Iflaviridae is a family of small non-enveloped viruses withmonopartite, positive-stranded RNA genomes of approximately9â€“11 kilobases. Viruses of all classified species infectarthropod hosts, with the majority infecting insects. Both beneficial and pestinsects serve as hosts, and infections can be symptomless  or cause developmental abnormalities (deformed wing virus),behavioural changes (sacbrood virus) and premature mortality (infectiousflacherie virus). The host range has not been examined for most members. Themost common route of infection for iflaviruses is the ingestion ofvirus-contaminated food sources. This is a summary of the InternationalCommittee on Taxonomy of Viruses (ICTV) Report on the taxonomy of theIflaviridae, which is available at www.ictv.global/report/iflaviridae. Virions are roughly spherical and exhibit icosahedral symmetry with a diameter of22–30 nm. Virions have no envelope and no distinctive surfacestructures , Fig. 1.Iflaviruses possess single-stranded, positive-sense, non-segmented RNA genomes with asingle open reading frame (ORF). The ORF is translated directly into a polyproteinthat is subsequently processed to yield structural, i.e. capsid and non-structural  proteins . The 5′ Replication occurs in the host cell cytoplasm. The coding regions for capsidproteins, arranged in the order VP2–VP4–VP3–VP1, are oftenpreceded by a region encoding a short leader protein (L) of unknown function that isremoved from VP2 before capsid assembly. VP4 is analogous to the VP4 present in somedicistroviruses and, in the case of infectious flacherie virus, is present as aminor structural component of the capsid. The non-structural proteins include an RNAhelicase, a 3C-like cysteine protease and an RNA-dependent RNA polymerase . EvidencIflavirus. However, phylogenetic analysis of the completetranslated genomes of iflaviruses shows that a number of distinct clades arepresent. These may be separated taxonomically into different genera in the nearfuture as more virus sequences become available.Currently, members of the family are placed together within a single genus,Apis species, as well as several Bombusspecies [Varroa and Tropilaelaps genera).Varroa and Tropilaelaps mites are also capableof serving as hosts for deformed wing virus and Varroa destructor virus 1. Deformedwing virus and Varroa destructor virus 1 are also vertically and sexuallytransmitted in honey bees. The most common route of infection for iflaviruses is theingestion of virus-contaminated food sources. Trophallaxis in social insectsfacilitates intra-colonial virus dispersal [All member viruses have been isolated from arthropods, primarily insects. Beyond theoriginal host descriptions, the host range of most members has not been examined.However, honeybee iflaviruses, deformed wing virus, Varroa destructor virus 1, slowbee paralysis virus and sacbrood virus have been shown to infect otherspecies . Deformeispersal . In addiispersal .www.ictv.global/report/iflaviridae.Full ICTV Online (10th) Report:


We analyzed blood samples from infants born with microcephaly and their mothers in Guinea-Bissau in 2016 for pathogens associated with birth defects. No Zika virus RNA was detected, but Zika virus IgG was highly prevalent. We recommend implementing pathogen screening of infants with congenital defects in Guinea-Bissau. In 2016, the health authorities in Guinea-Bissau reported 4 cases of Zika virus infection and 5 cases of microcephaly  . Most cases were found in the northern and eastern regions  of Guinea-Bissau  (Epidemiologic characteristics and diagnostics test results of infants with microcephaly and their mothers, Guinea-Bissau, 2016.


Giardia intestinalis is one of the most important zoonotic enteric parasites. As no information regarding prevalence and genotype of G. intestinalis in donkeys (Equus asinus) in China is available, 181 faecal samples from 48 donkeys from Jilin Province, from 104 from Shandong Province and from 29 from Liaoning Province were examined between May and December 2015.G. intestinalis-positive by nested amplification of the triosephosphate isomerase (tpi) gene. The prevalence in different regional groups varied from 10.42 to 18.27%. The prevalence in adult and young donkeys was 14.29 and 22.92%, respectively. Otherwise, the prevalence was 11.69% in summer and 18.27% in winter. However, no statistically significant differences were found in relation to region or age group. Sequence analysis of the tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci identified 4, 1 and 3 subtypes of assemblage B, respectively. Moreover, four novel multilocus genotypes (MLGs novel-1 to novel-4) were identified in assemblage B.Twenty-eight (15.47%) out of 181 donkey samples were tested G. intestinalis in donkeys in China indicates that further studies of nation-wide molecular epidemiology and geographical distribution of Giardia in donkeys are warranted. Effective strategies should be implemented to control G. intestinalis infection in donkeys, other animals and humans.This first report of The online version of this article (doi:10.1186/s13071-016-1936-0) contains supplementary material, which is available to authorized users. Giardia intestinalis is the only species of Giardia which is found in human beings [G. intestinalis [Giardia cysts in contaminated food or water [8 cases of human giardiasis are reported world-wide per year, and the majority of them are reported in developing countries [G. intestinalis infections have been reported in humans and a variety of animal species [Equus asinus).n beings –4. It isn beings , 6. Eighstinalis . Of thesstinalis , but assstinalis . Diarrhestinalis  and tranor water ; approxi species –16, therGiardia intestinalis infection in horses has been reported in many countries around the world including China [Equus. It is an important edible animal species and used in Chinese traditional medicine, and is closely related to horses. Because they are maintained in a close association with their owners and veterinary personnel, donkeys are the important reservoirs for transmission of pathogens (such as Cryptosporidium hominis and Toxoplasma gondii) to humans and other animals [G. intestinalis, we conducted a study on the prevalence and genotypes of G. intestinalis in donkeys in Jilin, Liaoning and Shandong Provinces, China.ng China –21. The  animals , 23. To A total of 181 donkey faecal samples  were collected from three provinces, in northeastern and eastern China, between March and December 2015. Each of the fresh faecal samples was collected into sterile gloves separately after its defecation onto the ground, placed into box with ice and transported to the laboratory immediately. Genomic DNA was extracted directly from each faecal sample using the Stool DNA kit  according to the manufacturer’s instructions. Genomic DNA was stored at -20 °C until PCR amplification.G. intestinalis were determined by the nested PCR amplification of the triosephosphate isomerase (tpi) gene, beta giardin (bg) gene and glutamate dehydrogenase (gdh) genes as described by Zhao et al. [The prevalence and genotypes of o et al. . The pritpi, gdh and bg gene sequences of G. intestinalis available in GenBank using the BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and computer program Clustal X 1.83.Positive secondary PCR products were sequenced by Genscript Company . Bidirectional sequencing was used to confirm the accuracy of the sequences. Sequences with mutations were considered as novel genotypes when confirmed from independent two PCR reactions on the same sample. To identify the assemblages and subtypes, nucleotide sequences were aligned with known reference G. intestinalis-infected donkeys and different variables including age, geographic origin and seasons were analyzed by Chi-square test using SPSS version 17.0  [P < 0.05. Odds ratios (ORs) and their 95% confidence intervals (CI) are also given.The relationship between prevalence of IL, USA) . The resG. intestinalis     out of 181 donkey samples were PCR-positive for G. intestinalis infection in donkeys was 15.47% , which was higher than that in horses in Xinjiang (1.5%) [G. intestinalis is more likely to be affected by climate (temperature and relative humidity) [G. intestinalis prevalence in different regions may be due to different local climatic conditions, as well as the detection methods, sampling time and sample sizes. Moreover, probably because of the smaller sample sizes, seasonal and age-related correlates previously found in cattle [The overall prevalence of g (1.5%) , Brazil g (1.5%) , foals ig (1.5%) . Previouumidity) , 27, so n cattle  and horsG. intestinalis in cattle [Budorcas taxicolor bedfordi) [Chinchilla lanigera) [G. intestinalis may be commonly occurring in China, and we should pay enough attention to. More importantly, assemblage B was also identified in raw urban wastewater in northern China [Assemblages A and B, responsible for the vast majority of human giardiasis , 28, andn cattle , have aln cattle . Howevern cattle , 28. In n cattle , horses n cattle , cattle n cattle , golden edfordi) , pet chianigera) , captiveanigera) , sheep [anigera)  and goatanigera) , suggestrn China . TherefoG. intestinalis genotypes have been recorded from a wide range of hosts worldwide [G. intestinalis infection [tpi, gdh and bg loci) has been developed and widely used for detection of G. intestinalis infection [G. intestinalis-positive samples were also genotyped based on bg and gdh loci. A total of 28 tpi, 22 bg and 16 gdh gene sequences were obtained, and analysis of these genes revealed only one assemblage (B); however, high genetic polymorphism was observed at these loci within this assemblage  was the most prevalent MLG, and responsible for 50% of all MLGs in the present study. These findings suggest a high genetic diversity of this prevalent genotype in donkeys in China, in agreement with previous conclusions that G. intestinalis isolates of the same assemblage may be grouped into distinct MLGs [Furthermore, 10 out of 28 positive isolates were successfully amplified at all three loci. These samples provided four novel MLGs in the assemblage B, namely MLGs novel-1 to novel-4 Table , Fig. 1.nct MLGs , 24.TablG. intestinalis infection in donkeys in China. Sequences analysis suggested that all the G. intestinalis isolates represented assemblage B, with four, one and three subtypes of assemblage B at the tpi gdh and bg loci, respectively. Moreover, four novel MLGs (MLGs novel-1 to novel-4) were identified within assemblage B. The results of the present study not only improve the information of the distribution of G. intestinalis genotypes in China, but also provide the foundation data for preventing and controlling G. intestinalis infection in donkeys, other animals and humans.The present study demonstrated the occurrence of


TTV has been detected in almost every human tissue type or body fluid reaching near 100% prevalence. Several studies report mother-to-child postnatal transmission of TTV in infancy but the risk of transplacental transmission of TTV is still unclear.The blood and plasma collected postpartum from 100 mother-child pairs were analyzed using TTV-specific qPCR. Samples were collected from the peripheral vein of the mother and the umbilical cord.4 copies/mL; range: 103 – 3 × 107). The TTV load in plasma was approximately 100 times lower than in whole blood. TTV was not detected in any of cord blood samples.Eighty four percent of pregnant women were TTV positive . The presence of the virus in infants may be associated with mother-to-child transmission through breast feeding or other routes of transmission. Torque teno virus (TTV) is a small virus of the Anelloviridae family with a circular ssDNA genome of negative polarity; it was first reported in 1997 in Japanese patients with non-A-G posttransfusion hepatitis , 3; the Mother-to-child transmission of viral infections can be transplacental, perinatal , or postnatal (through breast feeding or other routes). Transplacental transmission has been demonstrated for many viruses including the rubella virus, cytomegalovirus (CMV), herpes simplex virus (HSV), parvovirus B19 (B19V), varicella-zoster virus (VZV), West Nile virus, measles virus, hepatitis E virus, human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), enteroviruses, adenoviruses, and some other viruses .Several studies report mother-to-child postnatal transmission of TTV in infancy –14 but tThe study protocol was reviewed and approved by the Ethics Committee of the Pirogov Russian National Research Medical University (Protocol No.2016/64); the study was conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent. Blood samples obtained from the blood bank were anonymous.Blood and plasma samples were collected from 100 mother-child pairs over the period from January to November 2016 at Kulakov Research Center for Obstetrics, Gynecology and Perinatology . Mothers’ ages ranged from 20 to 47, with mean age of 31 years. The inclusion criterion was normal singleton pregnancy. The exclusion criteria were as follows: pregnancy complications of any type, premature delivery, multiple pregnancies, and caesarean section. Two separate aliquots of each sample collected postpartum from a peripheral vein of the mother and the umbilical cord were placed into 4 mL EDTA Vacutainers. All samples were stored at +4 °C for less than three hours before they were centrifuged for plasma separation first at 800 g for 5 min and then at 3000 g for 5 min. Then whole blood and plasma were immediately frozen at −80 °C until DNA extraction.DNA was extracted from 200 μl aliquots of thawed whole blood or plasma using a standard commercial silica-sorbent kit for DNA extraction from body fluids . To prevent exogenous contamination, DNA isolation was performed in a separate DNA extraction room (Zone 1). To prevent cross-contamination of the samples, all procedures were carried out in the UV-equipped PCR-box using sterile disposable tubes and aerosol-resistant tips.qPCR was performed using the DTprime Real-Time PCR Cycler  as described in , with teqPCR data were analyzed using the DTprime Real-Time PCR Cycler Software v.7.7 . Microsoft Office Excel  and Statistica 8.0  were used for statistical analysis.3 copies per milliliter of whole blood (test sensitivity), with a maximum of 3 × 107 and a median of 8 × 104 viral genomes per 1 mL  indicating the predominantly cellular localization of the virus.We have demonstrated that in 84% of pregnant women the TTV viral load is higher than 108 copies/mL, respectively).TTV was not detected in any of cord blood samples. The levels of human genomic DNA were pretty similar in maternal and cord blood samples . The TTV load in plasma was approximately 100 times lower than in whole blood indicating the predominantly intracellular localization of the virus.The TTV viral load of more than 1000 copies per 1 mL of whole blood was detected in 84% of pregnant women (with a maximum of 3 × 10Our data demonstrate the lack of transplacental transmission of TTV. The presence of the virus in infants may be associated with mother-to-child transmission through breast feeding or other routes of transmission.


Rattus norvegicus individual in the residential areas of Hainan Province in southern China.Wenzhou virus (WENV) is a rodent-borne mammarenavirus that was recently found to infect humans. In this study, we sequenced and analyzed the complete genome of a genetic variant of WENV, HMU  virus. The virus was harbored by a  Arenaviridae is classified into two genera, Mammarenavirus and Reptarenavirus. The genus Reptarenavirus represents arenaviruses from snakes, and at least 25 members of the genus Mammarenavirus whose primary natural reservoirs are rodents have been identified worldwide . Trinity version 2.0.6 was used for de novo assembly. Contigs were compared with sequences from the NCBI nucleotide database, and the WENV isolate Rn-242 (GenBank accession numbers KJ909795 and KJ909794) was identified as the most closely related genome to that of the HMU virus  and 7,147 bp (large segment [L]), with G+C contents of 44.21% and 42.66%, respectively. Both S and L segments encoded two viral proteins in an opposite orientation. The S segment encoded an envelope glycoprotein (GP) of 492 amino acids (aa) and a nucleocapsid protein (NP) of 567 aa, and the L segment encoded an RNA-dependent RNA polymerase (RdRP) of 2,230 aa and a zinc-binding protein (ZP) of 91 aa. In both of the segments, the genes were separated by a noncoding region of 62 bp for the S segment and 109 bp for the L segment, which were proposed to play roles in transcription termination. Sequence analysis indicated that the genome of the HMU virus was most closely related to that of WENV isolate Rn-242, with identities of 94% and 95% for the L and S segments at nucleotide level, respectively. They were also closely related to each other at their structural proteins . Phylogenetic analysis showed that HMU virus was closely related to WENV and LORV and formed an independent clade with them within the genus enavirus . Under tMF595889 and MF595888, respectively.The genome sequences of the S and L segments of the HMU virus have been deposited in GenBank under the accession numbers


Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10−4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of  Apple mosaic virus , Apple stem pitting virus  and Apple stem grooving virus  are distributed worldwide and constitute important virus pathogens for both apple  and pear (Pyrus spp.) crops [Pome fruit cultivation constitutes one of the most economically important tree cultivations in the world. .) crops –4. Their.) crops , 3, 5–8..) crops , 2, 9–11.) crops –18.The main control strategies as with any other plant viral disease rely on early detection, eradication and use of genetically resistant or tolerant cultivars. The use of certified planting material and germplasm for these three viruses along with other pathogens , is a prThe development of real-time quantitative PCR (qPCR) based methods led to superior sensitivity, speed, reproducibility and limited risk of contaminations compared to conventional RT-PCR. These characteristics often make it the method of choice in routine diagnostics. The possibility of multiplexing, allowing simultaneous detection of different targets in one sample, makes RT-qPCR even more appealing to diagnosticians and epidemiologists , 28. SinThe objective of this study was to develop a fast, sensitive and reliable assay for the simultaneous identification of ASPV, ASGV and ApMV in pome fruit trees.Apple chlorotic leaf spot virus (ACLSV), Apple scar skin viroid (ASSVd) and Pear blister canker viroid (PBCVd) from the collections of Benaki Phytopathological Institute  and CTIFL. The assay specificity was evaluated with healthy pear, apple and quince (Cydonia oblonga Mill.) tissues. Validation and comparative analysis was performed on 36 plant samples derived from commercial orchards located in different parts of Greece.Virus reference isolates were provided by the Centre Technique Interprofessionnel des Fruits et Légumes . An apple tree (B71) naturally triple-infected with ApMV, ASPV and ASGV was used along with reference isolates ApMV7, ASPV10 and ASGV13 for the assay development . The assTotal RNA was extracted from pear and apple leaves. Due to possible uneven virus distribution, leaf discs were collected from at least two different younger branches located at the upper one third of the tree. Leaf discs were mixed together and homogenized using liquid nitrogen.Considering the presence of compounds that bind or co-precipitate with the RNA of pome fruit trees  different extraction protocols and commercial kits were examined in pear tissues; TRI Reagent Solution (Ambion) was applied with the modification for polysaccharide contamination noted in the product manual, and a pre-treatment of crude plant extract in a dilution buffer described by Rowhani and co-workers . PurelinIn order to simulate composite sample examination, leaf samples from a naturally triple infected apple tree (B71) were mixed with virus negative tested pear leaf tissue in ratios of 1:25, 1:50, 1:100 and 1:150 before the extract preparation.™ P-Class P330 (IMPLEN). All RNA extracts were stored as 150 ng/μL aliquots at −80°C until use.Quality (OD 260/280) and concentration of nucleic acids were determined using a NanoPhotometerThe newly developed multiplex assay was compared to conventional RT-PCR assays. To obtain direct comparison between methods in copy number detection, primers for conventional PCR were selected to amplify products containing the same target sequence as the respective MGB probes .μL reactions with 2 μL of 5-fold diluted cDNA as template on a Veriti Thermal Cycler (Applied Biosystems) with Taq DNA Polymerase (Invitrogen).Reverse transcription of RNA was performed with MMLV Reverse Transcriptase (Invitrogen) following manufacturer’s instructions, at denaturation temperature 65°C for ASPV and ApMV, and 70°C for ASGV. Typically, PCR amplification consisted of an initial cycle of 95°C for 5 min, followed by 40 cycles of 94°C for 30 s, appropriate for each pair of primers annealing temperature for 30 s , 72°C for 60 sec and a final extension step of 72°C for 7 min. All PCR assays were performed in 25 in vitro and used as standards in the development of the RT-qPCR assays essentially as described previously [in vitro transcription reaction was done with TURBO DNase (Ambion) as described in manufacturer’s instructions. Standards were quantified using NanoPhotometerTM P-Class P330 (IMPLEN) and the number of standard copies per nanogram was calculated using the following equation: copies per nanogram = (NA x A) / (n x mw), where NA the Avogadro constant (6.02 x 1023 molecules per mole), A the amount of the standard in g, n the length of the standard in nucleotides, and mw the molecular weight per nucleotide (considering average molecular masses of 340 Da for one nucleotide of single-stranded RNA). All primers used for standards preparation are described in RNA transcripts were synthesized eviously ; basicalTaqMan MGB (Minor Groove Binder) probes and primers for RT-qPCR were designed using Primer Express Software v3.0.1 (Applied Biosystems). The most conserved region of the capsid protein (CP) of each virus was identified after alignment of all full-genome and CP sequences retrieved from NCBI GenBank database. Specificity of the designed primers and probes was tested using Basic Local Alignment Search Tool (BLAST). All primers and probes are listed in 2 in the reaction mix was optimized by additional use of 2 mM and the incubation of the reverse transcription step was performed at 42°C for 30 min. Primers and probes for all viruses were used at concentrations of 200 nM and 100 nM, respectively. For both multiplex and singleplex assays 20 μL reaction volumes were used and the amplification profile was 2 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min . All singleplex and multiplex assays were performed in duplicates and at least in two individual runs, using 150 ng of RNA template on a StepOnePlus Real-Time PCR System (Applied Biosystems). All appropriate controls were included, and ROX was used as passive reference dye.Concentration of MgCl2O (Merck) and mixed with 150 ng/μL RNA extracted from pear and apple samples testing virus negative. Serial 10-fold dilutions of RNA extracted from a naturally triple infected plant (B71) were also prepared in virus-free RNA at a final concentration of 150 ng/μL.Tenfold serial dilutions of the standards were prepared in RNAse-free type 1 Milli-Q H3 or 104 copies of one target and 107 copies of each of the other two targets, in the presence of 150 ng/μL virus-negative tested apple or pear RNA.Competition between amplicon detection in the multiplex assay was examined by using mixtures of 10Sensitivity, efficiency and linearity of the qPCR assays were estimated by constructing standard curves.260/OD280 from 1.9 to 2.7, OD260/OD230 from 0.1 to 1.5 and yields from 18.6 to 33ng/μL , resulted in a slight reduction of the cycle thresholds (Ct), and a higher reporter fluorescence. Thus, the addition of 2 mM MgCl2 was adopted in the assay protocol.Testing of all reference isolates by the new singleplex and multiplex assays showed that each set of primers and probe resulted in specific amplification of the target virus with no cross reactions being observed. Moreover, no detection was recorded when non-target viroid and virus isolates as well as virus negative tested apple, pear and quince tree species were analyzed by singleplex and multiplex RT-qPCR. The addition of extra 2 mM MgClSensitivity of RT-qPCR assay was examined by estimating the detection limit for each target using serially diluted i) standards and ii) RNA from triple infected B71 apple tree, into virus-negative tested apple and pear RNA.3 copies for ASGV, and 102 copies for each of ASPV and ApMV  ranged from 0.98 to 1. Efficiencies and R2 values using RNA extracted from the naturally infected B71 tree were 100% and 0.99, respectively for all virus targets. Standard curves constructed for each target are presented in Detection efficiencies of the multiplex assay using standards were 98.9%, 99.6% and 97.8% for ApMV, ASPV and ASGV, respectively, and regression coefficient values (R7 copies of each of the two targets were mixed with the detection limit copy number of the third target into apple derived virus-tested negative RNA (2 copies (104 copies of ASGV and 106 copies of ApMV and ASPV) indicating an inhibitory or competition effect of pear tissue in ASGV amplification  higher than 0.99 were obtained during simultaneous three target detection in a natural triple virus infection, demonstrating accuracy and linear response of the assay over a wide range of dilutions and performance that was not affected by competition among reaction components. These characteristics further demonstrated that the developed assay additionally to detection constitutes a reliable quantitative tool suitable for population or competition studies among the three viruses in mixed infections in a variety of pome tree hosts. Moreover, the low standard deviation of the mean Ct which was recorded in most cases proved the reproducibility of the method  serial dilutions of each standard in RNAs derived from tissues tested virus negative and ii) serial dilutions of RNAs extracted from tissue derived from a naturally triple infected plant. Amplification efficiencies of 100% and regression coefficient values  than those detected by the conventional protocols .In conclusion, a CTAB extraction protocol was combined with a multiplex single-tube RT-qPCR assay using TaqMan MGB probes, for the simultaneous detection of three important pome fruit viruses. Further experiments, in the form of ring tests including a larger range of reference virus isolates and pome tree species and cultivars, are required before the assay presented here could be recommended as a reference detection method. However, the multiplex RT-qPCR displayed high sensitivity, efficiency and improved reliability and has the capacity to assist large-scale surveys for disease management and certification programs. To the best of our knowledge this is the first report of a single-tube RT-qPCR assay for the simultaneous detection of three pome fruit tree viruses.S1 FigApple stem pitting virus (ASPV), Apple mosaic virus (ApMV) and Apple stem grooving virus (ASGV), and tenfold serially diluted total RNA extracted from a naturally triple infected plant . Serial 10-fold dilutions were prepared into RNA extracted from virus-negative tested apple tissue. L: 1 Kb Plus DNA Ladder (Invitrogen).Amplification plots of multiplex RT-qPCR  and product electrophoresis gels of conventional RT-PCR assays,  derived from assays applied on tenfold serially diluted standards  of (TIF)Click here for additional data file.S2 FigThe mixture was diluted into pear virus-negative tested RNA. Threshold line presented, corresponds to ASGV.(TIF)Click here for additional data file.S1 TableApple mosaic virus (ApMV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) reference isolates used for the multiplex RT-qPCR assay development.Description of (DOCX)Click here for additional data file.S2 Table260/OD280 and OD260/OD230) and integrity of the isolated RNA after measurements in a nanophotometer and gel electrophoresis. RNA was isolated from 70 mg aliquots of the same tissue homogenate derived from leaf disks of a pear tree.Evaluation of the extraction protocols tested in terms of yield, purity (ratios OD(DOCX)Click here for additional data file.


On April 3, 2017, two Florida residents consumed part of the same prepackaged salad before reportedly discovering the partial remains of a bat carcass in the salad. Bats are known reservoirs for rabies virus, which causes rabies disease in both animals and humans  recalled the affected lot of salads on April 8. CDC advised consumers to contact their local health department for PEP evaluation only if the consumer had eaten a recalled prepackaged salad and had found animal material in the salad. CDC was not notified of any other reports of dead bats in prepackaged salads.Tadarida brasiliensis mexicana), which is found throughout the southwestern United States. It is genetically distinct from T. brasiliensis cynocephala, which occurs in the southeastern United States  of mitochondrial DNA sequence data (Cytb and D-loop), the bat was identified as a Mexican free-tailed bat (d States  (5).The investigation determined that cutting and harvesting of greens for the recalled salad occurred in fields in the west and southwest United States before they were transported to a processing plant in Georgia. At the processing plant, the greens were washed with chlorinated water and packaged. Given the physical condition of the bat  and the geographic location of the fields and the processing plant, along with the genetic identification of the bat, investigators concluded the bat most likely came into contact with the salad material in the field during harvesting and cutting and was then transported to the processing facility.Several factors likely reduced the risk for rabies virus transmission to the two Florida consumers. No rabies virus was detected in the specimen, the bat’s cranium was intact, and the salad was rinsed before packaging, thereby diluting any potential virus. In addition, mucosal membrane exposures have rarely been proven to result in rabies disease, and rabies virus does not survive more than a few days outside a host (


In August and September 1967, an outbreak of a viral hemorrhagic fever occurred among laboratory workers in Marburg and Frankfurt, Germany, and Belgrade, Yugoslavia (now Serbia) who were processing kidneys from African green monkeys that had been imported from Uganda. (These kidneys were used in the production of polio vaccine.) Of 25 primary and 6 secondary cases, 7 were fatal.A new virus, named Marburg virus, was isolated from patients and monkeys, and the high case-fatality ratio called for the best biocontainment of the day . The Cen


Allotransplantation and xenotransplantation may be associated with the transmission of pathogens from the donor to the recipient. Whereas in the case of allotransplantation the transmitted microorganisms and their pathogenic effect are well characterized, the possible influence of porcine microorganisms on humans is mostly unknown. Porcine circoviruses (PCVs) are common in pig breeds and they belong to porcine microorganisms that still have not been fully addressed in terms of evaluating the potential risk of xenotransplantation using pig cells, tissues, and organs. Two types of PCVs are known: porcine circovirus (PCV) 1 and PCV2. Whereas PCV1 is apathogenic in pigs, PCV2 may induce severe pig diseases. Although most pigs are subclinically infected, we do not know whether this infection impairs pig transplant functionality, particularly because PCV2 is immunosuppressive. In addition, vaccination against PCV2 is able to prevent diseases, but in most cases not transmission of the virus. Therefore, PCV2 has to be eliminated to obtain xenotransplants from uninfected healthy animals. Although there is evidence that PCV2 does not infect—at least immunocompetent—humans, animals should be screened using sensitive methods to ensure virus elimination by selection, Cesarean delivery, vaccination, or embryo transfer. Allotransplantation can be associated with transmission of microorganisms which induce severe diseases in the recipient ,2. AmongThere are obvious differences between the transmission inside the human species during allotransplantation and transspecies transmission into a new species during xenotransplantation. Human microorganisms are adapted to humans and can be easily transmitted ,3,4,8,9.Even if porcine microorganisms can infect humans and replicate, it is still unclear whether they are pathogenic. For example, hepatitis E virus (HEV) genotype 3 coming from pigs mostly induces diseases in chronically ill and immunosuppressed humans, but not in healthy individuals , althougCircovirus of the family Circoviridae [orfs) have been recognized. Orf1 encodes the two replicases indispensable for viral replication (Rep and Rep’), and orf2 encodes the capsid protein Cap, which is the major structural protein [orf3, orf4, and orf5 encode proteins not essential for virus replication, but involved in the virulence and spread of the virus [The porcine circoviruses (PCVs) belong to the genus oviridae . Porcineoviridae . Other moviridae , and recoviridae ,21, dogsoviridae ,23,24,25oviridae ,27, and oviridae ). Two ma protein . Three ohe virus ,31. Cellhe virus ,33,34. Ahe virus . It was he virus , whereashe virus .PCV1 is apathogenic in pigs, but PCV2 is associated with severe diseases, among them PMWS, which is considered the most significant PCV2-related disease (PCVD) see . PCV2 isPCR is a sensitive method of choice to detect a circovirus infection in viremic animals and different PCR assays, including real-time PCR or quantitative PCR (qPCR) and digital droplet PCR (ddPCR) using specific primers for PCV1 and PCV2 have been developed and applied ,48,49,50rep gene and 91%–96% in the cap gene [Based on phylogenetic analysis, PCV2 is divided into different genotypes  . The fircap gene . They arcap gene . In rececap gene ,69. PCV2cap gene . Recombicap gene ,72,73.Since changes in the nucleotide sequence of genomic regions used as targets for PCR-detection of PCV may result in false-negative findings, the primers must be checked routinely by a Basic Local Alignment Search Tool (BLAST) search of GenBank for their fitness to detect new variants. If no highly conserved regions can be identified and problems related to genomic variation are anticipated, multiplex PCRs for different viral variants using more than one primer pair or next generation sequencing can be employed.Recently, a new virus, PCV3, with significant differences in the sequence when compared with PCV1 and PCV2, but more related to a bat-faeces associated circovirus, was described in pigs with cardiac and multi-organ inflammation . Since tPCVD was first detected in the early 1990s and has since then emerged as an economically important pig disease worldwide . The maiClinical signs of the disease include gradual wasting, fever, rough hair coat, dyspnea, pallor, diarrhea, and occasionally icterus. PCVD is characterized by lymphoid depletion, immunosuppression, and inflammation in affected organs. Morbidity varies from 2%–30%, but case fatality is high, approaching 80%. Occasionally, pigs may develop purple skin lesions and nephropathy, likely as an immune mediated sequel to viral infection, termed PDNS ,79. OccaWhen trying to infect human cell lines with PCV1 and PCV2, PCV1 persisted in most cell lines without causing any visible changes, while PCV2-transfected cells showed a cytopathogenic effect . Most imWhen humans were screened for antibodies against PCV, in an early study, antibodies to PCV were found in 30% of samples from hospitalized patients with fever of unknown etiology . These r5 or 106 particle-associated full-length PCV1 genomes were present in each dose of the contaminated vaccine [A large “experiment” testing the susceptibility of the human population to PCV was involuntary conducted when two vaccines against rotaviral gastroenteritis from two different manufacturers were found to be contaminated with PCV1 and PCV2 ,87,88. B vaccine ,87,88,89 vaccine ,87,88,91 vaccine . PCV1 DN vaccine . This co vaccine .However—and this is the main question in the context of xenotransplantation—up until now it is still unknown whether PCV is zoonotic in severely immunosuppressed humans.6 mg of DNA per reaction [In all of the clinical xenotransplantation trials documented in Paradise et al. , no screreaction . Islet creaction . In mostreaction . In addireaction . Recentlreaction . TestingIn future preclinical, as well as clinical trials, donor pigs—and, if necessary, also recipients—should be screened for the presence of circoviruses. When PCV is not found in the donor pig, no screening of the recipients needs to be performed.in utero or soon after birth by infectious colostrum in 29 of 100 cases [There is no specific treatment for pigs with PCVD. Anti-inflammatory agents and antimicrobials may help to suppress co-factors and secondary diseases associated with PCVD. All in/all out pig flow, thorough cleaning, and rigid disinfection between batches of pigs are measures that can help control the disease . In the 00 cases . When co00 cases . Success00 cases ,110.Since new PCV2 variants have emerged, the question of whether or not current vaccines can protect against new PCV2 variants that may be more virulent for pigs becomes a serious concern. Although it is still unclear whether the global switch from PCV2a to PCV2b and PCV2d was associated with higher fitness of PCV2, as reported ,69, rathde novo infection. Elimination means elimination from the herd, elimination from a single individual is impossible, since there is no treatment presently available. The efficacy of the vaccines should be improved and new vaccines against emerging variant virus strains should be developed. Since PCV2 is easily transmitted through the placenta and since colostrum was shown to be infectious, Cesarean section, and colostrum derivation are two promising strategies to eliminate PCV [For a safe xenotransplantation, elimination programs have been proposed for porcine viruses such as HEV , PCMV 11, porcinnate PCV ,107,113.nate PCV , which wnate PCV , confirmnate PCV .PCV2 is a very small virus, it is stable and resistant to some disinfectants, pH, and heat, and it induces severe diseases in infected pigs. PCV2 is an immunosuppressive virus and it is still unclear whether subclinical infections of pigs may decrease the functionality of the organs required for transplantation. Vaccination against PCV2 is able to prevent diseases, but in most cases is unable to prevent the transmission of the virus. Although PCV2 infects human cells and induces a cytopathic effect in vitro, no pathogenic effects were observed when PCV was transmitted by contaminated vaccines to children. It remains unknown whether PCV may infect severely immunosuppressed individuals. In conclusion, for all of these reasons, sensitive detection methods should be used to screen for the virus and improved vaccination, Cesarean delivery, colostrum deprivation, and embryo transfer should be used to prevent transmission of the virus.


To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection.We compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays.4 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine.Oligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 10We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results. Nucleotide mutations in the binding sites of primers and probes can affect the sensitivity.Here, we compare the sensitivity of published real-time RT–PCR assays and two new assays, which we designed to have less nucleotide mismatches with the current outbreak strains. We also present data on viral load profiles in blood and urine from infected patients, using one of the new assays. ,,,,,We compared nine different assays. We included all Zika virus real-time RT–PCR assays published until 1 April 2016. These assays target the membrane (M), envelope (E), nonstructural protein (NS) 1, NS2b, NS3 and NS5 genomic domains.http://www.european-virus-archive.com/Portal/produit.php?ref=1598&id_rubrique=9; Zika virus IVT II available at: http://www.european-virus-archive.com/Portal/produit.php?ref=1599&id_rubrique=9; Zika virus IVT III available at: http://www.european-virus-archive.com/Portal/produit.php?ref=1600&id_rubrique=9; Zika virus IVT IV available at: http://www.european-virus-archive.com/Portal/produit.php?ref=1601&id_rubrique=9; Zika virus IVT V available at: http://www.european-virus-archive.com/Portal/produit.php?ref=1602&id_rubrique=9; and uncRNA 1.0 available at: http://www.european-virus-archive.com/Portal/produit.php?ref=1603&id_rubrique=9.All controls are based on a current Zika virus outbreak strain (GenBank® accession number KU321639). As positive controls, we generated five assay-specific quantified in vitro transcripts (IVT) for the respective genomic target regions. Data on analytical sensitivity, including the standardized measure lower limit of detection, are not available for most of the published assays. To enable stoichiometrically exact analyses of the lower limit of detection for all of the assays, we joined all target domains into a quantative universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand ; Table 1The uncRNA was generated as described previously.A disadvantage of using a test control with a high concentration of viral RNA (e.g. from cell culture) is the potential for laboratory contamination, potentially causing false-positive test results. In contrast to natural viral RNA, potential cases of laboratory contamination with the uncRNA can be proven by two real-time RT–PCR marker assays we designed specifically to detect the uncRNA . These t,We purified viral RNA using the Qiagen Viral RNA Mini Kit  or the MagNA Pure 96 Viral NA Small Volume Kit  according to the manufacturer's instructions. Dengue virus RNA quantification and flavivirus typing were done as described previously.T values using different PCR cyclers and chemistry, we used the Bonn E- and NS1-based assays using either the Superscript III One-Step RT–PCR kit (Thermo Fischer) or the Qiagen® One-Step RT–PCR kit (Qiagen) on a Roche LightCycler® 480 and LightCycler 2.0, a Qiagen Rotor Gene HQ and an Applied Biosystems 7500 thermocycler. Reference conditions refer to the usage of Life Technologies SuperScript® III One-Step enzyme mix and a Roche LightCycler® 480 thermocycler as described above.For all of the experiments, except when assessing threshold cycle variation using different reaction conditions and thermocyclers, we quantified Zika virus RNA using the LightCycler® 480 Instrument II . Generally, 25 μl reactions were set up with 5 μl of RNA; 12.5 μl of 2 × reaction buffer from the Superscript® III one step RT–PCR system with Platinum® Taq polymerase ; 0.4 μl of a 50 mM magnesium sulfate solution (Thermo Fisher Scientific); 1 μg of nonacetylated bovine serum albumin (Roche), 600 nM of each primer and 280 nM of each probe and 1 μl of SuperScript® III RT/Platinum® Taq mix. Amplification involved 50 °C for 15 minutes, followed by 95 °C for 3 minutes and 45 cycles of 95 °C for 15 seconds, 56 °C for 20 seconds and 72 °C for 15 seconds. For comparison of CProbit regression analyses, to determine the lower limit of detection for all real-time RT–PCR assays, were done using SPSS V22  and eight parallel test replicates.We obtained clinical specimens from travellers for which routine medical investigation of either Zika or dengue virus had been requested due to compatible clinical symptoms or a travel history to affected countries. The travellers had acquired their infections in Brazil, the Dominican Republic or Suriname during 2015 and 2016.We spiked a Zika virus-negative human plasma and urine sample with defined quantities of the African Zika virus strain MR766 and an Asian lineage outbreak strain H/PF/2013. Spiked samples were serially diluted and two replicates of each dilution were individually purified using the MagNA Pure 96 Viral NA Small Volume Kit with an input volume of 200 μL and an extraction volume of 100 μL.6–109 RNA copies/mL) as a real-time RT–PCR template.All assay-specific IVT and the uncRNA allowed comparable quantification of Zika virus RNA with a mean twofold deviation of results (maximum deviation: sixfold), suggesting the ability to use these controls to generate comparable results even when different real-time RT–PCR assays are used in different laboratories. The two marker assays showed no detection of Zika virus RNA even upon using RNA from high-titred cell culture isolates . None of the published assays detected the co-circulating alphavirus chikungunya virus or any other flavivirus. As expected, an Asian Zika virus lineage-specific E-based assayWhen we determined the lower limit of detection using the uncRNA, all but two assays showed comparably high analytical sensitivities of around 5–10 Zika virus RNA copies/reaction . An NS2b2–106 copies/mL of either Zika virus representing the Asian lineage (strain H/PF/2013) or the African lineage (strain MR766). The results were consistent with lower limit of detections for the uncRNA. The assays with high analytical sensitivities could detect 103 Zika virus RNA copies/mL of blood or urine in at least one replicate experiment, whereas the assay with the lowest analytical sensitivity yielded negative results even at high Zika virus concentrations of 105 and 106 copies/mL  values – which could vary due to PCR instruments and reagents used – by testing our two new assays under diverse reaction conditions including reagents by different suppliers and different real-time RT–PCR instruments. Even a variation of only two variables could yield up to 4.3 cycles in difference in the CT values for the same virus target concentration, which corresponds to about 20-fold deviations in viral load results  and 2x104 RNA copies/mL of urine (range: 4x102–8x104). The viral loads in the 24 specimens sampled in the first 12 days after symptom onset were comparable to viral loads in serum from patients sampled during the Zika virus outbreak in the Federated States of Micronesia4 RNA copies/mL of blood (range: 1 × 102–4 × 105) and 5 × 103 RNA copies/mL of urine (range: 4 × 102–6 × 104). The samples were taken during comparable intervals: within 11 days after symptom onset for urine and within 12 days after symptom onset for serum samples. Two of these 41 samples contained viral loads of less than 5 × 102 RNA copies/mL . Accordingly, the estimated risk of false-negative results was several-fold lower for dengue than for the Zika virus (Many of the laboratories in countries affected by the current outbreak that are conducting Zika virus testing also have experience in detecting and quantifying dengue virus. To compare the risk of false-negative test results between these two viruses, we quantified 38 clinical samples positive for dengue virus,ka virus .This study provides guidance on the choice of method for diagnosing Zika virus infections and a molecular control that enables the comparison of results between different laboratories and studies. The low viral loads presented in this and a previous study,The results presented here suggest that some published real-time RT–PCR assays may be of limited use for clinical diagnostics during the current Zika virus outbreak. One NS3-based assay that was intended for virus typing should not be used for diagnostics because of its low sensitivity.,–,The low viral loads we detected in urine and blood samples are in agreement with two previous studies reporting quantitative data.,,–,–Although commercial diagnostic real-time RT–PCR reagents for Zika virus detection are available, laboratories in areas affected by the Zika virus outbreak often use non-commercial formulations because of resource constraints.Low viral loads in patients imply a high risk of false-negative test results. Until 7 April 2016, only 3% of 199 922 suspected Zika virus cases could be laboratory-confirmed in the PAHO Region.Endemic countries also need highly sensitive molecular Zika virus detection methods to ensure safe blood transfusions. The Zika virus has been detected in 3% of blood donors in previous outbreaks–The association of Zika virus infection and fetal malformations demand reliable Zika virus diagnostics for pregnant women.In conclusion, our data emphasize the need for highly sensitive assays in molecular Zika virus diagnostics. In addition to an appropriate choice of method, clinical sensitivity can be increased by testing several specimens per patient, by using more than one real-time RT–PCR target, optimizing RNA purification from clinical samples and by combining molecular and serological testing.


All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization.Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. African swine fever virus (ASFV) causes a hemorrhagic fever with a high case fatality rate in domestic pigs and wild boars that has a high socioeconomic impact in affected countries. The disease, African swine fever (ASF), is endemic or causes sporadic outbreaks in most sub-Saharan African countries and in Sardinia, Italy. Following the introduction of ASF to Georgia in the Caucasus in 2007, the disease spread to neighboring countries, including the Russian Federation, and from there to Eastern Europe, including the European Union countries Estonia, Latvia, Lithuania, Poland, and the Czech Republic –6. The c–ASFV is a complex large double-stranded DNA virus with a genome of 173 to 193 kbp, depending on the isolate. The variation in genome length mainly results from the gain or loss of genes belonging to one of five different multigene families (MGFs). The genome contains up to 167 genes, including those encoding enzymes for replication and transcription of the virus genome, which take place predominantly in the cytoplasm. Many genes are not essential for replication in cells, and some of these are known to code for proteins that inhibit host innate and intrinsic defenses , 9; memb10–15–21–Deletion of genes from virulent or already attenuated ASFV isolates has identified a number of nonessential genes. The impact of some of these gene deletions on virus replication in cells, virus virulence in pigs, and induction of protection against challenge with virulent virus has been studied , 25–28. 25–The DP148R gene is located close to the right end of the ASFV genome and encodes a protein of 254 amino acids in the Benin 97/1 isolate. In previous publications, a similarity to the ASFV MGF-360 genes was reported , 30; howd-arabinoside (AraC), an inhibitor of virus DNA replication and late ASFV gene transcription. The size of the band was consistent with a transcription start site upstream of the DP148R translation start and in the intergenic region between L11L and DP148R, thus confirming that the transcript detected was the mRNA for DP148R. The temporal expression pattern found, coupled with the effect of AraC, showed that DP148R is an ASFV gene expressed at early times postinfection. The DP71L gene, which is downstream from DP148R, read from the opposite strand and overlapping by 5 amino acids, is expressed at late times postinfection.The timing of transcription of the DP148R gene was analyzed by rapid amplification of cDNA ends (RACE) PCR using RNA collected at different time points after infection of macrophages . A band Fragments of ∼500 bp from the left and right flanking regions of the DP148R gene from the Benin 97/1 isolate were subcloned into a plasmid upstream and downstream of the β-glucuronidase (GUS) reporter gene under the control of the B646L gene promoter. Following transfection of this plasmid into porcine macrophages infected with the virulent Benin 97/1 isolate, cells infected with recombinant viruses were identified by expression of the GUS gene and purified from wild-type virus. The expected deletion was confirmed by PCR analysis using primers which amplified either the DP148R gene or the GUS gene  and by n6 50% tissue culture infective doses (TCID50)/ml at between 24 and 48 h postinfection. The results show that deletion of the DP148R gene did not significantly alter the ability of the virus to replicate in macrophages in culture  of 0.3 to determine if deletion of the DP148R gene affected the ability of the virus to replicate in these cells in culture. At different times postinfection , cell culture supernatants were harvested and the virus in these supernatants was titrated. The results showed no significant difference between the kinetics and the levels of virus replication of the BeninΔDP148R and parental Benin 97/1 isolates. Virus titers reached a plateau of approximately 10 culture .(a) Experiment 1. The effect of deleting the DP148R gene on virus pathogenesis in pigs and the effectiveness in inducing protection against lethal challenge were tested. In the first experiment, carried out at The Pirbright Institute, 5 pigs  were immunized intramuscularly with 103 50% hemadsorption doses (HAD50) of the BeninΔDP148R virus and boosted 21 days later with the same virus. After a further 3 weeks, pigs were challenged intramuscularly (i.m.) with 104 HAD50 of virulent parental virus Benin 97/1. In parallel, a group of 3 control pigs was challenged with the same dose of parental virus. Clinical signs were recorded daily from day 0 (immunization). Necropsies of control pigs showed gross lesions characteristic of acute ASF . Except (b) Experiment 2. In the second experiment, carried out at IRTA/CReSA, Barcelona, Spain, 1 group of 6 pigs was immunized intramuscularly with 103 HAD50 of the BeninΔDP148R strain (group C) and another group of 6 pigs (group D) was immunized by the intranasal route with 103 HAD50. Both groups were boosted 2 weeks later by the same route and with the same dose. Pigs were challenged intramuscularly 2 weeks after the boost with 104 HAD50 of virulent parental virus Benin 97/1. A control group of 3 nonimmune pigs was challenged by the same route at the same time as a control group for the inoculum.As shown in Macroscopic evaluations at necropsy of protected pigs vaccinated i.m. (group C) showed mild gross lesions . Pig C1 In the group of 6 pigs immunized by the intranasal route group D), five of the pigs had an increased temperature and reduced levels of food consumption postimmunization  and C. F, five ofAt necropsy, protected pigs displayed mild gross lesions . Pig D1 P < 0.03) in the animals infected with the parental Benin 97/1 virus (data not shown), confirming that the deletion of DP148R causes viral attenuation in vivo.In both experiments, the clinical scores and temperatures at days 3 and 4 postinoculation were compared between pigs immunized with BeninΔDP148R and control pigs. These were significantly higher ((a) Experiment 1. The levels of virus in blood samples from experiment 1 were measured by quantitative PCR (qPCR) to detect the virus genome in all immunized and control pigs. 5 and 1 × 106 genome copies per ml were detected from day 4 postimmunization. Genome levels reached a peak by day 7 of between 4 × 105 and 2 × 106 genome copies per ml. After this, the number of genome copies per milliliter gradually declined. At termination (day 58 postimmunization), levels of between 1.9 × 102 and 1.5 × 104 genome copies per ml were detected. This was at day 14 postchallenge. However, no increases in virus genome levels were detected following either boost or challenge, indicating that additional virus replication may not have occurred. In control nonimmune pigs, genome levels of up to 108 copies per ml were detected. At day 4 postinoculation , genome copy numbers were significantly higher (P = 0.0008) in control pigs (data not shown).5 and greater than 106 TCID50/ml. After day 7, the levels of infectious virus declined and became undetectable by day 28 at the latest. Thus, the levels of infectious virus were similar to the number of genome copies between days 4 and 10 postimmunization, but from day 15 to day 28, the levels of infectious virus detected decreased dramatically compared to the numbers of genome copies. Thus, infectious virus was detected up to 1 week postboost but was not detected postchallenge.The prolonged and gradual decrease in the level of the virus genome detected in blood from immunized pigs suggested that virus may not have been replicating throughout the period but, instead, may have been attached to red blood cells and gradually lost infectivity. A high percentage (>90%) of ASFV is found attached to red blood cells in blood from pigs infected with hemadsorbing isolates , 34; thi35–(b) Experiment 2. In experiment 2, pigs immunized with BeninΔDP148R at 103 HAD50 i.m. (group C) had detectable levels of the virus genome from day 3 postimmunization, reaching a peak at day 5 of between 105 and 107 genome copies per ml -specific enzyme-linked immunosorbent spot (ELISpot) assay and ELISA, respectively. Peripheral blood mononuclear cells (PBMC) collected prechallenge from pigs in group E (immunized by the i.m. route) were stimulated with virus or a mock inoculum, and the numbers of cells producing IFN-γ were measured by ELISpot assay . Mock stThe role of cytokines involved in the regulation of the immune response was also explored. The levels of IFN-γ and interleukin-10 (IL-10) in serum were measured at different days postimmunization and postchallenge (group E). A peak in the level of IFN-γ was detected in serum from all pigs at day 4 postimmunization , and smaThe lack of information on the function of many ASFV genes hinders the understanding of virus-host interactions and the development of vaccines. Genes that are not essential for virus replication in cells represent potential candidates for deletion so as to construct live attenuated vaccines. In addition, such genes may have roles in evading host defenses. Here we report the phenotype of an ASFV mutant with a deletion of a gene that encodes a protein, DP148R, without significant similarity to other known proteins. Although previously suggested to be a member of MGF-360, we found no significant similarity to genes in this family. We showed that deletion of the DP148R gene from the virulent Benin 97/1 isolate does not reduce replication of the virus in porcine macrophages, suggesting that the encoded protein is not essential. We determined the kinetics of mRNA transcription and showed that the DP148R gene is expressed early in infection.3 HAD50, and in the second experiment, one group of pigs was immunized by the intramuscular route and one was immunized by the intranasal route at a dose of 103 HAD50. In these experiments, 16 of the immunized pigs had an increased temperature, as well as lethargy and a loss of appetite, for 1 or 2 days. The timing of this increase in clinical signs was more variable for those immunized by the intranasal route than those immunized by the intramuscular route, possibly because the timing of peak virus replication was less synchronized. One pig (pig D4) immunized intranasally did not have an increased temperature but may not have been vaccinated since we did not detect an ASFV-specific antibody response in this pig. We have previously shown that intranasal immunization with a live attenuated vaccine can induce good levels of protection  at day 5 postimmunization compared to the control animals at day 5 postchallenge. The levels of the virus genome declined after this time, but it was detectable over an extended period. Extracellular ASFV particles are known to bind to the surface of red blood cells, and this is mediated by the CD2v protein . Some correlation between levels of protection and the numbers of IFN-γ-producing cells detected following virus stimulation has been observed previously, but this has not been observed in all studies , 39–41. 39–otection . The resotection . Howeverotection –46. IFN-tection –. These itection –. IFN-γ ptection –. Howeverin vivo. For example, deletion of the ERV1-ALR homologue 9GL gene affects virion maturation and viral growth in macrophages and attenuates the virus infection in pigs  or by immunofluorescence using antibodies against ASFV early protein p30/CP204L.The Benin 97/1 isolate was described previously . Virusesd-glucopyranosiduronic acid and purified by limiting dilution.Right and left genome fragments of approximately 500 bp flanking the DP148R gene were amplified by PCR and cloned into the pLoxPVP72GUSLoxP vector (Alveolar macrophages were infected with the Benin 97/1 isolate at a multiplicity of infection (MOI) of 1. RNA samples were collected at different times postinfection (p.i.) using an RNeasy minikit . DP148R transcripts were evaluated by RACE  according to the manufacturer's instructions. Briefly, 1 μg of RNA was reverse transcribed using the following gene-specific primer 1 (GSP1): GGAGCAGCAGTAAGAAGTC. After purification of the first-strand product, thymine-deoxyribosylthymine (TdT) tailing of the 3′ end of the cDNA was performed. The transcripts were amplified by PCR using a primer (GCTGCGAGCGTAGTTTGG) designed to be specific for the region upstream of GSP1 and the anchor primer provided in the kit.PBMs were infected at a multiplicity of infection of 0.3 with the Benin 97/1 or BeninΔDP148R virus. Cells and supernatants were collected at different times postinfection and subjected to 3 freeze-thaw cycles. Cellular debris was removed by centrifugation, and virus titers were determined by the immunofluorescence assay.3 HAD50 BeninΔDP148R and boosted 2 or 3 weeks later with the same dose of virus. Control and immunized pigs were challenged with the virulent parental Benin 97/1 strain 2 to 3 weeks after the boost , and experiment 2 was conducted at IRTA/CReSA  according to regulated procedures from the Animals (Scientific Procedures) Act UK 1986. At The Pirbright Institute, female Large White Landrace piglets were obtained from a high-health-status farm. At IRTA/CReSA, crossbred male piglets that were 8 to 9 weeks old and that had been vaccinated against porcine circovirus type 2 (PCV2) were obtained from farms with a high sanitary status where the animals tested negative for porcine respiratory and reproductive syndrome (PRRS) and Aujeszky's disease. In both experiments, pigs were immunized with 10he boost .After immunization (0 dpi), rectal temperatures and clinical signs were monitored daily and scored as described previously . Blood . Gross lesions were assessed during necropsies by following scoring methods based on previous standardized protocols . During DNA was extracted, using a MagMAX extraction system (Thermo Scientific) and a MagVet universal isolation kit (Life Technologies), from whole peripheral blood that had been collected in EDTA-containing tubes at different days postimmunization. qPCR was carried out on a Stratagene Mx3005P system  following a protocol modified from that of King et al. , 51 usin5 and 2.5 × 105 per well, in RMPI supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The cells were then incubated overnight in a final volume of 200 μl with 105 HAD50 of Benin 97/1, an equivalent volume of mock inoculum, or 2.5 μg/ml phytohemagglutinin as a positive control. Cells were lysed by incubation for 5 min in water and then washed with PBS. Biotinylated P2C11, followed by streptavidin conjugated to alkaline phosphatase, was used to visualize the spots, which were then counted using an ELISpot assay reader system (Autoimmun Diagnostika GmbH). The number of spots per well was converted into the number of spots per million cells, and the mean for duplicate wells was plotted  were purified from EDTA-anticoagulated blood using gradient centrifugation. ELISpot assay plates were coated overnight at 4°C with 4 μg/ml P2F6 in 0.5 M carbonate-bicarbonate coating buffer and then washed with phosphate-buffered saline (PBS). Cells were plated in duplicate at two different dilutions, typically, 5 × 10 plotted .The level of antibodies against ASFV-specific protein p72/B646L in serum was measured using a competition ELISA kit (Ingenasa PPA3 Comppac). The percentage of blocking was calculated using the following formula: [(negative-control OD − sample OD)/(negative-control OD − positive-control OD)] × 100, where OD is optical density.The levels of IFN-γ and IL-10 in serum were measured by ELISA according to the manufacturer's recommendations .t test was used.Statistical analysis was performed using GraphPad Prism (version 6) software. For clinical scores and temperatures, significant differences between groups were determined using two-way analysis of variance followed by Sidak's multiple-comparison test. For differences in genome copy numbers, an unpaired


Influenza A virus (IAV) membrane proteins hemagglutinin (HA) and neuraminidase (NA) are determinants of virus infectivity, transmissibility, pathogenicity, host specificity, and major antigenicity. HA binds to a virus receptor, a sialoglycoprotein or sialoglycolipid, on the host cell and mediates virus attachment to the cell surface. The hydrolytic enzyme NA cleaves sialic acid from viral receptors and accelerates the release of progeny virus from host cells. In this study, we identified a novel function of HA and NA as machinery for viral motility. HAs exchanged binding partner receptors iteratively, generating virus movement on a receptor-coated glass surface instead of a cell surface. The virus movement was also dependent on NA. Virus movement mediated by HA and NA resulted in a three to four-fold increase in virus internalisation by cultured cells. We concluded that cooperation of HA and NA moves IAV particles on a cell surface and enhances virus infection of host cells. Human influenza virus infects epithelial cells in the respiratory tract, the surface of which is covered by abundant cilia. Because a cilium is thin with a diameter of ~0.25 μm and its inside is fully occupied by microtubule bundlesInfluenza virus, Semliki Forest virus (SFV), and vesicular stomatitis virus (VSV) are enveloped viruses that are internalised into host cells by cellular endocytosis3−3 M115–1011-fold greater than the Kd of adhesive proteins on other viruses  or the Kd of several physiological ligands  moves on an artificial membrane surface by lateral diffusion of its receptor in a lipid bilayer ligands 131415161To investigate whether IAV movement occurs via the exchange of HA-receptor binding pairs, we coated the surfaces of glass coverslips with fetuin as a viral receptor and investigated the movement of the IAV strain Aichi/2/68(H3N2) (Aichi2) conjugated to a fluorescent probe, octadecyl rhodamine B (R18) on the fetuin-coated surfaces using total internal reflection fluorescence microscopy (TIRFM) combined with single particle tracking analyses . Using tQuantitative analyses of 100 virus particles were undertaken to adequately characterise the crawling and gliding motions of Aichi2 virus . Using tF(r) is the ECCDF of r, r is the displacement of virus particles per second, and P(r) is the frequency function of r. As the displacement elongated, its appearance frequency became lower. Therefore, virus motions with long displacements were difficult to analyse by the normal frequency function, P(r)  . ECCDF ion, P(r) . Here, won, P(r) . The freTo test for heterogeneity of virus motility, we examined the gliding frequency of individual viruses . All vir−2 μm2/s, which is similar to the previously reported values for the lateral diffusion of transferrin, LDL, insulin, and EGF receptors (2223The mean square displacement (MSD) of IAV particles indicated that the viruses moved two-dimensionally in a random fashion   that is eceptors 222324. OGenerally, influenza virus NA is thought to mediate progeny virus budding from host cells2627−2 μm2/s) was 3.4 times smaller than that of the wild-type virus (0.72 × 10−2 μm2/s)  . The NA 2 μm2/s) . The low2 μm2/s)  and S6. 2 μm2/s) . ImportaWe next used TIRFM to examine Aichi2 virus movement on human erythrocytes. We found that viruses moved around the cell surface . The NA NA inhibitors such as zanamivir and oseltamivir suppress virus infection26271To evaluate the inhibitory effect of zanamivir on virus internalisation, we infected A549 cells with Aichi2 virus and examined viral protein synthesis and plaque formation. The NA inhibitor decreased the percentage of cells producing viral proteins and virus plaque formation to approximately 25% compared with untreated controls . We alsoAnalysis of NA mutant virus infectivity indicated that limited motility was ineffective for virus infection. The NA R278K mutant virus had suppressed motility . InfectiA significant conclusion of this study is that IAV can move using the HA-receptor exchange mechanism . The virin vivo. Reportedly, NA inhibitors decrease the levels of infection in primary cultured respiratory epithelial cells by 20–500 foldResults of infection experiments using Aichi2 virus in the presence of the NA inhibitor or the PR8 mutant virus lacking NA activity (NA R103K) indicated that virus movement by this HA-receptor exchange mechanism accelerated virus entry into cultured cells by three to four fold . This efIn addition to intercellular migration, virus movement via HA-receptor binding exchange confers another potential advantage for virus infection compared with lateral movement of a receptor along the cell membrane. When using HA-receptor binding exchange, a virus would be able to move even if bound to immobile receptors. Attachment to immobile receptors would occur when membrane proteins and lipids are restricted from free lateral diffusion in lipid bilayers by membrane skeletons, membrane rafts, and cytoskeletons223234We further hypothesised that altering the ratio of crawling and gliding motions might influence IAV host specificity. IAV host specificity is dependent on virus receptor types. Human influenza HAs attach to oligosaccharides with terminal sialic acids (SA) linked to neighbouring galactoses  via α2–6 linkages , whereas avian virus HAs attach to oligosaccharides via SAα2–3 Gal linkages3738Our study demonstrated that IAV movement across the host cell surface was coordinated by its HA and NA proteins. This novel motility mechanism enables efficient access to target cells or cellular surface domains with high endocytosis activity. Until now, it was generally accepted that no viruses, including influenza, possess motile machinery. Our findings confirmed the presence of motile machinery employing HA and NA molecules in IAV. This virus motility mechanism is different from those known to exist in bacterial and eukaryotic cells. Further studies on this virus motility mechanism, particularly quantitative analysis of HA affinity and NA activity, might lead to a new paradigm for molecular mechanisms and energy transfer mechanics. Furthermore, systematic analysis and categorisation of the movement patterns of various viral strains could provide valuable information to understand the infectivity and host specificity of influenza viruses.Clostridium perfringens NA . These viruses were isolated from allantoic fluids at 3 dpi. All viruses were purified by differential centrifugation and sucrose density gradient centrifugation.The influenza Aichi2 virus was prepared from allantoic fluids of infected eggs at 2 days post-infection (dpi). The influenza PR8 wild-type and mutant viruses were generated by reverse genetics as described previouslyA549, MDCK, and 293T cells were subcultured using Dulbecco’s modified Eagle’s medium  containing 10% fetal calf serum. Zanamivir was provided by GlaxoSmithKline Research and Development Ltd. .IAVs were labelled with R18  as follows. Purified virus  was suspended in 1 ml phosphate-buffered saline , and R18 was dissolved in ethanol to a final concentration of 100 μM. The R18 solution (10 μl) was added to the virus suspension and mixed vigorously. The reaction mixture was then gently mixed for 1 h at room temperature and subsequently passed through a filter (0.22-μm pore size).For imaging analyses of virus movements, the glass bottoms of culture dishes  were coated with bovine fetuin  and bovine serum albumin  dissolved in PBS. In experiments to determine the dependency of virus motility on receptor density, the glass bottoms of culture dishes were coated with fetuin (0–1.0 mg/ml) and albumin (0–1.0 mg/ml) dissolved in PBS. Total protein concentrations of fetuin and albumin were adjusted to 1 mg/ml. The coating FR was defined asThe glass surface was incubated for 1 h at room temperature and then washed once with PBS and twice with distilled water to remove any unbound fetuin. Culture dishes were dried at room temperature for 30 min. This cycle of coating and drying was repeated three times. To examine virus behaviour, fluorescently labelled viruses diluted approximately 100–1,000 times by PBS were added to the fetuin-coated glass surfaces and observed by TIRFM  using a 150×  objective lens. Usually, fluorescence images were acquired every second for a total duration of 15 min. For high speed imaging, fluorescence images were acquired every 0.1 second for a total duration of 1 min.To explore virus movement on a cell surface, a monolayer of erythrocytes was prepared on the bottom surface of glass dishes. Human erythrocytes were washed three times with PBS and resuspended in PBS to a final concentration of 1% (v/v). The erythrocyte suspension was allowed to attach to poly-L-lysine-coated glass at 4 °C for 15 min. The glass was then washed with PBS three times to remove unbound erythrocytes. The movement of fluorescently labelled viruses on erythrocyte surfaces was observed by TIRFM as described above.http://rsb.info.nih.gov/ij/)42Images were analysed with Particle Tracker of ImageJ software  model. The MSD of viruses was fitted to the equation of two-dimensional random diffusion:2〉 is the MSD of viruses, D is the diffusion coefficient, and t is time.where R is the displacement of virus, which is obtained from the position where the virus particles initially attaches, 〈RN-acethylneuraminic acid 292 with MUNANA at a final concentration of 500 μM. The reaction mixtures (20 μl) were incubated at 37 °C for 30 min and stopped by the addition of 130 μl stop solution . Fluorescence of the released 4-methylunberiferon was measured with a Varioskan Flash fluorescence microplate reader (Thermo Fisher Scientific) using excitation and emission wavelengths of 355 and 460 nm, respectively.The NA activity of each purified virus was measured by a fluorometric assay using the fluorogenic substrate 2′-(4-methylunberiferyl)-α-D-NA expression levels were examined by western blot analysis. Viral proteins were separated by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) in a 5–20% gradient gel and then transferred to a nitrocellulose membrane. The membrane was stained with anti-NA mouse immunoglobulin G (IgG) and anti-M1 rabbit IgG , followed by staining with fluorescently labelled secondary antibodies, IRDye680CW goat anti-mouse IgG and IRDye800CW goat anti-rabbit IgG . Fluorescent intensities of NA and M1 bands were measured with an infrared imaging system Odesay CLx (LI-COR). Relative NA expression levels were evaluated using the fluorescence intensities of NA bands standardised to the intensities of M1 bands.Aichi2 virus was labelled with DiOC18 (Molecular Probes) and R18 using the same protocol described above for R18 labelling except that a probe mixture (6 μl) containing both DiOC18 (33 μM) and R18 (67 μM) was used instead of R18 alone. A549 cells cultured in glass dishes were incubated with fluorescently labelled viruses in the absence or presence of zanamivir (5 μM) for 30 min at 37 °C and then washed with PBS. Imaging was conducted using the dual wavelength imaging technique5 cells/well) and infected with Aichi2 viruses in the presence or absence of zanamivir (5 μM) for 30 min at 37 °C. The cells were washed five times with DMEM to remove residual zanamivir and then incubated in serum-free medium  for 6 h at 37 °C for virus production. The cells were then fixed with 1% paraformaldehyde in PBS, and viral antigens were detected by indirect immunostaining using a rabbit anti-human influenza A virus antibody  and Alexa Fluor 488-labelled goat anti-rabbit IgG . After staining with the secondary antibody, cell nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride. Using an image screening system , more than 1 × 104 cells were examined in each experiment, and cells producing viral proteins were counted.To examine Aichi2 virus infectivity, A549 cells were cultured overnight on a 24-well pate (1 × 105 cells/well) and then infected with purified wild-type or mutant viruses  in the presence or absence of zanamivir (5 μM) for 30 min at 37 °C. The cells were washed five times with DMEM and then incubated in VP-SFM for 6 h at 37 °C for virus production. After the incubation, staining and counting of cells were performed by the procedure described above.To examine PR8 wild-type and mutant virus infectivity, A549 and MDCK cells were cultured overnight on a 24-well pate (1 × 10Confluent A549 monolayers in six-well plates were infected with Aichi2 viruses in the presence or absence of zanamivir (5 μM) for 30 min at 37 °C. The cells were washed five times with DMEM to remove residual zanamivir and overlaid with VP-SFM containing 0.55% (w/v) agarose and 1 μg/ml acetylated trypsin. Plaques were counted at 1 dpi.How to cite this article: Sakai, T. et al. Influenza A virus hemagglutinin and neuraminidase act as novel motile machinery. Sci. Rep.7, 45043; doi: 10.1038/srep45043 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the field of forensic medicine, it is very difficult to know prior to autopsy what kind of virus has infected a body. We assessed the potential of the genome profiling (GP) method, which was developed in the field of bioengineering, to identify viruses belonging to one species. Two species in the same family, JC and BK viruses, were used in this study. Using plasmid samples, we compared the findings of molecular phylogenetic analysis using conventional genome sequencing with the results of cluster analysis using the random PCR-based GP method and discussed whether the GP method can be used to determine viral species. It was possible to distinguish these two different viral species. In addition to this, in our trial we could also detect the JC virus from a clinical sample.This method does not require special reagent sets for each viral species. Though our findings are still in the trial period, the GP method may be a simple, easy, and economical tool to detect viral species in the near future. Comparing the BKPyV isolates to the JCPyV isolates, the PaSS value among BKPYV isolates was 0.9632-0.9802 (0.9710 ± 0.0039) (Table 4). There was a statistically significant difference between the PaSS values among these two virus species (P<0.01). When the GP method was replicated, the PaSS values in the same virus strain were 0.9768–0.9893 (mean ± SD: 0.9841 ± 0.0036).For the GP method, the average number of 4). The JCPyV and BKPyV clusters were apparently independent. The JCPyV strains formed two major clusters. One of the clusters contained the SW-3, GR-3, GH-1, and CB-2 strains, while the other cluster contained the remaining strains. The results of cluster analysis after the GP method are shown in Fig.  shows the results of the GP method using the JCPyV positive urine sample. JCPyV specific spiddos were obtained in this sample. There were also numerous spiddos obtained from this sample. Those obtained spiddos were different from those of the plasmid DNA samples. Sequencing analysis was also performed on this sample, and this JCPyV genotype was CY. Fig. (6b) shows the results from the GP method using the JCPyV negative urine sample. There were no JCPyV specific spiddos obtained in this sample. Finally, we analyzed clinical samples. Fig. 6spiddos were much higher in the JCPyV. This is because the random primer attaches to and amplifies different DNA sequences. The homology between JCPyV strains is about 99%; however, several different spiddos are obtained. It also is considered that amplified DNA fragments might include vector DNA as well as virus DNA. However, the virus specific spiddos show that among those amplified fragments, virus DNA were surely amplified, and the number of the common spiddos is 2 or 3. Though these numbers are relatively small, if we consider the small size of virus genome, it is reasonable. Small differences, such as between the isolates in the same virus species, are calculated by the PaSS score. So the number of the spiddos is not used for the determination of virus species. The number of the spiddos is important for the calculation of PaSS. If the number of the spiddos is rich, we can get more reliable PaSS results. Because of the random PCR system, we cannot avoid the amplification of other contaminated DNA. Therefore specific spiddos are the most important for the diagnosis of those viruses.Though the homology between JCPyV and BKPyV is about 70%, the number of the spiddo pattern that represents a genome sequence. In addition, as around 70% of the genome sequence of BKPyV is the same as that of JCPyV, they can be distinguished using the GP method. However, because around only 1% of the genome sequence is different among the JCPyV or BKPyV isolates, it might be difficult to distinguish such small differences using the GP method. These small differences are very important for us to analyze population history not only in the studies using JCPyV and BKPyV [spiddos are selected manually, the researcher’s technique may affect the reproducibility of the results. In our previous study, when one highly skilled researcher performed the GP method 5 times using the same sample, the PaSS value was 0.9905 ± 0.0024. If the GP method had complete reproducibility, the PaSS value should have been 1.0. As a result, the difference of the PaSS value in the GP procedure was around 1% [spiddos using special software. If the spiddos are selected automatically, we will always be able to obtain the same spiddos; the PaSS values will be 1.0. Finally, the spiddos used in the calculation of PaSS might include the amplified fragment of the vector DNA. That dilute the difference among the each viral strains. Because of the small genome size of those viruses, the number of the specific spiddos is low. Therefore, we consider it might much more reliable to use all obtained spiddos for the calculation. The results of cluster analysis obtained using the GP method did not agree completely with those obtained using phylogenetic analysis. Although we could discriminate between virus species, we could not discriminate virus genotype with the GP method. We consider one of the main reasons for this to be that cluster analysis does not include molecular evolution aspects; it only classifies a similar nd BKPyV , 32-37 bnd BKPyV , 39. In In this study, the whole procedure required around 6 hours using the GP method and around 36 hours for the conventional method. Especially, checking the obtained sequence in the electropherograms generated by the sequencer requires a considerable amount of time. The GP method does not require such time-consuming procedures or expensive reagents and machines. The sensitivity of the GP method was also very high. However, the detection from plasmid DNA samples may different from the detection from clinical urine samples. We will try to examine the sensitivity using the clinical materials.spiddos were present only in JCPyV or BKPyV. These virus-specific spiddos represent virus-specific sequences. Therefore, if many kinds of virus genome are analyzed in advance with the GP method, it may be possible to identify virus species by detecting virus-specific spiddos.Some of the spiddos . In the JCPyV positive urine sample , there were also numerous spiddos obtained from this sample. These spiddos might have resulted from the amplification of human DNA contained in the urine. The number of human cells in the urine varies with the individual, and obtained spiddos were different from those of plasmid DNA samples. That might be due to the fact that most of the materials for amplification were used to amplify the human DNA. However, we could obtain the virus specific spiddos. In the JCPyV negative urine sample , there were no JCPyV specific spiddos obtained. Neither could we obtain many spiddos in this sample, indicating that the urine contain little DNA of human or of microorganisms. The amount of human DNA in the urine may be also affected by the urinary virus infection. With this result, we consider that we may be able to utilize the GP method to detect polyomavirus in urine samples. Of course, for this purpose it is necessary to validate this method with a large number of cases.In our trials using clinical samples, we could detect many In this study we showed that the GP method can be used to discriminate among different human polyoma-virus species. In addition, it may be used for other virus species. The GP method is a very simple and inexpensive tool to analyze virus genomes. It does not require virus-specific primers, so although it cannot distinguish virus genotypes, it may be used for virus screening. We demonstrated the possibility to utilize this method to detect urinary viruses using test clinical test cases. We plan to analyze various viruses in clinical samples in the near future.