Culicoides sonorensis (Diptera: Ceratopogonidae) is a vector of epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD) and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector.C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4–16 days post feeding (dpf). Midges with a virus titer of ≥102.7 median tissue culture infective doses (TCID50)/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14–16 dpf midges.To evaluate the susceptibility of 7 TCID50/ml) were virus isolation-positive, and starting from 10–16 dpf, 32% (35/109) of these virus isolation-positive midges were potentially competent (≥102.7 TCID50/midge). Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14–16 dpf. The WTD developed viremia and severe clinical disease.From 4–16 dpf, 45% (156/350) of midges that fed on WTD with high titer viremia (>10C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates that North America has a susceptible ruminant and vector host for this exotic, cattle-virulent strain of EHDV-7.This study demonstrates that  Orbivirus, family Reoviridae and is comprised of seven proposed serotypes worldwide. All clinical pathology assays were performed using standard protocols by the University of Georgia’s Clinical Pathology Lab . At the end of the study, the animal was euthanized with sodium pentobarbital (1 ml/5 kg IV), and a complete gross and microscopic postmortem examination was performed. In addition, tissue samples were collected in virus transport media for titration, including cerebrum, cerebellum, heart, lung, spleen, lymph node, skin, and epididymis. Serology was performed using agar gel immunodiffusion tests  and serum neutralization as previously described.21 and cattle pulmonary arterial endothelium cells (CPAE)  in a 24-well format[21 cells as previously described[50) were determined[2.3 TCID50/midge. Based on previous studies in domestic sheep with BTV and C. sonorensis, we considered midges with virus titers of ≥102.7 TCID50 to be potentially competent, as this was the virus titer of midges capable of efficient transmission to susceptible hosts[Midges were placed in virus transport media and were held at 4°C for up to 48 hours before being processed individually for virus isolation and titration. For virus isolation, individual midges were manually homogenized using sterile pestles and sonicated for 15 seconds using a sonicating water bath . Homogenized midges were then centrifuged at 4°C for 12 minutes at 1,500 x g. For virus isolation, supernatant (200 μl) was inoculated onto BHKl format. In addiescribed and endptermined. BecauseAll virus isolation and titration attempts from whole blood were performed using CPAE cells as previously described, and froFor the purpose of statistical analysis, midges were grouped into two categories according to the time elapsed from feeding: the 4–9 dpf group was compared to the 10–16 dpf group for midges fed on WTD with low-titer viremia and high-titer viremia. To statistically compare these values, a Chi-square test was performed. Statistical significance for the increase in the percentage of potentially competent virus-positive midges from 10–16 dpf was performed using the Cochran-Armitage Chi-square test for trend. All analyses were performed using COMPARE2 module in the WINPEPI statistical package. A p-val3.1 – 103.94 TCID50/ml) and two had high-titer viremias (107.03 – 107.6 TCID50/ml). There were three feeding trials on the two animals with low-titer viremias and five feeding trials on the two animals with high-titer viremias. Virus isolation and titration results from all midges are presented in TableC. sonorensis were susceptible to oral infection with EHDV-7, with a total virus recovery rate of 36% (47/130) from 4–9 dpf and 50% (109/220) from 10–16 dpf. Replication to titers higher than the competence threshold (≥102.7 TCID50/midge) occurred in 32% (35/109) of virus-positive midges between 10–16 dpf, with a mean virus titer of 103.21 TCID50/midge. During this time period, there was a statistically significant increase in the percentage of potentially competent virus-positive midges (p=0.001) . However, rather than considering only virus-positive midges in the above percentages, 12 % (12/100) of the 100 midges allowed to take a blood meal were potentially competent. Midge-to-deer transmission was demonstrated, as evidenced by a detectable viremia in the deer by 4 dpf, which peaked on 6 dpf. The viremia profile, clinical pathology findings, and rectal temperature from the WTD are presented in TableOf midges that originally fed on WTD with high titer viremia, 100 survived to 14–16 dpf. Before processing for virus isolation and titration, these midges were given the opportunity to take a blood meal from a naïve WTD in order to attempt transmission. Midges were sorted according to feeding status immediately after the feeding attempt and only 44 of the 100 midges (44%) had taken a blood meal. Virus isolation and titration results indicated that 27 of these 44 blood fed midges (61%) were infected  at the time of feeding. Of these 27 infected midges, titration results indicated that 12 (44%) had virus titers ≥ 1050/g) were variable: cerebrum, 104.03; cerebellum, 103.6; heart, <103.6; lung, 105.94; spleen, 106.03; lymph node, 105.1; skin, 105.8; epididymis, 105.8. Precipitating and neutralizing antibodies were detected by 9 dpf. Postmortem examination revealed widespread congestion and hemorrhage in a variety of tissues. Hemorrhages were common in the oral cavity, including the dental pad, palatal ridges, and buccal papillae. Bilateral, severe, diffuse, epididymal hemorrhages were noted, with multiple small foci of hemorrhage within the testicular parenchyma. Additionally, a subadventitial hemorrhage at the base of the pulmonary artery and a paintbrush hemorrhage within the wall of the pylorus FigureB and C wC. sonorensis is a potential competent vector for EHDV-7, as evidenced by the ability of the midge to become infected following ingestion of a blood meal from an experimentally infected WTD and subsequent bite transmission of EHDV-7 from C. sonorensis to a naive WTD. The observed viral dynamics, clinical abnormalities, and postmortem findings in the challenge animal were consistent with previous reports of HD in WTD, as reviewed by Howerth et al.[Our findings demonstrate that h et al.. Four dah et al.,29. Lymph et al.,29. The C. sonorensis has not been investigated, nor is a minimum infectious dose known. The bite from a single infected C. sonorensis has been shown to transmit BTV-1 to domestic sheep[Only 27 of the 44 midges that took a blood meal from the naïve deer were found to be infected at the time of feeding, thus it may be assumed that these 27 midges were responsible for the EHDV transmission. However, based on the titration data, only a portion of infected midges were likely to be competent . Thus, it is suspected that one or more of these 12 midges were responsible for transmission. While only 44 of 100 midges had a visible blood meal after the transmission attempt, we cannot rule out the possibility of probing without blood feeding by any of the 14 other potentially competent midges, which could have potentially contributed to virus transmission. The efficiency of EHDV transmission to WTD by ic sheep,31 and s4.0 TCID50/ml. This lower infection rate is not surprising, considering the poor virus recovery rate on 0 dpf  when midges were provided a lower-titer blood meal . It is known that WTD experimentally infected with EHDV can be viremic for as long as 56 days post-inoculation[7.0 TCID50/ml) which resulted in high infection rates in the midges. Previous studies show that peak viremia in WTD generally occurs 5–7 days post inoculation and blood virus titers of 105.0–6.0 TCID50/ml may be present for as long as seven days, whereas titers > 106.0 TCID50/ml are generally only present for 2–3 days early in the infection[Culicoides has not been explored, it is important to consider additional factors regarding transmission efficiency. For instance, the attack rate on WTD by Culicoides midges can be remarkably high. In a study by Smith and others, >10,000 C. debilipalpis (formerly lahillei) were aspirated from a WTD on five of six autumn mornings, with a maximum single collection of 20,840 midges [Our results suggest that, for EHDV-7, infection of blood fed midges is less efficient 4.6%, 6/130) when viremia was < 1030 when vCID50/ml. Our stuculation; howevernfection,32. Afteon 0 dpf %; 5/15 wC. sonorensis should be considered a potential vector of EHDV-7. Since EHDV-7 is exotic to North America, criteria 1 cannot be fulfilled. Although field research is scarce, previous studies have provided some evidence for both criteria 1[C. sonorensis serving as a vector of EHDV serotypes endemic to the US for WTD.The World Health Organization (WHO) has established criteria for the designation of an arthropod as a confirmed vector of an arbovirus: 1) recovery of virus from non-blood-fed wild-caught specimens, 2) demonstration of the ability to become infected experimentally, 3) demonstration of biological transmission, and 4) an accumulation of field evidence confirming an association of the infected arthropods and the appropriate vertebrate host. This stiteria 1,35,36 aniteria 1, as theyC. sonorensis is the primary vector of BTV for sheep and cattle throughout most of the US, the evidence is not as strong for EHDV and BTV transmission to WTD in wild habitat. With the exception of a previous study in Kentucky, where C. sonorensis was the predominate species trapped in captive WTD pens during an HD outbreak[C. sonorensis has been absent or present in very low numbers in subsequent field studies that have been performed in WTD habitat or on captive WTD facilities, using WTD as bait[C. sonorensis are likely to also be involved in EHDV and BTV transmission to WTD, specifically in the southeastern US. Based primarily on midge abundance, seasonal occurrence, and host preference, it has been suggested that C. debilipalpis, C. stellifer, C. obsoletus, C. paraensis, and C. spinosus warrant further investigation as potential vectors of EHDV and BTV to WTD in the southeastern US[C. debilipalpis has been shown experimentally to support replication of EHDV-2[Culicoides spp. other than C. sonorensis and C. insignis, both confirmed BTV vectors, have been listed as suspect or potential vectors of BTV to cattle and sheep in the southeastern US[Although there is significant field evidence to show that outbreak, C. sono as bait,37-39. Istern US,37,39 anf EHDV-2. Furtherstern US. The aboC. sonorensis was chosen in this study for multiple reasons: 1) it represents the only confirmed vector of EHDV in North America, 2) this species is widely distributed throughout much of the US and commonly feeds on domestic ruminants, thus its competence represents a large geographic risk for outbreaks and establishment following an introduction, and 3) C. sonorensis has been colonized, making it possible to test large numbers of insects that consistently feed in captivity.Despite gaps in our knowledge regarding EHDV and BTV transmission among wild ruminants in some portions of the US, Culicoides life cycle , virus replication within the midge, midge behavior, and midge distribution[Results from this study and previous work demonstrribution. Althougribution, multiplribution, and preribution,42 clearribution.The authors declare that they have no competing interests.MGR, DGM, DES, and EWH conceived and designed the experiments. MGR, DLC, ABA, and EWH conducted the experimental work. EK and MGR analyzed the data. MGR, DGM, DES, EWH, ABA, BSD, and EK contributed to the manuscript. All authors approved the final version for submission.


We performed a serologic investigation to determine whether orthobunyaviruses commonly infect humans in the Yucatan Peninsula of Mexico. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 146 (18%) of 823 persons tested. Further studies are needed to determine health risks for humans from this potentially deadly group of viruses. Orthobunyavirus , Kairi virus (KRIV), Cholul virus (CHLV), and South River virus (SOURV) from mosquitoes in the Yucatan Peninsula of Mexico , Quintana Roo (n = 8) and Campeche (n = 6). The study was approved by the Institutional Biosafety Committees at Iowa State University  and the Universidad Autónoma de Yucatán .All serum samples were examined at a dilution of 1:20 by plaque reduction neutralization test (PRNT) by using CVV (strain CVV-478), and PRNTs were performed as described (>90% reduction in the number of plaques (PRNT90). For etiologic diagnosis, the PRNT90 antibody titer for each virus was required to be >4-fold greater than that to the other viruses tested.Titers were expressed as the reciprocal of highest serum dilutions yielding Antibodies that neutralized CVV were detected in 146 (18%) of 823 study participants. The mean ages of patients with and without antibodies that neutralized CVV were 32.0 and 22.3 years, respectively. Logistic regression analysis showed that the risk for infection increased significantly with age (p = 0.0001).90 titer >4-fold than that to the other viruses tested received a conservative PRNT diagnosis of seropositive for SOURV or a SOURV-like virus. Because interserogroup crossreactivity of neutralizing antibodies to viruses in the BUN and CAL serogroups has not been seen, the 17 persons with antibodies that neutralized SOURV and >1 of the BUN serogroup viruses might have been exposed to >1 viruses from each serogroup.Serum samples from 50 seropositive patients were titrated and analyzed by comparative PRNT to identify the orthobunyaviruses responsible for these infections. Six persons were seropositive for CVV, 5 for CHLV, and 1 for SOURV or a SOURV-like virus; 38 had antibodies to an undetermined orthobunyavirus . BecauseCHLV and POTV share the same medium RNA segment, so antibodies for these viruses cannot be differentiated by PRNT. Furthermore, antibodies to CHLV and POTV cannot be differentiated by complement fixation test  of the study participants analyzed by comparative PRNT had antibodies to an undetermined orthobunyavirus. Most of these persons had low PRNTWe found 18% of the 823 Yucatan residents participating in our study had evidence of orthobunyavirus exposure. This number is presumably an underestimate; additional seropositive persons might have been identified if the initial PRNTs had not been restricted to CVV. In particular, additional seropositive persons likely would have been detected if SOURV was used in the initial PRNTs, because a screening algorithm that includes only a BUN serogroup virus would likely miss many CAL serogroup virus infections. Nevertheless, we provide evidence that orthobunyaviruses commonly infect humans in the Yucatan Peninsula.Previous serosurveys have provided information on the seroprevalence of orthobunyaviruses in humans in the United States. For example, antibodies that neutralized CVV were detected in 42/356 (12%) residents in Maryland and Virginia in 1961–1963 (All persons in our study cohort initially sought care for unspecified fever; however, we could not determine whether any of these febrile illnesses were a direct consequence of orthobunyavirus infection. The detection of acute orthobunyavirus infections is limited because no IgM-capture ELISA for orthobunyavirus diagnosis exists. PRNTs can be used to identify recent orthobunyavirus infections when paired acute and convalescent serum samples are available, but for our study, only single serum samples were available from each participant. Orthobunyavirus viremias in humans are transient and of low magnitude, which makes reverse transcription PCR ineffective for the detection of orthobunyavirus RNA in serum samples. However, a duplex reverse transcription PCR was recently developed for the detection of CVV RNA in human cerebrospinal fluid (In conclusion, we provide evidence that orthobunyaviruses commonly infect humans in the Yucatan Peninsula. These viruses are also a common cause of infection of livestock in this region (


To the Editor: From the end of August through the end of October 2011, a clinical syndrome involving adult cattle and the fetuses of pregnant cows emerged in the border area between the Netherlands and North Rhine-Westphalia, Germany , and 3 independent real-time PCR protocols were conducted to detect genomes of bovine viral diarrhea/mucosal disease virus, bluetongue virus serotype 8, and the novel SBV. Initial retrotranscription of the RNA genomes was followed by quantitative  PCR. The process was conducted by using our procedures (,Taken together, the above data suggest that, like other Simbu serogroup viruses, the new virus crosses the placenta, contaminates the bovine fetus, infects the fetus’ CNS, and causes necrosis and/or developmental arrest of the cerebral cortex. Unlike the viruses mentioned above (When calves from experimentally infected dams are infected with the closest phylogenetic relative to SBV, Akabane virus, porencephaly develops during gestational days 62–96 (


To the Editor: Since 1999, vaccinia virus (VACV) has been isolated frequently from dairy cattle and humans  and Torre de Pedra  counties, respectively. Between the 2 outbreaks, lesions were observed on the teats and udders of 10 lactating cows. The lesions appeared as macules, evolved into vesicles, pustules, and ulcers and healed after 2–3 weeks. Lesions developed on the hands and arms of the milkers after occupational contact with sick animals. The milkers also described headache, lymphadenopathy, and fever.Specimens from 7 scabs and 1 vesicle were collected for virus identification by laboratory assays. After DNA extraction , the samples were subjected to a specific orthopoxvirus PCR for the amplification of the A56R gene of vaccinia virus (www.ncbi.nlm.nih.gov/pmc/articles/PMC308517) by using MEGA3.1 (www.megasoftware.net) showed that a signature deletion was present in the sequences of several Brazilian VACV isolates . Optimal alignment of our samples and other orthopoxvirus A56R gene sequences with ClustalW (isolates . VACVs fWe describe a new zoonotic outbreak of bovine vaccinia in São Paulo State, Brazil. Our molecular data suggest that this outbreak was caused by a VACV that is genetically related to viruses isolated in previous years, including ARAV, which was isolated in 1999 (


In 2010, a HoBi-like pestivirus was isolated from clinically affected calves in Italy. This European virus reproduced a milder form of disease under experimental conditions and was genetically related to previously reported HoBi-like strains. Isolation of this novel virus from a clinical outbreak may have implications for cattle health and prophylactic programs. Pestivirus (family Flaviviridae) includes Bovine viral diarrhea virus type 1 (BVDV-1) and Bovine viral diarrhea virus type 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), and other pestivirus species detected in wild ruminants , cough, accelerated pulse and breath, seromucoid nasal discharge, and leukopenia. Most animals recovered progressively within 2 weeks after administration of supportive therapy. Two calves died, and necropsy indicated severe tracheitis and bronchopneumonia involving the apical lung lobes.3 to 5.48 × 105 copies/μL of template. The pestivirus strains  detected in the lung samples of 2 calves were successfully isolated on MDBK cells as shown by the positive results of an immunofluorescence assay by using an anti-NS3 monoclonal antibody pool . Other viral pathogens of cattle, as well as bacteria and parasites, were not detected in the examined samples, with the exception of the lungs of the dead calves, from which Streptococcus bovis and Vibrio spp. were isolated.The nasal discharge of 6 ill calves and the apical lobes of the dead calves were subjected to traditional or molecular assays to detect the main respiratory pathogens of cattle (www.ncbi.nlm.nih.gov) and EMBL (www.ebi.ac.uk) tools. By sequence comparison with reference sequences, strain Italy-1/10–1 had the same genomic organization of other members of the genus Pestivirus, consisting of a unique open reading frame of 11,700 nt flanked by 2 untranslated regions (UTRs). By sequence analysis of the near full-length genome, strain Italy-1/10–1 displayed the closest relatedness to atypical pestivirus Th/04_KhonKaen, whereas the nucleotide identities to BVDV-1 and BVDV-2 reference strains were much lower  of strain Italy-1/10–1, representative of the pestiviruses circulating in the herd, was determined through PCR amplifications and subsequent sequencing of overlapping fragments . Seroconversion was demonstrated by using the BVDV-Ab SVANOVIR ELISA  and virus neutralization (Pestivirus, but in the past few years, new putative members have been described worldwide. In 2004, an atypical pestivirus, D32/00_HoBi, distantly related to BVDV-1 and BVDV-2 was isolated from a batch of fetal calf serum collected in Brazil (Traditionally, 4 different species have belonged to the genus Pestivirus (,,More recently, another atypical pestivirus, Th/04_KhonKaen, was isolated from a bovine serum sample during an epidemiologic survey in Thailand (The results of our study show that atypical pestiviruses also are circulating on the European continent. Infection caused by this new virus was associated with overt disease in cattle. Although experimental infection of seronegative calves induced only mild disease, Koch’s postulates were fullfilled, as demonstrated by some clinical signs, viremia, viral shedding, and seroconversion.,–pro regions, atypical pestiviruses formed a monophyletic cluster that was approximately equidistant from BVDV-1/BVDV-2 and BDV/CSFV, whereas the dendrogram obtained from 5′ UTR showed a closer relatedness to BVDVs. Thus, phylogeny may not support naming HoBi-like strains as BVDV-3 because the proposed nomenclature (,At the genetic level, the HoBi-like strain from Italy was more strictly related to the viruses from Brazil than to the isolate from Thailand. Considering that the virus has been repeatedly detected in batches of fetal calf serum (,Antigenic differences have been found between HoBi-like strains and BVDV-1/BVDV-2 through cross-neutralization assays (


Theilovirus is considered to comprise four distinct serotypes, including Theiler's murine encephalomyelitis virus, Vilyuisk human encephalomyelitis virus, Thera virus, and Saffold virus. So far, there is no systematical study that investigated the genomic recombination of Theilovirus. The present study performed the phylogenetic and recombination analysis of Theilovirus over the complete genomes. Seven potentially significant recombination events were identified. However, according to the strains information and references related to the recombinants and their parental strains, four of the recombination events might happen non-naturally. These results will provide valuable hints for future research on evolution and antigenic variability of Theilovirus.At present,  Encephalomyocarditis virus (EMCV) and Theilovirus are two distinct species in the Cardiovirus genus of the family Picornaviridae  , which le in USA . Figure Theilovirus available in GenBank nowadays. Seven potentially significant recombination events were detected. However, four of the recombination events might happen non-naturally in the lab, which should be taken into notice in the future evolutionary analysis of Theilovirus. The other three recombination events were further analyzed using other algorithms in RDP software bag and some of them were confirmed by phylogenetic analysis. The recombination phenomena of Theilovirus will also be noted in the further research because this will be one pattern of virulence factor variation in Theilovirus.Recombination is a relatively common phenomenon in RNA viruses and understanding recombination will be helpful in unravelling the evolution of pathogens and drug resistance -25. In tThe authors declare that they have no competing interests.GS conceived the study. All authors performed recombination analysis, critically reviewed, and approved the final manuscript. GS wrote the paper. All authors read and approved the final manuscriptBOOTSCAN evidence for the recombination event 2, 3, 4, and 5. Analysis was based on pairwise distance, modeled with a window size 200, step size 20, and 100 Bootstrap replicates.Click here for file


During infection, many RNA viruses produce characteristic inclusion bodies that contain both viral and host components. These structures were first described over a century ago and originally termed “X-bodies,” as their function was not immediately appreciated. Whilst some inclusion bodies may represent cytopathic by-products of viral protein over-accumulation, X-bodies have emerged as virus “factories,” quasi-organelles that coordinate diverse viral infection processes such as replication, protein expression, evasion of host defenses, virion assembly, and intercellular transport. Accordingly, they are now generally referred to as viral replication complexes (VRCs). We previously used confocal fluorescence microscopy to unravel the complex structure of X-bodies produced by Potato virus X (PVX). Here we used 3D-structured illumination (3D-SIM) super-resolution microscopy to map the PVX X-body at a finer scale. We identify a previously unrecognized membrane structure induced by the PVX “triple gene block” (TGB) proteins, providing new insights into the complex interplay between virus and host within the X-body. In the process of host invasion, many plant viruses induce the formation of characteristic inclusion bodies that were initially termed “X-bodies” due to their unclear role ssRNA; Hull, Viral RNA (vRNA)-dependent RNA polymerases (“replicases”) are usually active as oligomeric arrays  and ∼500 nm along the focal axis  approaches have been developed that overcome the diffraction barrier that limits conventional light microscopy, enabling fluorescence imaging at resolutions smaller than the wavelength of the emitted light  is a widefield imaging approach that is amenable to most specimens suitable for confocal microscopy. In 3D-SIM, a diffraction grating is superimposed upon the sample, and rotated during image acquisition. Sub-diffraction information is contained in the shifting diffraction patterns, and can be extracted by mathematical transformation, permitting image deconvolution with a resolution of ∼100 nm in Potato virus X is a (+)ssRNA virus important for agriculture  encoding five viral proteins -GFP  of the virus coat is fluorescently labeled -GFP membrane marker (Boevink et al., In previous work we described the essential role of the TGB1 protein in generating the PVX X-body, and presented a model of the layered structure of this virus “factory” (Tilsner et al., Modification of host organelles and their redirection to, and involvement in, X-body organization is likely to be a vital event in the PVX infection process. One possible role of recruited host elements is to protect the virus from the host plant defense mechanisms by wrapping it in plant membranes and creating a unique isolated environment for the replicating virus in which it is more difficult for the plant to recognize and degrade the vRNA through the plant RNA silencing machinery. In addition, it is possible that the recruited host membranes enlarge the surface area for the replicating virus, making replication more efficient because of the production of increased concentrations of important viral components (Dunoyer et al., in vitro (Rodionova et al., The accumulation of encapsidated virions on the cytoplasmic side of the X-body (Oparka et al., 3D-SIM “super-resolution” has enabled us to gain new insights into the structural organization of the replication “factory” of a model plant virus and develop new hypotheses about its functions. This highlights the value of super-resolution approaches for the analysis of other viruses, including those that infect animal cells. The study of viral inclusions is an area within cell biology that lends itself to the practical application of super-resolution microscopy, bringing its powers to bear on important biological questions. To obtain 3D-SIM images does not require complicated embedding and sectioning techniques but only mild fixation and the use of antifade reagents, ensuring a low degree of sample disruption. Imaging was conducted on intact epidermal cells in single- and even multi-cell layer epidermal peels, showing the versatility of 3D-SIM for complex biological specimens. Due to their greater photostability, we found GFP fusions better suited to 3D-SIM imaging than RFP constructs, but the rapid development of new FPs is likely to overcome such limitations in the near future, and others have successfully imaged RFP fusions with 3D-SIM (Horsington et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Plant-Microbe_Interaction/10.3389/fpls.2013.00006/abstractThe Supplementary Material for this article can be found online at


Closteroviridae provides an overview on some representative members of this family. Most articles are reviews on different fundamental and applied aspects, but a few are original contributions focused on more specific issues. Even if biased toward closteroviruses, and more explicitly toward Citrus tristeza virus (CTV) due to its economic relevance, this compilation altogether results an attractive blend that we hope will attract the attention of the aficionados of this fascinating virus family.This e-Book on the virus family First, Dawson et al.  discuss Flores et al.  describeWhile one of the economically relevant CTV syndromes (decline) can be managed by using resistant/tolerant rootstocks, the other  cannot. Folimonova  reports In this same context, Lee and Keremane  elaboratAlso on a historical framework, Wang et al.  have exaNicotiana benthamiana with T36-based plasmids and results in systemic infection and production of enough CTV virions to infect citrus by slash inoculation. Using oncogenic Agrobacterium strains they have observed induction of tumors expressing GUS in different plant species, including citrus, but systemic infection only in N. benthamiana. Moreover, mechanical inoculation of CTV virions to N. benthamiana agroinfiltrated previously with a silencing suppressor resulted in systemic infection with T36, but not T318A, which replicates in protoplast of this plant to the same extent as T36. Finally, T36 was graft-transmitted from infected to healthy N. benthamiana plants agroinfiltrated previously with a silencing suppressor. These data indicate that extension of this genetic system still needs considerable improvement.Ambrós et al.  have triHarper , by examClosteroviridae. They conclude that the major factors that have shaped the genetic structure and diversity of this family of viruses comprise: (i) a strong negative pressure that seems responsible for the high genetic stability of some viruses, (ii) human transport of infected propagative material that has caused dispersion of genetically similar virus genotypes, (iii) recombination between divergent sequence variants resulting in generation of new genotypes, (iv) interactions between virus strains or between different viruses in mixed infections that may affect the final outcome, and (v) genetic drift caused by host change or insect transmission leading to changes in the viral population.Rubio et al.  deal witClosteroviridae has revealed that, in addition to genomic and subgenomic RNAs, infected plants often contain one or more double-stranded defective RNAs (dRNAs) of various sizes, most of which contain diverse internal deletions flanked by the two genomic termini. The roles and biological functions of dRNAs remain terra incognita, but one possibility is that these abundant double-stranded RNAs are used as a buffering system to protect the large and fragile viral single-stranded RNA genomes from being targeted by the RNA silencing defense of the host.Bar-Joseph and Mawassi  focus onBeet yellows virus (BYV), the type species of genus Closterovirus. Infection by eukaryotic viruses induces formation of membranous compartments, wherein replication occurs. Specifically, complexes from cell membranes of endoplasmic reticulum (ER) or mitochondrial origin appear in closterovirus infections. Computer-assisted analysis predicts several putative membrane-binding domains in the central region (CR) of the BYV polyprotein 1a. Transient expression in N. benthamiana of a hydrophobic segment of the CR results in reorganization of ER into ~1-μm mobile globules, suggesting that this segment may be involved in the formation of multivesicular complexes in BYV-infected cells.Gushchin et al.  report o(Cordyline fruticosa L.) in Hawaii. Besides confirming the presence of Cordyline virus 1 (CoV-1), sequence analysis has unveiled three additional closteroviruses (CoV-2 to -4), which based on the divergence of several viral proteins, represent four distinct closterovirus species. Phylogenetic reconstructions indicate that CoV-2, CoV-3, and CoV-4, together with Little cherry virus 1 and Grapevine leafroll-associated virus 7, form a distinct clade within the family Closteroviridae.Melzer et al.  have useGrapevine leafroll associated virus 2 is particularly illustrative.The chapter by Dolja and Koonin  summarizGrapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, which is regarded as the most important causative agent of grapevine leafroll disease (GLD). Complete genome sequencing of several isolates has revealed the existence of genetic variants, and characterization of the subgenomic RNAs has supplied insights into the replication strategy and the putative function of some viral proteins. Deep sequencing, apart from being a fine diagnostic tool, has furnished a more penetrating view of the complexity of viral infections and of the underlying plant pathogen interactions.Maree et al.  provide Almeida et al.  discuss Crinivirus with a bipartite genome, Kiss et al. (Lettuce infectious yellows virus (LIYV). LIYV RNA1 encodes proteins involved in replication, which results in formation of vesiculated membranous structures where most likely this process occurs (see above). Four of the LIYV RNA2-encoded proteins, CP (major coat protein), CPm (minor coat protein), Hsp70h, and p59 are virion structural components, and CPm is a determinant of whitefly transmissibility. P5 is a small protein encoded at the 5′ end of RNA2 and its ortolog in BYV is localized to the ER and plays a role in cell-to-cell movement. The other small protein, p9, is unique to members of the genus Crinivirus.Moving to the genus s et al.  review rin vitro and in situ localization of LIYV virions. Immunoblot analyses resulted in a virus detection limit comparable to that of DAS-ELISA, and in membrane feeding experiments they revealed that specific virion retention in whitefly vectors corresponded with successful transmission.A previous study using an AlexaFluor-based immunofluorescent localization assay has shown that retention of LIYV virions in the anterior foregut of its whitefly vector is required for virus transmission. Ng , by incoCrinivirus, most of whose members have been characterized in the last 20 years. Criniviruses have emerged as a major agricultural threat to important horticultural crops—including tomato, potato, lettuce, and cucumber—at the end of the twentieth century with the establishment and naturalization of their whitefly vectors, belonging to genera Trialeurodes and Bemisia, in temperate climates around the globe.Finally, the article by Tzanetakis et al.  provides


Oligoryzomys utiaritensis).To study the dynamics of wild rodent populations and identify potential hosts for hantavirus, we conducted an eco-epidemiologic study in Campo Novo do Parecis, Mato Grosso State, Brazil. We detected and genetically characterized Castelo dos Sonhos virus found in a species of pygmy rice rat ( Bunyaviridae, genus Hantavirus) distributed worldwide. In nature, these viruses are maintained in persistently infected rodents without disease manifestation. Hantaviruses are transmitted to humans through a respiratory route, mainly by inhalation of aerosolized, virus-infected particles in rodent excreta, such as feces, saliva, or urine. Hantavirus pulmonary syndrome (HPS) was first recognized in 1993 after an outbreak of acute respiratory distress syndrome associated with Sin Nombre virus occurred in the southwestern United States  G . Blood samples obtained from the rats were serologically screened by IgG-ELISA by using the Andes virus antigen as previously described  to study the dynamics of wild rodent populations and prevalence of hantavirus infection. Of 459 rodents captured during the project, 89 were classified as b mitochondrial DNA  with primers L14724 and Citb-rev by using standard PCR procedures and sequenced the samples with the same primers and an additional internal primer MVZ16 (O. nigripes (black-footed pygmy rice rat) and O. utiaritensis, the NJ analysis showed that O. utiaritensis pygmy rice rats are more closely related to Moojen’s pygmy rice rat (O. moojeni) (7.4% Kimura 2-parameter distance estimates) than to any other Oligoryzomys species  with ClustalW software  in BioEdit 5.0 (www.mbio.ncsu.edu/BioEdit/biodoc.pdf). The maximum-likelihood and Bayesian methods were implemented in PHYML and MRBAYES version 3, respectively, and used for phylogenetic reconstructions  . PartialAfter its isolation in 1995 from 1 patient with HPS in the Castelo dos Sonhos district of Altamira in southeast Pará State, CASV has only occasionally been detected in Pará and neighboring Mato Grosso states in Brazil’s Amazon region. However, a recent study performed among residents of 4 municipalities along interstate highway BR-163, which runs between southeastern Pará and northern Mato Grosso, has suggested continuous CASV circulation with occurrence of small outbreaks, sporadic HPS cases, and silent infections (Calomys (,Among Brazilian states, Mato Grosso has the fourth highest number of reported HPS cases. Cases have been associated with agricultural activities, mainly cultivation of soybean and grains, and occasionally corn and other beans. Previous studies in Mato Grosso have demonstrated the circulation of the Laguna Negra virus in the region as associated with rodents belonging to the genus The study of dynamics of rodent populations during the period identified the rodent population that naturally occurs in the rural zone of the municipality of Campo Novo do Parecis in Mato Grosso. Lung samples from 4 pygmy rice rats showed anti-hantavirus antibodies, yielding hantavirus RNA amplification. Sequencing and phylogenetic analysis indicated those rodents were infected by CASV, suggesting this species of pygmy rice rat as a potential host of CASV. Furthermore, this rodent species was previously unknown in the Amazon region. Pygmy rice rats may be a reservoir for CASV, and this hantavirus may be responsible for HPS cases in Campo Novo do Parecis and in neighboring municipalities located in the midwestern region of Mato Grosso state in central Brazil, near the border with Pará state, and also in the Castelo dos Sonhos district of Pará state.


Cerebrospinal fluid specimens from 57 patients diagnosed with meningitis were tested for Japanese encephalitis virus. Total RNA was extracted from the specimens and amplified. Two products had highest homology with Nakayama strain and 2 with Ishikawa strain. Results suggest that Japanese encephalitis virus causes some aseptic meningitis in Japan. Japanese encephalitis virus is one of the leading causes of epidemic encephalitis worldwide; 35,000–50,000 cases are reported each year with 10,000 deaths . During Japanese encephalitis virus causes meningitis as well as encephalitis . HoweverCSF specimens were obtained for diagnostic purposes from 170 patients with a clinical diagnosis of aseptic meningitis from August to October in each year from 1999 to 2002, in Hiroshima prefecture. These CSF specimens were sent to the Division of Microbiology II, Hiroshima Prefectural Institute of Health and Environment, for virologic examination. Viruses were isolated from 112 of 170 CSF specimens in cell cultures by using Vero cells, BGM cells, FL cells, Hep2 cells, and RD18S cells. Enteroviruses and mumps viruses were isolated from 96 and 16 CSF specimens, respectively . Thus, eTotal RNA was extracted from CSF by using ISOGEN-LS . The RNA pellet was resuspended in DEPC Treated Water . The RNA was reverse transcribed and amplified by using polymerase chain reaction (PCR) with AMV Reverse Transcriptase XL  and Tth DNA Polymerase  with the primer pair reported by Morita et al. . The PCRElectrophoresis demonstrated the band with expected size of 326 bp in 4 of 57 PCR products . Two nu.Patients 1–4 lived in Hiroshima prefecture . PatientEnteroviruses are the most frequent cause of aseptic meningitis in summer and autumn in Japan . In the Three Japanese encephalitis cases occurred in 2002 for the first time in 12 years, but anti–Japanese encephalitis virus IgM antibodies were detected in porcine serum samples every year in Hiroshima prefecture (data not shown). So we expected that Japanese encephalitis virus might be associated with aseptic meningitis, and CSF specimens from 57 cases during the past 5 years (1998–2002) had been stored at –30°C in Hiroshima Prefectural Institute of Health and Environment. Those specimens were used to detect Japanese encephalitis virus genome retrospectively. Four of 57 CSF samples were Japanese encephalitis virus genome–positive by nested PCR. We may find more febrile or meningitis cases caused by Japanese encephalitis virus if we routinely conduct serologic or molecular diagnostic tests for Japanese encephalitis virus. CSF samples were from infants and children. Most Japanese encephalitis patients in Japan are >55 years of age. Japanese encephalitis meningitis may also occur among elderly meningitis patients. The Japanese encephalitis vaccination coverage of children has been decreasing in Japan recently; <10 Japanese encephalitis cases are reported annually. If the general population recognizes that Japanese encephalitis virus causes aseptic meningitis at a higher rate than expected, the percentage of Japanese encephalitis vaccination may increase, and the emergence of Japanese encephalitis will be prevented. In 2000, anti–Japanese encephalitis virus IgM antibody was first detected in porcine serum samples in late July, in Hiroshima prefecture (data not shown). This finding suggests that Japanese encephalitis virus was active when these 4 meningitis cases occurred. Further, we found evidence that Japanese encephalitis viruses belonging to genotypes I and III were active in Hiroshima prefecture (data not shown). The results of this study suggest that Japanese encephalitis virus should be considered in the differential diagnosis of aseptic meningitis in areas where Japanese encephalitis is endemic or epidemic.


Nicotiana clevelandii and N. benthamiana whereas the milder isolates did not. Phylogenetic analysis of complete coat protein gene sequences of these isolates together with sequences obtained from 14 other field-collected samples from Kenya and Zanzibar, and reference sequences grouped them into two distinct clusters, representing the two species of cassava brown streak viruses. Put together, these results did not suggest the association of a hypervirulent form of the virus with the current CBSD epidemic in Uganda. Identification of the severe and milder isolates, however, has further implications for disease management and quarantine requirements.The genetic and symptom diversity of six virus isolates causing cassava brown streak disease (CBSD) in the endemic  and the recently affected epidemic areas (Uganda) of eastern Africa was studied. Five cassava varieties; Albert, Colombian, Ebwanateraka, TMS60444  and Kiroba (tolerant) were graft inoculated with each isolate. Based on a number of parameters including the severity of leaf and root symptoms, and the extent of virus transmission by grafting, the viruses were classified as either severe or relatively mild. These results were further confirmed by the mechanical inoculation of 13 herbaceous hosts in which the virulent isolates caused plant death in  Cassava brown streak disease (CBSD) is endemic in areas along the Indian Ocean coast of eastern Africa, from the northeastern border of Kenya across the Tanzanian border down as far as the Zambezi River in Mozambique, and it was widespread around the shore of Lake Malawi. In the endemic areas, CBSD was confined to altitudes below 1,000 metres above sea level . More reCassava brown streak virus (CBSV), and the highland epidemic virus as Cassava brown streak Uganda virus (CBSUV). Both species belong to the genus Ipomovirus, family Potyviridae  and CBSV-[MZ:Nam1-1:07] resulted in infections . The effAmongst the isolates, maximum number of cuttings were sprouted from the epidemic isolate CBSUV-[UG:Kab4-3:07] (96%) and the least number of cuttings from CBSV-[TZ:Nal3-1:07] (74%) . No signP < 0.05) were observed for virus-variety interactions on symptom score (data not shown).Mean maximum leaf symptom severity score of 3.8 was recorded for CBSV-[MZ:Nam1-1:07] and the mean minimum score of 1.9 for CBSUV-[UG:Kab4-3:07] . The sym−1 to 10−5 folds, virus was detectable at 10−5 dilutions only from CBSV-[MZ:Nam1-1:07] and CBSV-[TZ:Nal3-1:07] isolates. For the remaining four isolates virus was not detectable at 10−3 or greater dilutions.In a serial dilution of viral cDNA from 10Typical necrosis and dry rotting of infected tubers was recorded for all virus-variety combinations except for Kiroba infected with CBSUV-[UG:Kab4-03:07], which did not produce root symptoms .CBSD symptoms were variable but two recognisable patterns emerged, which are associated with the virus species involved.Initial symptoms of plants infected with CBSUV isolates appeared as faint yellow spots on the affected leaves which later developed into bright yellow patches of usually irregular to occasionally circular shape. The yellow patches were vividly defined especially in susceptible varieties . Of the plant species, N. clevelandii was most susceptible, showing symptoms on all plants between weeks 1 to 3.All six CBSD isolates infected nfection . All plaN. clevelandii and N. benthamiana. Plants infected with CBSV-[TZ:Nal3-1:07] and CBSV-[MZ:Nam1-1:07] were severely stunted and subsequently wilted by developing leaf necrosis  and CBSUV (440 bp), respectively . No amplcucumber vein yellowing virus (CVYV), squash vein yellowing virus (SqVYV), and sweet potato mild mottle virus (SPMMV). The most parsimony analysis grouped the CP aa sequences into two major clusters: CBSV and CBSUV (The deduced amino acid (aa) sequences for the complete CP of CBSV and CBSUV isolates consisted of 378 and 367 bases, respectively. These were used to estimate their genetic relationships together with the reference sequences of CBSV, CBSUV, nd CBSUV . AmongstUntil recently, research on CBSD diversity/severity has largely been restricted to observations in the field on cassava plants of different age, genetic makeup, and grown in different agroecological zones with varying environmental conditions and possibly infected with different virus strains, all of which can independently or in combination influence symptom development. This made the comparison of the field observations between the various studies particularly difficult and the question of whether a severe form of CBSD is associated with the latest epidemic in Uganda has remained unanswered. Inoculation of herbaceous host plants by various researchers provided somewhat uniform conditions for symptom diversity studies  but untiIn order to investigate this, a number of parameters were used to assess the severity levels between one epidemic and five endemic CBSD isolates including the symptoms on leaf and roots of five infected cassava varieties, the effect of virus on sprouting of cassava stem cuttings, the rate of graft transmission, and virus titres in infected leaves as well as symptom severity on herbaceous host plants. Amongst the isolates examined, the endemic isolates CBSV-[MZ:Nam1-1:07] and CBSV-[TZ:Nal3-1:07] produced the most severe symptoms with mean symptom severity scores of 3.7-3.8 on a five-point scale . In compN. benthamiana  and CBSV-[TZ:Nal3-1:07] produced symptoms early, caused severe stunting of infected plants, leaf necrosis, and often plant death. The remaining isolates including CBSUV-[UG:Kab4-3:07] caused various forms of leaf chlorosis, the symptoms were less severe and nonlethal  on cassava that may be responsible for the rapid spread of the virus in the field. The recent widespread introduction of CMD-resistant varieties that are particularly susceptible to CBSD or the combination of both could be a factor in disease outbreak. Recent surveys in Uganda indeed confirmed these possibilities, where more than 70% of the cassavas grown in 23 districts were CMD-resistant improved varieties, all of which are susceptible to CBSD. These varieties also support high whitefly numbers, in excess of 200 adults per top five leaves. Although such elite cassava has not been introduced in high quantities to the Lake Zone Tanzania, the high susceptibility of local land races grown in the region and the sudden development of unusually high whitefly populations on cassava there is ensuring the spread of CBSD [Put together, these collective observations on symptom diversity as well as genetic differences did not indicate the association of a severe form of CBSD in Uganda. These results are indeed consistent with studies on another epidemic isolate (Namulonge) from Uganda  and espe of CBSD , 7. Iden


Cryptococcus gattii genotype VGIIa who had no recent history of travel to disease-endemic areas. This strain was identical to the Vancouver Island outbreak strain R265. Our results suggest that this virulent strain has spread to regions outside North America.We report a patient in Japan infected with  Cryptococcus neoformans and C. gattii are closely related species of yeast; C. gattii was previously classified as C. neoformans var. gattii .,C. gattii was believed to be restricted to tropical and subtropical areas such as Australia, Southeast Asia, and South America  and VGIIb   in a patient from Japan who had no recent travel history to known disease-endemic areas.We report a case of cerebral cryptococcoma caused by In 2007, a 44-year-old man (sign painter) from Japan who was not infected with HIV came to the University of Tokyo Hospital with a 2-month history of headache and loss of right-sided vision in both eyes (right homonymous hemianopsia). He had had a diagnosis of hyperglycemia 3 years before admission but had declined to seek medical treatment. His medical history was otherwise unremarkable. He was an ex-smoker who rarely consumed alcohol and was not taking any prescription medications (including corticosteroids or other immunosuppressive drugs). He had traveled to Guam in 1990 and Saipan in 1999 and had no other history of overseas travel. He reported exposure to his dog, and he did not spend time in wilderness areas. The patient often worked near construction sites in urban locations.When hospitalized, the patient was afebrile and had stable vital signs. Physical examination showed agraphia (inability to write), anarithmia (inability to count numbers), right homonymous hemianopsia, and Romberg sign. Laboratory evaluations showed increased glucose (367 mg/dL) and hemoglobin A1c (10.5%) levels. Results of a complete blood cell count and hepatic and renal function tests were within normal limits. Levels of electrolytes and C-reactive protein were also within normal limits. Results of a test for antibodies to HIV-1/2 were negative.An enhanced brain magnetic resonance imaging scan showed a 4.4 × 4.1 × 3.3–cm lobulated mass in the left occipital lobe with surrounding edema. The lesion had low signal intensity on T1-weighted images, moderate signal intensity on T2-weighted images, and rim enhancement on T1-weighted images after administration of gadolinium . A chestCryptococcus spp. and led to a diagnosis of cerebral cryptococcosis.The brain mass was completely resected because it was suspected to be a tumor. Pathologic evaluation of the resected specimen showed that the mass was a large abscess containing encapsulated yeast. The yeast could be seen after staining with periodic acid–Schiff and Grocott methenamine silver stains. Specimen cultures were positive for The patient was treated with liposomal amphotericin B (4 mg/kg/d for 3 weeks) and flucytosine (100 mg/kg/d for 3 weeks). A cerebrospinal fluid (CSF) sample obtained by lumbar puncture soon after mass resection showed a slightly increased protein level but no leukocytosis or a low glucose concentration. Fungal cultures of the CSF showed negative results. Cryptococcal antigen titers were >512 in serum and 64 in CSF.After 3 weeks of induction therapy, the patient received consolidation and maintenance therapy  for 1 year. The pulmonary nodule decreased in size after antifungal treatment, suggesting that this lesion was a pulmonary cryptococcoma. At the 1-year follow-up visit, the cerebral cryptococcoma had not recurred, and the pulmonary nodule had disappeared.C. gattii by rRNA gene sequence analysis  analyzed by multilocus sequence typing, according to the method of Fraser et al. .C. gattii–endemic area. There have been only 2 reports of C. gattii infections in Japan; both infections apparently originated in Australia   (Japan has not been considered a C. gattii genotype VGIIa may have been present in the Pacific Northwest region of the United States long before the Vancouver Island outbreak (,,,C. gattii VGIIa genotype may be spreading. In North America, this genotype has been isolated from various environmental specimens such as tree surfaces, soil, water, and air (,,C. gattii in Japan are needed to determine likely reservoirs and to improve understanding of C. gattii epidemiology. Although many clinical laboratories in Japan currently do not differentiate between C. neoformans and C. gattii infections, identification of cryptococcal isolates to the species level, especially in apparently immunocompetent patients, is needed.Our results suggest that


Myotis nattererii) in Germany was differentiated from other lyssaviruses on the basis of the reaction pattern of a panel of monoclonal antibodies. Phylogenetic analysis supported the assumption that the isolated virus, Bokeloh bat lyssavirus, may represent a new member of the genus Lyssavirus.A virus isolated from a Natterer’s bat ( Lyssavirus within the family Rhabdoviridae contains 11 viruses: rabies virus (RABV), Lagos bat virus, Mokola virus, Duvenhage virus, European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2), Australian bat lyssavirus, Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus, and West Caucasian bat virus  associated with EBLV-1 (Myotis spp. bats (M. daubentonii and M. dascyneme) and has only sporadically been found in Europe and in Germany  were detected in Europe, but only 10 of the 45 known indigenous bat species tested positive for lyssavirus; most were serotine bats (h EBLV-1 . In Germh EBLV-1 . EBLV-2 M. nattererii). It was given mealworms and water ad libitum supplemented with minerals and vitamins. In February 2010, the bat began to act aggressively, directly approaching any moving object, vigorously trying to bite, and screaming ferociously. This agitated stage lasted for 7 days and was followed by general weakness, lethargy, and paralysis. After the first 3 days of the clinical course, the bat stopped drinking and eating. Ten days after recognition of the first clinical signs, the animal died.In November 2009, a bat was found on the ground in Bokeloh, Lower Saxony, Germany . The bat was morphologically identified as a Natterer’s bat  showed that BBLV differed from all other published lyssavirus sequences with the highest nucleotide identity to KHUV (80%), followed by ARAV (79%), EBLV-2 (79%), Australia bat lyssavirus (77%), EBLV-1 (77%), Irkut virus (76%), Shimoni virus (76%), RABV (73-75%) and Duvenhage virus (75%). Lagos bat virus (72-74%), Mokola virus (72%), and West Caucasian bat virus CBV (72%) showed the highest divergence to BBLV. Also, phylogenetic analysis based on concatenated N-P-M-G-L nucleotide sequences showed that BBLV is most closely related to KHUV, followed by EBLV-2  from a Natterer’s bat that died with rabies-like clinical signs. Initially, a distinctive pattern in the reaction with a panel of antinucleocapsid monoclonal antibodies indicated the presence of an antigenically atypical isolate. The differentiation from other lyssavirus species was confirmed by phylogenetic analysis .BBLV is pathogenic because it caused a fatal disease in the Natterer’s bat that was similar to the clinical picture of rabies seen in other bats. Viral antigen was present in many locations of the brain  but surpMyotis spp. bats  indicating that Myotis spp. bats play a key role in lyssavirus epidemiology. If one considers the history of bat rabies in Europe, it seems unlikely that BBLV had spread from a distant origin into central Europe or that the bat itself was translocated over long distances.Since the exact date of infection is unknown, the incubation period can only be estimated as >4 months. Whether the Natterer’s bat is the natural reservoir species of BBLV or whether it was a cross-species spillover remains a subject for further studies. However, closely related lyssavirus species were also isolated from Pipistrellus nathusii, Pipistrellus pipistrellus, and Plecotus auritus, have been found infected with EBLV-1 during this study , and this is the second discovery in recent years of a new lyssavirus species through routine passive bat rabies surveillance. Also, in 2007 a Daubenton’s bat found on the ground was taken to a rehabilitation center, where it died and subsequently tested positive for EBLV-2 . Germany is the only country where several bat species other than serotine bats, i.e., An encounter with a BBLV-infected Natterer’s bat could lead to a fatal outcome because bat lyssaviruses have caused several human cases of infection . In Euro


The better stability of SARS coronavirus at low temperature and low humidity environment may facilitate its transmission in community in subtropical area (such as Hong Kong) during the spring and in air-conditioned environments. It may also explain why some Asian countries in tropical area  with high temperature and high relative humidity environment did not have major community outbreaks of SARS.The main route of transmission of SARS CoV infection is presumed to be respiratory droplets. However the virus is also detectable in other body fluids and excreta. The stability of the virus at different temperatures and relative humidity on smooth surfaces were studied. The dried virus on smooth surfaces retained its viability for over 5 days at temperatures of 22–25°C and relative humidity of 40–50%, that is, typical air-conditioned environments. However, virus viability was rapidly lost (>3 log Severe acute respiratory syndrome (SARS), was a new emerging disease associated with severe pneumonia and spread to involve over 30 countries in 5 continents in 2003. A novel coronavirus was identified as its cause –3. SARS We and others have reported that infectivity of SARS CoV (SARS coronavirus) was lost after heating at 56°C for 15 minutes but that it was stable for at least 2 days following drying on plastic. It was completely inactivated by common fixatives used in laboratory , 10. Ano2 and were used for growing stock virus and for titration of viral infectivity [The SARS CoV strain used in this study is HKU39849. Foetal monkey kidney cells (FRhK-4) were cultured in minimal essential medium  with 10% foetal calf serum and penicillin streptomycin  at 37°C in 5% COStock virus was harvested when infection approximately 75% of the cell monolayer of a virus infected flask manifested cytopathic effect (CPE). Infected cells were subjected to one cycle of freeze and thaw centrifuged at 2000 rpm for 20 minutes to remove cell debris and the culture supernatant was aliquoted and stored at −80°C until use.μL of confluent FRhK-4 were infected with 100 μL of serial 10-fold of dilutions of stock virus in minimal essential medium with 1% FCS (maintenance medium) starting from 10−1 to 108. Titrations were done in quadruplicate. Infected cells were incubated for 4 days at 37°C. Appearance of CPE was recorded daily. TCID50 was determined according to Reed and the Muench method [96-well microtitre plates containing 100 h method . 7 TCID50 per mL of virus was placed in individual wells of a 24-well plastic plates and allowed to dry at room temperature (22~25°C) and relative humidity of 40–50% . One hundred microlitre of MM was used to resuspend the virus at 0 hr, 3 hr, 7 hr, 11 hr, 13 hr, 24 hr, and up to 4 weeks and the residual virus infectivity was titrated. Controls in closed screw cap eppendorf tube were included each time and treated similarly but without drying.Ten microlitre of maintenance medium containing 10The experiment was repeated at different temperatures  and relative humidities  for 3 hr, 7 hr, 11 hr, 13 hr, and 24 hr. A nebulizer under a controlled condition was used to generate high and relative low humidity environment. All the experiments above were conducted in duplicate and the residual viral infectivity was titrated. μL of confluent FRhK-4 cells. 100 μL of serial 10-fold of dilutions of virus in maintenance medium starting from 10−1 to 108 was added into FRhK-4 cells. The infected cells were incubated at 37°C for 4 days. Appearance of CPE was recorded daily. TCID50 was determined according to the Reed and Muench method [The infectivity of residual virus was titrated in quadruplicate on 96-well microtitre plates containing 100 h method . 7 TCID50 per mL of virus was placed in individual wells of a 24-well plastic plate (representing a nonporous surface) and dried. The dried virus was then incubated at different temperatures  at different relative humidity  for 3 hr, 7 hr, 11 hr, 13 hr, and 24 hr and the residual viral infectivity was titrated. A similar experiment was conducted at room temperature and relative humidity of about 40–50% (air-conditioned room) for up to 4 weeks. Virus dried on plastic retained viability for up to 5 days at 22~25°C at relative humidity of 40~50% with only 1 log10 loss of titre (Ten microlitre of 10of titre . After t10 loss of titre at 24 hr (10) when compared with high temperature (38°C) at a lower relative humidity 80–90%  at comparatively low temperature (28°C and 33°C) did not affect the virus infectivity significantly . High teat 24 hr . However Figures . Viruses do not replicate outside living cell but infectious virus may persist on contaminated environmental surfaces and the duration of persistence of viable virus is affected markedly by temperature and humidity. Contaminated surfaces are known to be significant vectors in the transmission of infections in the hospital setting as well as the community. The role of fomites in the transmission of RSV has been clearly demonstrated . SurvivaIn the present study, we have demonstrated that SARS CoV can survive at least two weeks after drying at temperature and humidity conditions found in an air-conditioned environment. The virus is stable for 3 weeks at room temperature in a liquid environment but it is easily killed by heat at 56°C for 15 minutes . This inOur studies indicate that SCoV is relatively more stable than the human coronaviruses 229E or OC43 and some other viral respiratory pathogens such as respiratory syncytial virus. These findings suggest that, while direct droplet transmission is an important route of transmission , the rolFaecal contamination of SCoV coronavirus may thus be an effective route of transmission of the disease. The outbreak in Amoy Garden in Hong Kong which affected over 300 residents in a single-apartment block with thought to have been transmitted by contaminated sewage. The stability of the virus on environmental surfaces and its presence in faeces indicates the potential that fecal contamination of fresh-food production may pose a threat for virus transmission; especially in countries with poor sanitation and sewage disposal systems and that studies to address this possibility are needed. In this study, we showed that high temperature at high relative humidity has a synergistic effect on inactivation of SARS CoV viability while lower temperatures and low humidity support prolonged survival of virus on contaminated surfaces. The environmental conditions of countries such as Malaysia, Indonesia, and Thailand are thus not conducive to the prolonged survival of the virus. In countries such as Singapore and Hong Kong where there is a intensive use of air-conditioning, transmission largely occurred in well-air-conditioned environments such as hospitals or hotels. Further, a separate study has shown that during the epidemic, the risk of increased daily incidence of SARS was 18.18-fold higher in days with a lower air temperature than in days with a higher temperature in Hong Kong  and othe