Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the the MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes Simplex Virus-1, Mumps Virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.the most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4 mg/ml, and a reduction of 3.5Log and 2Log, at 2 mg/ml and 1 mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1 mg/ml and 1Log at 4 mg/ml and 2 mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4 mg/ml. Herpes Simplex Virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis. Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan which consists of alternately repeating D-glucuronic acid and N-acetylglucosamine units. A huge variety of HAs, with different molecular weights, has been described, probably retaining distinct physicochemical and biological properties. HA is naturally present throughout all mammalian systems, especially synovial fluid, vitreous humour serum and many connective tissues . MoreoveInitially introduced in clinical practice as wound healing promoter, HA is currently used in many medical and cosmetic fields. Some examples of HA applications include eye drops for kerato-conjunctivitis, intra-articular injections for osteoarthritic joint pain, irrigations for bladder and vaginal chronic inflammatory disorders, tracheobronchial aerosolization for asthma, oral solutions for mouth care or for oesophageal-reflux and gastritis. Besides, HA is commonly used for cosmetic interventions, as a filler to counteract ageing and facial lipoatrophy, especially in HIV patients .in vitro. In particular, the replication of Herpes Simplex Virus type 2 [There is evidence showing the ability of HA to interfere with viral replication s type 2 , Respiras type 2  and retrs type 2  is inhibs type 2 . Such liin vitro effects of a high molecular weight HA against a wide group of viruses covering a large spectrum of structural features and replication strategies: ADV-5, Coxsackievirus B5 (COXB5), Herpes Simplex Virus type 1 (HSV-1), Human Herpesvirus-6 (HHV-6), Influenza Virus A/H1N1, Mumps Virus (MV), Porcine Parvovirus (PPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). We observed an antiviral activity against COXB5, HSV-1, MV, PPV and Influenza Virus encouraging the use of such compound as a medical tool in specific clinical circumstances.In this study we investigated the A high molecular HA (1.800 KD) in powder  was used. It was dissolved in Minimum Essential Medium (EMEM) at 8 mg/ml solution and sterilized by filtration through 0.45 μm filters.The following cell lines were used to cultivate the different viruses: two monkey kidney lines, VERO cells for ADV-5, COXB5, HSV-1, and MV and MARC145 cells for PRRSV; the human T-leukaemia lymphoblast line JJHAN for HHV-6; the canine kidney line MDCK for Influenza Virus; the pig cell line PK15 for PPV. VERO, MARC145, PK15 and MDCK cells were cultured in EMEM added with 10% (growth medium) or 5% (maintenance medium) foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml); RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) was used for JJHAN cells. All the cell lines were incubated at 37°C with 5% CO2.The viral strains of HSV-1, ADV-5, COXB5 and MV were clinical isolates, laboratory adapted through serial passages (>50) on VERO cells. The Influenza Virus strain used was the highly neurotropic cell culture adapted WSN33 strain (A/H1N1). For HHV-6, the U1102 strain (variant A) was employed, whereas the two swine viruses tested were reference strains: NADL-2 for PPV and the ATCC strain (Cat. N° VR-2402) for PRRSV. Batches of each virus were prepared, titrated on the suitable cell line and kept frozen at -80°C until they were used for the experiments.The MTT test  was usedTwenty-four hour growth VERO and MDCK cells in 96 well plates were exposed for 1 h to HA (4 mg/ml); then a cell lysis solution Triton X-100 was added to each well at a final concentration of 0.1% and 0.5%. After 5' for 0.1% concentration and 15' for 0,5% concentration, the lysis solution was removed and replaced with fresh medium. The cell viability was measured by MTT test and the survival of HA treated cells was compared with that one of HA untreated cultures. In each assay, cells, not exposed to lysis solutions, were used as controls (100% viability). Three experiments were carried out, each one with samples in triplicate.The antiviral activity was ascertained by means of virus yield assays. Twenty-four hour growth cell cultures were infected with the different viruses at the following multiplicity of infection values: for COXB5, ADV-5 and MV 0.1Tissue Culture Infectious Dose 50% (TCID50)/cell; for HSV-1, 0.1 Plaque Forming Unit (PFU)/cell; for WSN33 virus, 0.1 PFU/cell; for PPV, 0.1 TCID50/cell; for PRRSV, 0.1 TCID50/cell; for HHV-6, 0.1 TCID50/cell. After 1 hr adsorption at 37°C, the inoculum was removed, the plates washed with PBS and the different dilutions of HA, in maintenance medium, added to the cell cultures (each HA dilution was tested in triplicate). After 24 hr incubation for COXB5, ADV-5, MV and HSV-1, 48 hr for WSN33 and PRRSV, 72 hr for PPV and 6 days for HHV-6 the plates were frozen and thawed three times and the viral yield was titrated by end-point titration for ADV, COXB5, MV, PPV, PRRSV, and HHV-6 and by plaque assay for HSV-1 and WSN33. As far as end-point titration is concerned, 10-fold dilutions of each cell lysate were seeded on the 24 hr growth cells in a 96-well culture plate. After 3 days (6 days for HHV-6), the viral titre of each sample, expressed as TCID50/ml, was read taking into account the final dilution still showing the typical viral cytopathic effect and the results were elaborated using the Reed and Muench formula [Time course experiments were carried out with COXB5 and HSV-1 within a single replication cycle. HA (2 mg/ml) was added, at different time points, within 7 hr for COXB5 and 18-20 h for HSV-1 according to each viral replication cycle. In parallel wells, HA was added together with the viral inoculum (t = 0). The virus yield was assessed by end point titration (for COXB5) or plaque assay (for HSV-1). For each virus, 3 experiments were carried out, each in duplicate.The different viral inocula were exposed to HA at a final concentration of 4 mg/ml, for 30' at room temperature and then their residual infectivity was titrated on the suitable cells, as described above. A viral inoculum, treated with medium without HA was used as control for each virus. Two experiments for each virus were performed, each in duplicate.Initially, in order to rule out any direct cytotoxic effect of HA, dose-dependent experiments were performed exposing the five cell lines, employed in the experiments, to HA for different times  followed by MTT assay. We found modest cytotoxicity only at the highest HA concentration (4 mg/ml) which caused a OD reduction of about 20% in 4 of the 5 tested cell lines  were carried out. We found that both cell lines pre-treated for 1 hr with HA at 4 mg/ml were significantly more resistant to lysis than the untreated controls. In particular, for VERO cells in the HA pre-treated groups, the cell viability was close to 100% with the mild treatment (0.1% for 5') and 24.4% with the stronger one (0.5% for 15'), whereas in parallel groups, not pre-exposed to HA before lysis, the cell viability was reduced to 68.1% and 15.2%, respectively  after HA treatment, indicating no virucidal activity by HA against any of the tested viruses (data not shown).This paper provides a new information on the biological properties of HA, describing its antiviral in vitro activity against 5 viruses, with different structural and biological features.in vivo and, though to a lesser extent, also in vitro. The different cell system we used may account for the lack of replication enhancement observed in our experiments.HA is a non-sulfated glycosaminoglycan widely present in the extracellular matrix of soft tissues and in several biologic fluids . ExperimOur findings provide evidence on the effects of HA on a variety of RNA and DNA viruses, with or without the lipidic envelope, characterized by very different replication strategies. Therefore, we can speculate that HA antiviral mechanism(s) probably involves general/non-specific host cell-virus interaction at membrane level, such as virus entry or release, rather than restricted, virus-specific events occurring inside the cell. This speculation is supported by the kinetic results showing that both COXB5 and HSV-1 are growth inhibited irrespective of the time of HA addition during the virus replication cycle. In line with this hypothesis, we may suggest that HA, known to have a heavy ionic charge, may alter the electrostatic interactions between virus and cell receptors and/or other cell membrane components, thus in turn affecting virus entry and/or exit. Literature data substantiate this hypothesis. Dengue virus uses heparan-sulphate (HS) as a cell receptor: different types of HA, interacting with the virus envelope glycoprotein, responsible for virus attachment to HS, are able to inhibit virus attachment and entry . HSV-1 rin vivo.In conclusions, our study provides a wide spectrum demonstration of the antiviral activity by HA, opening new perspectives in prophylaxis and therapy of some viral diseases. The hypothesis of specifically counteracting cell-virus attachment and viral release, by local administration of HA, is very appealing especially for oral, genital and ano-rectal anatomical areas, where compound(s) can be administered as creams, gels or wash solutions. Many HA-based commercial products already available for topic use have HA concentrations much higher or equal to those we found active against different viruses: so it can be hypothesized that effective concentrations can be locally reached. The inhibitory activity observed against HSV-1 is particularly interesting since HA is the basic component of mouthwash solutions and ophthalmic drops, used as artificial tears. According to our findings, HA may be also considered/included as an antiviral agent in the treatment of HSV-1-associated stomatitis and kerato-conjunctivitis in this kind of preparations. Moreover, since Enteroviruses are often responsible for a childhood form of vesicular stomatitis as well as respiratory diseases, the present findings on Coxsackievirus inhibition may open new perspectives for oral administration of HA as natural atoxic medical treatment of newborns and babies. Similarly, the anti-influenza virus activity observed might be exploited in nasal sprays to locally reduce viral replication. Moreover, HA has been demonstrated to have pro-inflammatory activity. Low weight HA fragmentation products and, in the presence of IFNγ, even high molecular weight HA molecules can also activate innate immunity mechanisms through Toll-like Receptor 4 and CD44 ,22. ThisIn conclusion, our findings strongly support the use of this safe glycosaminoglycan in clinical practice as a potential antiviral compound, both for disease prevention and treatment. Further clinical trials on this topic are required to better understand the antiviral activity of this compound.ADV-5: Adenovirus-5; COXB5: Coxsackievirus B5; EMEM: Eagle Minimum Essential Medium; FBS: Foetal Bovine Serum; HHV-6: Human Herpes Virus-6; HSV-1: Herpes Simplex Virus-1; HS: heparan sulphate; MV: Mumps Virus; PFU: Plaque Forming Unit; PPV: Porcine Parvovirus; PRRSV: Porcine Reproductive and Respiratory Syndrome Virus; OD: Optical Density; SI: selectivity index; TCID50: Tissue Culture Infectious Dose 50The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.The authors hereby certify that all work contained in this paper is original work of CC, AC, MS, BC, RN, AA, EB, TI and BP. The authors claim full responsibility for the contents of the article. The authors contributed equally to this work. This article was not supported by grants.Coxsackievirus B5 - 16.1Mumps virus - 12.1Influenza A/H1N1/WSN33 - 11.9Herpes Simplex Virus-1 - 4.8Porcine Parvo - 3.6Selectivity indexes were calculated for those viruses inhibited by HA as the ratio between the toxic dose 50 and the inhibiting dose 50.


Liao ning virus is in the genus Seadornavirus within the family Reoviridae and has a genome composed of 12 segments of double-stranded RNA (dsRNA). It is transmitted by mosquitoes and only isolated in China to date and it is the only species within the genus Seadornavirus which was reported to have been propagated in mammalian cell lines. In the study, we report 41 new isolates from northern and southern Xinjiang Uygur autonomous region in China and describe the phylogenetic relationships among all 46 Chinese LNV isolates.-3 substitutions per site per year.The phylogenetic analysis indicated that all the isolates evaluated in this study can be divided into 3 different groups that appear to be related to geographic origin based on partial nucleotide sequence of the 10th segment which is predicted to encode outer coat proteins of LNV. Bayesian coalescent analysis estimated the date of the most recent common ancestor for the current Chinese LNV isolates to be 318  and the estimated evolutionary rates is 1.993 × 10The results indicated that LNV may be an emerging virus at a stage that evaluated rapidly and has been widely distributed in the north part of China. Liao ning virus (LNV) which is the member within the genus Seadornavirus family Reoviridae was composed of 12 double-stranded RNA (dsRNA). The Seadornaviruses include three species, Banna virus (BAV), Kadipiro virus (KDV) and LNV -5. Here,A total of 41 new LNV isolates were obtained from mosquitoes during 2006 to 2008 at sites in Xinjiang province  was found according to the Akaike information criterion. Both strict and relaxed  molecular clocks [http://tree.bio.ed.ac.uk/software/tracer/. The rate of nucleotide substitutions per site per year and the date of the time of most common ancestor (TMRCA) for these viruses were estimated. Maximum clade credibility (MCC) tree was constructed and converted to a graphics format using Figtree software version 1.2.2 http://tree.bio.ed.ac.uk/software/figtree/.The MrModeltest version 2.3 r clocks  were empr clocks . The dat-3 per site per year . Based on this nucleotide substitution value, the TMRCA of Chinese LNVs is estimated to be 318   Figure .Culex spp. in Kashi, southern Xinjiang in 2006; 7 from Culex spp. and Aedes dorsalis in Bayinguoleng, southern Xinjang in 2007; 3 from Aedes dorsalis in Aletai, northern Xinjiang in 2008(Figure Aedes dorsalis in Liaoning province in 1997 [Culex modestus in Qinghai province in 2007 [Culex pipiens and Aedes dorsalis in Shanxi province in 2007 [Phylogenetic analysis was conducted on 46 LNV isolates Table . Of thes08Figure . The oth in 2007  (Figure -4[-3 which is faster than other arbovirus. In addition, the TMRAC of LNV was estimated to be 381 years, indicating LNV is an emerging virus and at a stage that evaluated rapidly to accommodate the host and the environment now. Also, the nucleotide and amino acid homology rate showed high identity within each group and much divergent between the groups. The geographical characteristic implies that LNV could raise natural circle in the locality though the diverged time of each group was quite recent.Arbovirus undergoes substantially slower rates of evolution . For exa-4,10. Our In recent years, arbovirus surveillances have been done in many provinces. Japanese encephalitis virus, Tahyna virus, Getah virus and other arboviruses were isolated nationwide -13. But LNV seemed to be at a stage of evolving rapidly than most of the arbovirus. Also it is much divergent between each subtype. Can such virus genetic differences have pathogenicity differences? LNV is pathogenic to mice, and seasonal encephalitis in the absence of Japanese encephalitis occurs in the areas where LNV is found in China ,15, futuThe authors declare that they have no competing interests.ZL, XJL, QD, SZ, SXT, MHL and WJL collected the mosquitoes sample. ZL, SHF, MHL and WJL did virus isolation, RT PCR and sequencing studies. ZL and HL did the Bioinformatics analysis. ZL, HL, QT and GDL conceived the study and drafted the manuscript. All authors read and approved the final manuscript.


Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes – Dobrava, Kurkino, Saaremaa, and Sochi – that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans.Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus  Apodemus flavicollis, captured in Slovenia [Dobrava-Belgrade virus (DOBV) for this hantavirus species [Dobrava virus was isolated more than 25 years ago from a yellow-necked mouse, Slovenia . Later, Slovenia . Therefo species .Soon, reports on detection of DOBV in other European countries started to appear. DOBV nucleic acid was detected by RT-PCR and sequencing in Greek and Albanian HFRS patients . Using tA. agrarius, trapped on the Estonian islands Saaremaa and Vormsi [Surprisingly, DOBV was then detected also in striped field mice, d Vormsi  and subsd Vormsi . Other Dd Vormsi  and in Sd Vormsi , 73. Mead Vormsi , Denmarkd Vormsi , and othd Vormsi . Correspd Vormsi  and Eurod Vormsi .Apodemus flavicollis; Af) and striped field mouse -associated strains are sympatrically present, such as Slovakia [Saaremaa virus, SAAV [http://ictvonline.org/virusTaxonomy.asp).In several countries, both yellow-necked  Europe. DOBV sequences associated with yellow-necked mice have been reported from Slovenia [A. ponticus  were DOBV-antigen positive and from which virus could be isolated by cell culture with lung tissue homogenate as inoculum [Currently, mice of at least three Slovenia , 5, SerbSlovenia , 77, AlbSlovenia , 47, 48,Slovenia , 62, andSlovenia . IntriguSlovenia , 81, SloSlovenia , 83, HunSlovenia , 61 and Slovenia , 64. In inoculum , 79.DOBV belongs to the group of Murinae-associated hantaviruses. Its close relatives are Hantaan virus (HTNV), Seoul virus, and Thailand virus from Asia. The most closely related hantavirus currently is Sangassou virus, which is found in West Africa , 28.Phylogenetic analysis of the DOBV strains from yellow-necked and striped field mice occurring sympatrically in Slovenia  and SlovThe strict host-determined differentiation is particularly obvious in the sequence analysis of the M segment, which encodes the viral envelope glycoprotein Fig. B. HoweveAn alternative explanation based on different evolutionary rates of genome segments has been proposed , 59. AccHowever, the proposed host switch from yellow-necked mouse to striped field mouse has been called into doubt by others , 26. IndAdditional conflicts in tree topologies suggesting genetic reassortment during DOBV evolution have been observed for DOBV-Ap. In S-segment trees, DOBV-Ap forms a well-supported sister group to DOBV-Af but is an outgroup to all other DOBV strains in M- and L-segment trees . More coThe inference of S-segment phylogeny seems to be particularly problematic. Usage of various datasets and phylogenetic methods can result in different positions of the Saaremaa strains . NeverthA. agrarius as SAAV  regWe would like to propose a novel intra-species classification of DOBV, which is based on phylogenetic analysis of the S segment sequences. Due to the genetic basis of the classification, we propose to define virus genotypes. In agreement with the usual procedure in hantavirus terminology, genotype names should be derived from the geographical place where the first sequence of the genotype was detected.Following this concept, one can currently define four DOBV genotypes corresponding to the four above-listed lineages. The “Dobrava” genotype consists of DOBV-Af strains and is named after the prototype virus . StrainsDobrava-Belgrade virus (DOBV). They should neither be divided into DOBV and SAAV species nor do they represent four distinct species. This opinion is based on the following four facts. (i) The amino acid sequence differences between the genotypes are extremely small, not exceeding 5 % in case of N and RdRp proteins and 10 % in case of glycoprotein precursor (GPC)  infection; however, no molecular (nucleotide sequence) identification of the virus strains involved has been reported . Based oIt is highly interesting to note that the different genotypes of DOBV– despite their high genetic similarity – induce HFRS of different severity. The most severe clinical courses were observed in SE Europe, where human infections by the Dobrava genotype occur. The case-fatality rate (CFR) of clinical cases was 10-12 %, a rate that is similar or even higher than that known for HTNV infections in Asia , 49, 80.in vitro conditions, are associated with the genomic S and L segments of the viruses [It remains to be determined which genetic differences in the four virus genotypes are responsible for their different virulence. In initial investigations, we found that genetic markers associated with the divergent virulence of Kurkino (virus isolate SK/Aa) versus Dobrava (virus isolate Slo/Af), at least under  viruses .DOBV is the most virulent European hantavirus and is responsible for almost all fatal HFRS cases in Europe. Together with the more common, but less virulent, Puumala virus, it can be considered one of the two most important hantaviruses in Europe. Its unambiguous classification is therefore of significant benefit not only for the scientific community but also for hantavirus diagnostics, medical care, and public-health authorities. The different virulence of such closely related genotypes makes the virus particularly interesting for research. Understanding the mechanisms behind the different virulence properties of the DOBV genotypes could significantly advance the whole field of hantavirus pathogenesis.


The nucleotide sequences of the ten genomic segments of a Vietnam isolate of southern rice blacked-dwarf virus were determined. This complete genomic sequence will help to further understand the viral etiology  and phylogenetic relationships among fijiviruses. Fijivirus within the family Reoviridae  , the main vector of RBSDV (Southern rice black-streaked dwarf virus (SRBSDV) (or rice black-streaked dwarf virus 2) was first reported in China and was identified as a novel putative member of the genus oviridae –5. SRBSDHorváth) , 6 and oof RBSDV , 5.In Vietnam, a novel dwarf and leaf-twisting syndrome was first observed on rice plants in Nghe An Province in the summer 2009 crop. The disease rapidly spread among diverse agroecological regions in north and central Vietnam and also infected maize after rice was harvested. Although the disease was at first thought to be caused by a mixed infection of rice grassy stunt virus (RGSV) and rice ragged stunt virus (RRSV), transmission experiments and partial genomic data allowed us to identify the causal agent as SRBSDV . To dateFijivirus, in the family Reoviridae , which is 9 nt shorter than SRBSDV-GD or SRBSDV-HN but similar in organization to these two Chinese isolates and other group 2 fijiviruses that have been sequenced . Each ofoviridae . The newHQ731492 to HQ731501, corresponding to genomic segments 1 through 10, respectively.The complete genomic sequence of SRBSDV-V has been deposited in the GenBank/EMBL/DDBJ databases under the accession no.


The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies. Apis mellifera) populations, have been declining consistently worldwide due to a variety of causes Varroa destructor, Acarapis woodi, Tropilaelaps sp.), microsporidia (Nosema spp.), fungi , bacteria (American foulbrood (Paenibacillus larvae), European foulbrood (Melissococcus plutonius)), viruses, and pests  Honeybees provide both honey and key pollination services to much of the world PicornaviralesApis mellifera filamentous virus (AmFV), Apis iridescent virus (AIV); Arkansas bee virus (ABV) and Berkeley bee virus (BBPV) remain to be fully characterized molecularly Recently the role of pathogenic viruses gained particular interest since they have been suspected to be important drivers of colony declines. Honeybees are host to between 12–20 viruses, depending on classification Multiplex Ligation-dependent Probe Amplification (MLPA) is an amplification technique that allows simultaneous detection of up to 45 different targets with the use of a single primer set Mycobacterium tuberculosisPenicillium marneffeiThe detection of viral infections in honeybees is increasingly based on the detection of specific viral genomic nucleic acids. Since most (honeybee) viruses have RNA genomes, this means the detection of virus-specific RNA signatures. The most widely used method is reverse transcriptase quantitative PCR (RT-qPCR). Many individual RT-(q)PCR protocols have been described for the detection of specific honeybee viruses review  as well Replication in positive-strand RNA viruses, such as many honeybee viruses, proceeds via the production of a negative-strand intermediate. Strand-specific RT-PCR was first developed for detection of negative-strand RNAs of viruses Our report here shows the application of RT-MLPA for simultaneously detecting 10 targeted honeybee viruses. Two MLPA probe sets were developed which are able to detect selectively the positive strand RNA or the replicative negative strand RNA intermediate. The possibility to screen easily for replication will be valuable for studying virus replication and pathogenesis in naturally infected hosts. Because of its high sensitivity and specificity, the RT-MLPA assay is also a useful tool for prompt diagnosis and epidemiological studies of viruses in honeybee populations. Al last, we used this newly developed method in an epidemiological survey of honeybee viruses based on adult bee samples collected in Flanders during the summer of 2011.Varroa. Generally, each beekeeper sent two samples from their apiary. The bees were immediately frozen at −20°C until their shipment to the laboratory, where upon arrival they were stored at −80°C until RNA extraction. Excess bees were archived for long-term −80°C storage.All Flemish beekeepers were invited to participate in an epidemiological survey for virus screening; 170 beekeepers accepted the invitation and submitted a total of 363 samples of 30 adult bees, collected in July 2011 at the entrance of colonies that seemed healthy and that were not (yet) treated against To detect the positive strand viral RNA was isolated by using the QiaAmp Viral RNA mini kit (Qiagen). Individual whole adult bees were ground in a mortar in 1 ml ice-cold PBS per bee. The extract was centrifuged at 14 000× g and RNA was extracted from 140 µl of the liquid supernatant according to the manufacturer's instructions, eluting the RNA in a final volume of 50 µl. In the negative strand detection mode the total RNA was isolated using the RNeasy lipid tissue mini kit (Qiagen) starting from one complete honeybee. The tissue was homogenized by mechanical agitation in a TissueLyser (Precellys) for 90 sec at 30 Hz, in the presence of a pair stainless steel beads and 1 ml QIAzol lysis reagent. The total RNA was isolated according to the recommendation of the manufacturer's protocol, eluting the RNA in a final volume of 50 µl.For the Flanders virus survey, 10 bees per colony were homogenized in a total of 5 ml PBS by mechanical agitation in a TissueLyser for 90 sec at 30 Hz, in the presence of glass beads. The extract was centrifuged at 14 000× g and RNA was extracted from 140 µl of the liquid supernatant using the QiaAmp Viral RNA mini kit according to the manufacturer's instructions, as outlined above.www.ncbi.nlm.nih.gov). The primers for cDNA synthesis were positioned immediately adjacent to the MLPA probe, with no more than 15 nucleotides between the last nucleotide of the RT primer and the first nucleotide of the probe sequence, and with a maximum overlap of 7 nucleotides. The RT primers were designed with Primer 3 software (http://primer3.sourceforge.net). All primers and probes were synthesized by Integrated DNA Technologies . The RPO should be 5′ phosphorylated and synthesized with ‘ultramers’ quality. The RTprimers, MLPA half-probes (LPO and RPO) and PCR amplification primers used in the experiments are listed in MLPA probes and RT-primers were designed for 6 virus targets, covering the 10 most common honeybee viruses, and for two honeybee internal reference genes; β-actin and ribosomal protein 8 (RPL8), as positive controls for the quality of the RNA samples. For each virus or virus-complex a pair of probes was designed following the guidelines described in the manual “Design synthetic MLPA probes” . All probe pairs contain the same universal binding sites for the reverse PCR primer on the right probe oligo (RPO) and for the forward PCR primer on the left probe oligo (LPO). The probes were designed using the AlleleID ® software (PREMIER Biosoft) against the most conserved regions within each virus or virus family as determined by aligning all available gene sequences in the GenBank using Clustal X program. An additional selection criterium was the absence of mismatches within 5 nucleotides from the ligation site. The uniqueness of our selected probe sequences was inspected by BLAST analysis at the NCBI website .For the Flanders virus survey study the MLPA reactions were analyzed using 4% high resolution agarose gel electrophoresis.Fragments generated by the RT-MLPA reactions were desalted with MSB Spin PCRapace (Invitek) and subsequently cloned into pCR4-TOPO vector from TOPO TA Cloning Kit for sequencing  according to manufacturer's instructions. The cloned inserts were sequenced on a ABI 3130XL platform using the vector primers. Positive constructs were used as template in a standard PCR reaction to amplify the expected MLPA reaction products. The concentrations of the different products were determined using a nanodrop ND-1000 spectrophotometer (Thermo Scientific). The different products were mixed in equal amounts and 10 ng of each fragment was loaded on a gel as marker to simplify the interpretation of the results.The MLPA is a popular technique in the human genetics but, according to our knowledge, it has not yet been used in the field of veterinary virology. The multiplexing capacity of the technique is much higher than for PCR assays but far below the capability of microarrays. This medium-scale (1–40-fold) multiplexing ability makes MLPA extremely useful for the simultaneous screening of all honey bee viruses and its simplicity can facilitate widespread acceptance of the technique even in small size molecular laboratories.We designed an RT-MLPA approach to detect 6 targets simultaneously covering 10 common honeybee viruses: ABPV, BQCV, IAPV, KBV, DWV, KV, VDV-1, SBPV, SBV, CBPV. We opted for detection by agarose gel electrophoresis, although this part of the protocol can be easily transferred to other platforms, such as capillary electrophoresis or the Agilent bioanalyzer. A spacer in the RT-MLPA probes was included to adjust the final length of the specific RT-MLPA products so that they are separated by 7–9 nucleotide increments, for unambiguous identification of the fragments after electrophoresis . For DWVThe diagnostic capacity of the RT-MLPA-assay was tested on (RT-PCR) proven virus positive samples . All ampWe constructed an MLPA ladder by mixing equimolar amounts from the different expected RT-MLPA amplicons each corresponding to a specifc virus. These were amplified in a PCR reaction using the different pCR4-cloned RT-MLPA amplicons as templates. The use of this marker greatly facilitates the interpretation of the MLPA results after electrophoresis .The sensitivity of the MLPA was tested using synthetic templates for DWV and BQCV. A serial dilution of these templates in total RNA from a non-infected honeybee showed that as few as 1000 copies can be detected with clearly discernible signals. This detection limit is in accordance with the detection limit obtained in other studies Well-designed MLPA probes have the ability to discriminate between single-nucleotide polymorphism (SNP) “BeeDoctor” is optimized to detect various honeybee viruses simultaneously. MLPA is able to multiplex up to 45 targets. Probes used in MLPA usually range between 80 to 400 nucleotides in length. Accurate chemical synthesis of MLPA probes is possible up to a length of approximately 180 nucleotides All positive-strand RNA viruses replicate and express their genomes through negative-strand RNA intermediates that are used as templates for the production of positive-strand progeny RNAs that are then packaged in new virion particles. Therefore, the presence of negative-strand RNA intermediates is a reliable marker for active virus replication in infected honeybees. It is also a very effective means to distinguish between active infections and the non-infectious, passive presence of virus particles, which is an important epidemiological distinction. RT-MLPA is the ideal technique to selectively detect the positive strand genomic RNA or the negative-strand replicative intermediate RNA, since an amplifiable probe can only be generated in a strand-specific manner, through the ligation of the two half-probes hybridizing next to each other on a single-stranded cDNA target. Strand specific probes were developed  and testThe newly developed technique, “BeeDoctor”, was used in a survey of the prevalence and distribution of the targeted viruses in Flemish apiaries. This survey revealed that almost 80% of the samples were positive for at least one of the viruses screened for by “BeeDoctor” . No viruA. mellifera has also been reported in several other countries The most prevalent virus was DWV, with 69,4% of colonies screened being positive for the presence of this virus. The high occurrence of DWV in BQCV was detected in 13,5% of the colonies and is reported to have a variable prevalence in different colonies. The prevalence changes from 10 to 90% across Europe SBV was present in 19% of the Flemish colonies which is high in comparison with 2% in Hungary, 1.4% in England and 1.1% in Spain CBPV was detected in only 1,7% of the samples which is in correspondence with the findings of Tentcheva et al. in France, who found a maximum frequency in colonies of 4%. These low frequency rates can be explained by the finding that CBPV might persist at undetectable levels in healthy colonies The prevalence of the virus complex ABPV, IAPV and KBV is also very low, with only 3,3% of the Flemish colonies infected. These three viruses are closely related and were detected simultaneously. The infection rate of 3,3% for the ABPV family is low as each of the viruses separately have higher prevalences in other European countries. ABPV is present in 29% of the colonies sampled in England while KBV was not detected Slow bee paralysis virus could not be detected which confirms the low natural prevalence of SBPV across a large part of Europe In conclusion, in this study we developed an RT-MLPA approach to diagnose for the most common honeybee viruses in one single procedure. We were also able to develop a strand specific assay in which we can specifically screen for the negative strand intermediate as marker for effective virus replication. The multiplex power is an enormous advantage in comparison with other well established RT-PCR approaches. This RT-MLPA diagnostic tool is easy, cost effective, allows for very high throughput analyses and above all is extremely versatile. The “BeeDoctor” can easily be expanded with probes for additional pathogens and/or markers for honeybee health and disease. Moreover, the “BeeDoctor” and RT-MLPA in general also works well even with highly degraded RNA, since it requires only very short fragments of intact RNA, since the probe-specific RT primers can partly overlap with their corresponding probe and have to be elongated by only 50 nucleotides. Proper sample preservation is often difficult to achieve in practical beekeeping and sampling in the field, and often is a limiting factor for many other screening techniques The “BeeDoctor” assay was used to screen 363 apparently healthy colonies from randomly selected apiaries throughout Flanders. This survey showed that almost 80% of colonies are infected with at least one virus, and many with multiple infections, showing that virus infections in apiaries are quite common, even in the absence of clinical symptoms.


The objective of this innovative research study was to describe clinical shoulder complex range of motion (ROM) measures for a young, healthy, and physically active population. This investigation represents a cross-sectional experiment conducted at a military academy-based sports medicine center. Military cadets with no history of shoulder complex injury were assessed within two months of enrollment in the academy; 548 men  and 74 women  participated. Descriptive measures included cross-body adduction (CAD), flexion (FLX), external rotation (ER0) with the shoulder complex in adduction and elbow flexed to 90°, internal and external rotation  with the shoulder complex at 90° of abduction and elbow flexed to 90° as well as arc (ARC) of IR-ER using standardized clinical quantification techniques. Bilateral and sex differences were evaluated using dependent and independent t-tests, respectively. Percentiles by arm dominance and sex were also calculated for all ROM measures.Data were normally distributed. Active and passive ROM measures indicated significant bilateral differences (P < 0.05) except for ARC. Sex differences (P < 0.05) were noted for active and passive CAD, FLX and ER0 for the dominant arm as well as active and passive CAD, FLX and ARC for the non-dominant arm.These original data provide descriptive measures for shoulder complex ROM excursions, assisting sports medicine practitioners in potentially identifying clinical deficiencies and functional outcomes following shoulder injury. Clinical range of motion (ROM) assessment is often implemented to objectively evaluate shoulder complex excursion . VariousAs part of a larger prospective cohort experimental design , a crossWe implemented a reliable method  for measGroup means and standard deviations were calculated by arm dominance and sex for all ROM measurements. Probability plots were computed to determine if the data were normally distributed for purposes of satisfying necessary assumptions for t-test analyses. Bilateral and sex differences were evaluated using two-tailed dependent and independent t-tests, respectively. The significance level was set at P<0.05 a priori. Percentiles by arm dominance and sex were also calculated for all ROM measures.All data were normally distributed. Active and passive ROM measures indicated significant bilateral differences P<0.05) with the exception of ARC Table . Sex dif with theOur results demonstrate greater FLX and ER regardless of abduction angle as well as less IR in the dominant compared to non-dominant arm. These findings contrast those of Barnes et al.  that repContrary to a prior report , our datAlthough we identified similarities and differences for our study and previous reports, comparing our results with prior findings is difficult due to considerable differences in participant populations and methods. Notable sources of variability between our study and preceding investigations are mostly attributed to discrepancies in experimental design and participant demographics. Nonetheless, our study is novel in presenting clinically relevant descriptive data for healthy, young and physically active adults, which represents the population typically treated by sports medicine practitioners. Thus, while our findings are limited in generalizability to other populations, they are well suited for descriptions of young athletes.Determining descriptive values for an athletic population is essential to providing sports medicine practitioners with a frame of reference with which to interpret clinical measures obtained from respective patients. Such data are also necessary for evaluating patient progression through treatments to determine clinical intervention efficacy. Our investigation indicates that traditional shoulder complex ROM examinations and clinical interpretations, which do not account for arm dominance and sex factors, may be ambiguous in an athletic cohort. These findings suggest shoulder complex ROM differences, both bilaterally as well as among men and women, may be at least partially explained by arm dominance and sex in young, healthy and physically active adults. Therefore, influences of arm dominance and sex should be considered when shoulder complex ROM is clinically evaluated in a similar population.We observed statistically significant differences for several of the ROM measures examined in our study by arm dominance and sex, yet whether or not these differences are clinically important remains unclear. Due to the large number of observations and associated statistical power, it is likely that even small mean differences reached statistical significance in our study. In fact, the largest mean differences were no greater than 2.5cm for CAD or 5° for any of the goniometric measurements. It has been reported previously that mean differences less than 7°  or 8° 2 for goniOur study was associated with certain limitations. One-dimensional goniometric measurements do not concomitantly assess multi-planar joint excursions, which may contribute to the respective resultant net ROM measurement. Thus, more sophisticated, yet non-pragmatic, instrumentation, such as photogrammetry, may be better equipped to comprehensively capture ROM measures. We were also unable to estimate intra- and inter-rater reliability within and between examiners who assessed ROM using standardized procedures, which is an important limitation when interpreting the results of our study. However, we relied on prior reliability estimates ,9,26,27 ROM: Range of motion; CAD: Cross-body adduction; FLX: Flexion; ER0: External rotation with the shoulder complex in adduction and elbow flexed to 90°; IR: Internal rotation with the shoulder complex at 90° of abduction and elbow flexed to 90°; ER: External rotation with the shoulder complex at 90° of abduction and elbow flexed to 90°; ARC: Arc of internal and external rotation.We declare to have no competing interests.GLV contributed to the interpretation of data analysis, dissemination of findings as well as concept and construct of the related manuscript. MLD, BDO and KLC equally contributed to the experimental design, collection and analysis of data, dissemination of findings as well as review of the related manuscript. All authors read and approved the final manuscript.


A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection. Since February, 2013, a previously unrecognized novel avian-origin influenza A (H7N9) virus associated with human deaths has emerged in China A rapid and sensitive methodology for the diagnosis of H7N9 infection is urgently needed to facilitate clinical care, infection control, and epidemiologic investigations. Methods based on PCR are more rapid and sensitive than traditional techniques including virus isolation and serological assays. Real-time RT-PCR is at present the powerful molecular diagnostic method for the novel influenza A (H7N9) virus infection h), and excellent sensitivity and specificity make this RT-LAMP-LFD method more suitable for use in low-equipment setting laboratory.LAMP products can be detected by agarose gel electrophoresis, by the use of spectrophotometric equipment to measure turbidity Written informed consent for the use of the clinical specimens was obtained from all patients involved in this study. This study was approved by the Ethics Committee of the Jiangsu Provincial Center for Disease Prevention and Control.H7N9 virus isolates were cultured in Madin-Darby canine kidney (MDCK) cells using standard techniques. An isolated A/Nanjing/1/2013 (H7N9) virus was used as the reference virus. Other genetically and clinically related virus isolates including seasonal influenza viruses , 2009 swine-origin influenza virus A/H1N1, avian influenza viruses (A/H5N1 and A/H9N1), parainfluenza viruses , human coronaviruses  and respiratory syncytial viruses (types A and B) were used as control viruses to assess the specificity of the RT-LAMP assay. A total of 80 respiratory clinical specimens  were collected from suspected H7N9 patients in the acute phase of illness. All these isolates and specimens were stored at −80°C until use.The genomic viral RNA was extracted from 200 μl of samples by using a High Pure viral RNA kit  according to the manufacturer's instructions. The RNA was eluted in a final volume of 100 μl of elution buffer and kept at −80°C until further analysis.in vitro transcribed with T7 RNA polymerase  according to the manufacturer's instructions. The synthetic RNA transcripts were then purified, quantified, mixed in equal-molar amounts, and ten-fold diluted ranging from 107 to101 RNA copies/μl.The haemagglutinin (HA) and neuraminidase (NA) genes were amplified from the H7N9 virus strain A/Nanjing/1/2013 (H7N9) with primers containing T7 promoter sequence in the reverse sides Table 1 http://primerexplorer.jp/e/). The feasibility and specificity of the primers were subsequently checked by BLAST search against published genome sequences of the novel H7N9 viruses from the GISAID EpiFlu database and against NCBI database. Details of the final primers are shown in Two separate RT-LAMP assays (six primers each) were designed according to HA and NA gene sequences of the reference virus using a PrimerExplorer V4 program (RT-LAMP was carried out with a RNA Amplification Kit (RT-LAMP)  in a final volume of 25 µl containing 0.2 µM each of the outer primers F3 and B3, 2.0 µM each of the primers FIP and BIP, and 1.0 µM each of the primers LF and LB (labeled or not). The reaction mixture containing distilled water was used as negative controls. RT-LAMP amplification reactions were carried out at 60, 63, and 65°C for 60 min using a real-time turbidimeter . The RT-LAMP products were also detected by a LFD  as described previously To compare test sensitivity, RNA was extracted from the culture supernatants of H7N9 virus strain A/Nanjing/1/2013 (H7N9)-infected MDCK cells. Ten-fold serial dilutions of RNA were used as templates for RT-LAMP reactions at 63°C for 1 h. Besides real-time turbidity detection, The RT-LAMP products were simultaneously detected by LFD as described above.7 to101 RNA copies) were used to assess the detection limits of the HA and NA RT-LAMP-LFD assays. The specificities of the RT-LAMP-LFD assays were determined by analyzing the RNA extracts from various control viruses mentioned above and the reference virus.Ten-fold serial dilutions of synthetic H7N9 viral RNA transcripts of HA and NA genes .−7 dilutions, which were equivalent to their detection limits obtained by RT-LAMP-turbidity detection. Thus, the LFD method which can provide a result within 5–10 min after completion of the RT-LAMP reaction was as sensitive as real-time turbidity detection . After 60 min amplification, both the HA and NA RT-LAMP-LFD assays revealed a detection limit of 10 copies of synthetic RNA. To assess the potential for the RT-LAMP-LFD assays to cross-react with other related viruses which could cause similar symptoms, the HA and NA RT-LAMP assays were tested against viral RNA extracts from various control viruses. As shown in The analytical sensitivities of the HA and NA RT-LAMP-LFD assays were assessed using ten-fold serial dilutions of synthetic H7N9 viral RNA transcripts of HA and NA genes  virus which warrant the development of new methods to detect this virus. In this study, we describe a sensitive RT-LAMP-LFD method for the specific detection of H7N9 virus for the first time. The optimal LAMP reaction conditions are 63°C for 60 min, which is more suitable for low-equipment setting laboratory and for on-site testing.The use of the four specific primers and two loop primers targeting the HA or NA gene of H7N9 virus ensured high specificity of nucleic acid amplification. Both the HA and NA RT-LAMP assays showed 100% specificities for identification of H7N9 virus. Furthermore, loop primers could accelerate the LAMP reaction because they hybridize to the stem-loops, except for those loops that are hybridized by the inner primers and prime strand displacement DNA synthesis The concordance of high analytical sensitivity between RT-LAMP and the most sensitive molecular methods for detection of pathogens has been previously reported The RT-LAMP results can be determined by agarose gel electrophoresis, by the use of spectrophotometric equipment to measure turbidity, by naked eye for the presence of a white precipitate derived from magnesium pyrophosphate, or by visualization of the RT-LAMP products under natural light or UV irradiation after adding SYBR green I or calcein dyes. However, due to the use of several primers, RT-LAMP generates a complex mixture of DNA products, and thus these product detection methods cannot distinguish specific and non-specific amplicons. Furthermore, assessment of turbidity or color with the unaided eye is potentially subjective, and there is always the possibility that a sample may be somewhat ambiguous to the naked eye when the concentration of virus is low In conclusion, these data show that a reliable RT-LAMP-LFD assay has been developed for the detection of novel avian-origin influenza A (H7N9) virus causing the current outbreak, which would facilitate the clinical care, infection control, as well as epidemiologic investigations. The RT-LAMP-LFD assay is specific and sensitive, and does not require expensive equipment. The use of the LFD provides a rapid and objective readout of the assay's results and avoids cross-contamination. This RT-LAMP-LFD assay is especially useful in resource-limited situations such as primary care facilities.


Ranavirus, Novirhabdovirus, Betanodavirus, Isavirus, and some herpesvirus.The successful replication of a viral agent in a host is a complex process that often leads to a species specificity of the virus and can make interspecies transmission difficult. Despite this difficulty, natural host switch seems to have been frequent among viruses of lower vertebrates, especially fish viruses, since there are several viruses known to be able to infect a wide range of species. In the present review we will focus on well documented reports of broad host range, variations in host specificity, and host shift events hypothesized for viruses within the genera  The successful replication of a viral agent in a host is a complex process which consists of a number of interactions, most of them related to the coevolution of pathogen and host. This coevolution often leads to a species specificity of the virus and can make interspecies transmission difficult. Therefore, natural host range switches by viruses are rare events. However, when they occur the results can become severe because the viruses may then spread widely through non previously adapted, and therefore immunologically naïve host populations.Upon transmission to a new host species, viruses must usually adapt to a new genetic and immunologic environment in order to replicate and spread to other individuals within the species . The higInfluenza A is the paradigm of a virus capable of interspecies and interclass transmission. Those viruses are found in humans as well as in other animals, including swine, horses and birds, waterfowl being considered the natural reservoir . SubtypeA number of proteins have been implicated in determining host specificity of the virus. Influenza haemagglutinin binds to sialic acid linked to galactose on the surface of the targeted cell, and the differing nature of the sialic acid-galactose linkages in birds and humans provides an important barrier to host shift events. In this sense, a number of amino acid substitutions have been produced in influenza haemagglutinin to adjust to the different receptors -14. NeurSevere acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from humans (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor binding domain of the S protein that were potentially important for SARS progression and tropism. It has been demonstrated that the double substitution of two amino acid residues of pcSARS-CoV for those of huSARS-CoV made pcSARS-CoV capable of infecting human cells , suggestUnder certain circumstances, even a genetically stable DNA virus can gain the mutation required to adapt to a new host. That is the case of canine parvovirus (CPV) which emerged in 1978 as the cause of new enteric and myocardial diseases in dogs. The new virus spread globally in a pandemic and has since remained endemic in dogs throughout the world ,21. PhylRanavirus, Novirhabdovirus, Betanodavirus, Isavirus and several herpesvirus. However, the suspicion for interspecies transmission in other fish viruses remains.Viruses of lower vertebrates include a large number of viral agents, belonging to different viral families and genera, with RNA and DNA genomes, and displaying different host specificities. In fact, some viruses have a very narrow host range, whereas others are known to be able to infect a wide range of species. The wide host range suggests that, in any moment along the viral evolution, those viruses may have been involved in different host shift events. In the present review we will focus on well documented or hypothesized cases of host shift as well as variations in host range for the genera Iridoviridae. Among the five genera, two contain viruses of invertebrates (genera Chloridiridovirus and Iridovirus), whereas the remaining three genera  contain viruses that infect lower vertebrates [Iridoviruses are large double stranded DNA viruses with an icosahedral capsid ranging from 120 to 350 nm in diameter. Ranavirus is one of the five genera within the family tebrates . The famtebrates .Ranavirus, than to Lymphocystivirus. In fact, ranaviruses have become important pathogens for cultured and wild finfish, not only due to the severity of the diseases that they cause but also because of their rapid global emergence in recent years [Although the disease was known before , the lymnt years .Ranavirus infect vertebrates of three different taxonomic classes: amphibians, reptiles and fish [Bhole Iridovirus (BIV), Ambystoma tigrinum virus (ATV) and European catfish virus (ECV) are five closely-related viral species that share over 90% sequence identity within the MCP and other genes, but clearly differ from each other in host range and RFLP profiles. The sixth species is Santee-Cooper ranavirus (SCRV) that along with Singapore grouper iridovirus (SGIV), a tentative species, are the most divergent members of the genus. The MCP genes of these two viruses show approximately 80% and 70% sequence identity, respectively, with the MCP genes of the 5 other ranavirus species [Members of the genus and fish . Since tand fish  and the and fish , ranavirand fish , largemoand fish  and ornaand fish , as welland fish ,42. In aand fish -47. At pand fish  recognizand fish ,50, but  species ,52.Limnodynastes ornatus) tadpoles in Australia [Lates calcarifer) [Oreochromis mossambicus) [Rana temporaria with two ranavirus isolates obtained from diseased toads (Bufo bufo). In addition, apparently identical FV3 strains were isolated from dead or moribund free-living threespine stickleback fish (Gasterosteus aculeatus) and sympatric tadpoles of the red-legged frog (Rana aurora) [Esox lucius) [Several of these viruses have been demonstrated to have broad host specificity, suggesting the potential for interspecies and interclass transmission. A good example is BIV, which was isolated originally from diseased ornate burrowing frog  ,57. Moreambicus) ,58. A caambicus) , who inf aurora) , and FV3 aurora) . Moreove lucius) . A very  lucius) . These d lucius) -65.Pelophylax esculentus) in Denmark, which showed a 98.8% nucleotide identity in the MCP gene with EHNV [In a very recent and interesting study Jancovich et al.  obtainedith EHNV , would sIt has been suggested that after the divergence into the salamander virus and frog virus lineages a subsequent host specific evolution could have occurred that would have limited cross transmission between both hosts, at least in laboratory infections . HoweverBetanodavirus, within the family Nodaviridae [Piscine nodaviruses belong to the genus aviridae . Betanodaviridae . The affLates calcarifer and Dicentrarchus labrax), groupers , striped jack (Pseudocaranx dentex), parrotfish (Oplegnathus fasciatus), tiger puffer (Takifugu rubripes), and flatfish  [The disease typically occurs in an outbreak form in larval and juvenile fish, and several species have been shown to be specially affected such as sea bass (maximus) . The affBetanodaviruses are small (25-30 nm), nonenveloped, icosahedral RNA viruses. The genome consists of two single stranded, positive-sense molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA dependent RNA polymerase (RdRp) of approximately 100 kDa, also named protein A ,75. The Solea senegalensis [Sparus aurata and sea bass cultured in the Iberian Peninsula [Betanodaviruses have been classified into four genotypes, designated SJNNV (striped jack nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), RGNNV (red grouper nervous necrosis virus) and BFNNV (barfin flounder nervous necrosis virus), using a partial sequence of RNA2, the T4 region, which is a highly variable region of around 400 nt ,78. Thesgalensis ,80 as weeninsula . More reeninsula  reportedeninsula  observedeninsula  as a hoseninsula  and Ito eninsula . It is weninsula . In thisOther authors have also reported the existence of reassortants among betanodavirus isolates obtained from symptomatic sea bass harbouring an RNA1 segment of SJNNV type and an RNA2 of RGNNV type . These dSouto et al.  experimeNovirhabdovirus is one of the six established genera within the family Rhabdoviridae, and it is one of the two genera of this family known to infect aquatic animals . Two of the four recognised species of the genus are infectious haematopoietic necrosis virus (IHNV), the aetiological agent of infectious haematopoietic necrosis (IHN), and viral haemorrhagic septicaemia virus (VHSV), the causative agent of viral haemorrhagic septicaemia (VHS). Novirhabdovirus possess enveloped bullet-shaped virions. The viral genome consists of a linear non-segmented, negative-sense, single-stranded RNA of approximately 11 kilobases and contains 6 genes in the order 3'-N-P-M-G-NV-L-5' [-NV-L-5' . These vNovirhabdovirus and it is one of the most serious viral pathogens of salmonid fish, infecting wild [Oncorhynchus nerka, pink salmon O. gurbuscha, chinook salmon O. tshawytscha, chum salmon O. keta, and coho salmon O. kisutch,) as well as Atlantic salmon , and rainbow trout (O. mykiss) [IHNV is the type species for the genus ing wild  and culting wild . The vir mykiss) . However mykiss) -93. Garv mykiss)  have rep mykiss) . Isolate mykiss) ,94. In c mykiss) . If thisUntil the mid-1980s, VHS was regarded as a disease affecting only rainbow trout and a few other freshwater fish species in aquaculture in continental Europe. Since then, however, VHSV has been isolated from a large range of free-living marine fish species, either diseased or asymptomatic, throughout the northern hemisphere. So far, VHSV has been isolated from more than 70 different fish species .Different studies based on different gene sequences, including nucleoprotein (N), glycoprotein (N) and non-structural (NV) protein genes, have identified the existence of four genotypes of VHSV -101. GenThe use of phylogenetic tools has provided considerable genetic evidence indicating that rainbow trout pathogenic VHSV emerged from a genotype I-type marine ancestor ,101-103.Some phylogenetic studies indicate that VHSV may have been present in marine fish species in Europe for centuries and that the genotypes became separated a long time before fish farming was established in Europe and North America . HoweverIn spite of the lack of reports on the definition of molecular determinants involved in host specificity, some studies performing comparative analysis of the complete genome sequences provide clues to the possible involvement of a small number of nucleotides ,108. To Orthomyxoviridae, the only member of the genus Isavirus [Infectious salmon anaemia virus (ISAV), the etiological agent of infectious salmon anaemia (ISA), is an RNA virus of the family Isavirus . The genIsavirus  and a seIsavirus .Salvelinus alpinus, O. mykiss, O. keta, O. kisutch) [Clupea harengus, Gadus morhua) [ISA is characterized by high mortality, and natural outbreaks have only been described in farmed Atlantic salmon. However, ISAV has been reported in both wild salmonid and non-salmonid fish -115, andkisutch) -119 and  morhua) ,121.The HE surface glycoprotein is the molecule with the highest sequence variability, and is assumed to be of importance in determining virulence. Most of the variation in this molecule is concentrated on a small highly polymorphic region (HPR). It is widely assumed that the source of the virulent ISAV isolates is an ISAV variant- designated HPR0- without any deletion in the HPR gene. The non-virulent nature of HPR0 viruses was indicated by the lack of disease in vivo and by their failure to replicate in cell culture ,122. TheA recent study by Markussen et al.  has provHowever, Kibenge et al.  have poss of a large, linear, double-stranded DNA genome, an icosahedral capsid, a proteinaceous matrix (the tegument) and an envelope containing viral proteins [Herpesviruses (HVs) infect a wide variety of vertebrate hosts including mammals, birds, reptiles, amphibians and fish, and at least one invertebrate group, bivalve molluscs. HV share a characteristic virion structure, which consistproteins .Herpesviridae was raised to the order Herpesvirales and split into three families: Herpesviridae, which divides into the subfamilies Alpha, Beta and Gammaherpesvirinae, containing mammalian, avian and reptilian viruses; Alloherpesviridae containing fish and amphibian viruses; and Malacoherpesviridae containing one single virus Ostreid herpesvirus (OsHV-1). Table HV taxonomy has recently undergone a revision by the ICTV , in whicAcipenseridae (sturgeons), an ancient fish lineage and the sturgeon HV (AciHV1 and AciHV2) which are not sister taxa, with AciHV2 being the sister group of the ictalurid HV. Another example is provided by the eel HV (AngHV1), which grouped tightly with cyprinid HV. These authors suggested that the lack of cospeciation at deep nodes in the phylogenetic tree may indicate the existence of interspecies transmission.As a general rule, the natural host range of mammalian and avian HV is highly restricted, and most herpesviruses are thought to have evolved in association with single host species , but excCrassostrea gigas, a variant of OsHV-1 (termed OsHV-1var) was detected in the Manila clam Ruditapes philipinarum [Pecten maximus [There is clear evidence of interspecies transmission of OsHV-1 in marine bivalves ,133. Alt maximus .Aquabirnavirus is one of the four genera of the family Birnaviridae. The type species of the genus is infectious pancreatic necrosis virus (IPNV), the first fish virus isolated and characterised in cell culture [ culture . Aquabir culture .During the 1960's most of the reports on IPNV were associated with disease in juvenile salmonids. However, in the following years, isolations of aquatic birnaviruses were made from a large number of aquatic animals, most of them from animals with no evidence of disease, reaching 80 different species, including freshwater and marine species of fish and shellfish worldwide . AlthougSeriola quinqueradiata) [contributes to adaptation and replication in new hosts: natural reassortment. Thus, Romero-Brey et al. [Most aquabirnaviruses are antigenically related and belong to serogroup A, which includes nine serotypes (A1-A9), whereas a few isolates represent an antigenically unrelated serogroup (serogroup B) ,137. Sixradiata) . This geradiata) . The higy et al.  in an any et al. , reportey et al.  confirmeThe wide host range shown by many viruses affecting lower vertebrates is well known. In fact, for some of them -those historically most extensively studied- the list of susceptible species is surprisingly extensive. The best example are aquabirnaviruses. However, some others are only virulent to one or to a very narrow number of species. This diversity of host specificity patterns has not been well studied, and it is therefore poorly understood at present. In this sense, the status of knowledge varies dramatically among the different viral groups. In fact, for many of them only characterisation of field isolates has been performed, focusing on natural hosts, transmission pathways, genetic variation, etc. In a few cases, however, experimental studies have been conducted to document variations in host specificity between viral species (as for betanodaviruses) or between strains within a viral species (IHNV and VHSV). For some viruses, there is field data to support interspecies and interclass transmissions (ranavirus), for others, interspecies and interclass transmission is hypothesised based on phylogenetic relationships . For some, the molecular basis of host-specific virulence and/or host specificity has been investigated .Compared with the examples from mammalian viruses described in the introduction, there are no absolutely certain examples of host shifts in fish viruses, but there are some that have been hypothesised based on reasonable evidence. The viruses tackled in this review are the few for which some information has been documented and is available at present, and could be summarised as follows.Ranaviruses constitute a group of viruses with a broad host range, for which the interspecies and interclass transmission has been well documented; in addition, evidence for host shifts based on phylogenies and genome analyses is also available; however, to our knowledge, the molecular determinants and/or mechanisms for host-specificity have not been investigated.A variation in host-specificity among the four viral genotypes of betanodaviruses is well documented. In addition, molecular determinants for their host specificity patterns have been investigated using natural reassortants and chimeric recombinant viruses. From these studies, specific amino acid changes have been identified as putatively associated with differences in host specificity.In the case of novirhabdoviruses, variations in host specificity have been demonstrated among viral strains within both species -IHNV and VHSV-, although to a higher extent within VHSV. Although not scientifically demonstrated from a molecular basis, host shift/adaptation events could be hypothesised based on phylogenetic analyses. Moreover, whole genome sequence comparisons and infectious clones of IHNV and VHSV are now available, which are being used to study, and more deeply understand, host specificity determination in these viruses.Several studies on the molecular basis of virulence of ISA virus have indicated that changes located in the haemagglutinin (HPR) and in the fusion protein are associated with outbreaks in Atlantic salmon. Based on phylogenetic analysis, it has been hypothesised that these changes could have been involved in a change in host specificity.Phylogenetic analysis comparing fish herpesvirus and host lineages have revealed discordances that may suggest the existence of interspecies transmission. In addition, in mollusk bivalves there is field evidence of interspecies transmission of herpesviruses.Finally, regarding aquabirnaviruses little information other than broad host range and diversity of IPNV  genogroups, has been reported. Similarly, there is no demonstration of variations in host specificity among different viral strains, and no studies on host specificity are available; the recently demonstrated occurrence of natural reassortment among field isolates could have some implication in determining the host specificity and virulence of these viruses, and will probably be investigated in the future.Both authors carried out the compilation and analysis of references related to the subject, as well as the writing and edition of the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.