Newcastle disease virus (NDV) is the causative agent of the Newcastle disease, a severe disease in birds associated with substantial economic losses to the poultry industry worldwide. Sweden is situated along the Western European waterfowl flyway and applies a non-vaccination policy combined with directives of immediate euthanisation of NDV infected flocks. During the last decades there have been several outbreaks with NDV in poultry in Sweden. However, less is known about the virus prevalence in the wild bird population including waterfowl, a well-established reservoir of avian paramyxovirus type 1 (APMV-1), the paramyxovirus serotype that include pathogenic NDV.Anas platyrhynchos), trapped in the southern part of Sweden during autumn migration in 2010. These samples were screened for APMV-1 by real-time reverse transcription PCR, and viral strains from positive samples were isolated and characterized by sequence analysis of the fusion gene and by phylogenetic analysis.The survey constituted of 2332 samples from Mallards . The highest APMV-1 prevalence was detected in juvenile Mallards sampled in November =0.67%, 2.24%). Sequence analysis and evaluation of phylogenetic relatedness indicated that isolated APMV-1 strains were lentogenic, and phylogenetically most closely related to genotype Ib strains within the clade of class II viruses. The sampling system employed enabled us to follow APMV-1 infections and the shedding of one particular viral strain in one individual bird over several days. Furthermore, combining previous screening results with the APMV-1 detections in this study showed that more than 50% of Mallards that tested positive for APMV-1 RNA were co-infected with influenza A virus. Avulavirus genus in the Paramyxoviridae family. This virus family is composed of ten different serotypes (APMV-1 to APMV-10), most of which are exclusive to birds [Paramyxoviridae have a single stranded, non-segmented, negative-sense RNA genome containing six open reading frames (ORFs) that encode a nucleocapsid protein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase (HN), and the RNA-dependent RNA polymerase (L). HN and F proteins are involved in host cell attachment and membrane fusion, respectively, during infection [Newcastle disease is a highly contagious, infectious disease in birds with recurrent outbreaks in poultry all over the world. The infection is caused by the Newcastle disease virus (NDV), more formally termed avian paramyxovirus type I APMV-1), which is a member of the , which infection .The F protein is expressed as a precursor (F0) that needs to be activated by proteolytic processing into active F1 and F2 . The priThere is substantial genetic variation within the APMV-1 serotype, and viral strains are subdivided into two major clades termed class I and class II ,9. One fAnas platyrhynchos), in Sweden from 2002 to present [Wild waterfowl is also the main reservoir for influenza A virus (IAV). Naturally, there is a significant chance of birds being co- or superinfected with both AMPV-1 and IAV. The dynamics between these two viruses has been investigated experimentally in embryonated chicken eggs , but muc present ,18. SimiWild Mallards were captured in a live-duck trap at the Ottenby Bird Observatory, on the island of Öland, Sweden during October and November of 2010. The trap was emptied once a day and captured wild birds as well as sentinel ducks, held in a secluded compartment of the trap to attract the wild birds, were sampled for avian viruses including APMV-1 and IAV. Samples from individual birds were taken either by swabbing the cloacae or by collecting fresh faeces at the bottom of the single-use boxes, according to a well-established system that has been described previously . In addiRNA was extracted from 100 μl of samples using the MagNA Pure 96 isolation system and the MagNA Pure 96 DNA and Viral NA Large Volume Kit (Roche). For real-time reversed transcription PCR (rRT-PCR), 2 μl of each sample were screened for APMV-1 (amplifying 121 base pairs of the matrix gene) and influenza A virus (amplifying 101 base pairs of the matrix segment) by using the One-step RT-PCR kit (Qiagen), and established TaqMan-based real-time PCR protocols for each virus ,21. The In order to isolate virus, 100 μl of the positive swab samples, as determined by rRT-PCR, were inoculated in embryonated specific pathogen free (SPF) chicken eggs following established protocols . SuccessViral RNA for sequencing was extracted from allantoic fluid of inoculated eggs using EZ1 BioRobot system and the EZ1 Virus Mini Kit v2.0 (Qiagen). Viral RNA was eluted in a final volume of 50 μl. A 1595 base pair long fragment  of isolated APMV-1 strains were amplified with primers NDV+47Fw (5’-ATGGGCTCCAGACCTTCTACCAAGA-3’) and NDV+1671Rev (5’-TTGTAGTGGCTCTCATCTGATCTAGAGTAT-3’). Resulting amplicons were gel purified  and cloned into pGEM-T cloning vector according to the manufacturer’s instruction (Promega). Single bacterial clones encoding the APMV-1 F gene fragment were detected by PCR by using primers corresponding to the T7 and SP6 promoter regions of the cloning vector. Amplicons from multiple clones encoding the F gene of the same virus isolate were purified (see above) and sequenced using the T7 and the SP6 primer. Sequences generated in this study were deposited in GenBank with accession numbers KC631386- KC631395.The F gene nucleotide sequences were edited and assembled in Geneious version 6.0.6 , and aliConfidence intervals of a proportion, i.e. the virus prevalence, were estimated using the modified Wald method implemented in GraphPad Prism version 6.01 . In these analyses, the modified Wald method and a 95% confidence interval was used to quantify uncertainty . VariatiSince a given individual may appear several times in the cells of the contingency table, this could bias both the prevalence estimates (mean and CI) and the related test’s outputs. In particular, repetitions of a given individual for different co-infection may lead to overestimation of both the population prevalence and the correlation between APMV-1 and IAV infection. To be confident in the prevalence and test interpretation, 1000 random samples including only one observation per individual were generated. From these samples we computed the mean prevalence for each virus, the 95% CI of the means and the median of the p-values of the tests performed on each of these 1,000 samples.During October and November in 2010, 2332 samples from Mallards were collected and screened for APMV-1 by rRT-PCR , for which the overall virus prevalence was 4.2%, the odds of being infected were approximately three times higher in juveniles compared to adult birds. It is possible that the limited dataset in our study, especially the number of APMV-1 infected adult birds, affects the outcome of the statistical analysis.Comparisons of APMV-1 prevalence in Mallards of different age classes indicated that there is no significant difference in the probability of being infected. This observation does not agree with results obtained in a recent Australian study examining the relation between age and infection frequency . In the One advantage with the sampling system used at Ottenby, with daily sampling and subsequent release of sampled birds, is that it makes it possible to recapture individual birds and sample them several times during their stopover at the site. Among sampled birds in this study, four individuals that were positive on the first sampling occasion were later resampled within a two to four day period. Thus, in more extensive studies in the future, resampling systems may contribute to give a better understanding of the progression of infections in wild birds. Sequence analyses of the F gene sequence of isolates obtained from one of the ducks in this study seem to indicate that the same viral strain was shed during a four day period. To our knowledge this is the first observation of an APMV-1 infection in wild birds followed for several days.Virus isolates were generated from ten out of twenty APMV-1 positive samples. As indicated by the deduced amino acid sequence of the F0 cleavage site, none of the APMV-1 isolates seem to be of the velogenic type. Corresponding results from screenings of wild birds have been reported previously, both in North America, Japan and Europe ,37,38. HThe phylogenetic analysis of APMV-1 isolates showed that F gene sequences clustered with those of class II viruses. This is consistent with previous reports of a predominance of class II viruses in wild birds ,16,34,36Larus ridibundus), found in the Ia clade, sampled at the Ottenby Bird Observatory in 1994 [It is worth pointing out that the detection of predominantly genotype Ib viruses is different from observations in recent North American studies detecting predominantly genotype II viruses in wild birds ,16. Poss in 1994 .Superinfections and co-infections of the host with two or more pathogens may complicate studies of a certain pathogen’s effect on its host, but can also offer a valuable opportunity to explore particular properties unique to one of the pathogens during co-infection, and to evaluate the contribution of such factors to observed prevalence and virulence. Co-infections involving APMV-1 and IAV have been observed previously in North America and Europe ,34. In tAnother noticeable observation is the significant difference in APMV-1 and IAV prevalence in sampled ducks. APMV-1 and IAV have both negative-sense single-stranded RNA genomes with hemagglutinin and neuraminidase virion surface structures that attach to sialic acid moieties of host cell surface molecules. Also, for both viruses, waterfowl is a central reservoir for maintaining a constitutive circulation of virus. However, an important distinction between APMV-1 and IAV is their genome organization: The APMV-1 genome is consists of one continuous, linear RNA molecule, whereas IAV has a segmented genome consisting of eight linear RNA molecules. Possibly, the advantages with a segmented genome, including the capacity for genome reassortment, is a contributing factor to the higher IAV prevalence compared to that of APMV-1 in Mallards.In conclusion, approximately one percent of the Mallard population sampled during autumn migration at Öland, Sweden, was infected with class II APMV-1s. All virus isolates were of the lentogenic type and phylogenetically closely related to genotype I viruses from central and northern Europe, but clustered also with viruses isolated at live bird markets in the south eastern parts of China. Additional studies examining the circulation of APMV-1s in different wild avian species are essential in order to understand viral evolution and epidemiology.The authors declare that they have no competing interests.CT and AKH designed the experiments and carried out the rRT-PCR screening. CT carried out sequence analysis and phylogenetic analysis. CT and MW drafted the manuscript. AA contributed with statistical analysis of prevalence data. SZ was involved in experimental design, and in revision of the manuscript. JW was involved in planning the study and in revision of the manuscript. All authors have read and approved the final manuscript.


Camels were infected with this virus >10 years before the first human cases. Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein–specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most  camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most  serum samples had MERS-CoV–neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases. Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected patients  (Coronaviruses (CoV) are positive-sense RNA viruses. Viruses in the genera Insectivorous bats of the family Vespertilionidae were recently shown to carry viruses that are probably conspecific with MERS-CoV  serum samples were systematically sampled in Dubai, United Arab Emirates and the surrounding area in 2003  and in 2013 . The total number of camels in that area was 360,000 in 2010  and an input volume of 100 μL of serum or fecal material suspended 1:10 in phosphate-buffered saline buffer. The elution volume was 100 μL for serum and fecal suspensions. To identify CoV-specific nucleic acids, 2 generic CoV PCRs were performed as described  was compared against that of the human CoV-OC43 spike protein (OC43-S). As in our previous study , suggestA previously published microarray-based assay that used the receptor-binding S1 spike subunit of MERS-CoV (MERS-S1), human CoV-OC43 (OC43-S1), and SARS-CoV (SARS-S1) was also evaluated. In contrast to our previous studies . All rIFThe panel of camel serum samples was additionally tested in a commercially available IFA that used cells infected with MERS-CoV (vIFA) (EUROIMMUN AG). The use of whole virus provides additional structural and nonstructural protein antigens, including envelope, membrane, nucleocapsid, and diverse replicase proteins. However, because of conserved features of nonstructural proteins among even distantly related CoVs . DespiteTo confirm results from affinity assays with results from a functional test, we determined endpoint virus neutralization titers by using a microneutralization test against MERS-CoV and BCoV. In most animals MERS-CoV serum neutralization titers were higher than titers against BCoV (serum samples 4–11) . High IFOn the basis of the validation studies, we investigated 4 collections of serum samples from dromedary camels from the United Arab Emirates that were sampled in 2003 and 2013. For initial screening, we chose the rIFA because of its proven sensitivity and decreased chances of generating false-positive results. All 667 camel serum samples from the United Arab Emirates and Germany were initially screened at dilutions of 1:80. A total of 89.0%–100.0% of serum samples in 4 collections showed positive results . SeropreA confirmatory microneutralization test was conducted at dilutions of 1:640 and 1:1,280 for all IFA-reactive serum samples. These high dilutions were chosen on the basis of our observation of high levels of neutralizing serum activity in camels . In 18.4To rule out cross-reactivity and to study additional exposure of MERS-CoV–positive camels with BCoV . Of seruCoronavirinae–specific broad-range reverse transcription PCR (RT-PCR) and a highly sensitive RT-PCR specific for genus Betacoronavirus phylogenetic lineage C. Both assays were specific for the viral RNA-dependent RNA polymerase gene. Two positive fecal samples were identified by both assays. Sequencing of amplified cDNA fragments of 182 nt and 404 nt identified sequences 99% identical with BCoV strain Mebus (GenBank accession nos. KF894801 and U00735.2). To further confirm virus identity, we amplified a region within the spike protein gene (positions 24303–24702 in BCoV strain Mebus) by using RT-PCR and sequencing it. Amplicons from both animals were 97% identical at nucleotide level with BCoV strain Mebus, indicating the presence of BCoV in camels as reported . However, because MERS-CoV has a highly conserved receptor structure, we did not assign high priority to the hypothesis that the closely related camel species C. bactrianus, should be less susceptible than C. dromedarius camels to MERS-CoV  does not show cross-neutralization  comprises 2 subserotypes that show limited cross-reactivity but are considered 1 virus species. Transmissible gastroenteritis virus of swine shows more efficient cross-neutralization with 1 of these FCoV subserotypes than the other and is classified as 1 species with FCoV even though it is carried by a different host (Neoromicia, has been confirmed to be conspecific with MERS-CoV. Lineage C betacoronaviruses in other bat taxa have also been proposed to be related to MERS-CoV (Camels probably acquired MERS-CoV at some unknown time. Potential sources include bats of the family Vespertilionidae, in which a virus with a close phylogenetic relationship with MERS-CoV has been detected (Neoromicia, virus conspecific with MERS-CoV differs from human MERS-CoV, even if formally a member of the same species. The observed degree of sequence divergence between this virus and MERS-CoV makes any direct and recent transmission from bats to humans seem unlikely. Nevertheless, it cannot be excluded from available data that the virus source population in bats has not been detected. For example, a recent investigation of Rhinolophus bats in China identified viruses with close relationships to the bona fide ancestor of SARS-CoV, and viruses described in many studies yielded only conspecific yet less related viruses (In vespertilionid bats, including those in the genus If camelids should function as intermediary hosts in a similar manner, we should expect a virus in camelids that has a closer phylogenetic relationship with any bat-borne CoV and thus should be easily detectable with available RT-PCRs. Larger studies to confirm the presence of MERS-CoV in camelids should receive high priority so as to define the animal reservoir of MERS-CoV and possibly control it by such measures as vaccination or control of animal movement. However, before implementation of any control measures, whether camelids are a continuous source of infection for humans needs to be firmly established.


To determine if “early rupture of membranes” (early ROM) during induction of labor is associated with an increased risk of cesarean section in term nulliparas.The rate of cesarean section and the timing of ROM during the course of labor were examined in term singleton nulliparas whose labor was induced. Cases were divided into 2 groups according the timing of ROM: 1)“early ROM”, defined as ROM at a cervical dilatation<4 cm during labor; and 2) “late ROM”, ROM at a cervical dilatation≥4 cm during labor. Nonparametric techniques were used for statistical analysis.1) In a total of 500 cases of study population, “early ROM” occurred in 43% and the overall cesarean section rate was 15.8%; 2) patients with “early ROM” had a higher rate of cesarean section and cesarean section due to failure to progress than did those with “late ROM”  and this difference remained significant after adjusting for confounding variables.“Early ROM” during the course of induced labor is a risk factor for cesarean section in term singleton nulliparas. Induction of labor is one of the most common practices in obstetrics. More than 20% of pregnant women are delivered after the induction of labor, and the overall rate of induction of labor in the United States has become more than doubled from 1990 to 2006 For this reason, previous investigators have tried to identify risk factors for cesarean deliveries during the induction of labor. Nulliparity, advanced gestational age, increased birthweight, and use of cervical ripening agent have been reported as risk factors for cesarean delivery However, there is little information on the timing of ROM during induced labor at term as a risk factor for cesarean delivery. We have recently demonstrated that spontaneous early rupture of membranes (early ROM) is an independent risk factor for cesarean delivery in nulliparous women who delivered after the spontaneous onset of labor It is an important issue if there is a relationship between the occurrence of early ROM and the risk of cesarean delivery in induced labor, because prediction of the risk for cesarean delivery is not straightforward in clinical management of induced labor, which itself increases the risk of cesarean deliveryTo address this issue, we undertook this study to determine if spontaneous early ROM during induction of labor is associated with an increased risk for cesarean section in term nulliparas.In this retrospective cohort study, the rate of cesarean section and the timing of ROM during the course of labor were determined in term singleton nulliparas who were admitted for induction of labor between March 2002 and December 2007. Cases were classified into either an “early ROM” group or “late ROM” group according to the timing of ROM. Cases were extracted from the database of Seoul National University Hospital. Patients in whom the onset of labor or ROM occurred spontaneously before the induction of labor or those with an intrauterine fetal demise were excluded. The collection and the use of clinical data and the results of placental histologic examination for research purpose were approved by the institutional review board of Seoul National University Hospital.“Early ROM” and “late ROM” was defined as previously described Labor induction was performed with prostaglandins (dinoprostone or misoprotsol), intravenous oxytocin, or a combination of these agents. Oxytocin was administered intravenously as a dilute solution by infusion pump, and high-dose regimen is usually used; initial dose was started at 5.3 mU/min and it was increased every 15–30 minutes up to a maximum dose of 40 mU/min, with modification individualized to patient at the discretion of attending physician. Although the decision to perform an amniotomy was at the discretion of the attending physician, elective early amniotomy is not a routine practice in our institution. The diagnosis of failure to progress or fetal distress and the decision to perform cesarean section during the course of labor was made in accordance with the ACOG recommendations Acute histologic chorioamnionitis was defined as the presence of acute inflammatory changes on examination of a membrane roll and chorionic plate of the placenta; funisitis was diagnosed as the presence of neutrophil infiltration into the umbilical vessel walls or Wharton’s jelly, according to the criteria previously described in detail Proportions were compared with Fisher’s exact test and comparisons of continuous variables between groups were performed with the Mann-Whitney U test. To assess which variables were significantly associated with cesarean section, multiple logistic regression analysis with backward elimination was conducted. The confounding variables in multiple logistic regression analysis were selected according to the analysis of univariate analysis as risk factors for cesarean section (p<0.2) During the study period, there were a total of 5,481deliveries of singleton gestations. Among these, 4,603 cases were delivered at term, including 2,480 cases of nulliparas. Among these term singleton nulliparas, 565 cases underwent induction of labor before spontaneous onset of labor or ROM, without an intrauterine fetal demise. The timing of ROM was not available in the medical records of 10 cases. The remaining 555 cases were classified into either an “early ROM” group or “late ROM” group according to the timing of ROM, and additional 55 cases were excluded from analysis ). Finally, five hundred cases were eligible for study and “Early ROM” occurred in 43% (215/500) and “late ROM” occurred in 57% (285/500) of the patients. Among cases with cesarean section, 76% (60/79) of cesarean sections were performed due to failure to progress. The rate of cesarean sections due to failure to progress was also significantly higher in patients with “early ROM” than those with “late ROM”  and this difference also remained significant after adjusting for maternal age, use of intravenous oxytocin or regional analgesia, gestational age at delivery, birthweight, macrosomia, and presence of acute histologic chorioamnionitis and funisitis . However, the rate of cesarean section due to fetal distress and the rate of operative vaginal deliveries were not different between the two groups  and artificial ROM was performed in 103 cases (36%). The cesarean section rate was not different between cases with spontaneous ROM and those with artificial ROM . Even when confining the analysis to these cases with spontaneous ROM , the rate of overall cesarean section and the rate of cesarean section due to failure to progress was higher in cases with early ROM than those with late ROM . This difference remained significant after adjusting for confounding variables .The principal findings of this study were: 1) “early ROM” occurred in 43% in term singleton nulliparas after induction of labor; 2) patients with “early ROM” had a higher rate of overall cesarean section and cesarean section due to failure of progress than did those with “late ROM” (p<0.01 for each) and this difference remained significant after adjustment for confounding variables.Our study demonstrated that early ROM can be considered as a risk factor for cesarean delivery in induced labor, in addition to other risk factors including unfavorable cervix, advanced maternal age, epidural analgesia, intravenous oxytocin, macrosomia, and PROM2 test for trend). Thus, cases with early ROM had the highest cesarean section rate among the three groups .Among eligible populations, cases with pre-labor ROM during the course of induction were excluded (39 of 595 cases). The overall cesarean section rate in cases with pre-labor ROM was 17.9% (7/39), which was less than that in cases with early ROM and higher than that in cases with late ROM (p<0.001, χSeveral explanations for this relationship between early ROM and the risk of cesarean delivery in induced labor may be offered. First, early ROM itself may be reflective of dystocia with cephalopelvic disproportion, as suggested in our previous report Second, the absence of hydrostatic pressure of membranes after ROM may result in slow progress of labor and a corresponding increased risk of cesarean delivery. During labor with intact membranes, uterine contractile forces exert pressure on the fetal membranes, resulting in centrifugal force on the cervix and cervical dilatation Third, intra-amniotic infection or inflammation may result from a longer duration of ROM in cases with early ROM and are responsible for an increased risk of cesarean delivery because of inadequate uterine contractions due to uterine inflammation. It has been suggested that high virulence bacterial infections or chorioamnionitis are associated with dystociaIt was reported that several factors were associated with the increased risk of cesarean section during the induction of labor. First, previous studies have demonstrated that lower bishop score was associated with increased risk of cesarean delivery In the spontaneous onset of labor of nulliparas, early ROM has been demonstrated as a risk factor for cesarean delivery In conclusion, early ROM during labor induction is associated with an increased risk of cesarean delivery. Further studies on the possible mechanism of this association and on the development of new scoring system using early ROM for prediction of cesarean section in induced labor will be needed to enhance our understanding on the nature of early ROM in induced labor.


Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia.L) genes.RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus.This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia. Henipavirus in the subfamily Paramyxovirinae, family Paramyxoviridae, contains two highly pathogenic viruses, i.e., Hendra virus and Nipah virus. Hendra virus causes fatal pneumonia and encephalitis in horses and humans. The first case was identified in 1994 and Hendra virus disease still continues to arise sporadically in Australia[Pteropus vampyrus[Pteropus hypomelanus[Pteropus lylei[Pteropus poliocephalus, and Pteropus alecto[Pteropus vampyrus that are seropositive for both Nipah virus and Hendra virus are distributed nationwide[The genus ustralia. Nipah vustralia. Henipav vampyrus, Pteropupus lylei, Pteropuus alecto, which aus alecto-11. No htionwide,13. ThesRubulavirus of the Paramyxoviridae family, has been identified in pteropus bats from Australia[Rubulavirus, has also been isolated from Pteropus hypomelanus on the island of Tioman, Malaysia[Menangle virus, belonging to the genus ustralia. Menanglustralia. Tioman Malaysia. AlthougThe current study used molecular sequencing and phylogenetic analyses to identify RNA sequence from potential paramyxoviruses in fruit bats from Indonesia.Pteropus vampyrus was captured in Panjalu District (n = 26) and Lima Puluh Kota District (n = 20). Other pteropus bats captured in Popayato District (n = 4) and Paguyaman District (n = 25) were considered to be closely related to Pteropus hypomelanus, based on the shared nucleotide sequence identity of their 16S rRNA (96%) and cytochrome b (cyt b) (95%) with corresponding sequences from Pteropus hypomelanus (GenBank/EMBL/DDBJ entry AF069537 and AB062472). Acerodon celebensis was captured in Paguyaman District (n = 18). Dobsonia bats that were captured in Paguyaman District (n = 17) had high sequence similarity with 16S rRNA (96%) and cyt b (94%) from Dobsonia moluccensis (JN398196 and FJ218484). Information on the samples is summarized in Table A total of 110 fruit bats belonging to four different species were sampled from four locations in Indonesia  gene of the Paramyxovirinae subfamily, which includes Avulavirus, Rubulavirus, Respirovirus, Morbillivirus, and Henipavirus[Pteropus vampyrus specimens captured in Panjalu District. The size of PCR product detected in the positive sample (sample number IFBPV01/2010) was 584 bp, and the amplified viral sequence excluding the primer-derived sequences (530 bp) was deposited in GenBank (accession number AB691542). Positive results with amplification of the 530 bp viral sequence (excluding the primer-derived sequences) were also obtained for 4/25 (16%) Pteropus sp. captured in Paguyaman District, i.e., IFBPV25/2011 (AB691543), IFBPV32/2011 (AB691544), IFBPV39/2011 (AB691545), and IFBPV46/2011 (AB691546), and for 1/18 (6%) Acerodon celebensis specimens captured in Paguyaman District, i.e., IFBPV32/2012 (AB710472). No positive results were obtained for the 20 Pteropus vampyrus captured in Lima Puluh Kota District, the four Pteropus sp. captured in Popayato District, or the 17 Dobsonia sp. captured in Paguyaman District (Table RNA samples from each fruit bat spleen were screened using semi-nested broad spectrum reverse transcription PCR (RT-PCR), as described previously. The prinipavirus. Semi-neRousettus leschenaulti in China[BLAST search showed that all six amplicons shared less than 65% nucleotide identity with homologous fragments of paramyxovirus sequences previously deposited in GenBank. Deduced pairwise amino acid identities were then calculated to compare the homologous region with known paramyxovirus L proteins Table . IFBPV32in China. IFBPV32Henipavirus. IFBPV25/2011 and IFBPV32/2012 were most closely related to the genus Rubulavirus.A phylogenetic analysis was performed based on the deduced amino acid sequences (176 amino acids) from the six nucleotide sequences obtained Figure . The phyL gene by using different degenerate primer sets for MorbillivirusRespirovirusHenipavirus subgroup or RubulavirusAvulavirus subgroup[Henipavirus  and rabbit kidney RK13) cells because these cell lines are used for the isolation or propagation of various paramyxoviruses cells be,6,16,22.Four henipavirus-like and two rubulavirus-like nucleic acid sequences were detected in fruit bats from Indonesia. The phylogenetic analysis showed that these novel viral sequences possessed considerable sequence divergence, suggesting that a variety of paramyxoviruses are circulating in the Indonesian fruit bat population.Pteropus, partial paramyxovirus sequences were identified from fruit bats of the genus Eidolon, Rousettus and Epomophorus[Acerodon. This finding broadens the number of megabat genera which are associated with paramyxoviruses.In addition to fruit bats of the genus omophorus,23,24. TTuhoko virus, Tioman virus, Menangle virus, and Mapuera virus have been identified as fruit bat-associated rubulaviruses. Menangle virus causes central nervous system degeneration in pigs and it also infects humans,15. TherPteropus vampyrus, Pteropus hypomelanus and Acerodon celebensis), potentially representing three new henipaviruses and two new rubulaviruses. To the best of our knowledge, this is the first study to identify viral genome sequence from potential paramyxoviruses in the tissues of fruit bats from Indonesia. Local people consume bat meat in Indonesia, so further epidemiological and experimental studies are needed to determine the risk of fruit bat-associated paramyxovirus infection of humans in Indonesia.This study identified unique paramyxovirus sequence from three species of fruit bats ; Lima Puluh Kota District, West Sumatra Province during February, 2011 (n = 20); Popayato District, Gorontalo Province during February, 2011 (n = 4); and Paguyaman District, Gorontalo Province during February, 2011 and February, 2012 . All animal research was performed in accordance with the ethical guidelines of the Animal Care and Use Committee of Veterinary Teaching Hospital, Bogor Agricultural University. All of the spleen tissues from fruit bats were divided into two samples. Samples for RNA extraction were stored in RNAlater , followed by RNA extraction with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Samples for virus isolation were frozen at −80°C. These samples were exported with the permission of the Directorate General of Livestock and Animal Health Services, Ministry of Agriculture, Republic of Indonesia.′-AGAGGAGAYAAGTCGTAMCAAG-3′)[′-GTTTGCCGAGTTCCTTTTAC-3′)[b using the primer set Ptecytb-26 (5′-TTGTATTTCAACTACARGAAC-3′) designed in this study and H15149p (5′-CTGCAGCCCCTCAGAATGATATTTGTCCTC-3′)[Fruit bats were identified based on morphological characters and nucleotide sequence analysis of their mitochondrial 16S rRNA using the primer set 12l-f (5CAAG-3′) and 16q-TTAC-3′), and mitL gene of the Paramyxovirinae subfamily were as follows: for one-step RT-PCR, PAR-F1 and PAR-R; for semi-nested PCR, PAR-F2 and PAR-R[L gene of the Respirovirus-MorbillivirusHenipavirus subgroup or AvulavirusRubulavirus subgroup were amplified by using the following degenerate primer sets: RES-MOR-HEN F1, RES-MOR-HEN F2 and RES-MOR-HEN R, or AVU-RUB F1, AVU-RUB F2 and AVU-RUB R, respectively[RNA samples were screened for paramyxoviruses using semi-nested RT-PCR, as described previously. The prind PAR-R. The upsectively. PCR proThe nucleotide sequence encoding the GDNQ/GDNE motif from each sample was amplified using a SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Life Technologies). The inner primer PAR-F2 used in the semi-nested PCR was designed for a nucleotide sequence encoding this GDNQ/GDNE motif, so one-step RT-PCR was performed with the outer forward primer PAR-F1 and reverse primers specifically for each sample.The obtained nucleotide sequences and the deduced amino acid sequences were compared with those of known paramyxoviruses. Nucleotide and amino acid identity values were calculated using GENETYX software ver. 10 . Multiple sequence alignments were constructed based on the amino acid sequences deduced from the six nucleotide sequences obtained using the MEGA5 program. Phylogeg for 5 min and inoculated onto Vero cells and RK13 cells for 2 h at 37°C. Cells were washed with phosphate-buffered saline (−) and cultured with MEM containing 2% fatal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) and fungizone (2.5 μg/ml). All cells were subcultured every 5 or 6 days. After three serial passages, RNA samples were prepared from each culture supernatant using a High Pure Viral RNA Kit  and analyzed by semi-nested RT-PCR, as described above.Virus isolation was attempted in the biosafety level (BSL)-3 facility at the Research Center for Zoonosis Control, Hokkaido University. Frozen spleen tissues were homogenized  in MEM containing penicillin (100 units/ml), streptomycin (100 μg/ml), and fungizone (2.5 μg/ml) . The homogenates were clarified by centrifugation at 1000 × The amplified nucleotide sequences reported in this study have been deposited in the GenBank nucleotide database under accession numbers AB691542 to AB691546, AB748559 to AB748561 and AB710472. AB691543 and AB710472 are composed of three overlapping sequences that were amplified using three primer sets; PAR-F2 and PAR-R, AVU-RUB F2 and AVU-RUB R, and the primers for the amplification of GDNQ/GDNE motif.The GenBank/EMBL/DDBJ accession numbers of the amino acid sequences used in this study were ACB46872 (Avian paramyxovirus 3), NP_071471 (Newcastle disease virus), NP_054714 (Mumps virus), YP_001249278 (Mapuera virus), YP_415514 (Menangle virus), NP_665871 (Tioman virus), ADI80722 (Tuhoko virus 2), NP_056879 (Sendai virus), NP_604442 (Human parainfluenza virus 1), NP_067153 (Human parainfluenza virus 3), AAA75501 (Measles virus), YP_087126 (Rinderpest virus), NP_047207 (Canine distemper virus), NP_047113 (Hendra virus), NP_112028 (Nipah virus), and AAX86035 (J-virus), NP_056797 (Rabies virus), NP_478343 , NP_066251 (Ebola virus), and ACT79225 (Marburg virus).The authors declare that they have no competing interests.M. Sasaki, A. Setiyono, E. Handharyani, and T. Kimura designed research; M. Sasaki, A. Setiyono, E. Handharyani, S. Adiani, I. Rahmadani, S. Taha, M. Subangkit, I. Nakamura, and T. Kimura performed research; M. Sasaki, A. Setiyono, E. Handharyani, H. Sawa and T. Kimura analyzed the data; M. Sasaki, H. Sawa and T. Kimura wrote the manuscript. All authors read and approved the final manuscript.Phylogenetic analysis of amino acid sequences derived from partial L gene fragments.Click here for filePhylogenetic analysis of amino acid sequences derived from partial L gene fragments.Click here for file


Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20–30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes. In human genetic research, targeted resequencing of genomic nucleic acid is applied extensively in population studies to search for associations between sequence variants and diseases. The technique is also applied in diagnostic or prognostic tests. In this context, one is often confronted with samples of mixed sequence variants, some possibly present in minority: e.g. biopsies from cancer tissue usually contain a mixture of cancerous and non-cancerous cells. Therefore, one needs to distinguish the presence of a specific mutation, possibly in low abundance (minority), in a majority of ‘wild-type’ sequences. Several techniques are in use to resolve the sequence composition in mixed sequence samples, like allele-specific PCR , meltingIn this article, we focus on the case of HIV-1/AIDS. To reduce the morbidity and mortality world-wide, there is a high need for simple point-of-care genotyping tests to screen for key resistance mutations. However, the development of a simple genotyping test to screen for key resistance mutations is a technical challenge, due to high genetic variability of HIV-1 . The varIn this article, a new method based on microarrays hybridization will be introduced to perform targeted resequencing on nucleic acid samples. The idea to use hybridization for mutation detection is not new ,8, and iHIV-1 virus stocks were selected from the Janssen Diagnostics repository database, based on their known mutation profile in the region of codon 179 to codon 186 of the Reverse Transcriptase (RT) gene. This region was selected to cover key resistance mutations at position 179, 181 and 184. A mutation at position 179 or 181 causes resistance against non-nucleoside RT inhibitors (NNRTIs) , while nThe viral RNA extraction of virus stocks was carried out on an EasyMAG  according to the guidelines of the manufacturer, starting with 256 µl input material for plasma samples and 100 µl input material for virus stocks, both were eluted in 60 µl. A One-Step RT-PCR amplification  was used to generate a 2.3-kb HIV-1 fragment (containing the gag-protease-reverse transcriptase (GPRT) region) using the 3-RT (5′-CATTGCTCTCCAATTACTGTGATATTTCTCATG-3′) and 5-OUT (5′-GCCCCTAGGAAAAAGGGCTGTTGG-3′) primers. RNA input was 10 µl in a final volume of 35 µl. The complete amplification procedure was published by . The 2.3The raw microarray data were subjected to a primary quality control using the Agilent Feature Extraction Software (Version 10.7). For all arrays, the spot centroids in the four corners of the microarray have been located properly and consequently the grid was placed correctly. The QC values for homogeneity and those for checking the hybridization and washing steps, all fall within the good range according to Agilent guidelines. In the present application, we work with single color, hence there is no need of color normalization. In addition, the analysis in extended previous hybridization experiments ,15 showeTo define a manageable and relevant diagnostic problem, each unique sample from the database was selected and ranked in decreasing order of clinical frequency. The goal was to diagnose the top 100 unique sequences and mixtures of two sequences thereof. This corresponds to the coverage of 82% of the whole database. The sequence no.143 shown in The 15k custom Agilent array used in the experiment contains eriments  showed tI from different spots will be correlated according to equilibrium thermodynamics asA is a proportionality factor, c the target concentration in solution, R the gas constant, and T the temperature. For convenience in the rest of the article, hybridization free energies are shifted by the corresponding perfect match value. This amounts to use In the standard approach of hybridization-based targeted resequencing, the microarray contains probes that are perfectly matching to all possible variants of target sequences expected to be present in the biological sample. The brightest of all spots should indicate the sequence which is present in solution. Our approach is based on the analysis of the intensities of the brightest and also of many ‘dimmer’ spots which are expected to carry one or two mismatches with respect to the target . If a unique target sequence is present in solution, the fluorescence intensity Given the sequences of the target in solution and the surface-bound probes, the ur model . In thisribed in ,24.I versus I versus In a resequencing analysis, the target sequences are coming from a biological sample, and they are usually not known. One can, however, make a starting hypothesis about the sequence and compute the corresponding one case a, the hyone case b, the wrn is the iteration step), which are successive in silico hypotheses about the composition of the sample. The loop is repeated until convergence is reached. Two types of convergence are obtained. In some cases, after a certain number of iterations, the algorithm shows a collapsed nt as the sequence composition of the sample. In other cases, the algorithm converges to a two-cycle state i.e. nt and in silico hypotheses is the decision block (a) of the flowchart. At this point, the algorithm checks if the in silico hypothesis An important part for generating new in silico hypothesis: in silico hypothesis this is a G, while the actual target contains a T). Consider now all probe sequences in the microarray with an A at this position. The actual hybridization is a Watson-Crick AT pairing, while the in silico hypothesis estimates the in silico hypothesis differ. It is important to notice here that the probes in the different branches systematically differ from each other by specific nucleotides at specific locations. This systematic sequence deviation will be used to decide whether the in silico hypothesis denoted by The plot of nt and In principle, for a unique sequence sample, one can start with any hypothesis and iterate until a collapsed t1 corresponds to the sequence with highest clinical frequency  is selected. These are points that deviate the most from the expected thermodynamic behavior (dashed line). The nucleotide composition of this set is analysed: the right pane t1. In the algorithm, a threshold value of 70% is chosen as the minimum limit for the fraction of common mismatches per base positions between the selected probes and the current hypothesis (more details on the threshold selection and its influence on the output are presented in the Supplementary Section II). Based on this threshold, the selected probes are considered mismatching against the hypothesis t1 at base positions no. 6, 12 and 16, by nucleotides T, C and C, respectively. This information is used to generate a new hypothesis t2 by swapping nucleotides on hypothesis t1 so it becomes complementary to the three nucleotides detected in the mismatching base positions  from samples in which two or more different sequences coexist (mixed). For clinical purposes, it is important to identify early enough the rising of a resistant strain. Our microarray analysis is based on a large number of measured intensities not just from perfectly matching probes but also mismatching probes. Although in general the effect of the hybridization of a very low abundance sequence in a mixed sample can be small for each individual intensities, the correlated effect on a large number of different probes can be detected even from a target sequence at relatively low abundance. The analysis of , in whicin silico hypotheses for the sample composition and checked them against thermodynamic models. The algorithm identified correctly the sequences in the sample and was tested on seven clinical samples from the Janssen Diagnostics database. We showed how hybridization thermodynamics can resolve some intrinsic ambiguities of the Sanger sequencing. The results show the reliability of DNA microarrays and in principle any hybridization-based technology.Here, we presented an iterative algorithm that successively generates A method based on hybridization has the advantage that it is a simple test that can be miniaturized to a fully automated lab-on-chip, which can be used as point-of-care test. Indeed hybridization-based method is promising, and it has been a focus of interest in the recent years on either its fundamentals or applications . AlthougSupplementary Data are available at NAR Online, including [35].KULeuven [STRT1/09/042]; VITO [ZL39010200-401]. Funding for open access charge: VITO and Janssen Diagnostics.Conflict of interest statement. None declared.


The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). To counteract this innate defense, pathogens deploy effector proteins with a primary function to suppress PTI. In specific cases, plants have evolved intracellular resistance (R) proteins detecting isolate-specific pathogen effectors, leading to effector-triggered immunity (ETI), an amplified version of PTI, often associated with hypersensitive response (HR) and programmed cell death (PCD). In the case of plant viruses, no conserved PAMP was identified so far and the primary plant defense is thought to be based mainly on RNA silencing, an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and represses invasive nucleic acids such as transposons. Endogenous silencing pathways generate 21-24 nt small (s)RNAs, miRNAs and short interfering (si)RNAs, that repress genes post-transcriptionally and/or transcriptionally. Four distinct Dicer-like (DCL) proteins, which normally produce endogenous miRNAs and siRNAs, all contribute to the biogenesis of viral siRNAs in infected plants. Growing evidence indicates that RNA silencing also contributes to plant defense against non-viral pathogens. Conversely, PTI-based innate responses may contribute to antiviral defense. Intracellular R proteins of the same NB-LRR family are able to recognize both non-viral effectors and avirulence (Avr) proteins of RNA viruses, and, as a result, trigger HR and PCD in virus-resistant hosts. In some cases, viral Avr proteins also function as silencing suppressors. We hypothesize that RNA silencing and innate immunity (PTI and ETI) function in concert to fight plant viruses. Viruses counteract this dual defense by effectors that suppress both PTI-/ETI-based innate responses and RNA silencing to establish successful infection.  Innate immunity is an evolutionary ancient mechanism that protects plants and animals from a wide range of pathogens. Invading pathogens are recognized by diverse pattern recognition receptors (PRRs) ,2,3. In Arabidopsis thaliana, the Pep family members (AtPeps) are recognized by the RLKs PEPRs (Pep receptors) 1 and 2 which are structurally similar to FLS2 and EFR and also require BAK1 for transmitting Pep signals. Peps and other endogenous elicitors are collectively called danger-associated molecular patterns (DAMPs) [In addition to PAMPs, the PTI system can recognize plant endogenous peptide (Pep) elicitors that are thought to amplify the defense responses against invading microorganisms . In ArabTo counteract PTI and establish robust infection in susceptible hosts, the pathogens deploy effector proteins (virulence factors) into the host cell. Some of the effector proteins block MAPK cascades by targeting RLKs and BAK1 . In specAmong other factors, resistance to different pathogen types involves a regulation of the balance between salicylic acid (SA)- and ethylene/jasmonic acid (ET/JA)-dependent defense mechanisms . SA is ica. 160 in A. thaliana) could recognize thousands of different pathogens, a guard hypothesis (GH) was proposed [Most plant R proteins belong to the nucleotide binding site-leucine rich repeat (NB-LRR) family. Plant NB-LRRs are classified into two main classes: CC-NB-LRRs with the N-terminal coiled-coil (CC) domain and TIR-NB-LRRs with the N-terminal Toll-interleukin-1 receptor (TIR) domain, which have specialized functions in immune responses . To explproposed  stating proposed . The GH proposed .A. thaliana proteins and the bacterial and oomycete effectors reveal that independently evolved effectors converge onto hubs in the immune system network [In further support of GH, experimentally established interaction networks of the  network . By extrOne of the immune system hubs is the EDS1 (enhanced disease susceptibility 1) protein controlling both PTI-based responses that inhibit growth of virulent pathogens and ETI-based HR/PCD against avirulent pathogens. EDS1 forms two types of complexes, one on the plasma membrane (likely involved in PTI) and another with two TIR-NB-LRRs that ‘guard’ EDS1, and all these complexes are targeted by bacterial effectors ,18. InteEDS1 and SA act redundantly in immune responses , and CC-Plant R proteins functioning depends on the chaperon complex that includes SGT1 , HSP90 (Heat-shock protein 90) and RAR1 (Required for Mla12 resistance). This complex might facilitate a conformational change of R proteins inducing the immune signaling after recognition of pathogen effectors or modified host proteins targeted by pathogen effectors. Mutations of the genes encoding the chaperone components strongly affect stability of R proteins . InteresThe outcome of any plant-pathogen interaction depends on a relative contribution of susceptibility and resistance factors. In the zig-zag model, originally proposed by Jones and Dangl , PAMP a.MIR genes; they silence target genes through mRNA cleavage and/or translational repression. siRNAs of distinct size classes are processed by DCL4 (21-nt), DCL2 (22-nt) or DCL3 (24-nt) from dsRNA precursors produced by RNA-dependent RNA polymerases (RDRs) or from overlapping sense and antisense Pol II transcripts. RDR6-dependent, 21-nt trans-acting siRNAs (tasiRNAs) and secondary siRNAs silence genes post-transcriptionally (like miRNAs). RDR2-dependent, 24-nt heterochromatic siRNAs (hcsiRNAs) silence repetitive DNA transcriptionally through RNA-dependent DNA methylation (RdDM). RdDM involves plant-specific polymerases, Pol IV and Pol V, required for 24-nt siRNA production, amplification and action [Arabidopsis genome encodes 10 AGOs which are specialized in RdDM (AGO4/6/9), miRNA/tasiRNA pathways (AGO1/2/7) and other functions [RNA silencing is an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and defends against invasive nucleic acids such as transposons, transgenes and viruses ,24,25. Sd action . The Araunctions .Pseudomonas syringae  [P. syringae growth, which implicates auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling in resistance [Arabidopsis lines including dcl1 restore growth of effector-deficient P. syringae, thus suggesting that DCL1 and miRNAs play a key role in PTI [P. syringae effectors suppress biogenesis, stability, or activity of PAMP-responsive miRNAs [P. syringae [P. syringae [Growing evidence indicates that innate immunity and RNA silencing are closely linked. Various miRNAs and siRNAs have been implicated in innate immunity . E.g., myringae  . The bacsistance . Furthere in PTI . Notablye miRNAs . Most misyringae . Likewissyringae . Since AP. syringae on Arabidopsis rdr6 mutant plants [RDR1 known to be involved in production of secondary viral siRNAs  is induct plants .N. benthamiana [Arabidopsis ago4 mutant plants exhibit reduced resistance to avirulent P. syringae and increase susceptibility to virulent P. syringae, whereas rdr2 and dcl3 mutations that abolish RdDM had no effect on bacterial growth [Arabidopsis Pol V (but not Pol IV) contributes to JA-mediated resistance to fungi but counteracts SA-mediated resistance to virulent P. syringae [Emerging evidence implicates components of the nuclear silencing machinery in innate immunity. AGO4, in addition to its main function in RdDM, appears to have a distinct function in ETI in thamiana . Consistl growth . Furthersyringae .P. syringae [Recent studies reveal that a large proportion of NB-LRR genes are silenced by miRNAs of the miR482 superfamily ,38,39. mIn animals, the innate immune system is essential for initial detection of viruses and subsequent activation of adaptive immunity. Viral components, such as dsRNA, single-stranded RNA and DNA are sensed by three classes of receptors: retinoic acid-inducible gene I-like, Toll-like and nucleotide oligomerization domain-like receptors , with thAs noted above, no viral PAMP was identified so far and PTI-based antiviral responses can potentially be elicited by plant DAMPs. Additionally, ‘modified self’ products of the viral effector activity can be sensed by unconventional CC-NB-LRRs such as ADRs. Viral and non-viral pathogens induce similar immune reactions . Thus, lR genes are tobacco N (TIR-NB-LRR), potato Rx1 (CC-NB-LRR) and Arabidopsis HRT (CC-NB-LRR) which confer resistance to Tobacco mosaic virus (TMV), Potato virus X (PVX) and Turnip crinkle virus (TCV), respectively. The tobacco N mediates recognition of a helicase domain p50 of the TMV replicase p126/p180: interaction of the TMV effector with a chloroplastic protein leads to N protein activation and re-localization to the nucleus, which triggers PCD [For RNA viruses, the most studied gers PCD . TMV p12gers PCD . In the gers PCD ,46. Retegers PCD . Howevergers PCD . TCV CP gers PCD . TCV CP Y-1 gene (TIR-NB-LRR) confers resistance to the potyvirus Potato virus Y (PVY) [Arabidopsis plants infected with the potyvirus Turnip mosaic virus (TuMV) could sustain growth of non-virulent P. syringae, which was interpreted as a negative effect of the TuMV-encoded silencing suppressor HCPro on the miRNA pathway [N-mediated resistance to TMV, but, at elevated temperatures (inactivating N), enhanced susceptibility to TMV and other RNA viruses [The potato  Y (PVY) , but an  pathway . Intrigu pathway . HCPro-t viruses . These f viruses . Further viruses . Taken tGeminiviridae), the CC-NB-LRR gene CYR1 was recently implicated in resistance to Mungbean yellow mosaic India virus  [R gene was identified for pararetroviruses (family Caulimoviridae).Little is known about DNA viruses and plant innate immunity. In the case of geminiviruses (family a virus  . To our Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of Caulimoviridae, Caulimovirus and Tungrovirus, which differ in host range, insect vector and virion architecture [A. thaliana ecotype Tsu-0 is resistant to CaMV strain CM1841 but not W260 and that replacement of the N-terminal (Avr/Vir) domain of CM1841 TAV with the corresponding domain from W260 breaks the resistance [Nicotiana edwardsonii, with the TAV N-terminal portion being an Avr determinant [Nicotiana sp., it produces very mild infection on A. thaliana Col-0, unlike the strains W260 or CM1841 [itecture . Both viitecture ,58. The itecture ,60,61 anitecture ,63,64. Tsistance . Thus, ir CM1841 . This isA. thaliana lines NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility to CaMV strain JI [cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, or mutants deficient in ET/JA signaling displayed increased resistance to CaMV [Previous studies have implicated SA and ET/JA in regulation of defense against CaMV.However, they produced rather conflicting observations. SA-deficient train JI ,68, suggTAV has several interacting host partners that participate in TAV-mediated translation regulation, including TOR (target-of-rapamycin) kinase . NotablyRice tungro spherical virus (RTSV) [Rice tungro disease is caused by synergistic interaction between RTBV and the RNA pricorna-like virus s (RTSV) ,72. The s (RTSV) , but RTBRNA silencing is viewed as a primary antiviral defense system of plants . Most plCaulimoviridae and Geminiviridae accumulate 21, 22 and 24 nt vsRNAs. We and others have shown that all four DCLs (DCL1-4) produce DNA virus-derived vsRNAs [Arabidopsis dcl1/2/3/4 quadruple mutants, which carry a weak allele of DCL1 (caf1 or sin1) and null alleles of the other DCLs, do not exhibit increased susceptibility to CaMV [dcl1/2/3/4 lines which exhibit even stronger developmental abnormalities than dcl1 have not yet been tested for susceptibility to non-viral pathogens.Plants infected with DNA viruses of d vsRNAs ,79,80,81d vsRNAs . ArabidoCabbage leaf curl virus [Since DNA viruses do not replicate through dsRNA intermediates, precursors of vsRNAs could potentially be formed by antisense transcription, RDR activity or from secondary structures of viral RNAs. We have established that three functional RDRs , Pol IV, or Pol V are not required for vsRNA production from CaMV  or the grl virus  and thatrl virus ; reviewerl virus ).To counteract silencing-based defense, most viruses express suppressor proteins which act at different steps of the silencing process. Viral suppressors have evolved independently as they are structurally diverse and involved in a number of other basic functions in replication, movement and encapsidation. Suppressors of RNA viruses exhibit the following antisilencing activities ,84: a) bin vitro [Arabidopsis thaliana plants disrupts geminivirus infection [Interestingly, none of the above antisilencing strategies was reported for DNA viruses. In geminiviruses, AC2, AC4, V2 and betaC1 exhibit suppressor activities. AC2, the most studied geminiviral suppressor, is a transcriptional activator with its nuclear localization required for suppression, likely through transcriptional activation of endogenous silencing suppressors . Additioin vitro , suggestin vitro . The gemin vitro  and JA rin vitro ,93, provnfection . It remadrb mutants do not exhibit increased susceptibility to virus infection (unpublished data). Thus, TAV interaction with DRB4 may be unrelated to suppression of antiviral silencing. As discussed above, the interactions of a viral effector (such as TAV) with critical host proteins could be monitored by the innate immune system and lead to immune responses. Intriguingly, nuclear localization of TAV is required for silencing suppression: mutation in the TAV nuclear localization signal  interfered with TAV ability to block accumulation of tasiRNAs [In pararetroviruses, so far only CaMV TAV has been reported as a silencing suppressor. Our work  and othetasiRNAs .CaMV infection and TAV expression do not alter AGO1 protein levels . FurtherTungrovirus and Badnavirus do not possess a TAV homolog [A. thaliana, the 600 bp leader region spawns massive quantities of 21-, 22-, and 24-nt vsRNAs, comparable to the entire complement of host siRNAs and miRNAs, while other regions of the 8 kb CaMV genome spawn little vsRNAs [Some pararetrovirus genera including  homolog , suggest homolog ,100,101.e vsRNAs . This fie vsRNAs  indicatee vsRNAs . It is aCa. 600 nt 8S RNA has the same 5´ terminus as 35S RNA and ends near the gap in the minus strand DNA, suggesting that it is produced owing to abrupt termination of Pol II transcription on a fraction of viral DNA having the unrepaired gap. We assume that 8S RNA folds into viroid-like secondary structure that can be converted by Pol II into dsRNA. Indeed, plant RNA viroids are replicated by Pol II through dsRNA intermediates [The dsRNA precursor of CaMV leader-derived vsRNAs is composed of 8S RNA, which was previously identified in CaMV-infected turnip to cover the entire leader, and its antisense copy. Since genetic evidence excludes involvement of RDR1, RDR2, RDR6, Pol IV or Pol V in production of 8S dsRNA and vsRNAs , 8S dsRNmediates . Our finmediates . TAV genmediates  and ensuMoissiard and Voinnet  reportedIt remains to be established if a CaMV-like decoy strategy is used by RTBV and other plant pararetroviruses and perhaps animal pararetro- and retroviruses with structured leaders . InteresIn this review we have described growing evidence that innate immunity (PTI and ETI) and RNA silencing function in concert to defend plants against viruses and other pathogens. To counteract this dual defense and establish infection in susceptible hosts, pathogens have evolved effectors with a primary function to suppress both PTI/ETI innate responses and RNA silencing. In resistant host plants, effectors  are recognized by plant NB-LRR proteins, which results in ETI/PCD-based resistance. DNA pararetroviruses have evolved two types of effectors, a viral protein-based and a viral RNA-based. The pararetrovirus CaMV-encoded effector/Avr protein TAV interferes with RDR6-dependent secondary siRNA production likely required for amplification of antiviral silencing and, according to indirect evidence, suppresses PTI/ETI-based innate responses. Likewise, the pararetrovirus RTBV ORF IV of unknown function may suppress silencing and/or innate immunity. In CaMV, the pgRNA leader region spawns massive amounts of vsRNAs and the precursor of these vsRNAs, 8S dsRNA, serves as a decoy engaging critical components of the silencing machinery. The mechanisms of biogenesis and action of CaMV 8S dsRNA and its presumptive counterpart in RTBV and other pararetroviruses remain to be further investigated.


New measles virus genotype will increase epidemiologic and virologic surveillance in Africa. We report the first genetic characterization of wildtype measles viruses from Uganda. Thirty-six virus isolates from outbreaks in 6 districts were analyzed from 2000 to 2002. Analyses of sequences of the nucleoprotein (N) and hemagglutinin (H) genes showed that the Ugandan isolates were all closely related, and phylogenetic analysis indicated that these viruses were members of a unique group within clade D. Sequences of the Ugandan viruses were not closely related to any of the World Health Organization reference sequences representing the 22 currently recognized genotypes. The minimum nucleotide divergence between the Ugandan viruses and the most closely related reference strain, genotype D2, was 3.1% for the N gene and 2.6% for the H gene. Therefore, Ugandan viruses should be considered a new, proposed genotype (d10). This new sequence information will expand the utility of molecular epidemiologic techniques for describing measles transmission patterns in eastern Africa. Measles virus is a negative-sense, single-stranded RNA virus in the genus Morbillivirus within the family Paramyxoviridae. Infection with this virus is typified by high fever, maculopapular rash, conjunctivitis, cough, and coryza . Althoug––This virus is monotypic, but genetic variation in the hemagglutinin (H) and nucleoprotein (N) genes can be analyzed by molecular epidemiologic techniques to study transmission patterns. This molecular information, in conjunction with standard case reporting and investigation, is useful in assessing the effectiveness of vaccination programs  aimed at reducing the illness and death caused by measles virus. The strategies include increasing routine immunization coverage, conducting vaccination campaigns for children 6 months to 15 years of age, providing vitamin A, and initiating a case-based measles surveillance system. In October 2003, Uganda conducted a catch-up campaign for children 6 months to 15 years of age that reached ≈13.5 million children with measles vaccine, with a national coverage of 105%. This campaign resulted in a decrease in reported measles cases to 3,522 from January to August 2004, compared with 28,072 in 2003 and 33,633 in 2002 in the same period, with no deaths among confirmed measles cases as of August 2004. Case-based measles surveillance was established in 2003, and by August 2004, >80% of districts were investigating measles cases according to the guidelines.Some information is available on circulating measles virus genotypes in western, central, and southern Africa, but little is known about circulating strains in eastern Africa, including Uganda. The WHO Regional Measles Laboratory at Uganda Virus Research Institute (UVRI) undertook the present study to isolate and characterize circulating measles strains in Uganda.Specimens for virus isolation were collected from outbreaks in different districts of Uganda, sentinel sites, and Mulago hospital, the national teaching and referral hospital, in Kampala from 2000 to 2002. Staff members from the UVRI were part of the outbreak investigation team and responsible for collecting specimens. Urine and nasopharyngeal aspirate specimens were obtained <7 days of onset of a rash according to WHO procedures for laboratory diagnosis of measles viral infection (2 tissue culture flasks and observed daily for characteristic cytopathic effect (CPE). Infected cells were harvested when >75% of the culture showed CPE, and viral stocks were prepared and stored at –70°C in 0.5-mL aliquots. Specimens contaminated with bacteria or fungi were filtered through a 0.45-μm filter, and tissue culture additions were repeated. Isolates were shipped to the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, for molecular analysis.The specimens were added onto B95a cells  and Mulago Hospital (n = 28) from 2000 to 2002 were selected for genetic analysis. Isolates were obtained from 6 districts within Uganda , althoug,Sequence data from this report are available from GenBank under accession nos. AY923185–AY923212 and AY923213–AY92321. All sequences were derived from viral isolates that were passaged once in B95a cells. The region of the N gene that is routinely sequenced for genotyping is not affected by tissue culture passage ,. Most ofPhylogenetic analyses based on both N and H gene sequences showed that the Ugandan viruses were members of clade D. However, the sequences of the Ugandan viruses were not closely related to the sequences of any WHO reference sequences that represented the 22 currently recognized genotypes. The Ugandan sequences were closest to the sequence of the genotype D2 reference strain. The minimum nucleotide divergence between the Ugandan viruses and the D2 reference strain was 3.1% for the N gene and 2.6% for the H gene .,,,Bootstrap analysis of Ugandan H-gene sequences and the WHO reference sequences showed 100% confidence in the group containing Ugandan viruses. When N-gene sequences were compared with WHO reference sequences and contemporary genotype D2 (This is the first report of the genetic characterization of wildtype measles from Uganda and the second that describes characterization of viruses from eastern Africa. Although this study was successful, it highlights some of the difficulties in performing virologic surveillance in developing countries. The relatively low rate of virus isolation in this study was surprising considering that all the specimens were collected within 7 days of rash onset. However, many specimens were not collected properly, and many arrived at the laboratory in poor condition because of inadequate transportation. Better training and adequate specimen-collection equipment and supplies could prevent some of these problems. In addition, many of the specimens were contaminated, and the filtration step for removing the contaminants reduced the viral titer. Specimens that do not require reverse cold chain, such as blood spots dried onto filter paper, will improve the efficiency of virologic surveillance in countries such as Uganda.,,,,Our results show that the percentage sequence divergence between the N and H gene sequence of the Ugandan virus isolates and the sequences of the reference strains exceeds the recommended threshold for designation of a new measles genotype, which is 2.5% and 2.0% minimum nucleotide divergence for the COOH region of the N and H genes, respectively (–The Ugandan viruses have a unique genetic signature that clearly distinguishes them from other African viruses. During the time when d10 viruses were isolated in Uganda, genotype D4 viruses were circulating in nearby Kenya (The purpose of virologic surveillance is to establish the transmission pathways of measles virus. Both Uganda and Kenya have initiated accelerated measles control activities and successfully completed a baseline survey of viral genotypes. Molecular epidemiologic techniques will now be very useful in monitoring the transmission pathways in eastern Africa, contribute to the development of effective measles control strategies, and document the success of the measles vaccination program.


Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.Marburg virus (family  Rousettus aegyptiacus) that inhabited a Ugandan gold mine where miners were previously infected. Soon after, two tourists became infected with Marburg virus after visiting nearby Python Cave, a popular attraction in Queen Elizabeth National Park, Uganda. This cave also contained R. aegyptiacus bats . These events prompted a long-term investigation of Python Cave to determine if, 1) R. aegyptiacus in the cave carried infectious Marburg virus genetically similar to that found in the tourists, and 2) what ecological factors might influence virus spillover to humans. In the study, we found that, 1) approximately 2.5% of the bat colony is actively infected at any one time and that virus isolates from bats are genetically similar to those from infected tourists, and 2) specific age groups of bats (juveniles∼six months of age) are particularly likely to be infected at specific times of the year that roughly coincide with historical dates of Marburg virus spillover into humans.Marburg virus, like its close relative Ebola virus, can cause large outbreaks of hemorrhagic fever with case fatalities nearing 90%. For decades the identity of the natural reservoir was unknown. However, in 2007 Marburg viruses were isolated directly from Egyptian fruit bats ( Filoviridae), is the etiologic agent of Marburg hemorrhagic fever (MHF), a severe disease associated with person-to-person transmission and high case fatality. The virus was discovered in August 1967 when simultaneous outbreaks of MHF occurred in laboratory workers in Germany and Yugoslavia Cercopithecus aethiops; currently Chlorocebus tantalusMarburg virus (family Rousettus aegyptiacus (order Chiroptera: family Pteropodidae) which had the highest prevalence (20.5%) of antibody to the virus R. aegyptiacus to be positive for evidence of Marburg virus infection Since its discovery, the sporadic nature of Marburg virus outbreaks and the diverse history of human exposures have made it difficult to definitively trace the virus to its natural source, but mounting evidence has shown a recurrent link to caves or mines, leading investigators to suspect bats as a likely reservoir. In early February 1975, the second known outbreak of MHF occurred after two tourists traveled through Zimbabwe and reported sleeping in rooms with bats and visiting Chinhoyi caves in the days before developing symptoms R. aegyptiacus, from which five genetically diverse Marburg virus isolates were obtained from bats collected over an eight month period, demonstrating that R. aegyptiacus can naturally harbor infectious Marburg virus and that multiple lineages of virus can persist in a same bat colony for an extended period In July and September 2007, MHF re-emerged in gold miners, this time in southwest Uganda at the Kitaka mine which is approximately 1,280 km from Durba. Here, genetic evidence showed two independent virus introductions from the natural reservoir into humans. A mark-recapture study estimated the mine to populated by over 100,000 R. aegyptiacus colony upon which the snakes feed. The publicity from the Dutch MHF case resulted in the retrospective identification of a second, non-lethal, MHF case associated with Python Cave. This individual was an American tourist who visited the bat colony in late December 2007 and developed MHF symptoms soon after returning home to Colorado, USA A year later, in late June 2008, MHF again occurred in southwest Uganda. This case involved a Dutch tourist who became fatally infected following a visit to Python Cave in Queen Elizabeth National Park (QENP) R. aegyptiacus is a natural reservoir for Marburg virus. However, important questions remain such as how the virus naturally persists in these bats, and what ecological drivers cause occasional spillover from bats to humans. In the present study, we report a multi-year investigation of natural Marburg virus circulation among R. aegyptiacus in southwest Uganda, with emphasis on bats inhabiting Python Cave. Our data show a dynamic pattern of Marburg virus transmission that produces cyclical fluctuations in active infections associated with defined age cohorts of the bat population.Together, these epidemiologic and laboratory data indicate Rousettus aegyptiacus breed twice a year, becoming pregnant around November and May and giving birth in February and August, respectively (gestation period is approximately 105–107 days based on captive observations) R. aegyptiacus and a large number of smaller insectivorous bats (Hipposiderous spp.), the bat population at Python Cave was estimated to be at least 40,000 animals, and R. aegyptiacus was the sole chiropteran inhabitant of the cave.In response to the infection of the American and Dutch tourists, a series of four ecological investigations were conducted at Python Cave from August 2008 through November 2009. The goals of this study were to 1) determine if Marburg virus infected bats were present in the cave, and if so, what species of bat; and 2) determine what ecological factors, if any, may have led to the human infections. Python Cave is actually a tunnel open at both ends, and is approximately 15 meters (m) long and 12 m wide, formed by a subterranean stream that undercut a land bridge spanning a small gorge. The height of the interior is variable, ranging from 3.5 m to nearly 5 m due to the boulder strewn floor, and the cave contains numerous nooks, crevices and hidden chambers, with nearly every square centimeter of ‘hanging space’ used by the bats. The limited space forces bats to occupy sunlit ledges of the gorge on either side of the tunnel openings. Most juvenile bats were observed roosting in these more peripherally located pockets and ledges near the ground, both inside and outside of the tunnel proper while adults tended to occupy the darker interior. These juvenile bats were also observed roosting on the sides of the larger boulders and in holes on the cave floor.Python sebae), and several forest cobras (Naja melanoleuca). Also observed visiting the cave were African fish eagles , palm-nut vultures (Gypohierax angolensis), Nile monitor lizards (Varanus niloticus) and olive baboons (Papio anubis). Further, a variety of invertebrates were found, most notably argasid ticks (Family Argasidae) on the cave walls, nycteribiid flies (Family Nycteribiidae) in the bat pelage, and fresh water crabs (Crustacea: Decapoda) in the subterranean stream beneath the cave floor.In addition to the bats, other vertebrate fauna observed in the cave included at least two large African rock pythons , colon and rectum (n = 5), lung (n = 8), heart (n = 3), intestine (n = 3) and blood (n = 2). The array of virus-infected tissues indicates that R. aegyptiacus inhabiting Python Cave are probably in diverse stages of infection. Some bats, . One bat had Marburg virus-positive reproductive tissue (uterus/ovary) which, given the previous discovery of Ebola virus in reproductive tissue of infected humans Viral RNA extracted from pooled liver and spleen samples were tested for Marburg virus RNA using a real-time Q-RT-PCR assay designed to detect all known strains of Marburg virus R. aegyptiacus collection expedition in Uganda, including those at the Kitaka mine t = −0.023, p>.98), or likewise, from males  or females . Successful isolation of Marburg virus roughly correlated with samples that had Ct values of 30 or less (>2000 TCID50/ml).From the Q-RT- PCR positive bats at Python Cave, seven genetically distinct Marburg virus isolates  were obt50/ml), were additionally tested by IHC, but none were positive for Marburg virus antigen. There was no evidence of any pathology apparent during necropsies or IHC analysis that could be attributed directly to infection with Marburg virus. Moreover, there were no signs of overt morbidity or mortality witnessed during the capture or processing of the bats, including those actively infected with Marburg virus. However, the cave environment is such that dead or dying bats might not be visible for long periods of time due to predation, guano accumulation, and the large detritivore community living in the cave.Immunohistochemical analysis (IHC) was performed on formalin fixed liver and spleen tissues from all Q-RT-PCR positive bats and an approximate equal number of negative bats. Of the 40 Marburg virus positive bats, four (10%) were positive via IHC in liver, one of which (Bat #843) was additionally positive in spleen. All Q-RT-PCR positive heart, lung, kidney, colon and mid-gut tissues shown in R. aegyptiacus representing a bona fide long term reservoir species for the virus.Full-length genome sequences  were determined from all seven of the Python Cave Marburg virus bat isolates. Two isolates (164QBat Uga 2008 and 1328QBat Uga 2009) closely match the sequence of the virus isolate obtained from the Dutch MHF case  and Zimbabwe (OzoZim 1975) suggest that there is considerable animal movement over long distances and exchange of infectious virus through a network of R. aegyptiacus bats).In the initial 2007 Kitaka mine investigation t = 3.898, p<.001). When the active infection data from the Kitaka mine and Python Cave investigations are combined and sorted into three age categories, young juveniles, old juveniles and adults, a reproducible age-linked infection pattern emerges . Adults by contrast maintain a relatively constant level of active infection . Interestingly, no evidence of vertical transmission was found. In one instance, a Q-RT-PCR positive mother was identified with an Q-RT-PCR negative pup. Moreover, all pups from either Kitaka mine or Python Cave (n = 223) tested uniformly negative for active Marburg virus infection.Analysis of the Python Cave Q-RT-PCR data reveals a seasonal age bias among Marburg virus-infected bats which correlates with that observed at Kitaka mine  emerges . Levels nfection  ranging Together, these data present a dynamic picture of natural Marburg virus circulation in which juveniles are exposed to the virus at an early stage of their development following independence at three months of age and increasing up through their first six months of life. Once in the adult population after seven to eight months of age, the incidence of infection apparently drops off for reasons not currently understood and levels out to a more constant rate that is independent of season. We are currently developing reliable measures for sub-adult age classification, but until they are complete, tracking the younger age cohorts beyond six to seven months of age remains difficult.n = 20) were themselves antibody positive. It is unknown if maternal antibody is actually protective.The overall pattern of horizontal transmission is supported by serological data from the Python Cave bats in which Marburg virus-specific IgG antibody prevalence increases with age starting from 4.1% (10/242) among young juveniles and increases to 14.8% (26/175) among older juveniles and finally reaches 21.5% (214/993) in adults. The lower infection levels observed in young juveniles is likely due to lack of physical opportunity for exposure to other members of the population perhaps aided by maternal antibody protection for those pups born to antibody positive mothers. In our analyses, all pups of antibody positive mothers  from adults was witnessed with juvenile bats generally pushed to the periphery of the cave away from the center where it is darkest. At the periphery, juveniles were observed roosting tightly together primarily in small holes or on the sides of large boulders on the cave floor. Occasionally small groups of juveniles could be found low on the walls but outside the cave in filtered sunlight. The cave floor contains copious amounts of accumulated guano (feces and urine) that are continually refreshed by new deposits. Should virus be shed through bat excretions, the physical positioning of juvenile bats directly underneath the adult bats would make juvenile bats particularly susceptible to virus exposure. Unfortunately, testing of limited (<100 samples) urine and fecal samples for viral RNA has not yet yielded positive results, probably due to persistent Q-RT-PCR inhibitors that have thus far hindered our ability to detect Marburg virus RNA in experimentally spiked guano samples in the laboratory (data not shown). Nevertheless, finding of Marburg virus-positive kidney, colon/rectum, and intestine samples, suggests virus shedding through excreta may well occur.Rousettus aegyptiacus being a natural reservoir for Marburg virus.As the juveniles age and are recruited into the adult population or disperse to other caves or suitable sites, the low lying roosting areas are repopulated by the next pulse of newly weaned juveniles. These juveniles in-turn become infected, spreading the virus primarily amongst themselves until they too disperse or move into the adult population. This cycle continues season after season to perpetuate virus transmission within the colony. The pattern of continual circulation of the virus within the population coupled with the continued lack of any overt morbidity and mortality in infected bats is consistent with expectations for R. aegyptiacus, in the weeks just before the onset of MHF symptoms. The original 1967 outbreak was also included, and for that, a date was chosen that was one incubation period (three weeks) prior to the first shipment of infected monkeys that arrived in Frankfurt, Germany on 21 July 1967 (via London Heathrow airport) and further distributed within Germany (Marburg and Frankfurt) and to Belgrade, Yugoslavia R. aegyptiacus infections  standard for working with filoviruses in the field setting was used R. aegyptiacus were sampled over the course of four separate collections spanning one year and three months beginning in August 2008. Bats were captured and processed following procedures detailed in Towner et al. To maximize the chances of isolating virus, large numbers of Adult and nymphal argasid ticks (14 pools of 10–20) were collected from crevices in the rocks near bat roosting sites and immediately placed in chaotropic RNA extraction buffer. Collections of endoparasites occurred during necropsies and were identified as tongue worms of the phylum Pentastomida. These parasites were typically found on the liver and spleen.2 flasks at 37°C/5% CO2 for 1 hr. Media was then replaced with MEM/2% fetal calf serum and monitored for 14 days with a media change on day 7. All cultures were then tested by IFA for Marburg virus.Virus isolation attempts were carried out as described in Towner et al. ATCCTAAACAGGC“T”TGTCTTCTCTGGGACTT-3′ and 5′ Fam-ATCCTGAATAAGC“T”CGTCTTCTCTGGGACTT-3′ in addition to the amplification primers (forward) 5′-GGACCACTGCTGGCCATATC-3′ and (reverse) 5′-GAGAACATITCGGCAGGAAG-3′. The quencher BHQ1 was placed internally in the probes at the “T” locations. The nested VP35 RT-PCR assay is previously described 5′-GCTTACTTAAATGAGCATGG-3′, F3 (forward-inside) 5′- CAAATCTTTCAGCTAAGG-3′, R1 (reverse-outside) 5′- AGIGCCCGIGTTTCACC-3′ and R2 (reverse-inside) 5′- TCAGATGAATAIACACAI ACCCA-3′. The four primers used for the nested NP assay 5′-GTAAAYTTGGTGACAGGTCATG-3′, MBG719F2 (forward-inside) 5′-GGTCATGATGCCTATGACAGTATCAT, MBG1248R1 (reverse outside) 5′- CTCGTTTCTGGCTGAGG-3′, and MBG1230R2 (reverse inside) 5′-ACGGCIAGTGTCTGACTGTGTG-3′. The annealing conditions were 50°C for the first round (both assays) and 54°C (NP assay) or 50°C (VP35 assay) for the second round using high-fidelity one-step RT-PCR reagents (Invitrogen). Primer concentrations and amplification conditions used were as described by the manufacturer. Sequencing was performed using the appropriate amplification primers and standard di-deoxy sequencing methods.Q-RT-PCR, RT-PCR, and nucleotide sequencing, were all performed using reagents and procedures described in Towner et al. R. aegyptiacus (∼three months old). This age group was chosen because they were the cohort considered least likely to have evidence of previous Marburg infection based on data presented here and previously Briefly, IgG detection was performed essentially as described in Immunohistochemical analyses was performed following techniques described in All statistical analyses, Fisher's Exact and two-sided independent samples T tests, of the capture data were performed using PASW 18.0 .Sequencing of Marburg virus whole genomes and partial gene sequences (NP and VP35) were performed as previously described Table S1Suspected (extrapolated) exposure dates for 52 miners from the final Marburg hemorrhagic fever (MHF) patient list from the 1998–2000 outbreak in Durba, Democratic Republic of Congo.(DOCX)Click here for additional data file.Table S2GenBank accession numbers of all Marburg virus sequences analyzed.(DOCX)Click here for additional data file.