environment where HCMV cannot reactivate. We are not alone: there are several “competing” laboratories studying similar aspects of HCMV latency. I put competing in quotes because, in all fairness, these laboratories are run by very skilled scientists who I highly respect and who are incredibly supportive of each other. When you have a common enemy—the virus we are trying to cripple—you find that individuals with shared goals easily become some of your best friends. My friends and I are doing our best to make sure HCMV can’t kick you when you are down.