Three more isolates of Lagos bat virus were recently recovered from fruit bats in South Africa after an apparent absence of this virus for 13 years. The sporadic occurrence of cases is likely due to inadequate surveillance programs for lyssavirus infections among bat populations in Africa. Lyssavirus genus in the Rhabdoviridae family. Rabies virus (RABV) was first isolated as a unique virus within this group. However, after the isolation of rabies-related viruses in Africa and Europe in the mid-1950s, the Lyssavirus genus was created, and rabies virus (genotype 1) was designated as the type-species member of the genus. At least 7 different major Lyssavirus species (genotypes) are recognized (Lyssavirus species (genotypes) are recognized in Africa. Of these, RABV (genotype 1) occurs worldwide, but LBV (genotype 2), Mokola virus (genotype 3), and Duvenhage virus (genotype 4) have not been encountered outside of Africa. Although RABV infection of bats is well known in the Americas, this virus has only been associated with infections of terrestrial mammals on the African continent. Mokola virus has also been isolated only from various terrestrial species, never bats  infection in fruit bats were identified in South Africa, 1 each in 2003, 2004, and 2005. LBV is a member of the LBV was first isolated from a fruit bat in 1956 in Nigeria , includiEpomophorus wahlbergi carcass was recovered in Durban, KwaZulu-Natal, after the bat was caught by a domestic cat. In August 2004, a resident of Umbilo, Durban, found a dead E. wahlbergi fruit bat on her lawn one morning after hearing squeaking noises around the house during the night. The fluorescent-antibody test (FAT), performed on brain material, was positive for lyssavirus antigens, and virus was isolated in both cases when suckling mice died 9–14 days after intracerebral injection with brain suspensions. Antigenic typing was carried out with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies . These analyses identified both new isolates as LBV (genotype 2)  . AdditioIn June 2005, a caretaker/gardener at a communal outdoor sports complex in the Bluff, Durban, found a bat on the lawns of the complex. At the time, birds were picking at it, and on closer inspection, it was found to be an immobile adult animal with a pup attached to it. The caretaker collected both bats and placed them in a nearby tree. Later, the bats, still attached to each other, were again found on the ground, where eyewitnesses also saw a cat toying with it. The animals were then presented to a local bat rehabilitator. The adult animal died and was submitted for diagnostic testing, but results of FAT carried out on brain smears were repeatedly negative. The pup had at least 1 evident bite wound, presumably from the cat, but otherwise appeared healthy and was cared for by the rehabilitator. Although the pup was reported to be feeding and doing well, it suddenly died ≈4 days after being found, on June 21, 2005. By this time, RT-PCR and nucleotide sequencing assays, carried out as described above, showed LBV in brain material from the adult. Antigenic typing was not performed because the level of lyssavirus antigen in the brain matter was undetectable. In the meantime, the carcass of the pup was recovered, and brain material was subjected to FAT and diagnostic RT-PCR. Although the RT-PCR results were inconclusive, the FAT results were negative.DNA sequencing information from each case was compared with nucleoprotein sequence information for LBV and other lyssavirus species (genotypes) available in the public domain (GenBank). ClustalW was used to produce sequence alignments and generate a phylogenetic tree . A graphAlthough LBV is rare and has not been reported in South Africa in 13 years, a small-scale passive surveillance effort in KwaZulu-Natal, South Africa, enabled us to identify 3 new isolations of LBV in a relatively short time. This finding reemphasizes our lack of understanding of the true prevalence of lyssaviruses in Africa because of poor surveillance for non-rabies viruses  throughout the continent. Human infections with LBV have not been documented to date; however, this virus has been reported in domestic animals (2 cats  and a do


To the Editor:Zika virus (ZIKV), a member of the family Flaviviridae, genus Flavivirus, was first isolated from the blood of a sentinel rhesus monkey from the Zika Forest of Uganda in 1948  has conducted surveillance for acute fever to determine causes of the infection among patients who seek health care at local clinics in Cambodia. Patients were enrolled by the health clinic physician after they gave informed consent in accordance with an institutional review board protocol approved by NAMRU-2 and the National Ethics Committee for Human Research of Cambodia. At enrollment, the physician administered a questionnaire and collected specimens (blood and throat swabs). All items were transported to the NAMRU-2 laboratory in Phnom Penh, where testing was conducted for a variety of viral, bacterial, and parasitic pathogens. In August 2010, a blood specimen was collected from a 3-year-old boy at a health clinic in Kampong Speu Province, Cambodia. The child’s reported clinical symptoms included 4 days of fever and sore throat and cough and a headache for 3 days. A maculopapular rash was not observed, and the boy was not hospitalized. The clinic staff conducted a follow-up interview and reported that the patient recovered fully.–ZIKV infection was confirmed in this patient by using PCR, sequencing, and serology and through virus isolation. ELISA for chikungunya and dengue virus IgM and IgG antibodies on acute- and convalescent-phase serum was negative. A universal flavivirus real-time PCR screen that targets the nonstructural (NS) 5 gene (The most common signs and symptoms reported in confirmed ZIKV infections are fever, headache, malaise, maculopapular rash, fatigue or myalgia, and arthritis and arthralgia . In addiThe clinical characteristics exhibited by this case-patient are similar to those of shown in a small cluster of ZIKV infections described in Indonesia during 1977–1978 in which maculopapular rash was not observed (The clinical features of ZIKV infection are similar to those of dengue virus and chikungunya virus infections, and both arboviruses are found in Southeast Asia. In this region, laboratory-based confirmation is essential. The extent of ZIKV infections in Cambodia is unknown; further studies are needed to clarify the prevalence and geographic distribution of ZIKV infection in the country.


SARS coronavirus injected intratracheally into chickens, turkeys, geese, ducks, and quail, or into the allantoic sac of their embryonating eggs, failed to cause disease or replicate. This finding suggests that domestic poultry were unlikely to have been the reservoir, or associated with dissemination, of SARS coronavirus in the animal markets of southern China. An outbreak of severe acute respiratory syndrome (SARS) occurred in Guangdong Province, People’s Republic of China, in November 2002 and spread to patients in 30 countries in Africa, Asia, Australia, Europe, and North and South America , turkeys (turkey enteric coronaviruses), cats (feline infectious peritonitis virus and feline enteric coronavirus), dogs (canine enteric coronaviruses), swine , cattle (bovine enteric and respiratory coronaviruses), mice (Murine hepatitis virus), rats , rabbits (rabbit coronavirus), and humans (respiratory and enteric coronaviruses) , 3-week-old domestic Pekin ducks (Anas platyrhyncos), 4-week-old chickens , 3-week-old turkeys , and 5-week-old Japanese quail (Coturnix coturnix japonicus) were each injected intratracheally with 106.2 mean tissue culture infective doses (TCID50) of Vero E6 propagated Urbani SARS-CoV per bird in a volume of 0.1 mL. The inoculum was the third passage in Vero E6 cells from the original throat swab specimen of the patient. The chickens were specific pathogen–free from an inhouse flock. The other four species were conventional birds obtained at 1 day  or 5 weeks of age (quail) from commercial hatcheries and raised on site. Oropharyngeal and cloacal swabs were obtained on days 0, 1, 2, 3, 4 and 10 after injection from five birds per group for virus detection by real-time RT-PCR and virus isolation on Vero E6 cells. RNA for RRT-PCR was extracted with the Trizol LS reagent  in accordance with the manufacturer’s instructions. Two hydrolysis probe type real-time RT-PCR assays, both targeting the ORF 1b gene, were optimized and run on a Smart Cycler  with the superscript platinum taq one-step RT-PCR kit . Real-time RT-PCR tests included negative  and positive (Vero E6 propagated SARS-CoV) controls. Two injected birds of each species were euthanized. After necropsy, their tissues were collected for histopathologic examination  and virus detection  on days 2 and 4 after injection, and at termination on day 10 after injection. For determination of infection, serum was collected on days 0 and 10 after injection from all birds and tested by indirect enzyme-linked immunosorbent assay for anti-SARS-CoV antibodies. Antigen used to coat plates was tissue culture propagated Urbani strain of SARS-CoV inactivated by γ irradiation  (-7 dilution; the real-time RT-PCR test detected SARS-CoV in three of three replicates at 10-5 to 10-6 dilution, depending on primer sets. The real-time RT-PCR assay detected virus in oropharyngeal swab specimens from two chickens on day 1 PI (To establish the comparative sensitivity of virus isolation and real-time RT-PCR tests, serial dilutions of SARS-CoV propagated in E6 Vero cell culture were tested for virus reisolation in E6 Vero cells and detection of replicase ORF 1b gene by real-time RT-PCR (day 1 PI . Real-tiThese findings suggest that poultry were unlikely to have been infected during the recent SARS-CoV outbreak and were unlikely to have played any role as amplifiers in the animal markets of southern China. The low level of virus detected by real-time RT-PCR from the chickens and the failure to isolate virus from embryonating chicken and turkey eggs suggest that the detected virus was residual inoculum or nonviable virus and that substantial virus replication in the poultry was unlikely. In addition, this SARS-CoV was of low tissue culture passage, i.e., third passage in Vero E6 cell, which minimized the potential for increased cell culture adaptation and concomitant decrease in vivo replication. Using the original or second tissue culture passage would unlikely have resulted in substantial replication in poultry. However, the virus used in these experiments, the Urbani SARS-CoV, had a 29-nt deletion in the genome. Whether the GZ01 human virus or those from civet cats and raccoon dog containing the extra 29 nt would infect and amplify in poultry would be of interest for future research.


Tombusvirus (family Tombusviridae) contain an IRE within the polymerase coding region of their genomes and this RNA element participates in both genome targeting to sites of replication and replicase complex assembly. Here we propose that other members of the virus family Tombusviridae also possess comparable IREs. Through sequence and structural analyses, candidate IREs in several genera of this family were identified, including aureusviruses, necroviruses, carmoviruses, and pelarspoviruses. The results from subsequent mutational analysis of selected proposed IREs were consistent with a critical role for these structures in viral genome accumulation during infections. Our study supports the existence of IREs in several genera in Tombusviridae and points to previously unappreciated similarities in genome replication strategies between members of this virus family.Internal replication elements (IREs) are RNA structures that are present at internal positions in the genomes of different types of plus-strand RNA viruses. Members of the genus  Poliovirus . Tombusvirus RNA replication is initiated by association of p33/p92 with an IRE, termed RII(+)-SL , located in the p92 coding region of the genome , positions RII(+)-SL close to another important replication element in the 3′-terminus, termed RIV . These united elements form a bipartite RII(+)-SL/RIV RNA platform that allows viral and host proteins to assemble into functional viral RNA replicase complexes , which is also present in other members of this genus, forms an extended stem-loop RNA structure containing a CC mismatch that is a key determinant of p33 accessory replication protein binding and RNA genome replication -SL IRE in the tombusvirus type species ic virus , thus thThe criteria used to define new candidate IREs in the other tombusvirids included (i) mfold-predicted formation of the IRE in full-length genomes, (ii) base pair covariation within the IRE that maintained predicted RNA structures, and (iii) conservation of internal loop motifs similar to that in RII(+)-SL in tombusviruses. Additional support for candidate IREs in necroviruses and carmoviruses came from the observation that their locations correlated with previously reported RdRp coding regions that exhibited greater than expected synonymous site conservation . Such coFigure 1C). For aureusviruses, a central motif involving C residues was also evident; however, depending on the virus, one of two variations, involving either a C/CUC or C/CC internal loop, was present . Thus, potential IREs in tombusvirids with tombusvirus-like RdRps contain a common motif corresponding to an internal loop with mismatched C residues. A similarly structured motif containing a CC mismatch was also identified in luteovirus-associated RNAs .The proposed IRE for strain D-like necroviruses contained a central core structure that somewhat resembled that in tombusviruses, i.e., it contained an internal loop with a CC mismatch that was bounded by an upper stem and potential CG base pair below . The second motif (M2), CAUXCC/GGZAGG , was more extensive and formed an internal loop bounded by CG base pairs. M2 somewhat resembled the tombusvirus motif, i.e., the X and Z residues were bounded by an upper CG base pair and a potential lower UA base pair (versus CG in tombusviruses) and in most cases either X or Z was a C residue . M2 was also present in strain A-type necroviruses, which have carmovirus-like RdRps , and for both carmoviruses and strain A-type necroviruses, the predominant mismatch at the XZ positions was UC or AC . Additionally, members of the pelarspovirus genus, which also possess carmovirus-like RdRps, harbored a similar M2 with a UC at the XZ positions . The XZ positions diverged from either UC or AC in only two carmoviruses. In Angelonia flower break virus (AnFBV), XZ was a CA mismatch, and in Pelargonium flower break virus (PFBV), XZ was a GU wobble pair . Further differences in M2 were found only in Cardamine chlorotic fleck virus (CCFV), where the CG bounding the upper part of the internal loop was a UG wobble pair and the AG mismatch in the internal loop was a CG pair . Overall, M1 was maintained in carmoviruses and strain A-type necroviruses but not in pelarspoviruses, while M2 was maintained in all three of these genera.For the large carmovirus genus, two distinct conserved motifs were identified within their corresponding extended stem-loop RNA structures. The first motif (M1) was U/AU containing a UA base pair and an unpaired U located in an upper region of the structure , the aureusvirus Cucumber leaf spot virus (CLSV), and the carmovirus Turnip crinkle virus (TCV) were selected for mutational analysis. Substitutions were introduced into the infectious clone of each of these viruses at degenerate codon positions within the proposed IREs. These substitutions did not cause alterations in the amino acid sequences of the encoded RdRps; with the exception of the A-to-C substitution in CLSV, which was a conservative glutamic acid to aspartic acid substitution . The mutant genomes were then transfected into cucumber protoplasts and the relative levels of mutant genome accumulation, versus that for the wild-type virus, were determined . For TNV-D, which is more similar to tombusviruses, all substitutions within the conserved internal loop motif that modified the conserved CC mismatch completely inhibited virus accumulation . In contrast, mutations outside the core motif were tolerated, indicating a critical role for the CC mismatch in TNV-D accumulation . Similarly, changing one of the C residues in the core motif in CLSV to either A or G residues abolished genome accumulation, while a substitution at a more distal site was tolerated .Having identified candidate IREs in several different genera of Figure 3C). The tolerance of some changes at this C position indicates a degree of flexibility in the motif, which is consistent with the observed naturally occurring A and U, but not G, substitutions at this position in other carmoviruses . Therefore, unlike tombusviruses, aureusviruses and strain D-type necroviruses, all of which maintain a C at this 3′-position of the internal loop, carmoviruses appear to have diverged and are flexible with respect to this requirement (a concept further confirmed by verifying the stable maintenance of the C-to-A and C-to-U substitutions in progeny TCV genomes from infections via sequencing). This difference in carmoviruses suggests a greater functional role for other residues within M2 and/or M1. Moreover, the conservation of comparable motifs in strain A-type necroviruses and pelarspoviruses indicates similar functional requirements in these viruses. Collectively, the results of the mutational analysis are consistent with the proposed roles for the candidate IREs in genome replication and suggest that these viruses share aspects of their genome replication strategies with tombusviruses.For TCV, changing the C residue in the AC mismatch to either A or U resulted in approximately 50% reductions in genome accumulation, whereas substitution with a G residue essentially eliminated genome accumulation  in virus reproduction.The results support our hypothesis that IREs are prevalent in 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.


Bovine viral diarrhea virus (BVDV) is one of the most important pathogens in cattle. Previously, BVDV sub-genotypes of 1b, 1c, 1d, and 1 m were detected in China. However, isolation of BVDV type 1a from cattle has not been reported in China. In 2010, twenty nasal swabs and blood samples were collected from the cattle suspected BVDV infection in Henan province, China. A BVDV isolate was isolated using cell culture, and the pathogenesis of the virus isolate was studied.Virus isolation was performed on MDBK cells. The virus identification was conducted by RT-PCR, neutralization test and immunofluorescence assay. In order to determine the genotype of the newly isolated virus, the 5′ un-translated region (5′UTR) of the virus isolate was cloned, sequenced and phylogenetically analyzed. To evaluate the virulence of the virus isolate, four BVDV sero-negative calves were intranasally inoculated with the virus suspension. Rectal temperatures and clinical signs were recorded daily. Blood samples were analyzed for changes in white blood cell counts, and tissue samples were taken for histopathology analysis.A new isolate of bovine viral diarrhea virus (BVDV), named HN01, was isolated from the nasal swabs using MDBK cell culture. The HN01 strain caused cytopathic effect (CPE) in MDBK cell cultures after two passages. The virus specifically reacted to BVDV1-specific monoclonal antibody in an immunofluorescence assay. A fragment of 288 bp of genome from this isolate was amplified by the RT-PCR. Phylogenetic analysis of 5′UTR indicated that the virus was BVDV 1a. In the pathogenesis study, four calves experimentally infected with the BVDV strain developed depression, cough and other clinical signs. Calves showed high temperature over 40°C, and white blood cell counts dropped more than 40%.A new subgenotype 1a strain of BVDV was firstly isolated from dairy cattle in China. The experimental infection showed that the virus was moderate pathogenic to cattle and can be used as a BVDV challenge virus to evaluate the efficacy of BVDV vaccines in the target animals. Bovine viral diarrhea virus (BVDV) is a great economically pathogen in cattle and other ruminants in the world -10. The Flaviviridae. The genome of the BVDV consists of a single positive-stranded RNA, which usually have a length of 12.3 kb [pro or E2 gene, BVDV strains can be divided into two different genotypes, BVDV1 and BVDV2 [BVDV belongs to the genus pestivirus with classical swine fever virus and border disease virus in the family  12.3 kb . Two bio 12.3 kb ,17. Basend BVDV2 . Each gend BVDV2 ,18-24. Rnd BVDV2 . BVDV1 snd BVDV2 ,23,26,27nd BVDV2 ,28, partnd BVDV2 -31, Indind BVDV2 , and occnd BVDV2 ,32-35.Virulence is both important to understanding the mechanisms of pathology and selecting the challenge strains for evaluation of a vaccine. Variation in virulence among BVDV2 strains has been extensively reported -38, but To the present, many subgenotypes of BVDV1 have been isolated and detected in China ,40,41. BIn this study, one virus was isolated from nasal swabs of cattle using MDBK cell cultures, and identified as a BVDV isolate by the virus neutralization test, reverse transcriptase-polymerase chain reaction (RT-PCR) method and immunofluorescence assay. To investigate the genetic subgroup of the strain, the 5′UTR gene of the virus was sequenced and compared with other 13 reference BVDV strains by phylogenetic analysis. The pathogenesis of the virus was evaluated by intranasally inoculating to four susceptible calves to assess the potential endemic risk to the cattle herd in China.A virus isolate was isolated from the nasal swab samples after two blind passages on MDBK cells. The MDBK cells inoculated with nasal swab samples developed obvious cytopathic effects (CPE) as cells appeared aggregated and formed net-like in the monolayers within 48 hours of incubation. The non-inoculated control cells did not show the CPE. Specific fluorescent staining showed the appearance in form of granules distribution all over the cytoplasm in the infected MDBK cells, when an indirect immunofluorescence test was conducted using BVDV1 type-specific monoclonal antibodies . The control cells did not show any fluorescence . The homology for nucleotide of 5′UTR is 95.3%, and with the strain SH1060 (JN248741) was 89.2%. This result indicates that the newly isolated BVDV strain, HN01, belongs to subgroup BVDV 1a of genotype BVDV1 , and subsequently 3 of four infected calves showed asthma and excessive salivation. Calves in the control group had no clinical signs during the experiment course. The rectal temperature of cattle #655 was over 40°C from 6 dpi to 8 dpi with the highest temperature of 41.4°C. The rectal temperature of cattle #642 was over 40°C at 2, 5 and 6 dpi with the highest recorded temperature of 40.1°C. The rectal temperature of animal #675 was over 40°C from 5 to 7 dpi with the highest temperature of 41.1°C. The rectal temperature of cattle #666 was elevated to 40°C from 2 to 4 dpi with the higest temperature of 40.3°C  at day 0 to 3.6 (1000 per mm3) at 4 dpi, a 58% reduction to the initial number. 642# decreased from 7.2 (1000 per mm3) (0 day) to 3.0 (1000 per mm3) (6 dpi), a 58% reduction to the initial number. The cattle of 675# decreased from 10.3 (1000 per mm3) (0 day) to 4.8 (1000 per mm3) (2 dpi), a 53% reduction to the initial number. The cattle of 666# decreased from 10.1 (1000 per mm3) (0 day) to 4.6 (1000 per mm3) (6 dpi), a 54.5% reduction to the initial number. Average greatest decline of WBC counts among the control animal group was less than 20%, which were significantly less (P < 0.05) than those of the challenge group  and one mock calf (#799) were euthanized at 8 dpi for histopathology analysis. Gross pathological findings included hemorrhages in the spleen and mesenteric lymph nodes. Histopathologic changes included thymic and spleen atrophy, the lymph node was lymphadenopathy and hemorrhage, and lymphoid depletion of the Peyer′s patches in all the infected calves , and 1 ml DMEM , with 3.5% horse serum was added. The infected-MDBK cell plates were checked daily and appearance of cytopathic effects (CPE) was observed and recorded. If the CPE was not found, the cultures were frozen and thawed twice and the clarified supernatant was passaged three times in MDBK cells. Un-infected MDBK cultures were included as negative controls and MDBK cells inoculated with BVDV NM01 strain, which was previously isolated and identified by our laboratory, was used as positive control. After 3-4 days of incubation at 37°C, with 5% CO2 supply, the virus was confirmed by immunofluorescence on cell monolayers as described below.Twenty nasal swabs and blood samples were collected from the cattle that showed mild respiratory clinical signs, such as nasal discharge and cough in Henan province, China in 2010. The nasal swab samples were collected and put into a tube containing 2 ml DMEM  supplemented with 10% horse serum , 150 μg/ml gentamicin sulfate , 7.5 μg/ml fungizone , and Streptomycin at 100 μg/ml. Three milliliter (3 ml) blood samples were collected using an EDTA vacuum blood tube from the jugular vein. All the samples were kept at 2-8°C and quickly transferred to the laboratory. Nasal swabs were inoculated into MDBK cell monolayers in 24-well tissue culture plates for virus isolation. Briefly, following centrifugation at 1500 rpm for 10 min, the samples were filtered through 0.45 μm membrane  and then inoculated onto the MDBK cell monolayer cultured in 0.5 ml DMEM  supplemented with 6% horse serum  in 24-well cell culture plates, and incubated at 37°C, with 5% CO2 supply, the plates were observed for CPE to determine if the virus isolate was neutralized by the BVDV antisera. The antisera positive control and negative control were also included in this test.To identify the isolated virus, virus neutralization (VN) test was conducted using antisera raised in cattle against BVDV1a NM01, a vaccine strain in our laboratory. Briefly, virus fluids were mixed with the equal volume hyperimmune antisera (antibody VN titer ≥ 1∶64) and incubated at 37°C for one hour, and then the mixture was transferred into MDBK cell cultures in the 96-well tissue culture plates. After 4 days of incubation at 37°C, with 5% CO2 supply. Then, the supernatants were dumped and plates were fixed with the mixture of 80% acetone and 20% methyl alcohol; the fixed plates were incubated with monoclonal antibody specific to anti-BVDV1 . Finally, the rabbit anti-mouse fluorescein isothiocyanate (FITC)-conjugated immunoglobulin G (IgG)  was added to the plates, then incubated in a 37°C humid box for an hour. The plates were examined for fluorescence under a fluorescent microscope .To detect the BVDV in the infected-MDBK culture, an immunofluorescent assay (IFA) was conducted as follows. Briefly, cell lysate was added to each of four wells of a 96-well MDBK tissue culture plate. Positive virus (NM01 strain) and DMEM media (negative control) were also added to 4 wells respectively in the test. Then plates were incubated for 4 days at 37°C, with 5% COPrimers  for 5′UTR gene sequence of BVDV were designed according to the BVDV strain Singer (GenBank accession number L32875) and strain NADL (GenBank accession number M31182). Total BVDV RNA was extracted from infected MDBK cell fluids using Trizol reagent  according to the manufacturer′s instructions. The reverse transcription polymerase chain reaction (RT-PCR) was carried out using 5 μl of RNA extraction samples and 1 μl of BVDV reverse primer as reverse transcription primer in a 0.2 ml tube for 5 min at 70°C; and the tube was added with 1 μl (200U) M-MLV reverse transcriptase RNase inhibitor and 2 μl RNAse-free water, and then was incubated for one hour at 42°C. The amplification of cDNA by PCR was carried out in a total volume of 50 μl solution containing 1 μl cDNA, 5 μl 10 × Buffer, 3 μl 2.5 mmol/L dNTP, 0.5 μl each primer, 0.5 μl Platinum Pfx DNA polymerase and 39.5 μl sterile water. Amplification was performed as follows: 95°C for 5 min, and then submitted to 30 cycles of amplification. The conditions for the amplification were 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, then with a final extension at 72°C for 10 min. The PCR products were detected by electrophoresis through a 1% agarose gel stained with ethydium bromide and visualized under UV light. The target fragment of PCR products were purified and cloned into pMD18-T vector , and then transfected into E.coli DH5α cells. The positive clones were selected for sequencing by Shanghai Invitrogen Biotechnology Co., Ltd.The sequence analysis was performed by computer software DNASTAR  and MEGA5.1. The 5′UTR gene sequence of the virus isolate was evaluated for further sub-genotype classification and compared to 13 BVDV reference sequences .White blood cell (WBC) counts were conducted from 2 days pre-challenge through 8 days post-challenge and white blood cell (WBC) counts were conducted by a Vetscan HM5 veterinary hematology system . Deep nasal swab specimens were collected at 1 day prior to challenge through 8 days post-challenge. After collection, swabs were placed into a tube containing 3 ml of transport medium, consisting of DMEM  supplemented with 10% horse serum , 150 μg/ml gentamicin sulfate , 7.5 μg/ml fungizone and streptomycin at 100 μg/ml. The samples were kept at 2-8°C and quickly transferred to the laboratory. Upon arriving in laboratory, all swab specimens were stored at -70°C or below until they were cultured for virus isolation.On days 8 dpi, two calves in the infected group and one in the control group were euthanized; tissue samples of liver, spleen, lung, heart, kidney, intestine, mandibular lymph node, and mesenteric lymph node were collected and fixed in 10% buffered formalin for histopathologic analysis in Inner Mongolia agricultural university.The authors declare that they have no competing interests.WW participated in the design and conducted the majority of the experiments in the study and drafted the manuscript. XS and QT participated in the molecular genetic studies and the sequence alignment. YW and YJ took the animal experiment and collected samples. MX was performed the statistical analysis. WX and HW conceived of the study, and participated in its design. All authors read and approved the final manuscript.


Scheffersomyces segobiensis) with a derived nuclear genetic code where CUG codes for serine. The genomic architecture and phylogeny are consistent with infection by a double-stranded RNA virus of the genus Totivirus. We provide evidence of past or present infection with totiviruses in five species of yeasts with modified genetic codes. All but one of the CUG codons in the viral genome have been eliminated, suggesting that avoidance of the modified codon was important to viral adaptation. Our mass spectroscopy analysis indicates that a congener of the host species has co-opted and expresses a capsid gene from totiviruses as a cellular protein. Viral avoidance of the host’s modified codon and host co-option of a protein from totiviruses suggest that RNA viruses co-evolved with yeasts that underwent a major evolutionary transition from the standard genetic code.Among eukaryotes with modified nuclear genetic codes, viruses are unknown. However, here we provide evidence of an RNA virus that infects a fungal host ( Candida albicans and wood-digesting species such as Scheffersomyces stipitis) has been reassigned from leucine to serine such that the serine tRNA possesses both derived serine and ancestral leucine sequence motifs  and are characterized by an overlapping open reading frame between the capsid gene and the RdRp gene with a programmed ribosomal frameshift. The family (Totiviridae) that contains the totiviruses is ancient with a broad eukaryotic host range and extensive co-evolution in the fungi . Totivirstipitis . There aScheffersomyces segobiensis (Santa Maria & Garcia Aser) Scheffersomyces coipomoensis  Urbina & Blackwell, 2012 comb. nov. NRRL Y-17651 (Type strain). Thomas Jeffries  with an inoculum (single colony) of cells from streaked YPD agar plates.Freeze-dried culture stock was obtained from the USDA ARS Culture Collection for Jeffries  providedTotal nucleic acids  were extracted from whole cells . We puriS. segobiensis were isolated from a CsCl gradient fraction with a density of 1.40 g/cc .The tentative totivirus and satellite virus dsRNAs from .40 g/cc , which iDe novo RNA sequence assembly was carried out in CLC Assembly Cell 4.06 (http://www.clcbio.com) on an Apple Macintosh Mac Pro Xeon 64-bit workstation. The putative viral contigs were reassembled for mapping purposes using the reference assembly algorithm and the de novo contigs as references. A total of 409665 reads were mapped to the totivirus with an average coverage of 4404.80 times.We extracted total RNA from yeast cells using the Masterpure yeast RNA purification kit (Epicentre) and an RNAse-free DNAse treatment. RNA-seq was used to sequence the putative RNA virus, examine the tRNA expression of the host, test for host expression of known fungal viral sequences, and isolate host protein-coding sequences for bioinformatics analysis. Ribosomal RNA species were removed using the Ribo-zero Magnetic Gold kit (Epicentre). We then prepared an RNA-seq library using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre). Libraries were quantified using the Agilent 2100 Bioanalyzer RNA 6000 Pico Chip and submitted to the University at Buffalo Next Generation sequencing facility for RNA sequencing. The facility carried out RNA-seq using 50-cycle paired-end runs on two flow cells of an Illumina HiSeq 2000. http://www.geneious.com/).The putative viral sequence was confirmed by Sanger sequencing using random and specific primers for cDNA library construction. We used the Takara 5′-Full RACE Core Set to expand sequence. Sanger and next generation sequences were compared using Geneious version 5.6.3 created by Biomatters (available from http://www.454.com) sequencing with GS FLX Titanium series reagents of a DNA library from Scheffersomyces coipomoensis. This form of sequencing also permitted multigene bioinformatics analysis of the host protein coding genes. Strain identity of the assembly was confirmed by BLAST analysis , yielded 14.7 Mb of aligned bases (488 contigs) with an average peak depth of 12X.Because endogenous RNA viruses of fungi can be fragmented and differ greatly in their nucleotide sequences from known viruses , PCR proanalysis  of the nS. segobiensis), and segoSvLgap3R GTACACCAAGGTTAGTAGACAAG (RdRp of virus from S. segobiensis). cDNA synthesis was performed at 48 °C for 30 min, followed by 15 min at 94 °C for reverse transcriptase deactivation and Taq activation. DNA only reactions were added to the thermal cycler 2 min before the end of the previous 94 °C step to activate the Taq polymerase. PCR amplification was done for 35 cycles of 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 1 min. A final extension at 72 °C for 10 min was performed. New sequences from this study have the following Genbank accession numbers: KC610514, KC616419-KC616429.To establish that the virus was coded by exogenous RNA and not by the DNA of the host, we compared RT-PCR and PCR products. For RNA templates, DNase-treated extracts were exposed to RT-PCR using the Qiagen one step RT-PCR kit. For DNA templates, nucleic acid extracts were exposed to PCR by excluding reverse transcriptase from the RT-PCR protocol. We amplified a fragment of the single copy xylose reductase gene as a positive control for the PCR of DNA. Primers used were: segoxylF CTGTTCTGAACAGATCTACCGTGC (xylose reductase), segoxylR AAGTATGGGTGGTGTTCAACTTGC (xylose reductase), SvLgap3F CGCAATACGACCAGGAGATTG (RdRp of virus from Saccharomyces cerevisiae virus L-BC(La) as queries and E_values <1e−05. We searched the non-redundant (nr) peptide sequence database  and the Department of Energy Joint Genome Institute (J.G.I.) genome browser for matches. Fossil or paleoviral copies of Totivirus-like genes were identified by significant BLAST tblastx hits  of relevant NCBI databases using the sequences of Saccharomyces cerevisiae virus L-BC(La). Duplicated capsid gene copies adjacent to the complete integrated viral genomes in the assemblies of S. stipitis and D. hansenii were assumed to be paralogs (I) + gamma parameter for among-site rate variation (G) was the best fit under a Bayesian Information Criterion (BIC). For reliability estimates we used SH-like approximate likelihood ratio tests  and satellite virus of S. cerevisiae  of similar length to the dsRNA on the gel. Assemblies from Sanger sequencing of the RNA virus agreed with the assembly using Illumina RNA sequencing, but the 5′ UTR was complete only in the Illumina assembly. The assembled virus had the genomic architecture of totiviruses with overlapping capsid and RdRp open reading frames flanked by 5′ and 3′ UTRs (S. segobiensis (S. segobiensis) genome, but RT-PCR did amplify an RNA copy of the viral gene. The results support the existence of an exogenous RNA virus in S. segobiensis with the genomic architecture of a totivirus.Because the fossil viruses in yeast have a similar architecture to dsRNA totiviruses , we carr 1.2 kb, . Viral p 1.2 kb, . No such 3′ UTRs . We iden 3′ UTRs . The fivobiensis , suggestobiensis . HoweverS. segobiensis. A BLAST blastp analysis of the RdRp-like ORF yielded a conserved domain match (E = 1.56e−58) to RdRP_4, a viral RNA-directed RNA-polymerase family that includes “RdRPs from Luteovirus, Totivirus and Rotavirus”. The best expect value (E = 2e−137) obtained was the totivirus, Saccharomyces cerevisiae virus L-BC(La), with an identity of 37% of residues. The RdRp gene phylogeny (Scheffersomyces segobiensis virus L within the totiviruses and most closely related to Saccharomyces cerevisiae virus L-BC(La). Support values are strong enough to rule out random error as an explanation for the evolutionary position of Scheffersomyces segobiensis virus L within the totiviruses. The less conserved capsid gene tree  that had a significant match to the killer satellite K2 virus of S. cerevisiae. An RTPCR with specific primers confirmed that the contig was not coded in the DNA genome of the yeast. The existence of a satellite virus bolsters the evidence for a totivirus in the CTG clade.As with other totiviruses, we found evidence that Scheffersomyces segobiensis virus L originated from exogenous totiviruses that use a standard genetic code. The RdRp permits the deepest assessment of evolution, and it reveals a derived position of the Scheffersomyces segobiensis virus L at the tip of the standard code totivirids. The known paleoviruses in the CTG clade are distantly related to Scheffersomyces segobiensis virus L and lack the ability to produce the fusion gene product typical of totiviruses  is most closely related to the fossil virus in the CTG yeast S. stipitis rather than the virus of another genus of basidiomycete, Xanthophyllomyces. Horizontal transfer of paleoviruses could be a source of some evolutionary noise, but this process appears rare in the CTG clade. The timescale of the totivirus evolution is difficult to estimate because of the dearth of reliable fossil calibrations for fungi  is underrepresented compared to the standard code yeast  versus third position base composition , Scheffee codons . In S. calbicans , and seeScheffersomyces segobiensis virus L is consistent with adaptation to offset the functional effects of the genetic code shift in the host. But, our results also indicate that the endogenization of viral genes by host yeasts of both genetic codes is more common than previously thought. Presently, it is unknown if these endogenous non-retroviral genes function as proteins or are merely transcriptional noise. Here, we show that at least one of these genes derived from a totivirus is expressed as a protein. Isolation of proteins migrating between 73 kDa and 92 kDa from S. stipitis yielded approximately 535 proteins, as estimated from the equivalent size range of proteins in S. cerevisiae. The mass spectroscopy analysis was able to unambiguously identify the capsid protein (cap) of Saccharomyces cerevisiae virus L-A in the S. cerevisiae control and 153 proteins in S. stipitis. By this method, we were able to detect one of the four Non-retroviral integrated RNA virus (NIRV) capsid polypeptides from S. stipitis. S. stipitis capsid4 and Saccharomyces cerevisiae virus L-A cap. All of the S. stipitis virus-like peptides had probabilities of 0.999 or greater. Our inability to detect the remaining cap proteins from S. stipitis may be due to the lack of sensitivity of the method used. We were able to detect a single tryptic peptide from Saccharomyces cerevisiae virus L-BC(La) cap, which is present at about one tenth of the concentration of Saccharomyces cerevisiae virus L-A cap. Our results indicate that the co-option of a non-retroviral RNA viral protein has occurred as with retroviral proteins  or untreated (lane 2) with an RNAse cocktail. Lane 3 is a DNA size marker labeled in kilobases. The gel was electrophoresed on a 1.4% agarose gel and stained with ethidium bromide.Click here for additional data file.10.7717/peerj.50/supp-2Fig. S2S. segobiensis of density 1.40 g/cc, comprised of the same nucleic acid band sizes (L and M) isolated by dsRNA CF11 chromatography. The DNA marker in lane 2 is labeled in kilobases.The first lane shows the total nucleic acids extracted from CsCl purified viral particles from Click here for additional data file.10.7717/peerj.50/supp-3Fig. S3Scheffersomyces segobiensis shows conserved residues, whereas the paleoviral copies of capsid proteins (below the dashed line) reveal weakened conservation of these residues. Gray shading indicates a residue proposed to be functionally important from structural evidence. Dark shading indicates residues that function in decapping as evidenced by mutagenesis experiments. His154 is thought to be part of the active site of the cap-snatching activity.The virus in Click here for additional data file.10.7717/peerj.50/supp-4Fig. S4S. segobiensis, and primers targeting a 365 bp region of the RdRp gene from the viral RNA. Lane 2 contains RT, Taq polymerase, no nucleic acid template, and the viral RdRp primers used in lane 1. Lane 3 contains Taq polymerase, total nucleic acid from S. segobiensis, and the same primers as lanes 1 and 2. This is a control against amplification of the RdRp target from the genomic DNA of S. segobiensis. Lane 4 contains Taq polymerase, whole nucleic acid from S. segobiensis, and primers targeting a 504 bp region of the host gene XYL1, xylose reductase. Lane 5 contains DNA markers. Band size labels are in base pairs.Lane 1 contains M-MLV reverse transcriptase (RT), Taq polymerase, DNase-treated nucleic acids from Click here for additional data file.10.7717/peerj.50/supp-5Fig. S5S. segobiensis is from the present study and the other sequences are from reference NCBI genomes.The tRNA shows strong conservation and a chimeric composition of serine identifiers with the CAG anticodon. The sequence of Click here for additional data file.10.7717/peerj.50/supp-6Fig. S6(A) Sequence of a totivirus capsid-like protein (cp4) isolated from Scheffersomyces stipitis. Tryptic peptides demonstrated in S. stipitis proteins are shown in bold and underlined. (B) Sequence of the capsid protein from Saccharomyces cerevisae virus L-A. Tryptic peptides demonstrated are shown in bold and underlined.Click here for additional data file.10.7717/peerj.50/supp-7Table S1Fungal strains and NCBI accession numbers used for isolating protein sequences for the bioinformatics analyses of fungi.Click here for additional data file.10.7717/peerj.50/supp-8Table S2Click here for additional data file.


Charlie Calisher is a great storyteller, and he probably has more stories to tell than anyone who has worked in the field of arbovirology. Throughout his 30-year distinguished virology career beginning at the Centers for Disease Control and Prevention to his current role as professor emeritus at the Colorado State University, Calisher has been a prolific researcher, writer, teacher, and mentor. The title phrase “lifting the impenetrable veil” originally appeared in a letter written in 1900 by Dr. Walter Reed to his wife, when he realized that he and his colleagues had shown that yellow fever was caused by a virus that was transmitted by mosquitoes. In his book, Calisher has recounted his version of arbovirus history and discovery in his unique blend of academic rigor and humorous personal interpretation.Established as a field in the late 1950s, arbovirology is the study of arboviruses (arthropod-borne viruses). Lifting the Impenetrable Veil is organized into an introduction, 16 chapters, and an appendix that provides 25 biographic profiles of arbovirology researchers. Throughout the book the reader catches glimpses of the many colorful characters who defined arbovirology, some in shaded insets that Calisher uses to provide humorous anecdotes, usually involving himself.I enjoyed the stories behind the many viral discoveries, especially the many lessons learned from the expansion of Venezuelan equine encephalitis virus into the Americas, the importation of West Nile Virus in the United States, and dengue reemergence worldwide. I appreciated learning about the pivotal role of the Rockefeller Foundation, founded in 1912, in professionalizing international health. I valued the clear explanations about the development of various laboratory procedures and the role they played in discovering and characterizing new viruses identified from the early 1900s to 2012. I even valued reading about the early development and current state of viral taxonomy. Most of all, I appreciated learning about fundamental epidemiologic and ecologic observations that helped elucidate aspects of arbovirus disease transmission, such as virus overwintering, transovarial transmission, and virus transmission by migrating birds. The later chapters read somewhat like a parade of researchers; however, it was still intriguing to read about these public health professionals and their pursuit of viral hemorrhagic fevers, bat-borne viruses, and Schmallenberg virus.I once had the good fortune of meeting Dr. Calisher and feel that reading his book is not unlike listening to him in person; i.e., you can’t help but smile and learn from his informative, opinionated, yet always down-to-earth storytelling. The experience is eclectic and memorable. Be warned: this book has minor shortcomings that are typical of self-published books, such as uneven editing. However, I strongly recommend this book for anyone interested in the history of virology, particularly researchers in the field of arbovirology.


Human cells are known to express many chimeric RNAs, i.e. RNAs containing two genes' sequences. Wondering whether there also is trimeric RNA, i.e. an RNA containing three genes' sequences, we wrote simple computer code to screen human expression sequence tags (ESTs) deposited in different public databases, and obtained hundreds of putative trimeric ESTs. We then used NCBI Blast and UCSC Blat browsers to further analyze their sequences, and identified 61 trimeric and two tetrameric ESTs (one EST containing four different sequences). We also identified 57 chimeric, trimeric or teterameric ESTs that contained both mitochondrial (mt) RNA and nuclear RNA (nRNA), i.e. were mtRNA-nRNA fusions. In some trimeric ESTs, the downstream partner was fused to the poly-A tail of the upstream partner, which, together with the mtRNA-nRNA fusions, suggests a possible new mechanism for RNA fusion that occurs after both transcription and splicing have been terminated, and possibly outside the nucleus, in contrast to the two current hypothetical mechanisms, trans-splicing and transcriptional-slippage, that occur in the nucleus. The mt-sequences in the mtRNA-nRNA fusions had pseudogenes in the nucleus but, surprisingly, localized mainly in chromosomes 1 and 5. In some mtRNA-nRNA fusions, as well as in some ESTs that were derived only from mtRNA, the mt-sequences might be cis- or trans-spliced. Actually, we cloned a new cis-spliced mtRNA, coined as 16SrRNA-s. Hence, mtDNA may not always be intron-less. Fusion of three or more RNAs to one, fusion of nRNA to mtRNA, and cis- or trans-splicing of mtRNA should all enlarge the cellular RNA repertoire, in turn enlarging the cellular functions. Therefore, future experimental verification of the existence of these novel classes of fusion RNAs and spliced mtRNAs in human cells should significantly advance our understanding of biology and medicine. The swift spread of high throughput RNA sequencing technology in recent years has led to identification of tens of thousands of putative chimeric RNAs, which generally are considered as RNA molecules containing two genes' sequences. While a few hundreds of these chimeras are known to be transcribed from fusion genes that are formed due to genetic alterations, such as chromosomal rearrangements and genomic DNA deletion or amplification A significant percentage of putative chimeric RNAs encompass a short homologous sequence (SHS) shared by the two partners Since two RNA molecules could fuse to one, we wondered whether three RNAs could also fuse to one. In this study, we identified some expression sequence tags (ESTs) that contained three genes' sequences, thus coined as trimeric ESTs or trimeras. We also identified some ESTs that contained both mitochondrial (mt) RNA (mtRNA) and nuclear RNA (nRNA). In some ESTs, the downstream partner was fused to the poly-A tail of the upstream partner. Since mtRNA are not transcribed in the nucleus and polyadenylation occurs after both transcription and splicing are terminated, these findings are inklings of the existence of a previously unsuspected mechanism for RNA fusion, besides the hypothetic trans-splicing and transcriptional-slippage. The possible existence of trimeras and mtRNA-nRNA fusions in human cells suggests that the whole cellular RNA repertoire may be much larger than what we have known, which in turn enlarges cellular functions.We designed simple computer code to screen preliminarily EST and mRNA collections in the NCBI database and obtained hundreds of putative trimeric ESTs. We then used the NCBI Blast and UCSC Blat browsers to analyze the sequences of these ESTs and identified 61 trimeric ESTs , such asSome trimeras or tetrameras, such as BQ638079, contained two or three nRNA elements that were derived from the same chromosomal locus but were not linear, i.e. not a cis-spliced product ; likely,We also identified 57 ESTs that were nRNA-mtRNA fusion, i.e. contained both nRNA and mt-sequence . In mostMost of these mt-sequences were better matched to the mtDNA than to their nuclear counterparts, as exemplified by BF306729 , and thuSome chimeric and trimeric ESTs listed in In four mt-only ESTs, the mt-sequences were not linear along the mtDNA, thus likely being trans-spliced products. Besides the light (L) strand containing BE898652 shown in In a fusion RNA, the two neighboring partners have three different relationships , i.e. 1)Intriguingly, in some ESTs the gap was actually a poly-A sequence, or a poly-T when the upstream partner was reverse-complementary to the mRNA, indicating that the downstream partner was fused to the poly-A tail of the upstream partner, such as in the AU142287 in which the mt-sequence was fused to the poly-A tail of the upstream nRNA . The polth nt region of the mtDNA, deleted from the 16S rRNA, thus coined as 16SrRNA-s (We performed reverse transcription (RT) and polymerase chain reaction (PCR) followed by T-A cloning and sequencing to verify those trimeric or terameric ESTs shown in 6SrRNA-s . The sam6SrRNA-s . BecauseThis study reports, for the first time, the existence of trimeric and tetrameric ESTs. Although our RT-PCR studies failed to confirm the expression of seven of these fusion RNAs in HEK293 cells, it cannot be excluded that these seven and other trimeras and tetramers are expressed in other situation or in other cell types. How the three or four different sequences are fused into one is unknown. It remains possible that the mechanism for formation of chimeras, trimeras and tetrameras is similar, no matter whether the fusion occurs as a cellular event or as a technical artifact.This study is also the first report on ESTs that are mtRNA-nRNA fusions. If this type of fusion truly exists and if the involved mt-element is truly derived from mitochondria, but not from its nuclear counterpart (i.e. NUMT), it suggests that two different pools of RNA, i.e. nRNA and mtRNA, can join together to constitute a previously unaware mechanism to enlarge the cellular RNA repertoire. Since most part of the transcript from the L-strand is known to be non-coding, it is possible that some parts of the L-strand transcript may be used as a resource for mtRNA-nRNA fusion to enlarge the ensemble of the cellular RNA, which is supported by a recent report of the existence of mtRNA antisenses www.ornl.gov/sci/techresources/Human_Genome/project/info.shtml), it seems that mtRNA has a much higher frequency than nRNA to form fusion RNA, no matter whether the fusions are genuine or spurious. This may partly be because a set of processed mtRNAs are not polyadenylated Considering that mtDNA encompasses only about 16.5 kb whereas the human genome contains 3164.7 million base-pairs (bp)  and UCSC Blat browsers (http://genome.ucsc.edu/) to analyze the sequence of those putative trimeric ESTs. Since some of the chimeric, trimeric and tetrameric ESTs contained mt-sequences and since mtDNA was highly polymorphic, different mtDNA sequences (with access number provided if not the UCSC reference) were used as the reference to get the best match during sequence alignment. Therefore, the exact nt numbers and locations might slightly differ from different publications.We wrote simple computer code and used it to screen preliminarily the EST and mRNA collections in the NCBI database to identify putative trimeric RNAs. We then used NCBI Blast (5′-GGTGATAGCTGGTTGTCCAA-3′) as the forward primer and mtR3071 (5′-GAACTCAGATCACGTAGGAC-3′) as the reverse primer. All PCR products were fractioned in 0.9–1.2% agarose gel and visualized by ethidium bromide staining. The band of interest was excised out and purified using a simple method we described before http://www.genewiz.com) for sequencing. The details of these methods were described before We cultured HEK293 and Hela cells in the routine fashion and extracted total RNA from whole cell lysates with Trizol as described before


Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer.FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV) and 3 strains of border disease virus(BDV) in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b.we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China) using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE), indirect immunoperoxidase test (IPX) and electron microscopy(EM). The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer. Bovine viral diarrhea virus, a single-stranded RNA is found in cattle and other ruminants worldwide -4. The ppestivirus within the family Flaviviridae. BVDV is closely related to the classical swine fever virus (CSFV) and the ovine border disease virus (BDV) [pro, C, E0 , E1, E2, NS2/3, NS4a, NS4b, NS5a and NS5b by viral and cellular proteases [The Bovine viral diarrhea virus (BVDV) belongs to the genus us (BDV) . The pesroteases . E0 protAlthough BVDV from the skia deer has been seriously concerned, there are only a few articles with respect to the prevalence of BVDV investigations and BVDV clinical sign , ParticuThe significant cytopathic effects (CPE) were observed in MDBK cell infected with virus 24h-48h. The MDBK cells generate obvious cell lesion and net between cells in comparison to the control cells, which is consistent with CPE of BVDV on MDBK cell Figure . The IPXThe MDBK cell infected with CCSYD were positive by the RT-PCR assays, and the expected sizes of the PCR products, 706 bp for E0 of CCSYD, were observed as clear electrophoretic band Figure . The obtPestivirus from cattle, swine and ovine in GenBank were used to construct phylogenetic trees. Figure To better understand the relationship of CCSYD to other 9 strain of BVDV, 7 strains of CSFV and 3 strains of BDV variants co-circulating in the word, genetic sequences of In the present study, CCSYD strain was isolated from skia deer in Changchun city. By employing a series of biochemical and biophysical methods, we have firstly identified that CCSYD might be BVDV. The BVDV can be classified into biotypes and genotypes . Biotype0 protein of CCSYD construction a DNA vaccine might induce cellular immune response and antibody responses specific for BVD from skia deer. Therefore, isolation and identifcation of a skia deer bovine viral diarrhea virus (CCSYD) contributes development new BVDV vaccine to prevent and control of BVDV in sika deer.The protection conferred by conventional inactivated BVDV vaccines is strongly correlated with genetically and antigenically of specific BVDV strain. In addition to, an increasing frequency of skia deer with elevated antibody titers to BVDV suggests that exposure to field strains has not been diminished despite the use of both management and vaccine . This suThe present study described the isolation and identifcation of a skia deer bovine viral diarrhea virus (CCSYD) isolated from sick skia deer for the first time in China. This study provided a detailed analysis of the genetic and evolution of CCSYD which is likely to be helpful to guide efficient diagnostic, preventive and control strategies against BVD from skia deer in China.24V as positive control.Field samples came from skia deer at field of Changchun (China), which might infected with BVDV according to clinical sign such as diarrhea and miscarriage and stillbirth. Skia deer liver was collected from an aborted fetus deer. A total of 1 g of fresh liver tissue was homogenized in 4 ml of phosphate-buffered saline  and then repeated freeze thawed 3 times, and centrifuged at 5000 rpm for 30 min. A certain liver extract was then inoculated onto MDBK monolayer cultures, 25 cm flasks with a 1 ml inoculum  and the total volume was 5 ml. The MDBK cultures were observed for 6 days with presence or absences of CPE recorded. The cultures were frozen at 70°C, thawed and supernatants collected after centrifugation with subsequent storage at 70°C. Uninoculated cultures were included as negative controls and inoculated C2, 37°C. Plates were fixed and dried, then stained with immunoperoxidase as described by Meyling [BVDV was detected by indirect immunoperoxidase test (IPX) and electron microscopy technique (EM). For IPX, the test was carried out on 96-well plates using low-passage MDBK cells. Serum 20 μl) was added to each of four wells before the addition of 100 μl of cell suspension. Positive and negative control sera were run on each plate. The test plates were incubated for 4 days in 5% CO μl was aTotal RNA was isolated from infected MDBK cell using TRIzol reagent (Invitrogen China) according to the manufacturer's protocol and as described in the online supplement. In short, 200 μl infected MDBK cell was incubated with 1 ml TRIzol for 5 min at room temperature (RT). Cell debris was removed by centrifugation  and 0.4 ml chloroform was added. After vortexing the mix was incubated for 5 min at RT. The phases were separated by centrifugation  and the aqueous phase was transferred to a new tube. 0.6 × volume of isopropyl alcohol and a 0.1 × volume of 3 M sodium acetate were added to this aqueous phase and incubated for 10 min at 4°C. The precipitated RNA was pelleted by centrifugation  and after the removal of the supernatant the RNA pellet was washed twice with 70% ethanol. After drying, the RNA was resuspended in 30 μl DEPC-treated water. Using mRNA as template, single-stranded cDNAs were generated by Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's directions. The E0 primer sequences were as follows: sense prime: 5'-CCGGATCCACCATGGAAAACATAACACAGTGG-3'; anti-sense prime: 5'- GCCTCGAGTTAAGCGTATGCTCCAAACCACGT -3'. The PCR conditions were 94°C for 3 min, followed by 30 cycles of DNA amplification  and 8 min incubation at 72°C. PCR products were separated by electrophoresis at a constant voltage (2 V/cm) in a 1.2% (w/v) agarose gel. The full-length E0 gene subjected to DNA sequencing.The sequence data were analyzed with computer programs such as DNAMAN and DNASTAR. Phylogenetic analysis was done by the distance-based Neighbor-joining method using software EGA 4.1. (DNAStar Inc.).BDV: Border Disease Virus; BVD: Bovine viral diarrhea; BVDV: Bovine Viral Diarrhea Virus;CPE: Cytopathic Effects; CSFV: Classical Swine Fever Virus; DNA: Deoxyribonucleic Acid; EM: Electron Microscopy; IPX: Indirect Immunoperoxidase; MDBK: Bovine Kidney; PCR: Polymerase Chain Reaction; RNA: Ribonucleic Acid.The authors declare that they have no competing interests.YG and PZ participated in the design and conducted the majority of the experiments in the study and drafted the manuscript. RD and SW contributed to the interpretation of the findings and revised the manuscript. QW and NW edited the manuscript. CS performed analyses of data. All authors read and approved the final manuscript.


It typically causes an acute febrile illness, with joint pain and a skin rash. Chronic arthropathy may develop , severe finger joint and muscle pains, mild headache, and a skin rash, and he sought treatment at the Prince of Wales Hospital (PWH) Infectious Diseases Clinic on the second day of his illness. Physical examination showed a generalized erythematous rash over the trunk and limbs and petechiae over the lower limbs. Mild finger joint stiffness was observed, but no joint swelling. No lymphadenopathy or eschar was detected. Level of C-reactive protein was elevated at 10.4 mg/L. Results of screens for malaria and dengue were negative. Results of other routine assessments were unremarkable. His symptoms subsided gradually within a week.Serum specimens taken on days 2 and 6 were positive for chikungunya virus RNA by in-house reverse transcription (RT)-PCR at the Public Health Laboratory Service (PHLS)  and PWH laboratory (targeting both NSP-1 and the envelope glycoprotein [E1] gene). An additional serum sample taken on day 8 of illness, received by PHLS only, was also positive for chikungunya RNA. Both laboratories confirmed RT-PCR results by sequencing. At PWH, phylogenetic analysis was performed to determine the likely origin of the virus. In-house immunofluorescent slide serologic assays developed at PHLS found chikungunya immunoglobulin G (IgG) titers of <10, 160, and 320 in the serum samples taken on days 2, 6, and 8 of illness, respectively, and detected chikungunya IgM in the day 8 serum. The acute cytokine immunologic response to this virus was also tested (Appendix).Sequencing and phylogenetic analysis was consistent with an imported infection, almost certainly originating from the current chikungunya outbreaks in the Indian Ocean. Phylogenetic analyses of the NSP-1 and E1 regions, indicated that this virus is most closely related to previous African rather than Southeast Asian chikungunya viruses . The perThe most striking finding from the cytokine analysis  is the h,,Interpretation of the significance of these cytokine results is necessarily speculative. Some comparison can be made with other viral infections. In severe acute respiratory syndrome–associated coronavirus (SARS-CoV) (–The function of CXCL10 is to act as a chemoattractant for Th1 cells in the activation of cell-mediated immune response. Its expression can be up-regulated by the Th1 cytokine IFN-γ during acute inflammation. CXCL10 has been implicated in the pathogenesis of SARS-CoV and H5N1 influenza infections, in which persistently high CXCL10 concentrations seem to correlate with disease severity and progression (


Dengue virus infection is a major concern in several countries, and more than 50 million people are infected worldwide each year. Thailand is one of the countries where people are susceptible to infection due to favourable geographical and environmental conditions. In this retrospective study, we reported the changing pattern of dengue virus serotypes during the period between 2004 and 2010. The following percentage prevalence showed different serotypes of dengue virus (DENV) predominant in respective years: DENV1 in 2004 (56.41%), DENV4 in 2007 (50%), DENV1 in 2008 (57.41%), and DENV3 in 2010 (38.7%). Moreover, the major serotypes were not stable as they showed a shift from one serotype to another. We also found co-infection with two different serotypes and reported the clinical manifestations, which were not different from infection with a single serotype. Co-infection with various serotypes may not necessarily cause more severe disease. Dengue virus (DENV) is the most important arbovirus requiring high treatment cost. The virus also contributes to high mortality rate, particularly in tropical and subtropical countries. It has been a public-health concern for several years with an estimated 50 million infections worldwide per year, of which 250,000–500,000 cases turn to dengue haemorrhagic fever (DHF), leading to 25,000 deaths . BesidesFlavivirus. It is an enveloped particle containing a 10.7 kb single-stranded RNA genome of positive polarity  to dengue shock syndrome (DSS) according to the World Health Organization criteria . For exaMorbidity from dengue virus infection can be attributed to any serotype, with some reports indicating that multiple infections with different serotypes may contribute to increased disease severity . DENV1-3As Thailand is one of the countries that reported dengue virus infections, enhancing our knowledge on viral dissemination patterns would be beneficial. In this study, we have investigated infection with various serotypes in Thai patients between 2004 and 2010. Moreover, we report the co-infection of dengue virus with information on clinical symptoms. We hope that our report on the changing pattern of dengue virus serotypes, along with co-infection, may provide important information on epidemiology, prevalence, and virulence of dengue infection.The protocol of the study was reviewed and approved by ethics committee of the Institutional Review Board (IRB), Faculty of Medicine, Chulalongkorn University, Thailand. We also included data from two previous reports ,9. All tSerum samples were collected from patients within 1-7 day(s) after the onset of fever. The samples were collected from patients who had fever, headache, rash, or myalgia/arthralgia-positive tourniquet test result, thrombocytopenia or plasma leakage since they were likely to be infected by dengue virus according to WHO criteria . We haveTM Reverse Transcriptase .RNA was extracted from serum samples, using the Viral Nucleic Acid extraction kit  according to the manufacturer's protocol. The extracted RNA was reverse-transcribed into cDNA by ImProm-IIoC for 2 minutes, the amplification reaction comprised 40 cycles of denaturation at 95 oC for 25 seconds, annealing at 50 oC for 35 seconds and extension at 72 oC for 1 minute and was concluded by a final extension step at 72 oC for 5 minutes and a final hold step at 25 oC for 5 minutes. The primer targeting the 3’ non-translated region has been previously described . After initial denaturation at 95 escribed . The estThe PCR products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV light. The PCR products of the expected sizes were excised from the gel and purified using the gel purification kit . The purified DNA was subjected to direct sequencing , and the obtained nucleotide sequences were analyzed using the BLAST program available at the GenBank database. Serotypes were compared based on the relationship between genomic RNA of dengue virus and the serotype .The results were compared statistically by using chi-square . The p value of <0.05 indicated statistically significant differences. Each serotype was determined independently. We calculated the p value of each pair in different years, such as between 2004 and 2007, 2004 and 2008, and 2004 and 2010. Yearly data for 2004, 2007, 2008, and 2010 were also compared.In 2004, a total of 39 positive samples were analyzed showing that the 2004 outbreak included DENV1 , DENV2 , DENV3 , and DENV4 . Two years later, in 2007, the results included DENV1 , DENV2 , DENV3 , and DENV4 . When compared DENV1 between 2004 and 2007, the percentage of DENV1 in 2007 was significantly lower compared to the 2004 outbreak (p<0.05). However, when compared DENV1 between 2007 and 2008, the result showed an exponential increase to 57.1% (n=48) (p<0.05) and then a decrease to 25.8% (n=8) in 2010.When compared between 2004 and 2007, the DENV4 percentage increased significantly from 10.26% (n=4) to 50% (n=16) (p<0.05). After that, in 2008 and 2010, DENV4 percentage declined to 11.9% (n=10) and 6.45% (n=2) respectively.In 2004, DENV2 was 28.21% (n=11), which is quite similar to the proportion of DENV2 in 2007 . DENV2 still showed lower proportion in 2008 . In 2010, however, the percentage of DENV2 considerably increased up to 38.7% (n=12) (p<0.05), which also seemed to be predominant during this year, although there was no obviously-predominant serotype amounting to 50% in 2010.The DENV3 proportion in 2004 was 5.13% (n=2) and then increased to 21.9 % (n=7) in 2007 and showed insignificant changes in 2008 and 2010 with 21.4% (n=18) and 29.03% (n=9) respectively. The details are shown in 3, N 23%, L 52.9%, M 17.4%, B 6.1%, atypical lymphocyte 3.9%, and decreased platelets on blood smear. She recovered and was discharged after 3 days of hospitalization. Dengue fever was diagnosed based on detectable IgG and IgM specific to dengue virus through ELISA. The diagnosis was confirmed by detection of DENV RNA with semi-nested multiplex RT-PCR. The results showed evidence of co-infection with two serotypes of dengue virus—DENV3 and DENV4  that mediates the entry of dengue virus into the host cell and can lead to a severe condition, particularly cross-reacting antibodies. Besides, the major sites of DENV replication are monocytes and macrophages, and when the immune system cross-reacts, it might mediate entry of virus into the Fc-bearing cell, and eventually increase replication of virus . Dengue Co-circulation and co-infection with various dengue serotypes are common as has been reported from India and Brazil . Our stuA single serotype of dengue virus may not be able to circulate in any given endemic area. The predominant DENV serotype has been continuously shifted from year to year depending on the herd immunity to DENV and competition among serotypes. Co-infection with various serotypes of dengue virus may not necessarily lead to a more severe condition or DHF/DSS.This study was supported by the Center of Excellence in Clinical Virology, Chulalongkorn University, CU Centenary Academic Development Project, King Chulalongkorn Memorial Hospital, the National Research University Project of CHE (HR1155A) and the Ratchadaphiseksonphot Endowment Fund. We would like to thank the staff and nurses of Chumphae Hospital, Khon Kaen province, Thailand, for collecting the clinical data and specimens. Besides, we would like to express our gratitude to Ms Petra Hirsch for reviewing the manuscript.


After the recent summary of World Health Organization–authorized research on smallpox, several clinical issues remain. This policy review addresses whether early hemorrhagic smallpox is disseminated intravascular coagulation and speculates about the cause of the high mortality rate among pregnant women and whether ocular smallpox is partly the result of trachoma or vitamin A deficiency. The joint destruction common in children with smallpox might be prevented by antiviral drugs, but intraarticular infusion of antiviral drugs is unprecedented. Development of highly effective antiviral drugs against smallpox raises the issue of whether postexposure vaccination can be performed without interference by an antiviral drug. Clinicians should consider whether patients with smallpox should be admitted to general hospitals. Although an adequate supply of second-generation smallpox vaccine exists in the United States, its use is unclear. Finally, political and ethical forces suggest that destruction of the remaining stocks of live smallpox virus is now appropriate. These problems included high rates of adverse events associated with most strains of vaccinia virus; our inadequate understanding of the pathophysiology of smallpox; lack of a good animal model of the disease; difficulty of rapid laboratory diagnosis, including the poor ability of most standard laboratory tests to distinguish between orthopoxviruses; lack of an effective antiviral drug; and rudimentary knowledge about the genetic makeup of the virus.After the World Health Organization (WHO) declared smallpox eradicated in 1980, several problems remained concerning the disease and its causative virus, variola major virus–Although many observers wished to destroy the remaining stocks of variola major virus in 1980, several respected researchers wanted to use the live virus to help answer some of these remaining questions. In 1999, WHO agreed to a research agenda, with oversight by a WHO committee, to continue research with live variola major virus until substantial progress was made on these questions. The WHO oversight committee has now declared that satisfactory (if in some areas imperfect) progress has been made toward developing improved vaccines, better laboratory diagnostics, a reasonable nonhuman primate animal model, effective antiviral drugs, and good understanding of the genetics of orthopoxviruses. Reports of this progress have been published . Patients had widespread hemorrhaging in the skin during the early septic phase, usually before a rash developed. Bleeding occurred from multiple orifices. Although necropsy evidence is minimal, internal bleeding likely affected many organs. Results of studies of bleeding time, clotting time, and tourniquet tests in patients with early hemorrhagic smallpox were consistent with what might be expected with DIC  is theoretically possible. Inserting minor modifications into the genome of a well-characterized strain of vaccinia virus should be even easier. Orthopoxviruses are large, stable, DNA viruses that are fairly easy to manipulate genetically . It requires syringes and needles and must be administered by someone trained to give injections. First-generation and second-generation vaccines are lyophilized and can be reconstituted and administered in the field with bifurcated needle scarification by persons with minimal training. Because MVA does not produce a visible lesion or scar, rapid determination of who has been already vaccinated is difficult. Optimal immunity with MVA requires 2 injections. In contrast, single injections are fully protective for first-generation and second-generation vaccines.In the absence of a perfect animal model for smallpox, and because it is impossible to test these vaccines against smallpox, we have only laboratory evidence of their efficacy. Given these limitations, third-generation vaccines may be best for persons who anticipate possible exposure, such as military personnel or laboratory personnel working with orthopoxviruses. In an actual outbreak, ACAM2000 should be used for field vaccination. However, its use increases the risk for development of progressive vaccinia or eczema vaccinatum; these adverse events would then need to be treated with ST-246.WHO is planning on creating a modest real, and substantial virtual, stockpile of vaccines. There are ≈60 million doses of vaccine in this stockpile and plans to increase it to ≈200 million doses. How much of this stockpile should be second-generation or third-generation vaccines that may not yet have been licensed widely? What should be the rules for release of such vaccines from the stockpile? Widespread use of first-generation or second-generation smallpox vaccines in era of AIDS and iatrogenic immune suppression by cancer chemotherapy or for transplant surgery seems unlikely unless there are actual cases of smallpox. In that situation, we might respond by using first-generation and second-generation vaccines with proven efficacies.–The WHO-approved research agenda with variola major virus has been largely fulfilled (Discussions about destruction of the remaining variola major virus stocks should not be limited to the scientific points set forth in the US Institute of Medicine report (


Some potential competing interests in relation to this work were not declared in the article. The authors apologize for this omission and would like to disclose the following information: Drs Blackwell, Makarova, Molinaro, Suppiah and Ly have a U.S. patent (PCT/US2010/050633) and pending international patent application (WO 2011/041350 A2) on compositions and methods for analyzing and treating a disease, disorder, or condition associated with Xenotropic Murine Leukemia Virus-Related Virus infection.


Dear Editor,Occupational exposure to blood-borne viruses (BBVs) is a major concern in the health care setting. The BBVs of primary concern are the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). Health care workers (HCWs) can be exposed to these viruses through needle-stick and other injuries through contaminated material with blood and other infectious body fluids. Nurses, health technicians, cleaning personnel and physicians are potentially targets for such accidents. The Center for Disease Control and Prevention (CDC) estimates that hospital-based healthcare personnel sustain an estimated 385,000 percutaneous injuries (needle-sticks and other sharp-devices)  annually


Avian tembusu virus (TMUV), which was first identified in eastern China, is an emerging virus causing serious economic losses in the Chinese poultry industry. Here, we report the complete genome sequence of goose tembusu virus strain JS804, isolated from Jiangnan white geese with severe neurological signs. The genome of JS804 is 10,990 nucleotides (nt) in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids. Research of the whole sequence of tembusu virus will help us to understand further the molecular and evolutionary characteristics and pathogenesis of this virus. Since April 2010, a novel contagious disease in ducks and geese, with egg drop, feed uptake decline, and neurological signs, caused by a newly emerged virus, has spread throughout eastern China. The infection rate and morbidity of shelducks were as high as 100%, and mortality varied from 5% to 30%, possibly due to secondary bacterial infections , but theHerein, we report a TMUV strain isolated from Jiangnan white farm geese with severe neurological signs in Jiangsu Province, China. First, in order to determine the nucleic acid type, a convenient method called DNase-SISPA (DNase-Sequence independent single primer amplification) was utilized; after that, three fragments highly homologous to the envelope (E) gene of tembusu virus were obtained and named S1, S2, and S3. Then, based on the sequence of flavivirus Bagaza virus (GenBank accession no. AY632545), 5 pairs of nucleotide primers were designed to amplify the other regions and bridge the gaps between amplified regions. At last, all of the amplified products were purified and cloned into the pMD18-T vector (TaKaRa) and sequenced in triplicate. Sequences were assembled using the SeqMan programs (DNAStar and Lasergene) to produce the final genome sequence. The isolated virus was identified as TMUV and named JS804. The full-length genome sequence of JS804 was 10,990 nucleotides (nt) in length, with a typical flavivirus genome organization, and the 5′ and 3′ untranslated regions (UTR) were 94 and 618 nt, respectively. Additionally, the coding region of JS804 included a single open reading frame (ORF)  that encoded a polypeptide of 3,425 amino acids (aa), three structural proteins , and five nonstructural proteins .N-linked glycosylation sites (NLGlys) were also found in this virus, with a distribution of two in the PrM protein, two in the E protein, three in the NS1 protein, two in the NS4B protein, and three in NS5. We found that there is one more N-linked glycosylation site in the E protein of JS804 than in WJ-1 (GenBank accession no. JX549382).Comparison to previous avian tembusu virus strains isolated from different breeds of ducks and geese in China showed that JS804 was 99.6% identical to BYD virus (GenBank accession no. JF312912) and 99.2% identical to ZJ-6 virus (GenBank accession no. JF459991) at the nucleotide level. This indicates that all avian tembusu-related virus polyprotein sequences possess high identities at the nucleotide level. A phylogenetic tree based on the whole polyprotein sequence demonstrated that strain JS804 is more closely related to Bagaza virus, which is an African flavivirus isolate that causes disease in both humans and animals (Therefore, the study of the whole genome sequence of goose tembusu virus profits further investigation of the epidemiology and evolution of avian tembusu virus; it also contributes to the mechanisms of virus replication and pathogenesis.JF895923.The complete genome sequence of the goose tembusu virus isolate has been submitted to GenBank under the accession no.


A surveillance system to detect West Nile virus (WNV) was established in Cuba in 2002. WNV infection was confirmed by serologic assays in 4 asymptomatic horses and 3 humans with encephalitis in 2003 and 2004. These results are the first reported evidence of WNV activity in Cuba. Serum and cerebrospinal fluid specimens were shipped at 4°C to the Tropical Medicine Institute "Pedro Kouri." Human sera were screened for WNV IgM and IgG by using commercial IgM and IgG ELISA kits  according to manufacturer's instructions. Hemagglutination-inhibition (HI) tests were also undertaken with WNV and Saint Louis encephalitis virus (SLEV) antigen  with WNV , SLEV , and dengue virus . PRNT was performed to confirm WNV-specific antibody and was carried out as described previously , blue jays (Cyanocitta cristata), herring gulls (Larus argentatus), yellow-faced grassquits/olive finches (Tiaris olivacea), and northern parulas (Parula americana). None of the birds were positive for WNV RNA by RT-PCR. We attempted to isolate WNV from most samples, but virus was not cultured. Most dead birds were received in good condition for testing.Most (58%) of the 1,217 birds tested were resident species of Cuba, primarily chestnut manakins (Nineteen (9.0%) of the 210 horses had serum specimens with antibodies to flaviviruses . Four anSerum specimens from 13 patients with encephalitis were tested for possible WNV infection. Two of these patients (M.M. and K.R.) were identified as part of WNV surveillance in 2003. Both acute-phase and convalescent-phase serum specimens from these patients were positive for flavivirus antibody by IgM and IgG ELISAs . CerebroSerum specimens from the 10 remaining patients were negative for WNV IgM but were positive for flavivirus IgG by ELISA; most of these were also positive by HI assay . One perWe report the first evidence of antibodies to WNV in horses and humans in Cuba. The fact that human and horse infections have been detected strongly suggests that a local amplification cycle has been established in Cuba. The mode of entry of the virus into Cuba is unknown.–In North America, avian death from WNV infection has been well documented (Aedes aegypti and dengue prevention, may be required to respond to this new public health threat.This study also provides evidence that suggests WNV and SLEV may co-circulate in Cuba. Further studies are required to confirm these observations and to characterize the transmission cycles involved. Finally, expansion of existing mosquito control programs in Cuba, which currently focus on


Numerous viruses are responsible for respiratory infections; however, both their distribution and genetic diversity, in a limited area and a population subgroup, have been studied only rarely during a sustained period of time.A 2-year surveillance program of children presenting with acute respiratory infections (ARIs) was carried out to characterize the viral etiology and to assess whether using gene amplification and sequencing could be a reliable approach to monitor virus introduction and spread in a population subgroup.Using multiplex RT-PCR, 15 different respiratory viruses were detected within the 486 nasopharyngeal positive samples collected among 817 children aged <9 years old who presented with ARI during October 2006 to September 2008. A single virus was detected in 373 patients (45.7%), and two to four viruses in 113 patients (13.8%). The most frequent causative viruses were respiratory syncytial virus (RSV) (24.7%), human bocavirus (24.5%), and human rhinovirus (HRV) (15%). RSV was more prevalent in winter and among young infants. Cases of seasonal influenza A and B viruses were reported mainly in January and August. An increase in adenovirus infection was observed during the spring of the second year of the study. Sequence analyses showed multiple introductions of different virus subtypes and identified a high prevalence of the newly defined HRV-C species. A higher viral incidence was observed during the winter of 2008, which was unusually cold.This study supports the usefulness of multiplex RT-PCR for virus detection and co-infection, and for implementation of a molecular monitoring system for endemic and epidemic viral respiratory infections.


Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined.Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV.We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health. Lyssavirus genus presently comprises 12 species and two unapproved species with different antigenic characteristics. Rabies virus is detectable worldwide; Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus, and Ikoma lyssavirus circulate in Africa; European bat lyssavirus types 1 and 2, Irkut virus, West Caucasian bat virus, and Bokeloh bat lyssavirus are found in Europe; and Australian bat lyssavirus has been isolated in Australia. Only Aravan and Khujand viruses have been identified in central Asia. Bats are recognized as the most important reservoirs of lyssaviruses. In China, all lyssavirus isolates in previous reports have been rabies virus, mainly from dogs; none has been from bats. Recently, however, at least two bat-associated human rabies or rabies-like cases have been reported in northeast China. Therefore, we conducted a search for bat lyssaviruses in Jilin province, close to where the first bat-associated human rabies case was recorded. We isolated a bat lyssavirus, identified as an Irkut virus isolate with high pathogenicity in experimental mice. Our findings suggest that public warnings and medical education regarding bat bites in China should be increased, and that surveillance should be extended to provide a better understanding of Irkut virus ecology and its significance for public health.The  Lyssavirus genus presently comprises 12 species: rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus types 1 and 2 (EBLV-1 & 2), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), West Caucasian bat virus (WCBV), and Shimoni bat virus (SHIBV) Bats are the most important reservoirs of lyssaviruses A 1998 survey in Philippines found that 22/231 (9.52%) serum samples from insectivorous bats contained neutralizing antibody (VNA) to ABLV In 2002 and 2010, two bat-associated human rabies cases were reported in northeast China, in Tonghua county and Longjing city respectively Since 2002, efforts have been made to obtain human specimens in hospitals and bat specimens in the field. Human specimens have not been obtained, however, since bat-associated human rabies is rare, even though there have been several reports of bat bites Guideline on the Humane Treatment of Laboratory Animals stipulated by the Ministry of Science and Technology of the People's Republic of China (MOST) All animals experiments described in this paper have been conducted according to the Plecotus auritus (n = 12), Murina leucogaster (n = 104), Myotis daubentonii (n = 66), Rhinolophus ferrumequinum (n = 59), and Myotis ikonnikovi (n = 20), were collected in Tonghua county, Jilin province  and identified by both morphological traits and amplification and sequencing of the cytochrome B (cytB) gene as described previously In April 2012, 261 bats of five species, Rabies virus BD06 (GenBank #EU549783.1), isolated in our laboratory in 2006 from a rabid dog in China, was maintained in dog brain and could cause ≥80% mortality via i.m. injection in unvaccinated dogs in previous experiments 2 humidified incubator.Mouse neuronal-2a (N2a) cell line was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin sulfate at 37°C in a 5% CO5.0–105.5 TCID50/mL.To compare the pathogenicities of lyssavirus isolate and BD06 at the same dose in mice, both kinds of these viruses were cultivated in N2a cells as described previously in vivo pathogenicity assay. Virus isolation was conducted using the mouse inoculation test (MIT) method as described previously One-day-old suckling and 8-week-old adult Kunming mice were purchased from the Changchun Institute of Biological Products, and respectively used for lyssavirus isolation from the DFA-positive samples and 1–104 TCID50; 1 dilution per group).For pathogenicity, serial 10-fold dilutions of the viruses (second passage in N2a cells) were prepared in PBS/2% horse serum and injected i.m.  into 4 groups of 10 adult mice  according to the supplier's instructions.For amplification and analysis of the viral genomes, a total of 9 pairs of primers of overlapping fragments covering the full length of the viral cDNA were designed based on the similar sequences identified using BLAST. Fragments containing the 3′ and 5′ termini of the viral genome were obtained using a 3′ and 5′ Full RACE Kit . The PCR products were purified, and nucleotide sequencing was performed on both forward and reverse strands of each fragment by Genscript Nanjing Co., China.4 model was favored for all datasets. The percentage identities and similarity scores of the viral protein sequences were calculated using DNAstar Lasergene software. .Phylogenetic tree was constructed using the Maximum Likelihood method in MEGA 5.10 Complete genome sequences of each lyssavirus species were used as follows: RABV (16 sequences), LBV (4 sequences), MOKV (2 sequences), DUVV (4 sequences), EBLV-1 (5 sequences), IRKV (3 sequences), EBLV-2 (2 sequences), ABLV, ARAV, KHUV, WCBV, BBLV and SHIBV (1 sequence each).Murina leucogaster bat was found positive by DFA. From this sample, a 255-bp sequence was obtained using nested RT-PCR. Using BLAST, its sequence was found to be highly similar (92%/98%) to that of both IRKVs, especially isolate Ozernoe. All the eight inoculated suckling mice with lyssavirus isolate developed neurological signs between days 7–9 post-inoculation, at which point they were euthanized. Each mouse brain sample was DFA positive. During the next intracranial passage, the incubation period was reduced to 3–4 days in suckling mice. All the other bat brain samples were DFA negative.Of all 261 bat specimens tested, only a single brain sample from an adult male As originally proposed by Kuzmin et al., N gene nucleotide identities provided unequivocal separation of all lyssavirus species with an identity threshold of 82% 4 TCID50, with 7/10 dying from BD06 injection at the same dose. At lower doses (102–103 TCID50), some animals survived in every group; however, given 102 TCID50 or more, the mortalities in each IRKV-THChina12 group were higher than that in BD06 groups at the same dose.Following i.m. injection of adult mice at the same doses, rabies-like symptoms were observed 6–10 d post-injection of IRKV-THChina12, whereas the incubation period of BD06 was 8–11 d. All injected adult mice died 24–48 h following appearance of rabies-associated symptoms such as lethargy, ruffled hair, muscle weakness, loss of body weight and progressive paralysis of one or both hind limbs. The genome of IRKV-THChina12 was determined to be 11980 nucleotides (nt) in length with a G+C content of 44.72%, similar to the G+C content of 44.49% of Irkut virus and 44.72% of Ozernoe virus . The genAs shown in IRKV-THChina12 and the two other IRKV isolates all provide independent origins for the species IRKV. However, according to the branch length, IRKV-THChina12 has undergone more changes since the three isolates diverged from their common ancestor; i.e., the evolution rate in the lineage leading up to IRKV-THChina12 is greater than that leading up to the isolate Ozernoe. It has been previously suggested that complete genomes provide the best tools for genotyping Murina leucogaster bats, other considerations suggest that this bat species should receive special attention concerning its association with IRKV. The greater tube-nosed bat (Murina leucogaster) is a non-migratory species, distributed throughout India, Mongolia, Siberia and the Far East of Russia, China, Korea, and Japan Murina leucogaster) is the principal vector of the virus. IRKV may circulate in bats of this species across all this vast territory.The identification of IRKV-THChina12 represents the first isolation of a bat lyssavirus in China. Although it was found in only a single sample (1/104) from The overall genomic organization of the isolate IRKV-THChina12 is similar to those of IRKVs and other lyssaviruses At the genus level, the N gene is the most conserved protein-coding region, followed in order by L>M>G>P with minor variations In previous studies, the pathogenicity of strain BD06 was the highest of all rabies virus isolates stored in our laboratory, including Chinese ferret badger isolate Figure S1Tonghua county and surrounding regions in Russia and China. Black dots indicate the location of IRKV isolates and/or IRKV-associated human rabies cases within China and Russia. The red dot identifies where IRKV-THChina12 virus was isolated.(DOC)Click here for additional data file.Table S1Primers used for amplification and sequencing of the IRKV-THChina12 genome.(DOC)Click here for additional data file.Table S2Lyssavirus sequences used in the present study.(DOC)Click here for additional data file.


Hepatitis E virus (HEV) is a zoonotic virus for which pigs are the primary animal reservoir. To investigate whether HEV occurs in mink in Denmark, we screened feces and tissues from domestic and wild mink. Our finding of a novel HEV variant supports previous findings of HEV variants in a variety of species. Hepeviridae) is a main cause of acute liver inflammation in humans. It is a nonenveloped RNA virus with a positive-sense genome of ≈7.2 kb. In 1997, HEV was discovered in pigs  has been characterized. It is widespread globally and has been proposed to comprise 3 genotypes . The sequences were 98%–100% identical, with only 1 nonsynonymous mutation, resulting in a neutral amino acid change from isoleucine to valine  variant; characterization of the 4 HEV-positive samples; and alignment of the 4 mink positive for HEV variant sequences obtained by the nested PCR.