After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT–PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT–PCR). After SBV infection both animals in the study showed viraemia (qRT–PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4–7 post-inoculation) but concentrations were relatively low . Viable SBV was only isolated from blood samples and not from semen or genital tissues.To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 10 During 2011 and 2012 Europe experienced the emergence of a new arbovirus of domesticated ruminants, namely Schmallenberg virus (SBV) , 4. SincOrthobunyavirus within the Bunyaviridae family, a large family comprising hundreds of viruses able to infect a broad range of vertebrate and invertebrate hosts. Bunyaviruses are enveloped viruses and have a segmented single-stranded RNA genome of negative or ambisense polarity. The viral genome comprises three RNA segments referred to as small (S), medium (M) and large (L) which encode four structural proteins: the nucleocapsid protein (N); two glycoproteins (Gn and Gc); and the viral polymerase. Members of Orthobunyavirus encode two additional non-structural proteins, NSs and NSm. NSs is encoded by an open reading frame in the S segment overlapping the N gene while NSm is encoded by the M segment.Phylogenetic analysis revealed that SBV belongs to the genus Bunyaviruses are significant pathogens both in humans and animals and cause a range of diseases including febrile illnesses (Oropouche virus), encephalitis (La Crosse virus) and haemorrhagic fevers  . SBV cluThe SBV outbreak caused major trade issues. In particular exports of live sheep and cattle and genetic products of sheep and cattle from Europe to countries outside Europe were blocked by February 2012. In December 2012 it was reported that SBV RNA was detected in bovine semen produced in European countries . In an eTo obtain a better insight in SBV excretion in bovine semen after SBV infection, two bulls were infected experimentally and excretion of SBV in semen was studied over a period of 3 weeks. In addition at 24 days post-infection (p.i.) the bulls were necropsied and genital organs were tested for SBV.et al. [Culicoides midges by filtering the air at inlets. Both animals were inoculated subcutaneously on day 0 with Vero-cell cultured SBV which was isolated from the blood of an acutely infected cow in August 2011 in The Netherlands . This Vero cell-cultured virus was shown to be virulent by inoculation in sheep and comparison with an SBV viraemic blood inoculum. Viraemia and clinical outcomes were the same for both inocula (data not shown).The animal experiments were approved by the Ethics Committee for Animal Experiments of Utrecht University, in accordance with legislation of The Netherlands and the European Union. The two bulls (identification nos. 6361 and 6488) were 15-month-old Holstein Friesians, kindly provided by the CRV International Enterprise in the Field of Cattle Improvement. Both bulls were tested to be free of SBV-specific antibodies by virus neutralization test according to Loeffen et al. . From thg, 10 min). All samples were stored at −70° C until testing. At day 24 p.i. both bulls were necropsied and genital tissues were sampled. This included prostate glands; testicles ; bulbo urethralis glands; epididymis  and mesenteric lymph nodes, ileocaecal lymph nodes and inguinal lymph nodes .The two bulls were clinically examined daily until day 21 p.i. Every day rectal temperatures were measured and feed uptake and overt clinical signs were recorded. Blood samples were collected three times a week post-infection and semen samples were collected daily by allowing the bulls mount a dummy and using an artificial vagina. Semen samples were directly transferred to at least three different tubes: undiluted aliquots, 1:10 diluted semen in Tris (Tris-hydroxymethyl-aminomethane), 20% egg-yolk extender aliquots, and 1:10 diluted semen in fetal calf serum (FCS) aliquots. Used FCS originated from Brazil and was collected in 2010/2011 before the introduction of SBV in Europe. Blood was collected in tubes with and without EDTA to obtain whole blood as well as serum samples . Semen samples with extender (1:10 Tris/20% egg yolk) and without extender  were tested in triplicate. Samples showing a Ct value of ⩽40·0 in two or three amplification runs were regarded as positive. SBV-specific antibody development was tested using the virus neutralization test as described by Loeffen et al. [et al. [All samples were tested for SBV RNA by qRT–PCR as described by Bilk et al. , with mi [et al. , with min et al. . All sam [et al. . The samβ-actin) was not detected or showed a relatively high Ct value. After tenfold dilution and retesting, the inhibitory conditions in these samples diminished. To give an impression of the variability of the test results standard deviations are included in Experimental SBV infection resulted in mild clinical disease in both inoculated bulls. Both of the animals showed temperatures >39°C (days 3–7 p.i.) and some diarrhoea (days 5 and 6 p.i.) in the first week after infection . SBV RNATo obtain a good insight in SBV excretion in bovine semen after SBV infection, two bulls were experimentally infected, and excretion of SBV was studied over a period of 3 weeks. Only two bulls were studied but testing of produced semen resulted in SBV RNA detections in both animals with highest (semi-quantitative) levels at days 5–6 p.i. , Fig. 3.Although semi-quantitative qRT–PCR testing showed the highest levels of SBV RNA excretion in semen in the first week after inoculation, scattered low concentration positives were found in weeks 2 and 3 after inoculation in single amplification runs. This latter observation may have been due to the fact that the concentrations of RNA in the tested semen samples were very low and close to the detection limit of the assay. Moreover, a variation in the level of virus excretions may have promoted this observation. In case such variation really occurs this might conceal intermittent excretions of low levels of RNA in semen. On the other hand this would mean that intermittent excretion cannot be excluded based on this study. In the PCR amplification reactions of a few undiluted samples, qRT–PCR inhibitory conditions were observed despite the fact that the qRT–PCR protocol was validated (using spiked semen samples) for semen with and without extender. After tenfold dilution and retesting, the inhibitory conditions in these samples diminished. By testing all semen samples with extender (1:10 Tris/egg yolk 20%) and without extender in triplicate, the variability in the test Ct values, especially around the detection limit of the assay, could be anticipated. In this way reliable outcomes could be obtained for all samplings.et al. [Virus isolations in Vero cell cultures did not result in propagation of SBV from any of the semen samples or the tissue samples, whereas the virus was isolated from blood samples shortly after inoculation. This means that the inoculation was effective but viable SBV could not be detected in any of the semen samples or tissue samples. In a comparable study by Parsonson et al. , eight bSurprisingly, SBV RNA was not detected in the genital organs at 24 days p.i., but virus RNA was detected in lymph nodes in the proximity of the genital organs. This could mean that SBV is not targeting genital tissue cells or, in the experiment, had been cleared completely from these cells at 3 weeks p.i. qRT–PCR analyses of sperm cells and seminal fluids separately showed higher levels of SBV RNA in the cell fraction (data not shown) compared to seminal fluids, but this could simply be due to attachment to cells instead of infection of these cells. Different types of cells were not tested separately in this study. Positive RNA detections in lymph nodes might represent degraded SBV cleared via the lymph system.Both of the semen-producing bulls in the study showed a clear SBV-specific antibody development as demonstrated by virus neutralization test. In both of the animals SBV-specific antibody could be detected by virus neutralization test at 10 days p.i. One bull (6488) showed a relatively high titre at 17 days p.i.; however, this is not out of the ordinary compared to titres of other animals after infection.The observations from this study indicate that SBV RNA can be excreted in bovine semen. This suggests that bovine semen might be contaminated with viable virus. Although viable virus was not detected in semen in this study, in an earlier study by others, infectivity of semen from SBV RNA-positive straw was reported after subcutaneous injection in calves, even in specimens with relatively low concentrations of SBV RNA . In view


To the Editor: Ngari virus (NRIV) is a single-stranded RNA virus belonging to the family Bunyaviridae, genus Orthobunyavirus. The genome comprises 3 segments, the small (S), medium (M), and large (L) segments, which encode the nucleocapsid (N) protein, the 2 glycoproteins Gn and Gc, and the RNA-dependent RNA-polymerase, respectively. Sequence analysis showed that NRIV is a reassortant between Bunyamwera virus (BUNV) and Batai virus (BATV), both from the genus Orthobunyavirus. S and L segments derived from BUNV, and the M segment derived from BATV   and 28 (sample 65), respectively. Both samples showed similar melting peaks at ≈78.2°C and shared the identical partial nucleotide sequence of the S segment. The sequence belongs to the Bunyamwera serogroup, but the short partial sequence was not sufficient for accurate virus determination and identification. For this reason, both serum samples were used to inoculate cell monolayers of Vero E6 cells that were assayed for virus replication. Only sample 51 displayed a cytopathic effect after 72 h and was further analyzed. We isolated the viral RNA from cell culture with TRIzol reagent  and used it to prepare a sequencing library according to a recently published protocol . Phylogenetic analysis of complete genome sequences indicated that the virus belongs to the Ngari virus group and showed high homology to previous NRIV isolates in all 3 segments . As for This evidence supports the extension of the range of NRIV infection to goats (complete sequences already had been derived from a human and from mosquitoes [The possible clinical importance to livestock and the circulation of NRIV among mosquitoes, livestock, and humans needs to be clarified. No further information about clinical signs of sampled animals or reports of human NRIV cases is available. Because infection with both RVFV and NRIV induces hemorrhagic fever, affected humans also should be tested for NRIV infection. Further development of specific molecular and serologic diagnostic tools for NRIV should be pursued to obtain more information about NRIV distribution in humans and livestock in Mauritania and other African countries.


The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit “R4368” and its 2014 replacement “R4415.” We also provide characterization of the master stock used to create “R4415.” The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results. Ebola virus disease (EVD) is a frequently lethal human viral hemorrhagic fever caused by four distinct ebolaviruses . EVD occurs sporadically and usually affects no more than several hundred people during an outbreak (reviewed in ). In 201Zaire ebolavirus in the genus Ebolavirus  [Taxonomically, EBOV is the only member of the species viridae) . The EBOviridae) , 4. For viridae) ) differsviridae) , 7) and viridae) ) by lessviridae) .in vitro experiments [The development, evaluation, and final licensure of medical countermeasures (MCMs) against EVD in the US is critically dependent on standardized animal models of filovirus infection and standardized assays and reagents –12, incleriments , 13. Eboeriments . Unfortueriments .GP genes to access three partially overlapping open reading frames (ORF) [GP gene that typically consists of seven consecutive uridylyls (7U). Read-through results in the transcription of a cognate 7 adenylyl mRNA (7A mRNA) and thereby in the expression of pre-sGP, a protein precursor that is proteolytically processed to a secreted homodimeric glycoprotein (sGP) and a probably monomeric secreted peptide (Δ-peptide). Stuttering of the ebolavirus RNA-dependent RNA polymerase (L) at the editing site results in transcription of mRNAs that contain various numbers of adenylyls. The majority of non-7A mRNAs contain eight adenylyls, leading to the expression of the homotrimeric ebolavirus spike glycoprotein GP1,2, or six or nine adenylyls, leading to expression of the small soluble glycoprotein (ssGP) [1,2:ssGP = 75%:25%:5% [All ebolaviruses make use of co-transcriptional editing of their glycoprotein-encoding es (ORF) , 16. Edin (ssGP) –20. The n (ssGP) . Editing%:25%:5% , 22.in vitro and in vivo environments [GP gene editing sites. For instance, during serial passage of a 7U EBOV/Yam-May isolate in Vero E6 cells, a viral population evolved that predominantly contained an 8U editing site. The opposite occurs in vivo: 8U EBOV/Yam-May populations converted to 7U populations in guinea pigs [1,2 and/or sGP. Preliminary evaluations indicate that a 7U→8U mutation in EBOV does not alter pathogenesis in guinea pigs or nonhuman primates [1,2 in different environments . In addition, a recent study reported subtle, but significant, differences in disease course and severity between nonhuman primates exposed to 7U or 8U virus stock variants [EBOV, Sudan virus (SUDV), and possibly other ebolaviruses adapt to different ronments , 23–25. nea pigs . These cvariants . All of Here, we report the coding-complete genome sequence  was obtained from an EBOV-infected 65-year old female patient during an EVD outbreak that occurred in 1995 around Kikwit, Zaire . The patient’s disease onset was recorded as April 29, 1995. She was hospitalized on May 1, 1995, and died on May 5, 1995. The clinical specimen, most likely plasma or serum, was obtained from the patient on May 4, 1995. How the patient became infected and what medical care she may have received is unclear. Unfortunately, chain-of-custody records that further detail the origin of 9510621 or its shipment to the CDC are not available anymore, and EBOV titration was not attempted from this specimen prior to cell-culture passage.Chlorocebus aethiops) Vero E6 cells (ATCC #CRL-1586). The multiplicity of infection (MOI) used for this passage is unknown. Virus was harvested after a 6-day incubation period on May 19, 1995, but the method of harvest  is not indicated in the available records. A second passage of virus, was conducted at the CDC, again using Vero E6 cells, and resulted in VSP 807224 (MOI unknown). Virus was harvested after an 8-day incubation, using a “freeze/thaw” method, on May 29, 1995. A titer of 3.2E+06 was determined for “VSP 807224” in January 2008 using the TCID50/Reed-Munch viral titration method. “VSP 807224” was transferred from CDC between late May and late June 1995 to USAMRIID.The first passage of EBOV/Kik-9510621, designated “virus seed pool (VSP) 807223” , was conet al. using classical dideoxynucleotide sequencing. “134” (passage 3) is cell culture-adapted, consisting predominantly of viruses with an 8 uridylyl (8U) glycoprotein (GP) gene editing site. A coding-complete genome for “134” was deposited on July 24, 2003, in GenBank under accession #AY354458. “135” (passage 2) was amplified at USAMRIID at least three more times (and in the process was depleted), leading to three separate NHP challenge stocks: “R4367” , “R4369” , and “16502” . The genomic characterization of stock “16502” (passage 3) and its evolution in a nonhuman primate study over several days was previously described [At USAMRIID, “VSP 807224” was stored frozen at -70°C and designated there as “135” (passage 2). After an additional passage in Vero E6 cells, the virus, now designated “134” (passage 3), was sequenced by Chain escribed  Fig 1)..et al. uescribed . Each ofIndependently at UTMB, “VSP 807223” was passaged one time on Vero E6 cells, harvested after a 10-day incubation period on February 21, 2012, and stored in 1.0-ml aliquots as “WRC000121” . A titer of 1.8E+07 pfu/ml was determined (titration method unknown). On May 1, 2012, UTMB transferred “WRC000121” to USAMRIID, where the virus was stored at -70°C and designated as “R4414” (passage 2). This material was passaged at an MOI of 0.001 on Vero E6 cells to prepare NHP challenge stock “R4415” (passage 3), which was harvested on June 11, 2013, after a 12-day incubation period (time to develop 2–3+ CPE) with a titer of 1.31E+06 pfu/ml determined by agarose-based plaque assay [Vials (1 ml) of EBOV/Kik-9510621 challenge stocks were stored and maintained at USAMRIID at -70°C. “R4368” (passage 4) was stored since July 24, 2011, and thawed on March 2, 2012; “R4414” (passage 2) and “R4415” (passage 3) were stored since June 11, 2013, and were thawed in January 2014. An aliquot of 100 μl of each thawed virus stock was placed in 3:1 TRIzol . Nucleic acids were isolated from TRIzol-treated material, and genome sequence was determined on Illumina technology (MiSeq or HiSeq) with EBOV-specific oligonucleotides following sample preparation performed as described in [ATTACCAGAGTTGATTAGTGTG; 5’ RACE (inner primer): TTAAATAACGAAAGGAGTC; 3’ RACE (outer primer): TGAATCTCCAATCCTCTAAGTA; 3’ RACE (inner primer): AAGGGATTTTCAACTGAGCACACT. Amplification primer removal, duplicate removal, low average quality exclusion (≤ Q30), and quality trimming was performed. Viral assemblies were completed in DNAStar Lasergene nGen  with ≈4×105 reads. Only single nucleotide polymorphism (SNPs) present in the population above the 2% threshold are presented in this report . Considering this threshold, a target depth of 200 requires an SNP to have ≥ 4 supporting reads prior to being called a SNP. The depths are reported in the tables presented in this text for all samples . A consensus change is defined here as a change relative to the published sequence for EBOV/Kik-9510621 “134” (GenBank accession # AY354458) present in ≥50% of the population. Below that threshold, SNPs are considered subclonal substitutions and part of a minority subpopulation of the virus.For rapid amplification of cDNA ends (RACE), “R4415” (passage 3) RNAs were extracted with Zymo Direct-Zol  from cell-culture supernatant in TRIzol according to the manufacturers' instructions. SMARter RACE 5’/3’ kit  was used to amplify both 5' and 3' untranslated regions (UTRs) of the virus genome from the extracted RNAs. The kit's two-stage nested PCR protocol was found to be optimal. The gene-specific primers for each RACE experiment are as follows 5’ RACE (outer primer): Research has been reviewed for compliance with dual-use guidelines and approved for publication by the USAMRIID Institute Biosafety Committee (IBC) and the Operational Security office.Compared to EBOV/Kik-9510621 “134” (passage 3), “R4368” (passage 4) acquired only one consensus-level substitution . Six intrahost single nucleotide variants (iSNVs) (≥2% of the population) were detected and are reported in in vitro and in vivo Ebola virus experimentation, including pathogenesis studies and candidate medical countermeasure evaluation. “R4415” (passage 3) replaced “R4368” (passage 4) in December 2014 and has been used for most pathogenesis and medical countermeasure evaluation research since then. This work provides a framework for genomic comparison between past experiments as challenge stocks are replaced to address propagation issues and depletion. Characterization of these NHP challenge stocks was completed to the level of “Coding Complete” plus population-level characterization in the case of “R4368” (passage 4) and “Finished” in the case of “R4415” (passage 3) [We have provided here a concise report on the history and genomic characterization of the USAMRIID Ebola virus/H.sapiens-tc/COD/1995/Kikwit-9510621 NHP challenge stocks “R4368” (passage 4) and “R4415” (passage 3), as well as the “R4415” (passage 3) predecessor, “R4414 (passage 2).” “R4368” (passage 4) was used between July 2011 and December 2014 in both ssage 3) . This chssage 3) .


Haptolina ericina and Phaeocystis pouchetii with CeV-01B or PpV-01B dsDNA viruses, respectively, and assessed the extent to which host cellular responses resemble programmed cell death (PCD) through the activation of diagnostic molecular and biochemical markers. Pronounced DNA fragmentation and activation of cysteine aspartate-specific proteases (caspases) were only detected in virus-infected cultures of these phytoplankton. Inhibition of host caspase activity by addition of the pan-caspase inhibitor z-VAD-fmk did not impair virus production in either host–virus system, differentiating it from the Emiliania huxleyi-Coccolithovirus model of haptophyte–virus interactions. Nonetheless, our findings point to a general conservation of PCD-like activation during virus infection in ecologically diverse haptophytes, with the subtle heterogeneity of cell death biochemical responses possibly exerting differential regulation on phytoplankton abundance and diversity.The mechanisms by which phytoplankton cope with stressors in the marine environment are neither fully characterized nor understood. As viruses are the most abundant entities in the global ocean and represent a strong top-down regulator of phytoplankton abundance and diversity, we sought to characterize the cellular response of two marine haptophytes to virus infection in order to gain more knowledge about the nature and diversity of microalgal responses to this chronic biotic stressor. We infected laboratory cultures of the haptophytes  Emiliania huxleyi–Coccolithovirus system, for which seasonal algal blooms are consistently terminated by virus infection (Marine phytoplankton account for a large proportion (∼50%) of global primary production and have a strong influence on global nutrient cycling  or infecnfection . PCD-liknfection , these fE. huxleyi–EhV paradigm is relevant for other marine haptophytes for which similarly large, double-stranded DNA containing viruses (Phycodnaviridae) have been isolated and characterized. We investigated cultures of the single-celled marine haptophytes Haptolina ericina, a cosmopolitan non-blooming phytoplankter  (P. pouchetii (PpV-01B) (We performed experiments to determine the extent to which the Haptolina ericina (Parke & Manton) and P. pouchetii  virus lysate to give a multiplicity of infection (MOI) of ∼10 viruses per host cell. Cultures were incubated for 4–5 days until lysis was apparent. Viruses were harvested from culture lysates by two rounds of centrifugation at 5445 × g at 4°C for 20 min, followed by passage through a sterile 0.45-µM pore-size cellulose acetate syringe filter to remove cellular debris and bacteria. Half of the volume of fresh virus lysate was boiled for 10 min to inactivate virus particles. This heat-killed virus lysate was used as a negative control inoculum for virus infection. All virus lysates were stored at 4°C for no longer than 1–2 days prior to use in infection experiments.The double-stranded DNA viruses CeV-01B , demonstrating that cultures were actively growing prior to infection. We infected healthy cultures only at the start of the day period in an attempt to emulate culture synchronicity at the time of virus exposure. Cultures were inoculated with either fresh active virus lysate (MOI > 5), heat-killed virus lysate or sterile IMR/2 medium, with each amendment mixed into the culture by gently swirling before dividing equally into three replicate culture flasks. H. ericina cultures were incubated at 15°C with ∼180 µmol photons m−2 s−1 on a 14:10 light: dark cycle, while P. pouchetii was incubated with similar continuous irradiance (180 µmol photons m−2 s−1) at 8°C. Virus, phytoplankton and bacterial cell counts were performed on a FACSCalibur flow cytometer (Beckton Dickinson) according to the methods of Thyrhaug et al.  according to kit instructions. After elution in 70°C elution buffer, RNA was removed from samples by the addition of 20 U RNAse A  and incubation at 37°C for 30 min. DNA was then purified using the Clean and Concentrator kit  and eluted in a final volume of 20 µL of 70°C distilled water. Four microliters of 6× DNA loading buffer (Promega) was added to each sample, and samples were stored at 4°C in the dark until analysis by agarose gel electrophoresis at 4°C in 1% (w/v) agarose at 100 V for 2 h in 40 mM Tris–Cl, 20 mM acetic acid, 1 mM EDTA, pH 8.0. Gels were stained in 1X SYBR Gold  at room temperature in the dark for 1–2 h prior to visualization using a GelDocXR .Electrophoretic examination of genomic DNA extracted from addering  occurs dH. ericina and P. pouchetii for changes in caspase-like catalytic activity during virus infection by measuring cleavage of the flurogenic, canonical caspase-8 substrate isoleucyl-glutamyl-threonyl-aspartic acid-7-amino-4-methylcoumarin  (Supplementary data). The kinetics of fluorescence were measured using a Spectra Max Gemini XS plate reader  and normalized to total protein [relative fluorescence units (RFU) mg protein−1 hr−1], as previously described (Activation of host cysteine aspartate-specific proteases (caspases) is another hallmark of PCD . We examin vitro inhibition of caspase activity with the pan-caspase inhibitor z-Val-Ala-Asp-fluoromethyl-ketone  . Fresh z-VAD-fmk inhibitor was dissolved immediately before use in molecular biology grade dimethyl sulphoxide (DMSO) to a concentration of 20 mM. Pooled control or virus-infected cell extract samples were pre-treated with 20 µM z-VAD-fmk (−1 hr−1) for z-VAD-fmk-treated extracts were normalized to the activity for untreated cell extracts and are expressed as a per cent reduction in IETDase cleavage activity. Total caspase inhibition (100%) with z-VAD-fmk was confirmed on recombinant human caspase-8 (data not shown).The same cell extracts were used to verify E. huxlyei  during infection by the EhV virus has been shown to play a critical role in the regulation of virus-mediated cell lysis in  huxlyei . Ten-mil huxlyei .H. ericina or P. pouchetii were pre-treated with 20 µM z-VAD-fmk prior to infection by their respective viruses, in order to determine whether z-VAD-fmk impairs virus production  relative to control cultures (black circles) at all time points after T0 Fig. , indicatH. ericina  DNA fragments at 1-day post-virus addition. Characteristic DNA fragmentation in virus-infected cultures was not apparent once cultures had completely lysed , a DNA topoisomerase I inhibitor known to induce replication arrest and PCD .Pre-treatment of In situ addition of z-VAD-fmk to virus-infected cultures of H. ericina did not significantly impact host cell growth or CeV production relative to the DMSO control  lipids in CeV-01B-infected cultures only  in control cultures relative to virus-infected cultures  found in E. huxleyi strains  stress-phase induction of DNA laddering upon both virus infection and camptothecin treatment suggests that DNA fragmentation may be activated earlier in these haptophytes than in classical metazoan models, in which caspase-initiated DNA fragmentation occurs during later stages of apoptosis  after infection by the LPP-1 virus . The relatively weak intensity of the DNA laddering patterns on agarose gels further suggests that DNA fragmentation only occurs in some host cells, or that only a fraction of host cell chromatin is accessible for endonucleases. It is important to note that H. ericina and P. pouchetii cultures were not synchronous sensu stricto with regard to timing of virus infection, and therefore experiments likely consisted of multiple rounds of infection prior to cumulative culture lysis ∼72 h after virus addition. The persistence of large-molecular weight DNA in control as well as infected cultures may be indicative of un-infected algal cells with intact genomes, infected algal cells with partially fragmented genomes, nascent viral genomes and/or bacterial DNA.The difference in DNA fragmentation patterns between virus-infected cultures of Chlorella sp. genome upon virus infection  EhV production, indicating a dependence on this activity for productive infection and invoking a Red Queen host-virus co-evolutionary dynamic around this activity and PCD  is the enzyme that catalyses the first and rate-limiting step in the biosynthesis of GSLs . The authors are unaware of any studies of algal host–virus systems in which the photophysiological effects of viral infection have been shown to be due to a fundamental disruption of the thylakoid membranes in algal host cells.During CeV infection of Supplementary data). Analysis of the draft CeV-01B proteome indicated only two ORFs containing a putative caspase cleavage site (LEVD), whereas the draft proteome of PpV-01B contains 15 predicted protein-coding genes, most of which had highest sequence similarity to hypothetical viral proteins in the NCBI database with known tetrapeptide caspase cleavage motifs. Two of the predicted PpV-01B proteins containing caspase cleavage motifs had highest similarity to predicted proteins in the EhV genome with identical caspase cleavage motifs  and “HAPTODIV” (nr. 190307) projects, and by the European Research Council Advanced Grant ERC-AG-LS8 “Microbial Network Organisation” . J.L.R. received a FRIBIO overseas research fellowship from the Norwegian Research Council. K.D.B. and B.V.M. were supported by funding from the United States National Science Foundation (OCE-1061883). Funding to pay the Open Access publication charges for this article was provided by the Bergen Open Research Archive (BORA) through the University of Bergen Library.Funding to


These birds are highly susceptible to strains circulating in Europe and, thus, may serve as surveillance sentinels. Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains’ virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows ( Flaviviridae) transmitted by mosquitoes, uses birds as its primary vertebrate reservoir host. WNV has an extensive geographic range that includes Europe, Africa, the Middle East, southern Asia, and Australia (Corvidae comprises the most highly susceptible species to WNV (Corvus brachyrhynchos) have been used to track the spread of the virus across many parts of North America (West Nile virus (WNV), a flavivirus , which are ubiquitously present across Europe, by injecting them with selected strains of WNV circulating in Europe and with the prototypic NY99 strain. In addition, we inoculated American crows with a selection of these viruses to assess and compare the virulence of WNV strains from Europe in a bird species known to be highly susceptible to WNV. Susceptibility was assessed in terms of death, survival time, magnitude and duration of viremia, and spread of virus to different organs.Experimental infection of American crows with the North American genotype of WNV (NY99) has shown that the strain has a highly pathogenic phenotype: viremia titers exceeded 9 logFive different WNV strains were used in this study: lineage 1a strain NY99-4132 (NY-99)  at the Colorado State University Animal Disease Laboratory.American crows were captured by using cannon net traps in Bellvue, Colorado, USA; the National Wildlife Diseases Program, Animal and Plant Health Inspection Service, United States Department of Agriculture, assisted with the captures. The crows were banded and transported to Fort Collins, Colorado, where they were housed in 1-m50) of virus per 0.1 mL of serum-free Dulbecco’s Modified Eagle Medium (DMEM) . Carrion crows (8 per virus) were injected with WNV strain NY99, Greece-10, FIN, Ita09, or 578/10. American crows were inoculated with NY99 (n = 6), FIN (n = 5), or Ita09 (n = 5). Approximately 0.1 mL of blood was collected from carrion crows at 2-day intervals, up to 8 days postinoculation (dpi), and 0.2 mL of blood was collected from American crows at the same time points and added to 0.9 mL of serum-free DMEM. Coagulated blood from carrion crows was centrifuged at 1,300 × g for 5 min in MiniCollect vials  to separate serum, and coagulated blood from American crows was centrifuged at 3,700 × g for 10 min to pellet clotted cells. Serum samples were stored at −80°C until further use.Crows were subcutaneously inoculated in the thigh or breast region with 2,000 50% tissue culture infectious doses  and a MagNA Pure LC automated nucleic acid robotic workstation (Roche) according to the manufacturer’s instructions, and subsequently stored at −80°C. RNA copy numbers were quantified by using unmodified primers as previously described (10) RNA copies.We used quantitative real-time reverse transcription PCR (qRT-PCR) to determine virus titers in serum and tissue samples and TCID10 titration of serum samples on Vero E6 cells, cytopathic effect was determined at 5 dpi and TCID50 infectious titers were calculated by using the Spearman–Kärber method . Sections were subsequently incubated overnight at 4°C with polyclonal goat anti-WNV NS3 protease  or isotype control  and then detected with polyclonal rabbit anti-goat IgG/HRP (Dako) antibody. Sections were counterstained with Mayer hematoxylin, mounted with Kaiser glycerin-gelatin, and analyzed by using a light microscope.Paraffin sections (4-µm thick) of sagittal organ were processed for streptavidin–biotin–peroxidase immunohistochemical detection of nonstructural protein (NS) 3 antigen. Sections were deparaffinized in xylene, rehydrated in descending concentrations of ethanol, and incubated for 10 min in 3% HSurvival curves were analyzed by using the log-rank (Mantel-Cox) test. Statistical analyses between >2 groups were performed by using Kruskal-Wallis 1-way analysis of variance; any significant differences were more closely analyzed between the groups by using the Mann-Whitney U test. A Bonferroni correction was applied to each p value, according to the number of comparisons . For all comparisons, each group had 6 crows, except for American crow groups that received FIN or Ita09 (n = 5).Signs of illness  were observed among most crows within 9 dpi. All 6 carrion crows inoculated with Greece-10 or Ita09 died, and 5 (83%) of the 6 inoculated with NY99 or 578/10 died. All 6 carrion crows inoculated with strain FIN survived . Surviva8.7 RNA copies/mL of serum , and the median peak infectious virus titer was 107.4 TCID50/mL of serum (range 101.8–108.8); these values include 1 bird in which detectable viremia did not develop during the entire course of infection. The median peak viremia titer for Greece-10–infected birds was 1010.3 RNA copies/mL of serum (range 109.8–1011.7) and 107.8 TCID50/mL of serum (range 107.3–109.8). FIN-infected birds had median peak viremia titers of 102.7 RNA copies/mL of serum (range 101–105.9) and 101.8 TCID50/mL of serum (range 101.8–102.5); however, viremia was detectable in only 3 of 6 birds, and infectious virus could be isolated from only 1 bird. The median peak viremia titers for Ita09-infected birds were 109.7 RNA copies/mL of serum (range 108.0–1010.0) and 107.6 TCID50/mL of serum (range 106.3–108.8). Birds infected with strain 578/10 had median peak viremia titers of 108.4 RNA copies/mL of serum (range 106.0–1010.1) and 105.1 TCID50/mL of serum (range 102.8–108.5).WNV viremia profiles were determined in terms of viral RNA  and infeStrain Greece-10–infected birds had median peak viral RNA titers significantly higher than those for NY99-infected (p = 0.004), FIN-infected (p = 0.005), and 578/10-infected (p = 0.004) birds. Greece-10–infected birds also had median infectious virus titers significantly higher than those for FIN-infected birds (p = 0.003), but FIN-infected birds had RNA and infectious titers lower than those for Greece-10–infected , Ita09-infected , and 578/10-infected  crows.9.6 RNA copies/mL of serum (range 109.1–1010.1) and 107.2 TCID50/mL of serum (range 104.7–107.2). Detectable viremia developed in only 2 of the 5 FIN-infected birds, resulting in median peak viremia titers of 101.0 RNA copies/mL of serum (range 101–106.9) and 101.8 TCID50/mL of serum (range 101.8–102.7). Median peak viremia titers for Ita09-infected birds were 108.8 RNA copies/mL of serum (range 108.0–109.1) and 106.7 TCID50/mL of serum (range 106.0–107.5). American crows infected with strain NY99 had the highest median peak viral RNA and infectious virus titers, and FIN-infected birds had the lowest median titers .Viremia profiles were also determined for American crows infected with 3 of the 5 different WNV strains . NY99-inVirus loads were determined in the heart, liver, spleen, kidney, bone marrow, and brain of all birds. To assess the spread of virus to the different organs at the approximate peak of viremia, we euthanized 2 birds per group at 4 dpi. Virus was detected in all organs from these birds. On average, the highest viral RNA titers were detected in the liver, followed by the bone marrow, spleen, kidney, and heart; the lowest titers were found in the brain . BetweenBirds euthanized because of illness had virus present in all organs; in most cases, the spleen, liver, and bone marrow contained the highest average viral RNA load, followed by kidney and heart; the lowest average viral RNA titers were in the brain. Viral RNA titers in organs of Greece-10–infected birds were higher than those in organs of birds infected with the other viruses, but this observation was not statistically significant .3.1 RNA copies/g of tissue) and in at least 3 of the 6 organs from FIN-infected birds (median virus load of 102.0 RNA copies/g of tissue), showing that these birds did undergo productive WNV infection.The 1 NY99-infected and 3 FIN-infected survivor birds that were free of viremia throughout the 8 days of blood sampling underwent necropsy at 14 dpi. Of interest, virus was present in all organs of the NY99-infected bird , kidney (80%), bone marrow (80%), and spleen (78%); tissues least consistently positive for WNV antigen were heart (50%) and brain (10%) . HoweverIn this study, we assessed the susceptibility of carrion crows to different strains of WNV. First we demonstrated that carrion crows are susceptible to WNV infection by using the North American strain NY99, which has previously been shown to be highly virulent in American crows  and Israel (Is98) also suggested that carrion crows are susceptible to infection with WNV in a strain-dependent manner  and with strain NY99 from North America. Similar to what was seen with carrion crows, American crows infected with Ita09 had high peak viremia titers, and all succumbed to the infection, whereas those infected with FIN had low viremia titers, and all survived infection. Furthermore, it was demonstrated that the Greece-10 strain used in this study was also 100% lethal in American crows . In fact, American crows infected with Greece-10 (vs. the other strains used in this study) had the highest median peak viremia titers in terms of RNA and infectious virus (data not shown). These results show that in American crows, WNV strains (apart from FIN) from Europe are as virulent as the prototypic NY99 strain from North America.Corvidae. Studies in North American and European corvids are ongoing in order to test the relevance of the T249P substitution and several other mutations when introduced into the genome of WNV-FIN.The fact that susceptibility of birds to WNV can be strain-dependent was clearly demonstrated by the attenuated virulence phenotype of WNV strain FIN in carrion and American crows (this study) and in European jackdaws  was less susceptible to lethal WNV infection  to WNV may also prove to be insightful, as this is the more predominant corvid species in eastern and southeastern Europe, where WNV is more common. The susceptibility of European birds to WNV has been demonstrated in multiple studies (We have shown that carrion crows, a species of bird ubiquitously found across Europe, are highly susceptible to WNV strains currently circulating in Europe. These birds could therefore potentially be useful as part of dead bird surveillance in the early detection of WNV in Europe. Future studies assessing the susceptibility of the closely related hooded crow (Methods for detecting preexisting West Nile virus neutralizing antibodies in crows captured for study.


Myocarditis is often cited as the cause of fatalities associated with foot-and-mouth disease virus (FMDV) infection. However, the pathogenesis of FMDV-associated myocarditis has not been described in detail. The current report describes substantial quantities of FMDV in association with a marked mononuclear inflammatory reaction, interstitial edema and cardiomyocyte degeneration in the myocardium of two pigs that died during acute infection with either of two different strains of FMDV. Despite similar clinical progression, there was a marked variation in morphological characteristics of myocarditis with a significant difference in intensity of myocardial inflammation between the two cases. Phenotypic characterization of leukocyte populations revealed that in both cases, the inflammatory infiltrate consisted mainly of combinations of CD172a+, CD163+ and CD44+ cells, with a distinct subset of CD8+ cells, but with consistent lack of detection of CD3+ and CD21+ cells. This suggests that the FMDV-associated acute myocardial inflammation in the two observed cases consisted mainly of leukocytes of monocyte lineage, with a distinct population of CD8+ cells which, based on lack of CD3 detection in serial sections, are likely to represent NK cells. Foot-and-mouth disease (FMD) is a highly contagious vesicular infection of cloven-hoofed animals caused by FMD virus (FMDV), a single stranded positive sense RNA virus of the Picornavirus family. FMD is generally a disease that causes high morbidity and low mortality. However, there are sporadic reports of FMD outbreaks with unusually high mortality rates ,2]], wi], wi2]],], wi], wAlthough commonly reported as the cause of fatalities associated with this disease, detailed investigations into the pathogenesis of FMDV myocarditis are lacking. The current report presents virological and histopathological findings in the myocardium of two pigs that died during acute infection with FMDV strains A24 Cruzeiro (case 1) or O1 Campos (case 2). The association between the pigs’ deaths and the presence of FMDV in myocardial tissue samples was confirmed through virus isolation, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunomicroscopy. Myocarditic properties of the two FMDV strains were investigated through routine histopathological evaluation in combination with phenotypic characterization of inflammatory infiltrates.This report is based on data obtained from two pigs  which were associated with separate experimental studies that have been described in detail elsewhere ,8]]. Th]. Th8]].]. Th]. TThe two pigs developed severe clinical FMD at four (case 1) and two (case 2) days after virus exposure (dpe), with fever (>40°C), lameness and vesiculation at lesion predilection sites . Both pigs died with clinical signs of congestive heart failure (respiratory distress and tachycardia) on the morning of the second day of clinical FMD infection . Postmortem examinations of both pigs revealed multifocal regions of pallor on the epicardial surfaces, which extended into the myocardium when sectioned. No other abnormalities were observed.Cardiac tissue samples were harvested during necropsy examinations, and were split into aliquots that were either frozen on liquid nitrogen for virus isolation and qRT-PCR , embedd10 FMDV RNA copies/μl. The highest quantity of viral RNA detected in myocardial tissue samples from this pig was 7.73 log10 viral RNA copies/ mg. Virus isolation on LFBK αvβ6 cells [[Serum samples collected one day prior to death (4 dpe) from case 1 (infected with FMDV A24 Cruzeiro) contained 7.62 log6 cells  was posHigh quantities of FMDV capsid protein VP1 were detected within cardiomyocytes by MIF analysis of cryosections Figures A-B, 4A-D10 viral RNA copies/μl, and the highest viral load found in myocardial tissue samples was 6.46 log10 RNA copies/mg. Virus isolation on LFBK αvβ6 cells was positive for both serum and tissue samples. Myocardium had regions of myocyte degeneration and interstitial edema, comparable to that observed in case 1  and total numbers of inflammatory cells (P = 0.0035) compared to case 1  from case 2 (infected with FMDV O1 Campos) contained 5.50 log dpe fromThe current report presents a descriptive investigation of the myocarditic properties of FMDV, based on analysis of myocardial tissue samples from two pigs that died during acute infection with FMDV A24 Cruzeiro or FMDV O1 Campos. There was a difference in the time elapsed from inoculation to death between the two cases (3 and 5 days respectively). However, both pigs died during the second day of clinical FMD. There are several potential contributors to the differences between the cases including variability of the viral strains used, host immune responses, and manner of exposure to FMDV (IOP inoculation vs. contact exposure). High levels of FMDV were detected by qRT-PCR and virus isolation in the myocardium of both pigs. For both pigs, myocardial levels of FMDV RNA exceeded those measured in serum although there was no serum available for analysis from the day of death for case 1. Histopathological evaluation of tissue sections from both cases indicated comparable extents of myocardial tissue damage, although with a substantially greater quantity of inflammatory cells in samples from the pig infected by FMDV A24 Cruzeiro. Since the cardiomyocyte degeneration is present with and without inflammation, this finding suggests that a substantial portion of the tissue damage may be a direct consequence of virus replication rather than a result of the host-response to infection. There was also a distinction in the morphology of inflammatory cells, with a greater abundance of large macrophage-like cells in case 1, in comparison to a relatively larger proportion of elongated cells (suspect dendritic cells) in case 2 . The CD8+/CD3-/CD21- phenotype, small mononuclear cells detected in myocardial infiltrates of both cases described herein is consistent with NK-cells . The laPicornaviridae [[Variation in the intensity of myocardial inflammation between different virus strains has previously been reported for Cocksackie B virus, a related virus within viridae . Previoviridae , suggesviridae . This sOverall, the two cases of FMDV-associated myocarditis described herein had exceedingly similar clinical and pathological characteristics. There was substantial histomorphologic variation between the cases, but the mechanistic differences underlying this disparity could not be determined in this purely descriptive analysis. There is a need for more comprehensive studies to further investigate the pathogenesis of FMDV-associated myocarditis. Analysis of specific host-virus interactions involved in the onset and progression of myocardial injury, including comparative studies of related picornaviruses with more consistent cardiotropism, could help to further elucidate the mechanisms of this atypical, yet important variation of FMDV pathogenesis.The authors declare that they have no competing interests.CS planned and performed immunofluorescence assays and imaging, executed the animal experiment with FMDV A and drafted the manuscript. JP executed the animal experiment with FMDV O, oversaw laboratory analyses, and edited the manuscript. MB & LR contributed to study design and provided critical revision of the manuscript. JA designed and coordinated the studies, revised manuscript. All authors read and approved the manuscript.


On March 20, 2015, 30 days after the most recent confirmed Ebola Virus Disease (Ebola) patient in Liberia was isolated, Ebola was laboratory confirmed in a woman in Monrovia. The investigation identified only one epidemiologic link to Ebola: unprotected vaginal intercourse with a survivor. Published reports from previous outbreaks have demonstrated Ebola survivors can continue to harbor virus in immunologically privileged sites for a period of time after convalescence. Ebola virus has been isolated from semen as long as 82 days after symptom onset and viral RNA has been detected in semen up to 101 days after symptom onset .On March 14, 2015, a woman from Monrovia aged 44 years (patient A) developed headache, weakness, joint pain and nausea. She went to a hospital on March 19, and was triaged as a suspected Ebola patient to a nearby transit center . On March 20, Ebola was confirmed by reverse transcription–polymerase chain reaction (RT-PCR). Genomic sequencing of Ebola virus from her blood specimen identified six mutations not found in 25 other genomes sequenced from Liberia –8. The iPatient A did report unprotected vaginal intercourse on March 7, 2015, with an Ebola survivor (survivor A), a man aged 46 years from another community in Monrovia. Survivor A had experienced onset of symptoms consistent with Ebola, including fever, anorexia, and headache on September 9, 2014, and was admitted to an Ebola treatment unit on September 23. His first test by RT-PCR on September 28, 2014, was indeterminate . A second specimen was negative by RT-PCR on October 3, 2014. Survivor A was discharged from the Ebola treatment unit on October 7, 2014 and reported no subsequent illness or symptoms.Survivor A had multiple family members with whom he lived or interacted with confirmed or suspected Ebola during the same period as his symptoms and Ebola treatment unit admission . His oldA new blood specimen was collected from survivor A on March 23, 2015, as part of patient A’s case investigation. The specimen was negative for Ebola virus by RT-PCR. Enzyme-linked immunosorbent assays for Ebola virus glycoprotein- and nucleoprotein-specific immunoglobulin G (IgG) antibodies were positive; immunoglobulin M (IgM) was undetectable. A semen specimen, collected from survivor A on March 27, 2015, was positive by RT-PCR with a cycle threshold of 32. Complete genome sequencing of the viral RNA from survivor A’s semen has not been possible to date given the low level of detectable viral nucleic acid. However, the partial sequence obtained so far (28% of the genome) closely matches the sequence from patient A. A rapid diagnostic test was conducted to evaluate human immunodeficiency virus (HIV) as a possible reason for long-term viral shedding. The HIV test was negative.In addition to patient A, survivor A reported recent unprotected vaginal intercourse with a woman aged 45 years (contact A) with no history of illness. Intercourse with contact A occurred on three to five occasions between the last week of February and March 15, 2015. A blood specimen collected from contact A on March 27, 2015 was negative for Ebola virus–specific IgG and IgM.Since January 21, 2015, all new confirmed cases of Ebola in Liberia have been epidemiologically linked to a single transmission chain . Ebola viral RNA from three of the 22 confirmed cases in this transmission chain  were sequenced and compared with the genetic material from patient A. None of the sequences from these isolates shared the mutations observed in patient A’s isolate.Available epidemiologic and laboratory findings indicate that patient A may have been exposed to Ebola virus through sexual contact with survivor A, whose semen was PCR-positive 199 days  after his likely Ebola onset. Although the diagnostic RT-PCR in September was indeterminate, survivor A’s positive enzyme-linked immunosorbent assays, specifically against the viral nucleoprotein, indicate previous Ebola virus infection. His clinical course and epidemiologic links suggest that he had Ebola in early September 2014. The diagnostic tests were performed 18 and 24 days after symptom onset, and the results may have reflected convalescence. Although less likely, it is also possible that his Ebola virus infection occurred later and the indeterminate test result reflected the absence of Ebola virus in September 2014.Ebola virus RNA in survivor A’s semen in March 2015 does not prove the presence of infectious virus. However, the absence of patient A’s genetic signature in sequenced RNA from three patients in Liberia’s last known cluster of epidemiologically-linked cases makes it unlikely that patient A was infected from unrecognized, ongoing community transmission. Culture of survivor A’s semen specimen for Ebola virus is planned to determine whether viable virus was present.It is not possible to definitively ascribe Ebola infection in patient A to transmission from survivor A, and another sexual partner or other source cannot be excluded. However, the timing of intercourse between survivor A and patient A, the subsequent illness in patient A, the presence of viral RNA in survivor A’s semen, matching genetic sequences (where coverage has been obtained) in isolates from survivor A and patient A, and the lack of other known exposures suggest possible sexual transmission. Enrichment methods are being applied to survivor A’s semen sample to amplify existing Ebola virus RNA and complete genomic sequencing. Other limitations of the investigation include 1) the relatively small number of sequenced genomes from Ebola patients in this epidemic, which limits an assessment of the generalizability of the molecular findings; and 2) incomplete laboratory results and Ebola treatment unit and hospital records for some of survivor A’s family members, preventing confirmation of Ebola and exact dates of death.Previously, CDC and WHO recommended abstinence or condom use for at least 3 months following recovery from Ebola. However, to prevent transmission of Ebola, contact with semen from male survivors should be avoided. If male survivors have sex , a condom should be used correctly and consistently every time until further information is known. Used condoms should be handled and disposed of safely to avoid contact with semen. After handling of condoms, or following any physical contact with semen, skin should be washed thoroughly with soap and water. Based on information from this investigation, CDC, the World Health Organization, and the Government of Liberia issued updated recommendations for survivors ,9,10.Investigations of several other recent Ebola cases in West Africa have suggested sexual transmission from survivors but have not been confirmed . Additional studies are planned to determine clearance, persistence, and shedding of Ebola virus in body fluids of survivors and to evaluate possible sexual transmission of infection. Use of RT-PCR testing of semen  might be a useful tool for assessing and counseling male survivors on measures they should take to prevent transmission of Ebola virus. CDC and other public health partners are reviewing existing data to determine the validity and feasibility of potential recommendations.What is already known on this topic?Ebola virus persists in seminal fluid following recovery, but the duration of viral shedding and the likelihood of sexual transmission are not known. Earlier studies have demonstrated that the virus can be isolated from semen as long as 82 days after symptom onset, and that semen can be positive by reverse transcription–polymerase chain reaction, indicating presence of viral RNA, up to 101 days after onset. Possible sexual transmission was reported in 1968 for Marburg virus, a related filovirus, but has not been clearly documented for Ebola.What is added by this report?Ebola virus can persist in the seminal fluid of convalescent men for longer than previously recognized and can potentially lead to sexual transmission of Ebola.What are the implications for public health practice?Until more information is known, contact with semen from a male survivor should be avoided. If male survivors have sex , a condom should be used correctly and consistently every time. Additional studies are planned to examine Ebola virus persistence in body fluids of male and female convalescent patients and the likelihood of sexual transmission.Transmission of Ebola in West Africa has diminished over the past few months. However, awareness of possible sexual transmission from survivors to partners and the importance of prevention measures is needed. Sufficient supplies of condoms and counseling to promote their correct and consistent use should be provided as part of the response in Ebola-affected countries. In addition, efforts should be undertaken to prevent the possibility of sexual transmission from stigmatizing survivors.


Infections caused by canine parvovirus, canine distemper virus and canine coronavirus are an important cause of mortality and morbidity in dogs worldwide. Prior to this study, no information was available concerning the incidence and prevalence of these viruses in Cape Verde archipelago.To provide information regarding the health status of the canine population in Vila do Maio, Maio Island, Cape Verde, 53 rectal swabs were collected from 53 stray dogs during 2010 and 93 rectal swabs and 88 blood samples were collected from 125 stray dogs in 2011. All rectal swabs  were analysed for the presence of canine parvovirus, canine distemper virus and canine coronavirus nucleic acids by quantitative PCR methods. Specific antibodies against canine distemper virus and canine parvovirus were also assessed (2011 n = 88).From the 2010 sampling, 43.3% (23/53) were positive for canine parvovirus DNA, 11.3% (6/53) for canine distemper virus RNA and 1.9% (1/53) for canine coronavirus RNA. In 2011, the prevalence values for canine parvovirus and canine coronavirus were quite similar to those from the previous year, respectively 44.1% (41/93), and 1.1% (1/93), but canine distemper virus was not detected in any of the samples analysed . Antibodies against canine parvovirus were detected in 71.6% (63/88) blood samples and the seroprevalence found for canine distemper virus was 51.1% (45/88).This study discloses the data obtained in a molecular and serological epidemiological surveillance carried out in urban populations of stray and domestic animals. Virus transmission and spreading occurs easily in large dog populations leading to high mortality rates particularly in unvaccinated susceptible animals. In addition, these animals can act as disease reservoirs for wild animal populations by occasional contact. Identification of susceptible wildlife of Maio Island is of upmost importance to evaluate the risk of pathogen spill over from domestic to wild animals in Cape Verde and to evaluate the associated threat to the wild susceptible species. Over the past few years, efforts have been made towards a better understanding of the health status of animal populations, particularly regarding viral infections. Due to their high mutation rate and replication strategies, viruses are responsible for recently recognized emerging diseases, posing a danger not only to domestic and wild animals, but also to humans ,2.The high density of domestic and stray animals in urban areas enables viral dissemination and maintenance in these populations. Consequently, these animals can act as reservoirs of diseases, with the possibility of transmission to wildlife populations through occasional contact.Canine parvovirus (CPV) was first identified in the late 1970s and was responsible for severe hemorrhagic gastroenteritis and myocarditis in dogs . ParvoviCanine distemper virus (CDV) is the etiological agent of canine distemper, a highly contagious disease, responsible for high mortality rates in dogs worldwide. Sequence analysis of CDV strains originated in different geographical areas from several animal species, showed that the hemagglutinin gene has undergone a genetic drift according to the geographic location ,8. Phyloper si a major infectious agent responsible for several epidemics  x Avogadro's number  detection was only performed for 2011 serum samples for CPV and CDV using an indirect ELISA kit (Ingezim Canine Parvo 15.CPV.K1® and Ingezim Moquillo 1.5.CDG.K.1®-Ingenasa), for specific IgG detection, according to the manufactures instructions.This method allowed the quantification of the Ab titer for CPV, using a formula provided by the kit, although it did not specify the correspondence with the hemagglutination inhibition assay for quantification of anti-CPV antibodies. The values were organized in three classes: 1) 100–1000 ELISA Units (EU); 2) 1000–10000 EU and 3) > 10000 EU.For CDV the OD values had correspondence with the indirect immunofluorescence (IIF) method, and the values were divided into three categories: 1) low titer , 2) medium titer  and 3) high titer .2) with IBM SPSS Statistics 19.0 software. A P value <0.05 was considered significant.A possible association between serological findings and the age and gender of the animals was determined by chi-square statistical test (χThe authors declare that they have no competing interests.PC, AD and LT were responsible for the conception and the study design and actively participated in the analysis and data interpretation. PC and AD were also involved in the drafting and revision of the article. SG and CC were involved in the data analysis. MM and SV were responsible for the organization of the field work and sample collection.


We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 10 The 2014 Ebola virus outbreak is the largest in history, with widespread transmission in multiple West African countries and sporadic cases in Europe and North America ,4. The mRapid, point-of-care detection of the Ebola virus could enable early quarantine and halt future epidemics and pandemics ,6. WhileEbola virus is a class A select agent filovirus that was first identified in Zaire in 1976 and named after the River Ebola in Zaire ,9. MatheCurrent approaches to Ebola identification include antigen-capture ELISA testing, IgM ELISA, RT-PCR, virus isolation, electron microscopy, and serologic testing for IgM or IgG antibodies. Quantitative measurement of the viral load also has prognostic importance, with a much higher case fatality rate in patients with viral loads over 10 million copies per milliliter . Current2, which is an order of magnitude lower than detection limits for other marker-free systems such as optical surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) devices [Our group has developed a point-of-care biosensor that employs surface acoustic waves for label-free pathogen detection without the need for any sample preparation ,13,14. T devices .With this sensor platform, viral detection is rapid , and the entire detection protocol (from collecting the test sample to obtaining a positive or negative test result) requiring about 5–10 min. Such technological benefits would be ideal for rapid, point-of-care identification and diagnosis of the Ebola virus, especially in environments with minimal or overloaded infrastructure, such as in public health or emergency response situations. Here, we discuss initial modifications of the biosensor to demonstrate detection of Ebola and its potential utility to combat this recurrent epidemic.3) wafers by lithographic deposition and patterning of inter-digital transducers (IDT) and waveguide layers as previously described [3 wafers were patterned with four IDT patterns with sets of transducers and delay lines using negative tone photoresist AZ2020 . Then, a metallization step was performed with 5000 Å aluminum using an electron-beam evaporator . The wafer was placed in an acetone bath to lift off the photoresist and any excess aluminum, followed by an acetone spray as needed and by the following rinses in methanol, isopropyl alcohol, and deionized water. This process was repeated for ground metallization, bus lines, and contact pads. Next, a waveguide layer of 5000 Å silicon dioxide (SiO2) was deposited as a film on the wafer using lift-off plasma enhanced chemical vapor deposition . The oxide was coated with hexamethyldisilazane in a vacuum oven, and positive tone photoresist AZ4330  was used to form a photoresist mask on the wafer having exposed portions. The exposed portions of SiO2 was etched with reactive ion etching to access electrical contact pads. Finally, the resultant wafer was diced to form individual chips, in which photoresist from dies was removed by rinsing in acetone, methanol, and isopropanol.The sensor chips were prepared using lithium tantalate  . Typically, two lanes were functionalized with antibodies specific for the target analyte, and two lanes were functionalized with isotype control antibodies; the latter are referred to as reference lanes.Antibodies were individually patterned on sensing lanes as previously described . Briefly6 rads total dose) on dry ice, and sonicated. Culture cell debris were removed by centrifugation. The virus was confirmed non-viable (killed or inactivated) by inoculation of cell culture (10 days on Vero cells) followed by a second passage (10 days on Vero cells). The concentration of the Ebola virus antigen sample was provided in plaque-forming units (PFU/mL) by the supplier, indicating the concentration of viable viral particles prior to inactivation, and allowing for comparison across different animal models and experimental conditions [The Ebola virus antigen sample  consists of inactivated and highly disrupted viral particles and was used as provided. Fully intact Ebolavirus is highly pathogenic and designated BSL-4 material. For research purposes, the supplier provides inactivated and disrupted Zaire (Mayinga) Ebola virus , which was prepared as follows. As stated by the supplier, Zaire (Mayinga) Ebola virus from infected Vero E6 pellets was suspended in 50 mM sodium borate, gamma irradiated  to roughly determine the size distribution of the degraded viral particles. Ebola samples were stored in aliquots of 50 µL in screw cap safety microtubes at −80 °C until ready for use.2HPO4·2H2O, 2.0 mM KH2PO4, pH 7.4 with HCl). All Ebola antigen samples were prepared fresh for each experiment.When ready to be tested, the samples were thawed on ice and supplemented with phosphate buffered saline  were recorded with a custom acquisition program developed using Visual Studio (Microsoft) as previously described . In brieEbola antigen samples of various concentrations were prepared in 100 µL of PBS and applied to the biosensor. Binding of viral antigens to the sensor surface resulted in a net phase shift of the signal Δφ (expressed in degrees on the y-axis) along the time plot (on the x-axis). While phase shift differences associated with specific Ebola signal are apparent and stable within seconds of sample addition, we continue measuring up to 5 min after sample addition to assure signal stability and reproducibility. All measurements represent multiplicates of at least three, and data points are represented as means ± standard error of the mean (SEM).We present here the initial development and characterization of a biosensor for point-of-care, quantitative detection of the Ebola virus. This device enables log-linear, concentration-dependent detection of disrupted viral particles in the clinically relevant range. Overall sensitivity and limit of detection are dependent on the mass of the species that is bound to the sensor surface; this biosensor is likely to be orders of magnitude more sensitive for intact, infectious Ebola viral samples .. The piezoelectric substrate propagates horizontally polarized surface shear waves, and such waves are induced by applying an alternating voltage to the IDTs at a high frequency . The surface shear waves are characterized by a particular resonant frequency that is sensitive to changes on the sensor surface.The biosensor was adapted to allow for sensitive detection of the Ebola virus. The biosensor has a planar, piezoelectric substrate containing inter-digital transducers (IDTs) ,13. The Specificity for the target bioagents is obtained by functionalizing the surface of the substrate a. Here, i.e., the Ebola virus in this study), as well as a reference lane including a control IgG1 antibody  Ebola virus , which is available for research purposes under non-BSL-4 conditions. Thus, transmission electron microscopy studies were conducted to identify the size distribution of the fragmented viral particles. Representative images are provided in TEM of the inactivated virus sample shows highly disrupted, fragmented particles that form aggregates a, rather4 PFU/mL to 3.0 × 106 PFU/mL prior to virus inactivation. The lowest concentration is below the average viremia level of 3 × 104 RNA copies per mL observed in Ebola patients on the first day of disease symptoms [In analogy to our previous report on the detection of HIV-1 and HIV-2 , we funcsymptoms .Representative phase shift data are provided in 4 PFU/mL to 6.5 × 106 PFU/mL (R2 of 0.92. The linear range and correlation coefficients compare favorably to those recently reported for qRT-PCR [4 PFU/mL was calculated for Ebola virus by linear regression and using the average background noise Δφ value of 0.31. These results indicate that the prototype SAW biosensor rapidly detects the Zaire strain of Ebola antigens in a defined buffer with detection limits below the average viremia level at onset of clinical symptoms.Detection of Ebola virus resulted in a concentration-dependent increase with Δφ values ranging from 0.20 ± 0.04 to 4.46 ± 0.86, corresponding approximately to 1.6 × 106 PFU/mL . There w qRT-PCR . A limitBacillus anthracis bacteria simulant, Bacillus thuringiensis [Francisella tularensis bacteria [We have demonstrated an adaptable, label-free sensing system for the rapid detection of bioagents and show its use to detect the Ebola Zaire virus. We have previously shown the effectiveness of this system in detecting the ngiensis ; the Coxngiensis ; the Sinngiensis ; the Frabacteria  and the bacteria . This stAs fully intact Ebola virus is highly contagious, we used an inactivated virus in order to conduct this study under non-BSL-4 conditions. However, in a first responder or real world scenario, the sample would not necessarily have to be inactivated and could be tested directly, providing the sensor was contained in a controlled environment.The SAW biosensor described here provides the basis for a rapid and specific response to Ebola outbreaks and other emerging diseases. This point-of-care sensor will provide rapid diagnosis and improved infection control, dramatically decreasing the human and economic costs of this disease. Further work will focus on portability and optimization for field use. A disposable cartridge can be employed to house the piezoelectric substrate and fluidic channels to guide the test sample to the substrate. The user interface can be readily simplified to provide a simple positive/negative result to a field worker on the front lines of the next epidemic.Sample preparation is not required for this label-free sensing methodology, simplifying use in field conditions without centralized laboratories or refrigeration. While additional testing can be conducted to verify the detection limits for Ebola Zaire in complex media, such as blood, serum, or saliva , we beli


Warmer temperatures are associated with decreased populations in soil and on trees. Cryptococcus gattii infection among humans and animals. To identify key biophysical factors modulating environmental concentrations, we evaluated monthly concentrations of C. gatti in air, soil, and trees over a 3-year period. The 2 study datasets were repeatedly measured plots and newly sampled plots. We used hierarchical generalized linear and mixed effect models to determine associations. Climate systematically influenced C. gattii concentrations in all environmental media tested; in soil and on trees, concentrations decreased when temperatures were warmer. Wind may be a key process that transferred C. gattii from soil into air and onto trees. C. gattii results for tree and air samples were more likely to be positive during periods of higher solar radiation. These results improve the understanding of the places and periods with the greatest C. gattii colonization. Refined risk projections may help susceptible persons avoid activities that disturb the topsoil during relatively cool summer days.Vancouver Island, Canada, reports the world’s highest incidence of  Cryptococcus neoformans, are common causes of death and illness among persons with compromised immune systems. C. gattii is a related fungus that can cause serious illness. Specific genotypes  are isolated more commonly from immunocompetent persons, and other genotypes  are isolated more commonly from immunocompromised persons. In 1999, a C. gattii genotype that had previously been reported in Brazil and Colombia was first documented on Vancouver Island in the province of British Columbia, Canada  most strongly influence C. gattii concentrations. For example, which is the primary driver of airborne C. gattii levels in southern Australia: temperature, dryness, or both? More frequent C. gattii measurements and longitudinal statistics can help distinguish between competing processes. Most long-term studies documented C. gattii dynamics on trees; however, seasonality of C. gattii may differ in the soil and air  C. gattii dynamics from the air, trees, and soil on Vancouver Island, Canada. The first research question examines specific plots from which repeated measurements were made during 2003–2004, and the second question examines only newly sampled C. gattii plots during 2002–2004. Based on environmental samples, these investigations were designed to provide insight into the periods with the greatest C. gattii area concentrations. This study expands on previous research in the area by studying changes over time, using representative weather stations, considering more biophysical conditions, and using statistics that control for autocorrelation.Our goal with this study was to determine the relative strength of associations between biophysical conditions and monthly C. gattii in the environmental soil, air, and trees were collected by previously described standardized methods  previously collected by different sampling strategies: repeatedly measured and newly sampled. The first strategy sporadically resampled a geographic plot after a positive C. gattii sample was obtained for this site during 2003–2004. This dataset is similar to the permanently colonized sites analyzed in an ecologic habitat study   . The secC. gattii dynamics. This analysis broadly considered biophysical conditions over the previous and current day, previous week, and previous month (past 30 days). For each sampling date, C. gattii observations for each plot were aligned with the corresponding weather conditions of the surrounding study area.There is minimal research to support the selection of periods over which biophysical conditions most strongly influence C. gattii studies may reanalyze data collected for different purposes, such as surveillance and detection. C. gattii was rarely sampled continuously from the same plots. More commonly, repeated measurements were sporadically taken from the same plots. For example, tree A might have been sampled in January–March and August–October, tree B in April–July and November–December, and tree C in April–October. Although no tree was continuously sampled throughout the year, standardizing and pooling the sporadic samples can collectively yield seasonal C. gattii information. The analysis maximized the information available from the sporadic samples by use of hierarchical generalized linear and mixed effect models (GLMMs) that control for repeated measurements and clustered sampling  or C. gattii presence/absence (trees). Poisson GLMMs with a random effect for each study observation accounted for overdispersion for the soil and air samples. Logistic GLMMs were used to analyze the tree samples. The analysis was conducted in R version 2.15.3 with use of the LME4 package (http://www.R-project.org/). In the first analysis of longitudinal samples, hierarchical random effects controlled for repeated plot measurements and plots nested within study areas.GLMMs were used to investigate association of weather conditions with monthly C. gattii were observed <20 times in trees of a given genus, genera were further aggregated into families or lumped into the “other” category. To control for residual spatial autocorrelation, we considered latitude and longitude as candidate independent variables in the analysis. We also controlled for seasonality with fixed-effect indicator variables for winter (November–February), spring (March–May), summer (June–July), and fall (August–October). The GLMM results were reported when the postvariable selection model residuals were not significantly autocorrelated. Residual autocorrelation was tested by autocorrelation and partial autocorrelation functions that were adjusted for missing data.The random effect in the second analysis accounted for plots nested within study areas. Both analyses controlled for tree genus . If C. gattii levels for a given month may be strongly related to the previous month’s values. Monthly C. gattii samples may exhibit a more complex temporal correlation structure. If the GLMM residuals were significantly autocorrelated, we conducted the analysis on a reduced dataset. For the first analysis, the reduced dataset included plots sampled in sequential months from the plots with >4 longitudinal samples. For the second analysis, the reduced dataset included all first samples in a study area, provided that the study area was sampled in the previous month. Thus, the GLMM controlled for seasonality, plot, or study area–specific random effects, and first-order autoregressive terms for each plot  or study area . The autoregressive term was the natural logarithm of average C. gattii concentration plus 1  or proportion of positive C. gattii samples (tree) in the previous month. In the reduced dataset, study areas in which C. gattii were observed <20 times were lumped together in the “other area” category.Intuitively, the C. gattii association, weekly or monthly absolute humidity was not tested in the next stepwise iteration. There are minor to moderate differences in the magnitudes of weather conditions across the study area. The statistical results therefore reflect time periods and geographic areas in which weather systematically influences C. gattii levels. Weather conditions in the study area were not further standardized to retain the interpretability and biological plausibility of weather conditions for C. gattii population dynamics.Because of the relationships among weather conditions, a forward stepwise variable selection procedure involving the Akaike information criterion was used to select the multiple variable models. After a weather variable entered the model, the selection procedure did not consider other temporal aggregations of the same variable. For example, if daily absolute humidity exhibited the most significant C. gattii concentrations and sample size for the soil and air samples and the proportion of positive tree swab samples. On a plot level , weather systematically influenced soil and airborne C. gattii levels . Average wind speeds were weakest in the study areas surrounding Courtenay and Errington. During October–April, area-averaged (≈14 km2) monthly wind speeds were <2 m/s.i levels . The soiC. gattii levels for a given month were not associated with those of the previous month. Therefore all plots sampled >4 times were included in the analysis. Similar to the trend for the soil samples, there was an increasing eastward trend of C. gattii across the island. Solar radiation intensity was positively associated with airborne C. gattii concentrations. The most daily solar radiation is received in the southerly areas  and during May–August. Wind speeds exhibited a more complex, nonlinear relationship to airborne propagules. Moderate daily wind speeds (1.5–3 m/s) may be more likely than less windy days (<1.5 m/s) to entrain C. gattii propagules into the air. However, C. gattii concentrations were lower on very windy days than on relatively tranquil days. Temperature was not associated with airborne concentrations.Airborne C. gattii sample in a given month was more likely to be positive in the following month. Thus, results are reported from the reduced dataset of trees sampled in sequential months . As with the soil samples, geographic areas and periods with warmer temperatures were associated with reduced frequency of C. gattii isolation. Similar to the air samples, solar radiation and wind speed were positively associated with frequency of C. gattii isolation. C. gattii isolation was more likely in southern study areas and during May–August, which had the most solar radiation.Of note, temperature, wind speed, and solar radiation strongly influenced C. gattii exhibits specialized habitat preferences. It thrives in the area of the Vancouver Island rain shadow , where winter temperatures are predominantly above freezing and summer temperatures are not too hot   than in temperate and tropical regions (C. gattii–endemic areas were slightly cooler (9.8°C–11.4°C). Outbreaks of C. gattii infection in humans or animals in Western Australia, Mediterranean Europe, and North America have been characterized by dry summers or dry winters with warm but not hot monthly temperatures (<22°C)  and trees (7.7%) on Vancouver Island is remarkably high  than are British Columbia and Washington .The aversion of the Cryptococcus spp. produce melanin. Melanin may increase the integrity of C. neoformans cells and make them less susceptible than nonmelanized cells to temperature extremes  are greater than concentrations deeper (15–30 cm) in the soil . According to our results, the highest airborne C. gattii concentrations occur during August–October on sunny days with moderately windy conditions. The greatest risk for exposure to C. gattii from the soil is during relatively cool June and July summer days. Although these associations are consistent, until more research provides information about the infectious dose for humans, the study results characterize the risk for exposure associated with environmental factors, rather than disease risk. Weather and airborne concentrations of C. gattii should be associated with human cryptococcosis incidence; however, onset of documented cryptococcosis cases in British Columbia does not vary by season or month (C. gattii transmission.The primary route of human Cryptoccoccus gattii Populations, Vancouver Island, Canada, 2002–2004.Supplemental introduction, methods, and references for study of Climatic Influences on