Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases. Palatine tonsils and adenoids are prominent secondary lymphoepithelial organs associated with the upper respiratory tract, where inhaled antigens first come in contact with host defense cells . Nevertheless, systematic studies of viruses in adenoids and tonsils have been scarce, especially in association with chronic hypertrophic tonsillar disease. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases we have performed a cross-sectional study in surgical tissue samples and cavity washes from patients with recurrent tonsillitis and chronic adenotonsillar hyperplasia. To the best of our knowledge, this has been the most extensive study of its kind to date. The results revealed high rates of virus detection in adenotonsillar tissues, with divergent virus preponderance in different sampling sites, possibly owing to different routes of infection and/or tissue composition. The high rates of detection of respiratory viruses in adenoids and palatine tonsils of patients with chronic adenotonsillar disease bring new insights about the transmission of respiratory viruses and their role in pathogenesis of chronic adenotonsillar diseases.Recovery of adenovirus and herpesvirus from tonsils and adenoids has been reported since the 1950s and 1960s A total of 121 children (63 boys) 1 to 14 years of age (mean age 5.9±3.2 years years) were enrolled in the study, all with indication for tonsillectomy due to tonsillar hypertrophy (60) or recurring tonsillitis (61), and in the absence of symptoms of acute respiratory infection.This cross-sectional study was carried out from May 2010 to August 2011 and it enrolled patients who underwent tonsillectomy at the Division of Otorhinolaryngology of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil. Exclusion criteria were: presence of symptoms of acute respiratory infection at the time of surgery and antibiotic treatment within one month prior to surgery.Patients were grouped according to palatine tonsil size and history of prior tonsillar infections as follows:RT group: Included 11 patients (9.1%) with 4 to 14 years of age (mean age 5.91 years) with recurrent tonsillitis (RT) according to the Paradise criteria: 7 episodes in the last year, 5 episodes/year in the last two years, or 3 episodes/year in the last 3 years; plus tonsils that occupied less than 50% of the oropharyngeal airway (Brodsky tonsil size ≤2).TH group: Included 49 patients (40.5%) with 2 to 13 years of age (mean age 5.85 years) with only tonsillar hyperplasia (TH) with enlarged tonsils occupying 50–100% of the oropharyngeal airway (Brodsky tonsil size ≥2), without history of recurrent tonsillitis.RT/TH group: Included 50 patients (41.3%) with 1 to 14 years of age (mean 6.33 years) with recurrent tonsillitis and tonsillar hyperplasia (RT/TH).Non RT/TH group: Included 11 patients (9.1%) with 3 to 10 years of age (mean of 7.09 years), with normal size tonsils and no history of recurrent tonsillitis. These patients presented tonsils that occupied less than 50% of the oropharyngeal airway (Brodsky tonsil size ≤2), but they had OSAS - obstructive sleep apnea syndrome.According to adenoid size, as determined by lateral X-ray and/or nasal endoscopy, the 119 patients who underwent adenoidectomy (2 patients who underwent tonsillectomy did not have adenoids removed), were grouped as follows:Group 1: Included 15 patients (12.6%) with 3 to 13 years of age (mean of 7 years), with less than 50% airway obstruction.Group 2: Included 58 patients (48.7%) with 1 to 14 years of age (mean of 6.3 years) with 50–75% airway obstruction.Group 3: Included 46 patients (38.7%) with 2 to 14 years of age (mean 5.3 years), with airway obstruction greater than 75%.Samples obtained during surgery were: a) nasopharyngeal secretions (NS), consisting of a combination of nasopharyngeal washes with swabs from mucosal surfaces of adenoids and palatine tonsils, all three collected from all patients; b) fragment of tissue from removed adenoids and palatine tonsils; c) peripheral blood. Tissue fragments were placed in Eagle’s minimal essential medium (MEM) with 10% fetal bovine serum and 15% antibiotics-antimycotic solution   and all samples were transported on ice within less than four hours to the virology laboratory, where they were processed and tested for the respiratory viruses.In the laboratory, the tissue fragments were washed twice with MEM to remove excess of cells debris and blood, and cut into portions of approximately 0.5 cm x 0.5 cm using RNAse free surgical material. These fragments were macerated in Trizol  and in one preservative solution  for further nucleic acids extraction. All the secretions were treated with antibiotics and antimycotics  for 1 hour and aliquoted in an equal volume of freezing medium  and in the triple volume of Trizol .After processing, all samples were aliquoted and kept at −70°C until further testing.The project was based on analysis of clinical samples from patients sent for tonsillectomy, and all obtained tissues would normally excised from all patients. The study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Review Committee of the Hospital das Clínicas de Ribeirão Preto (Number 10466/2008) and a written informed consent was obtained from all parents and guardians prior to enrollment.Nucleic acids were extracted from 200 µL of nasopharyngeal secretions using the QIAamp Min Elute Virus Spin Kit ; from ∼30 mg of tissue using the AllPrep DNA/RNA mini kit ; and from 1 mL of peripheral blood using the QIAamp RNA and DNA blood mini kit , all following manufacturer’s instructions.). qPCR for β-actin and RNAseP housekeeping genes was done in all samples. The qPCR assays using two sets primers for different clusters of HRV types, HRSV subgroups A and B, and HCoV types OC43 and 229E, were done in duplex format. The remaining qPCR assays done with one set of primers per reaction for HAdV, HBoV, HMPV A, HMPV B, FLU A, FLU B, HPIV 1, HPIV 3, RNAseP and β-Actin were done separately. For the RNA viruses  reverse transcription was done prior to qPCR using 1 µg of extracted RNA primed with random hexamers, with Multiscribe reverse transcriptase  following protocol proposed by the manufacturer. Duplex qPCR assays were done in a final volume of 15 µL with 3 µl of cDNA, 10 µM forward and reverse primers, 5 µM probe, 7.5 µl of TaqMan master mix , with the following parameters: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The qPCR assays with a single pair of primers per reaction were done in a final volume of 10 µL with 3 µl of cDNA, 10 µM forward and reverse primers, 5 µM probe, 5 µl of TaqMan master mix, with the following parameters: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute, except when the target was FLU A, HEV or HMPV A and B, when the parameters were: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 60°C for 1 minute. For the DNA viruses, qPCR was done with 300 ng of extracted DNA in a final volume of 10 µL, containing 10 µM forward and reverse primers, 5 µM probe and 5 µL of TaqMan master mix, using the same cycling parameters as for the RNA viruses. Applicable measures to prevent contamination of PCR reactions were taken in this study, including sample handling and reaction mix preparation in separate rooms. In addition, all qPCR plates included appropriate negative controls, matched to every step of clinical sample treatment. The negative controls were RT-qPCR products from RNA or DNA extracted from cultures of uninfected Hela cells; RT-qPCR product from ultrapure water; and just qPCR product from ultrapure water.Samples were tested by TaqMan real-time PCR (qPCR) for the presence of genomes of human bocavirus (HBoV), human adenovirus (HAdV), rhinovirus (HRV), enterovirus (HEV), human respiratory syncytial virus (HRSV) A and B, metapneumovirus (HMPV) A and B, human Influenza virus (FLU) A and B (including H1N1 2009), parainfluenza virus (HPIV) 1 and 3, and coronavirus (HCoV) OC43 and 229E. All qPCR assays were done on Thermocycler 7300  with specific primers and probes . qPCR fP value of ≤0.05 was chosen for significance.Chi-square and Exact Chi-square tests were used to compare patient groups and viral detection rates in relation to different parameters analyzed. Agreement between samples was tested using Kappa test, followed by the calculation of odds ratios. Analysis of presence of viruses in relation to ages and clinical conditions were done with Mann-Whitney test. Analyses were conducted using the SPSS software , version 17(SPSS Inc.). A This study included nasopharyngeal secretions (NS), fragments of tissue from adenoids and palatine tonsils and peripheral blood from a total of 121pediatric patients with indication of tonsillectomy due tonsillar hyperplasia or recurring tonsillitis. The overall frequency of virus detection was very high: 118 of 121 patients (97.5%) had at least one virus detected in at least one clinical sample . Only 3 Considering the results of virus detection altogether, HAdV was the most often detected agent, in 57 (47.1%) patients, followed by HEV in 49 (40.5%), HRV in 46 (38%), HBoV in 36 (29.8%), HMPV in 21 (17.4%), HRSV in 19 (15.7%), FLU in 7 (5.8%), HPIV in 6 (4.9%) and HCoV in 4 (3.3%) . FrequenThe detection of specific viruses notably varied among sample sites, indicating that respiratory viruses have different tissue tropisms, even when we compared pharyngeal and palatine tonsils. HAdV and HBoV were detected in adenoids more often than in others sites, whereas HEV and HRV were more frequently detected respectively in palatine tonsils and NS. Of the 119 patients from whom samples of all sites were obtained, 59 (49.6%) had at least one virus detected simultaneously at three sample kinds. In only 15 patients (12.6%) viruses were detected in only one sample .The virus most frequently detected in NS was HRV, in 42 (34.7%) patients, followed by HAdV (29.8%), HEV (18.2%), HBoV (11.6%), and all other viruses were detected in frequencies lower than 10%. In the palatine tonsils, HEV was found in 45 samples (37.2%), followed by HAdV in 29 samples (24%), HRV in 15 samples (12.4%), and all other viruses were detected in frequencies lower than 10%.Overall virus detection rates were notably higher in adenoids, where the most frequent virus detected was HAdV in 42.9%, followed by HEV in 37 (30.6%), HRV in 30 (24.8%) and HBoV in 28 (23.1%) patients. The only virus detected by qPCR in the peripheral blood was HBoV, found in two patients .HAdV was more frequently detected in the adenoids, although simultaneous detection in other sampling sites was very frequent. Only 6 of all 57 HAdV-positive patients had the virus detected only in sites different from the adenoids . The kapSimilarly, HBoV was also more frequently detected in adenoids than in other sites, and 52.8% of patients had HBoV detectable only in the adenoid tissue. Detection of HBoV in adenoids was associated with simultaneous detection of that virus in palatine tonsils  and NS . In just 2 patients HBoV was detected only in the palatine tonsils, but not in the adenoids.NS was the only sample positive for HRV in 34.8% of patients, and in only 4 patients HRV was detected exclusively in samples other than NS . DetectiWhile HAdV and HBoV were more frequently detected in adenoids, enteroviruses were significantly more frequent in palatine tonsils. Of 49 patients positive for HEV, 45 (92%) had positivity in palatine tonsils, which was strongly correlated with their presence in adenoids  and nasopharyngeal secretions . Thus, the detection of HEV in palatine tonsils increased the chance of finding HEV in adenoids by 48.13 times  and in NS by 65.63 times . Interestingly, in 11 of 45 (24.5%) patients with HEV detectable in palatine tonsils, that site was the only one positive for HEV, as opposed to 4 (8.8%) patients in whom HEV was detected solely in sites other than palatine tonsils .For all remaining viruses the rates of detection were lower, hampering comparisons between sampling sites.High rates of viral co-infections were obtained in this patient group. Of 118 patients positive for any respiratory virus, 82 (69.5%) had more than one virus. Of the 82 patients who had more than one detectable agent, 47 (57%) had 2, 26 (32%) had 3, 8 (9.7%) had 4, and 1 patient had five simultaneously detectable viruses . The virThe rates of detection of more than one virus per sample site were lower in palatine tonsils than in adenoids (44.5%) and NS (31.4%) . In onlyAlthough the frequencies of viral co-infections for specific viruses varied among sample sites, DNA viruses (HAdV and HBoV) were more often detected as part of co-infections than RNA viruses . HBoV waThe virus co-infections observed in this study generally involved DNA viruses in combination with some RNA virus, most notably HRV and HEV . The mosFrequencies of specific combinations of co-infecting viruses varied among sample sites. In adenoids, co-infections more frequently involved HAdV + HEV, HAdV + HRV, and HAdV + HBoV + HRV, respectively in 8 (6.7%), 6 (5%) and 5 (4.2%) patients. The most frequent virus associations in palatine tonsils and NS were, respectively, HAdV + HEV, and HAdV + HRV.All patients enrolled in this study underwent tonsillectomy due to adenotonsillar hypertrophy or recurring tonsillitis. Of 121 patients, 16 (12.4%) also had otitis media with effusion, 61 (50.5%) had apnea, and 33 (27.3%) reported underlying conditions, including asthma in 5 and symptoms of allergy in 19.2 = 4.61, p = 0.04).The presence of allergic symptoms, bronchial asthma and recurrent otitis media with effusion were not significantly associated with the presence of specific respiratory viruses, except for an association of HRSV in adenoids with reported allergy , when that analysis was done for specific agents, significance was noted only for HBoV .2 = 3.01, p = 0.10) in children with recurrent tonsillitis and/or severe tonsillar hypertrophy (>70%), as compared to children without recurrent tonsillitis and in those with lower degrees of palatine tonsil hypertrophy (45.45%).Virus detection, both overall and agent-specific, was not associated with recurrent tonsillitis . In fact2 = 3.58, p = 0.06). However, there was an association between higher virus detection rates with larger palatine tonsils in the specific case of HEV, since this agent was detected in palatine tonsils from 40 of 98 (40.8%) patients with higher hypertrophy degrees (Brodsky ≥3), and in 5 of 22 (22.7%) patients with less severe hypertrophy (Brodsky <3) . Rates of detection of other viruses were similar between patients with larger and smaller palatine tonsils  and small palatine tonsils (Brodsky ≤3)  in patients with virus detected in the adenoidal tissue  than in patients without virus in adenoids . Patients with virus in adenoid tissue had a chance 3.4 times higher of having a high degree of adenoid hypertrophy (75–100%) than those without virus detected there . Although HEV was frequently detected in adenoids from patients with nasopharyngeal obstruction ≥75% , no single virus was individually associated with large adenoids .Overall rates of virus detection in adenoids were higher in patients with more severely enlarged adenoids . The proTo the best of our knowledge, this has been the most comprehensive study of respiratory viruses in chronic adenotonsillar diseases of children so far conducted. The study was based on sensitive molecular assays to detect respiratory viruses in sets of samples prospectively collected from children with a full spectrum of chronic adenotonsillar diseases requiring tonsillectomy. The observed frequency of respiratory virus genomes detected in 97.5% of the children is remarkably high. Such staggering observation is novel and should be regarded in the light of the high prevalence of adenotonsillar diseases, pondering the possible roles that persistence of common respiratory virus may play on their pathogenesis.Testing the nucleic acid extracts obtained from macerated fragments of upper respiratory lymphoepithelial tissues by qPCR revealed an array of respiratory viral genomes in over 85% of adenoids and close to 70% of palatine tonsils. Considering that the children had no symptoms of acute respiratory infections, these proportions of detection in tissues are by all means extremely high.Lymphoid tissues associated with the upper respiratory tract have been recognized as sites of latency/persistency of such agents as Epstein-Barr virus, human herpes virus 6, HIV, measles virus, picornaviruses, human bocavirus and human papillomavirus-16 While detection of DNA virus genomes (HAdV and HBoV) in association with lymphoepithelial organs is not novel, such is not the case of detecting respiratory RNA viruses, for which the existence of persistency and latency is far from clear. Therefore, the most surprising findings were the high frequencies of detection of the picornaviruses HRV and HEV, both detected in approximately 40% of the children, in the absence of discernible clinical features of acute respiratory infection. When HRV and HEV were taken together, the frequencies of picornavirus detection in adenoids and palatine tonsils were respectively 55.4% and 49.6%, showing that these agents together can be present in lymphoepithelial tissues in up to one half of subjects with chronic tonsillar hypertrophy. However, when considering HRV and HEV separately, as made possible with specific primers and probes by qPCR, interesting distinct features become evident.Detection of HRV in nasopharyngeal secretions was highly frequent (34.7%), and was associated with an increase of 30–40 times in the chance of finding it also in tonsils. Indeed, in only 4 of 46 HRV-positive patients (8.7%), the virus was detected only in tonsils, without concurrent detection in nasopharyngeal secretions. In contrast, detection of HEV in palatine tonsils was significantly more frequent than in adenoids and nasopharyngeal secretions and, conversely, in only 4 of 49 HEV-positive patients (8.2%) the virus was detected in sampling sites either than palatine tonsils. A tropism of HEV for palatine tonsils is illustrated by this site being the only HEV-positive site in 25% of all 45 patients with HEV in palatine tonsils.The frequent finding of picornaviruses in adenoid and palatine tonsil tissues in the present study expands on findings of a previous study done in Finland Not surprisingly, HAdV was the most frequently detected agent, present in near one half of all patients when results from all sample sites were taken together; and in over 42% considering only macerated adenoid tissues. In fact, HAdV was first identified in association with adenoid tissue HBoV is a recently identified parvovirus, found in association with respiratory and gastrointestinal disorders This is the first report of viral co-infection in chronic adenotonsillar diseases, and it is notable that two thirds of the patients had more than one respiratory virus detected simultaneously, even in the absence of symptoms and signs of acute respiratory infection. The majority of viral co-infections resulted from combinations of HAdV, HBoV, HEV and HRV, and co-infections were more frequent when detections of viruses both in tissues and nasopharyngeal secretions were considered. When only adenoids and palatine tonsils were analyzed, rates of viral coinfections were close to 45% and 21%, respectively.The presently reported findings on co-infections are similar to what has been known for acute respiratory infections, which can yield as much as 40% to 70% of virus co-detection in situ hybridization in younger patients with higher degrees of palatine tonsil hypertrophy than in older patients with a lower degrees of hypertrophy.No clear associations were found between any particular virus and clinical features. However, it should be pointed out that there was a high frequency of virus detection, especially HEV, in highly hypertrophic tissues. This is in keeping with results published for EBV , and that could be caused by viruses, such as the case of human coronavirus, which has a nonstructural protein that inhibits apoptosis triggered by the mitochondrial antiviral signaling adaptor (MAVS) While mechanisms that might underlie a link of the presence of prolonged viral infection in tonsils with chronic tonsillar diseases, it is tempting to speculate that virus products may function as pathogen-associated molecular patterns (PAMPs), that would maintain inflammation by the stimulation of the innate immunity, with continuous processes of damage and repair, thus leading to hyperplasia and hypertrophy of tonsils, whose enlarged sizes would cause clinical features of chronic tonsillar disease. This finds support on histological studies of enlarged tonsils, which reveals enlargement of follicles, indicating hyperplasia with marked increase in the numbers of lymphoid cells in the germinal centers In summary, the present study has shown that a very high proportion of patients with chronic adenotonsillar diseases harbor at least one respiratory virus, and that these agents are frequently detected in adenotonsillar tissues. The patients were sampled only once, which hampers determination of the duration of virus presence in them. Nevertheless, significant proportions of such patients had different combinations of viruses simultaneously detected at different sites, suggesting that some of them might be persistent. It is remarkable that not all respiratory viruses were detected at similar frequencies in adenoids and palatine tonsils, nor were uniformly shed in nasopharyngeal secretions. Specific viruses seem to preferentially infect some tissues, such as human enteroviruses that were more often detected in palatine tonsils than in adenoids, and were shed in secretions much less frequently than all others. Taken together, these data suggest that respiratory virus persistence/latency may play roles in pathogenesis of chronic adenotonsillar diseases, and that these tissues may serve as inter-seasonal virus reservoirs, with possible impact in transmission in the community.Table S1Primers and probes used for qPCR.(DOCX)Click here for additional data file.


Based on epidemiological data, it is believed that human-to-human transmission plays an important role in Nipah virus outbreaks. No experimental data are currently available on the potential routes of human-to-human transmission of Nipah virus. In a first dose-finding experiment in Syrian hamsters, it was shown that Nipah virus was predominantly shed via the respiratory tract within nasal and oropharyngeal secretions. Although Nipah viral RNA was detected in urogenital and rectal swabs, no infectious virus was recovered from these samples, suggesting no viable virus was shed via these routes. In addition, hamsters inoculated with high doses shed significantly higher amounts of viable Nipah virus particles in comparison with hamsters infected with lower inoculum doses. Using the highest inoculum dose, three potential routes of Nipah virus transmission were investigated in the hamster model: transmission via fomites, transmission via direct contact and transmission via aerosols. It was demonstrated that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. These findings are in line with epidemiological data which suggest that direct contact with nasal and oropharyngeal secretions of Nipah virus infected individuals resulted in greater risk of Nipah virus infection. The data provide new and much-needed insights into the modes and efficiency of Nipah virus transmission and have important public health implications with regards to the risk assessment and management of future Nipah virus outbreaks. Understanding how viruses are transmitted plays an important role in our ability to intervene in virus outbreaks. Over the last decade, Nipah virus has caused multiple outbreaks in Malaysia, India and especially Bangladesh. Fruit bats form the natural reservoir for Nipah virus; from the bats the virus is introduced into the human population, either directly or via an intermediate host. Epidemiological data suggest that upon introduction into the human population the virus has the ability to spread from person-to-person. We performed experimental studies in a hamster model to investigate if we could mimic human-to-human transmission and to determine the route of transmission through which Nipah virus spread between people. We discovered that Nipah virus-infected hamsters predominantly shed virus via excretions from the nose and lungs. In transmission experiments, we showed that Nipah virus is efficiently transmitted via direct contact. Fomite transmission was inefficient and transmission via aerosols did not occur. The elucidation of the mode of Nipah virus transmission has important public health implications because it allows a targeted and experiment-based assessment of intervention strategies and surveillance for emerging Nipah virus strains better adapted to human-to-human transmission. Henipavirus genus in the Paramyxoviridae family. Nipah virus first emerged in humans in Malaysia in 1998–1999, during a large outbreak of encephalitis and respiratory disease in humans, causing 276 cases of encephalitis, with 106 fatalities Nipah virus is a member of the In this study we assessed the potential of human-to-human transmission of Nipah virus in the Syrian hamster model. The Syrian hamster has been shown to replicate both the respiratory and neurological symptoms seen in humans All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories (ASP #2011-03), and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC-approved facility.Virus and cells. Nipah virus  was kindly provided by the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, Georgia, United States and propagated in VeroE6 cells in DMEM (Sigma) supplemented with 10% fetal calf serum , 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco).tm:AURA, Harlan Laboratories) were inoculated intranasally with 103, 105 or 107 TCID50 of Nipah virus in a total volume of 100 µl. On days 2 and 4 post inoculation 6 animals were euthanized and lungs, trachea and nasal turbinates were collected for virologic and histopathologic analysis. Daily nasal, oropharyngeal, urinogenital and rectal swabs were obtained from the remaining 6 hamsters. Swabs were collected in vials containing 1 ml DMEM supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. Hamsters were euthanized on day 14 post inoculation or earlier upon signs of severe infection.To study the relation between inoculation dose and Nipah virus shedding, 3 groups of 18 6–8 week old female Syrian hamsters . Homogenates were centrifuged to clear the homogenate before inoculating cells.Virus titrations were performed by end-point titration in VeroE6 cells. VeroE6 cells were inoculated with tenfold serial dilutions of swab medium or tissue homogenates. One hour after inoculation, the inoculum was removed and replaced with 200 µl DMEM supplemented with 10% fetal calf serum , 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). Three days after inoculation, cytopathic effect (CPE) was scored and the TCIDHistopathology and immunohistochemistry was performed on hamster tissues. Anaesthetized hamsters were euthanized by exsanguination. Necropsies and tissue sampling were performed according to a standard protocol approved by the Institutional Biosafety Committee. After fixation for 7 days in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (H&E) staining and an immunohistochemical method using a rabbit polyclonal antiserum against the Nipah virus nucleoprotein 50 equivalents in the samples.RNA was extracted from swab samples using the NucleoSpin 96 Virus Core kit (Macherey-Nagel) and a Corbett Robotics model CAS 1820 automatic RNA extractor. RNA was eluted in 100 µl. 5 µl RNA was used in a one-step real-time RT-PCR targeted at the NP gene using the Rotor-Gene™ probe kit (Qiagen) according to instructions of the manufacturer (primer sequences are available on request). In each run, standard dilutions of a titered virus stock were run in parallel, to calculate TCID50 of Nipah virus was added. After 1 hr at 37°C, this mix was added toVeroE6 cells. Three days after inoculation, wells were scored for CPE. The virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibits Nipah virus replication.Two-fold serial dilutions of heat-inactivated hamster sera were prepared in DMEM containing 2% fetal calf serum, 1 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and 100 TCIDImmuno-globulin G antibody responses were measured in an enzyme-linked immunosorbent assay (ELISA) using Nipah virus Malaysia. Nipah virus-containing cell culture supernatant was concentrated and purified by spinning two hours at 21000 rpm over a 20% sucrose cushion. The pellet was resuspended in PBS and triton X-100 was added to a final concentration of 1%. This suspension was then used to coat immuno 96 microwell maxisorp plates (NUNC) at 4°C overnight. Subsequently, plates were blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1.5 hours at 4°C. After 3 washes with PBST, 50 µL of diluted serum samples were added, and the plates were incubated for 1 hour at 37°C. Bound antibodies were detected after 3 washes using an anti-hamster secondary antibody conjugated with horseradish peroxidase . Following incubation for 1 hour at 37°C, bound HRP was detected using the ABTS® Peroxidase Substrate System (KPL). The absorbance at 405 nm was measured using a microplate spectrophotometer. Sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera.3, 105 or 107 TCID50, to select the dose that resulted in the highest amount of Nipah virus shed from the nose, throat, urogenital tract and rectum. Upon intranasal inoculation of hamsters with 103 TCID50, limited shedding of Nipah virus was observed in nasal, oropharyngeal, urogenital tract and rectal swabs in five out of six hamsters  and 12 dpi .Upon inoculation with 10culation . Virus s3 or 105 TCID50 of Nipah virus, only oropharyngeal swabs were positive  for 103 TCID50 vs. 4.958  for 105 TCID50 vs 7.44  for 107 TCID50, 1-way ANOVA p<0.0001).Virus titrations were performed on all PCR-positive swabs. For all infectious doses, urogenital and rectal swabs were negative upon virus titration. Upon inoculation with 10positive . With a solation . Compari7 TCID50 could Nipah virus be isolated in all three tissues of all three hamsters. The inoculum of 107 TCID50 of Nipah virus showed significantly higher virus titers for 2 dpi and 4 dpi compared to the other 2 inoculum doses .3 TCID50 of Nipah virus did not show any pathological changes at 2 dpi  inoculation with a dose of 107 TCID50 not only shed significantly more Nipah virus particles as determined by realtime RT-PCR than hamsters inoculated with lower doses, but more importantly also shed more viable Nipah virus as determined by virus titration. With the lower inoculum doses limited shedding of viable Nipah virus was observed, indicating that the inoculum dose may very well affect the ability of Nipah virus to transmit efficiently, through an effect on virus shedding. Our results indicate the importance of nasal and oropharyngeal shedding and transmission and are in line with previous experimental Nipah virus infections in pigs where virus excretion was also observed in inoculated and contact pigs, although the mechanism by which the transmission occurred was not investigated Our data suggest that hamsters inoculated with 10Interestingly, epidemiological data gathered during the Nipah virus outbreak in Malaysia have not identified human-to-human transmission in this outbreak, although four potential cases of nosocomial transmission have been reported Evidence for vertical transmission was observed in a cat model of Nipah virus disease. In a pregnant cat, infectious virus was detected in placental tissue and Nipah virus genomic RNA was detected in fetal tissue The experimental data on the route of transmission of Nipah virus presented here have important public health implications with regards to the risk assessment and management of future Nipah virus outbreaks. In addition, this novel transmission model can be used to evaluate the efficacy of outbreak intervention strategies, such as vaccination and antiviral therapies. Whereas current intervention strategies are predominantly focused at post exposure treatment, the ability to efficiently block transmission and thereby spread of the outbreak is currently not assessed in antiviral or vaccination treatment strategies Figure S1Shedding of Nipah virus RNA in inoculated hamsters. Groups of 6 hamsters were inoculated intranasally with 103 (black bars), 105 (grey bars) or 107 TCID50 (white bars) Nipah virus. Nasal (A), oropharyngeal (B), urogenital (C) and rectal (D) swabs were collected daily for 14 days and viral load in the swabs was determined as TCID50 equivalents by real-time RT-PCR. Geometric mean viral loads are displayed; error bars indicate standard deviation.(TIF)Click here for additional data file.Figure S2Loss of bodyweight and survival in hamsters inoculated with Nipah virus. Loss of bodyweight (A) and survival (B) after intranasal inoculation of with 103 , 105  or 107  TCID50 Nipah virus are plotted. Hamsters were weighed daily, and the percentage of body weight was calculated relative to the weight at time of inoculation. The percentage of mice surviving the infection is shown as a function of time.(TIF)Click here for additional data file.


The etiology of reptilian viral diseases can be attributed to a wide range of viruses occurring across different genera and families. Thirty to forty years ago, studies of viruses in reptiles focused mainly on the zoonotic potential of arboviruses in reptiles and much effort went into surveys and challenge trials of a range of reptiles with eastern and western equine encephalitis as well as Japanese encephalitis viruses. In the past decade, outbreaks of infection with West Nile virus in human populations and in farmed alligators in the USA has seen the research emphasis placed on the issue of reptiles, particularly crocodiles and alligators, being susceptible to, and reservoirs for, this serious zoonotic disease. Although there are many recognised reptilian viruses, the evidence for those being primary pathogens is relatively limited. Transmission studies establishing pathogenicity and cofactors are likewise scarce, possibly due to the relatively low commercial importance of reptiles, difficulties with the availability of animals and permits for statistically sound experiments, difficulties with housing of reptiles in an experimental setting or the inability to propagate some viruses in cell culture to sufficient titres for transmission studies. Viruses as causes of direct loss of threatened species, such as the chelonid fibropapilloma associated herpesvirus and ranaviruses in farmed and wild tortoises and turtles, have re-focused attention back to the characterisation of the viruses as well as diagnosis and pathogenesis in the host itself.1. Introduction2. Methods for working with reptilian viruses3. Reptilian viruses described by virus families3.1. Herpesviridae3.2. Iridoviridae3.2.1 Ranavirus3.2.2 Erythrocytic virus3.2.3 Iridovirus3.3. Poxviridae3.4. Adenoviridae3.5. Papillomaviridae3.6. Parvoviridae3.7. Reoviridae3.8. Retroviridae and inclusion body disease of Boid snakes3.9. Arboviruses3.9.1. Flaviviridae3.9.2. Togaviridae3.10. Caliciviridae3.11. Picornaviridae3.12. Paramyxoviridae4. Summary5. Acknowledgements6. Competing interests7. References The etiology of reptilian viral diseases can be attributed to a wide range of viruses occurring across different genera and families. Thirty to forty years ago, studies of viruses in reptiles focused mainly on the zoonotic potential of arboviruses in reptiles and much effort went into surveys and challenge trials of a range of reptiles with eastern and western equine encephalitis as well as Japanese encephalitis viruses -3. In thGenerally, diagnosis of reptilian viruses can be approached like all other viruses. Histopathology can give the initial indication of a viral infection and most infections are described alongside the pathological changes they induce. Some viruses have been isolated, characterized and used in challenge trials or to produce specific antisera for immunological tests, but like all other fields of virology, reptilian virus researchers are venturing into both serological surveys and molecular tools for diagnosing viral infections ,14-20.Bothrops alternate, followed by Japanese encephalitis virus from snakes, calicivirus from snakes, a flavivirus-like agent from tortoises and West Nile virus from alligators using either mammalian derived cell lines at 37°C or mosquito cell lines at 28°C [Isolation of viruses in culture has the advantage of amplifying the agent for easier identification and characterisation, but also for use in transmission studies. Specific reptilian cell lines are available that will support viral growth and for some viruses display cytopathic effect, where others do not cause CPE or do not grow at all in known cell culture systems. For those cell lines that support propagation of a particular virus, the temperature regime for both cell lines and viral growth will be different from mammalian systems due to the poikilothermic nature of reptiles. Zoonotic viruses have been isolated from reptiles since 1939 when Rosenbusch isolated Western Equine Encephalitis from a  at 28°C -25. Seve at 28°C ,27. Clar at 28°C ,30 and rWhere electron microscopy (EM) used to be the ultimate diagnostic tool for viruses, polymerase chain reaction (PCR) has revolutionized our ability to identify infectious agents with generic and specific primers used for diagnosis. Subsequent sequencing of the viral genome opens possibilities for fast characterization, insight into their phylogenetic relationship and epidemiological investigations. The drastic decrease in the cost of sequencing has turned the diagnostic attention to molecular methods, which has the added advantage of accurate diagnosis and the subsequent possibility of reproducing the antigenic part of the virus, irrespective of our ability to culture the viral pathogen in vitro. However electron microscopy still has a diagnostic role since agents identified under the EM scope may direct the diagnostician toward selecting the best primers to use.With the current trend and affordable sequencing costs, researchers will keep looking for and finding viruses in reptiles at a great rate and over the next decade we are likely to see the clades being populated with new findings, known isolates being placed into taxonomic relationships and possibly revealing genetic virulence markers.The type of genome and the presence of an envelope are important in the replication strategy of viruses and subsequently the possibilities of diagnosis, prevention and control of the associated disease. The viral families in this review are presented with like groups as indicated in Table Herpesviridae together with mammalian and avian herpesviruses. Chelonid herpesvirus 5 and 6 in marine turtles are unassigned species in the Subfamily Alphaherpesvirinae and although other reptilian herpesviruses are still unassigned by the International Committee on Taxonomy of Viruses (ICTV), molecular characterisation of available isolates places them within the Subfamily Alphaherpesvirinae in the proposed genus Chelonivirus [Herpesvirus infections appear to manifest as acute signs which may turn latent and be quiescent for the rest of the animal's life, or until the host becomes sufficiently stressed for the virus to reappear as a disease . Reptilionivirus -34. OrigChelonid fibropapilloma-associated herpesvirus (CFPHV) (Chelonid herpesvirus 5), is associated with the development of fibropapillomas and fibromas in marine turtles in all tropical waters, both externally on the epidermis, eyes, carapace and plastron, and in severe cases on the serosal surface of internal organs -40. FibrAlthough many biotic factors such as leeches, mites, other viruses and algal blooms as well as abiotic environmental factors and adjacent land use are associated with FP of wild turtles ,34,46-49The lung-eye-trachea disease-associated virus (LETV) (Chelonid herpesvirus 6), was first described in 1 year old green turtles raised in mariculture. As the name implies, the virus affects the eyes and respiratory tract of the turtles and has a clinical course of 2-3 weeks. The virus was cultured in green turtle kidney cells and initially visualized by EM . The abiChelonia mydas) was reported to cause mortalities in 5-20% of the severely afflicted animals [Grey Patch Disease (GPD)-associated virus (Chelonid herpesvirus 1) affecting cultured green turtle hatchlings  were identified by PCR in lesions of moribund animals [Two herpesviruses from loggerhead turtles  ,62-64. PNaja naja kaouthia) with reduced venom production [Elapid herpesvirus (Indian cobra herpesvirus) was associated with degeneration and focal necrosis of columnar glandular epithelial cells in the venom gland of Siamese cobras . Previous accounts of iridovirus causing systemic infections were later classified as ranavirus in many cases. Ranavirus has received increasing attention due to its involvement in serious diseases of fish and amphibians to the extent that epizootic haematopoietic necrosis virus (EHNV) in fish and ranavirus in general for amphibians meet the criteria for listing by the World Organization for Animal Health (OIE) [th (OIE) . Ranavirth (OIE) -80 and hth (OIE) ,81,82. TTestudo hermanni [Bopherus polyphemus) [Trachemys scripta elegans) with a ranavirus isolate from Burmese Star tortoise. The red eared sliders developed clinical signs and lesions consistent with those observed in the Burmese tortoises. Virions were observed in lesions by EM and ranavirus was re-isolated from challenged animals [Until recently, overt diseases in reptiles caused by systemic iridovirus were only rarely reported: namely in a spur-tailed Mediterranean land tortoise, hermanni  and a goyphemus) . Howeveryphemus) ,85-87, fyphemus) , and faryphemus) . Retrosp animals .Ranavirus infections of reptiles appear to target multiple organs including the stomach, oesophagus, lungs, spleen, liver and kidney, although some isolates may have a propensity for infecting the respiratory tract ,85,87. ICytoplasmic inclusions in erythrocytes of amphibians, fish and reptiles have long been known to be of iridoviral origin due to light and EM -96. The Chamaeloe hoehnelii) were found to be highly pathogenic to crickets (Gryllus bimaculatus) [Although viruses in this genus mostly occur in invertebrates, they do occasionally occur in reptiles  and may culatus)  showing Caiman sclerops and Caiman crocodilus fuscus [Crocodylus niloticus, C. porosus and C. johnstoni [Testudo hermanni) that succumbed to broncho-pneumonia [Chamaeleo dilepsis) [Tupinambis teguixin) with poxviral dermatitis that healed spontaneously over four months have also been reported [Poxviruses have been identified in skin lesions of s fuscus -102Crocoohnstoni -107. Theneumonia , a co-inilepsis)  and a tereported , but genreported  places cAdenoviruses cause respiratory infections in many vertebrate species. Infections have been diagnosed in crocodiles, ,113, snaMastadenovirus and Aviadenovirus genera which traditionally covered mammalian and avian adenoviruses respectively [Ictadenovirus which hosts a single member from a fish, namely the White sturgeon adenovirus 1. Atadenovirus was named according to the very high A+T genome base composition in some of the early viruses in this group, which encompass viruses from reptilian, avian and mammalian hosts. Until recently, all reptile adenoviruses belonged to the Atadenovirus genus indicating strong co-evolution of the host and virus. Siadenoviruses have a very small genome (30 kb) and a putative sialidase gene at the terminus of the genome, which gave rise to the name of this genus [Indotestudo forsteni) by sequence analysis as a novel adenovirus of the genus Siadenovirus which currently makes it the only reptile virus in this genus [A great amount of work has gone into the phylogenetic analysis of adenoviruses, which has resulted in the proposal for creation of three new genera recently in addition to the ectively . The latis genus . In 2009is genus . The cheis genus  does notPlatemys platycephala) exhibited symptoms of circular papular skin lesions on the head and forelimbs [Two side-necked turtles (orelimbs . Histoloorelimbs . Drury eorelimbs .Lacerta viridis) by Raynaud and Adrian [Papilloma-associated viruses were also identified via EM of benign papillomas from Green lizards (d Adrian . The vird Adrian . Althougd Adrian . The othChelonia mydas) and loggerhead turtle (Caretta caretta) with fibropapillomatosis [Lately, papillomavirus was reported in samples from sea turtles, green turtle  and from the brain of a rattlesnake exhibiting neurological symptoms [Elaphe obsolete), re-isolate the virus and induce similar symptoms in another black ratsnake [Lacerta viridis [Reoviruses can cause severe and often fatal disease in reptiles typically presenting as pneumonia and neurological disorder -137. Reosymptoms ,136. Thesymptoms . Lamiran viridis . Reoviru viridis . Identif viridis .Boa constrictor) was inoculated into young Burmese pythons (Python molurus bivittatus) with a resultant IBD development [Retroviruses often appear as incidental findings with no disease reported in turtles, crocodiles, tuataras, a Komodo dragon and snakes ,139-142.elopment . This dielopment .Python curtus but not in other boid species, irrespective of them displaying the symptoms of inclusion body disease which further clouds the etiological origins of this disease [With the advance of molecular techniques, endogenous retroviruses are identified in many vertebrates, including reptiles . Huder e disease .Arboviruses are arthropod borne viruses that multiply in both the arthropod vector and the vertebrate host . Many arAmeiva ameiva and classified by EM [Trionyx spinifer emoryi) during a survey of reptiles in Texas in 1970 and 71 [Chaco, Timbo and Marco rhabdoviruses were isolated from the lizard ed by EM . They gred by EM . Despiteed by EM . Likewis0 and 71 . There iElaphe rufodorsata) and in 2001, Drury et al. isolated a flavivirus-like agent from a tortoise [Antibodies to St Louis encephalitis virus and Japanese encephalitis virus (JEV) have been reported from turtles and snakes -160. Leetortoise . Much ofSero-surveys detected antibodies to West Nile Fever virus (WNV) in farmed Nile crocodiles in Israel, farmed crocodiles in Mexico, wild alligators in Florida and free-range American alligators in Louisiana ,153,161.This group of viruses includes members such as Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis . AlthougExperimental infection of snakes and tortoises show them to be highly susceptible  with virBothrops alternata, but not from the blood, showing that blood may not be the best organ for viral isolation, and that the virus may replicate in other organs during periods of low or no viremia [Isolation of togaviruses from reptiles has been attempted predominantly from blood samples with variable results ,165. Thi viremia .Culex tarsalis) was sufficient to transmit the infection to a garter snake [One incidence of feeding by an WEEV-infected mosquito  were asymptomatic and isolation was obtained by rectal swab. The other eight isolates were obtained at necropsy of animals found dead in their cages. These included four Aruba Island rattlesnakes, two Bell horned frogs (Ceratophrys orata), one rock rattlesnake (C. lepidus) and one eyelash viper (Bothrops schlegeli). Histopathology revealed a variety of inconsistent lesions in the necropsied animals. The isolates grew in Vero cells at 37°C and were identified by physicochemical characteristics as belonging to the Caliciviridae. The 16 isolates were antigenically indistinguishable and the strain was designated as reptilian calicivirus Crotalus type 1. These isolates were compared to isolates from feral pinnipeds  and were found to be closely related at the serological and genomic level [Sixteen isolates of calicivirus were obtained from four species of poikilothermic animals in a zoological collection . Eight Aic level ,171, furic level .The only record of picornavirus in reptiles is by Helbstad and Bestetti . A boa cBothrops atrox) causing up to 87% mortality in individual rooms [Crotalus lepidus) and several viper species [Several epizootics in snake collections have been attributed to a paramyxovirus infection. The first record was from Switzerland, with a respiratory disease of farmed Fer-de-lance snakes  with pneumonia and identified in faeces of farmed Nile crocodiles [Electron microscopy of in vitro propagated virus showed the virions to be pleomorphic, spheroidal or filamentous particles budding from plasma membranes or as mature enveloped particles in the cytoplasm ,188,190.ocodiles ,191. HowFerlavirus within the subfamily Paramyxovirinae with Fer-de-Lance virus as the type species [On a genomic level, paramyxoviruses from snakes and lizards are closely related, while the one isolate from the tortoise was in the same cluster but more distant to the other isolates . The sam species ,192.A multitude of viruses exists in reptiles, some of which are described above, and no doubt many more will be described in the future. Some, but not all cause disease and most of them are not very well studied in terms of virulence, pathogenicity, phylogeny and fulfilment of Koch's postulate, which requires cultivation and challenge trials. For viruses like CFPHV that cannot be cultured in vitro, the association with fibropapillomatosis is very strong, but apparently other cofactors are needed before it becomes pathogenic. Transmission may in some cases be via vectors such as arthropods or leeches, whereas others by direct contact presumably, but most transmission routes are unknown. The risk of transfer of viruses between reptiles and humans is negligible due to the thermoregulatory differences between reptilian and mammalian hosts which would limit the suite of pathogens able to grow in both temperature regimes. However, some zoonotic potential exists when certain reptiles act as reservoirs for arboviruses that are pathogenic to humans such as West Nile fever virus.Compared to fish, poultry and mammalian viruses that are of high importance in our society for either commercial or humanitarian reasons, very little is known about reptilian viruses. Crocodile, turtle and snake farms only exist on a relatively small scale and reptiles still feature as rare and exotic pets although individual specimens may be quite valuable. Specialised veterinary clinics and research scientists take an interest in reptilian viruses though and future directions for research and diagnostic tools are likely to venture further into sero-surveys. Hopefully these will become commercially available, thereby facilitating testing of reptiles prior to transfer between zoological collections to protect valuable specimens from known diseases. As for all other veterinary fields, the future diagnosis of reptilian viruses lies with molecular tools for identification, taxonomy, phylogeny and epidemiology.The author declares that they have no competing interests.


However, biochemical studies that shed light on mechanisms of drug action and resistance are hampered primarily due to technical problems associated with enzyme expression and purification. In contrast, the orthologous bacteriophage RB69 polymerase gp43 has been crystallized in various forms and therefore serves as a model system that provides a better understanding of structure–function relationships of polymerases that belong the type B family. This review aims to discuss strengths, limitations, and opportunities of the phage surrogate with emphasis placed on its utility in the discovery and development of anti-herpetic drugs.Viral polymerases are important targets in drug discovery and development efforts. Most antiviral compounds that are currently approved for treatment of infection with members of the  HSV1Herpes Simplex Virus 1HSV2Herpes Simplex Virus 2VZVVaricella Zoster VirusEBVEpstein–Barr VirusHCMVHuman cytomegalovirusHHV6Human Herpesvirus 6HHV7Human Herpesvirus 7KSHVKaposi’s sarcoma-associated herpesvirusPFAPhosphonoformic acidPAAPhosphonoacetic acidACVAcyclovirGCVGanciclovirCDVCidofovirherpesviridae family are important human pathogens. In all, there are eight different human herpesviruses; Herpes simplex virus 1 and 2 (HSV1: HHV1 and HSV2: HHV2), varicella zoster virus (VZV: HHV3), Epstein-Barr virus (EBV: HHV4), Human cytomegalovirus (HCMV: HHV5), Human Herpes virus 6 and 7 (HHV6 and HHV7) and Kaposi’s sarcoma-associated herpesvirus . Human herpesviruses cause a spectrum of diseases ranging from relatively benign cutaneous lesions to serious conditions like encephalitis and cancer. Viruses that belong to the herpesviridae family are characterized by their ability to establish lifelong, latent infections. Thus, a substantial proportion of the global population is seropositive for one or more herpesviridae viruses. Although individuals with a functioning immune system can generally keep the virus suppressed, the ability to form latent infections, and the fact that the virus is widespread in the human population means that herpesvirus reactivation is a major source of disease and morbidity in immunocompromised individuals. The eukaryotic viruses of the herpesviridae. The nucleoside analog acyclovir (ACV) and its pro-drug valacyclovir are utilized to treat infection with HSV1, HSV2 or VZV, while the nucleotide analog ganciclovir (GCV)  and cidofovir (CDV) are approved to manage HCMV infection. The pyrophosphate analog phosphonoformic acid  provides an option to treat HSV1, HSV2, VZV and HCMV, if first-line drugs have failed to lower the viral burden. Like all current antiviral treatments, long-term treatment can lead to the development of drug resistance. Severe side effects and complicated treatment schedules represent other problems in the management of herpesvirus infection.The majority of approved antiviral drugs have been shown to inhibit the herpesvirus-specific DNA polymerase, reducing viral DNA replication, and, in turn, viral load . Howeverherpesviridae DNA polymerases are difficult to overexpress in heterologous expression systems and have limited solubility. Hence, it has been difficult to characterize structural and functional details of these polymerases .,33.32,33Several mutations remain to be confirmed as resistance-conferring amino acid substitutions . Becauseherpesviridae polymerases. Most importantly, the phage enzyme is not inhibited by approved drugs that bind to the polymerase active site in either post- or pre-translocational states. Chimeric enzymes composed of a RB69gp43 backbone and important elements of the active site of herpesviridae DNA polymerases can potentially address this problem. These findings warrant further investigation in such enzymes as novel tools in future drug discovery and development efforts. RB69gp43 provides an excellent model for the study of structure–function relationships of B family polymerases. However, there are limitations for the study of orthologous


Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the Dt (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci  in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of  MIRNA genes that are transcribed by RNA polymerase II. In plants, the primary transcript (pri-miRNA) of a MIRNA gene is processed by the RNase III-like enzyme DICER-LIKE1 (DCL1) into a hairpin structure, miRNA precursor or pre-miRNA, which is further cleaved by DCL1 to produce a miRNA/miRNA* duplex from the stem region of the hairpin. The miRNA/miRNA* duplex is assembled into a RNA induced silencing complex (RISC). Once incorporated into the RISC, the mature miRNA binds to complementary sites in its target genes through base pairing and causes degradation and/or translational repression of the target mRNAs, which depends on the level of complementarity between miRNA and its targets microRNAs (miRNAs) are 20–24 nucleotides (nt) long small noncoding RNAs and are processed from Arabidopsis have been shown to either positively or negatively regulate elicitor flg22-induced callose deposition, a signature of the PTI defense response Arabidopsis plants elicited by flg22 showed induction of MIR393 transcription and down-regulation of miR393 targets, including three F-box auxin receptors TIR1 (Transport Inhibitor Response 1), AFB2  and AFB3, and consequently increased resistance to Pseudomonas syringaeR) genes N gene from Nicotiana benthamianaN-targeted miRNAs in N. benthamiana attenuates N-mediated resistance to TMV N. benthamiana. Further bioinformatic mining identified several miRNAs, including miR482 and miR2118, which target members of different R-gene families in tomato, potato, soybean and Medicago truncatulaNBS-LRR defense genes. Using a transient assay system, it has been shown that N. benthamiana mRNAs encoding NBS-LRR proteins could be silenced by tomato miR482 Pathogen infection triggers transcriptional reprogramming in host plants. Plants have evolved multiple layers of mechanisms to protect themselves from pathogen attacks, including non-host specific resistance, PAMP (pathogen-associated molecular pattern)-trigged immunity (PTI) and effector-triggered immunity (ETI) R-genes. This has been verified in tomato and M. truncatulaNBS-LRR gene has been shown to target mRNA encoding another defense-related protein NBS-LRR genes can possess the characteristic of trans-acting siRNAs (ta-siRNAs) miR482, miR2118 and nta-miR6019 belong to a specific type of miRNA that is 22-nt long and generated from pre-miRNAs containing asymmetric bulges in the miRNA/miRNA* duplex. This type of miRNA has been demonstrated to be the trigger for production of phased 21-nt secondary small RNAs from their target transcripts through the RDR6/DCL4 pathway Gossypium spp.) consists of 50 species, including 45 diploids and five allotetraploids. Two A-genome species (G. arboreum and G. herbaceum) and two AD-genome species (G. hirsutum and G. barbadense) were independently domesticated and cultivated for their fibers G. arboreum or G. herbaceum and a D-genome-like Central American species similar to modern G. raimondiiG. hirsutum, one each in G. arboreum and G. herbaceum, and four in G. raimondii). This paucity is largely due to the lack of cotton genome sequences and use of only a narrow range of tissues in miRNA identification. This has changed with the release of the draft genome assembly of G. raimondii, a D-genome diploid cotton species that is the closest living relative of the ancestral Dt-genome in tetraploid cotton Verticillium dahliae infection V. dahliae is a fungal pathogen causing the soil-borne vascular disease verticillium wilt (VW), one of the major cotton diseases worldwide. The small RNA populations in V. dahliae-inoculated cotton (G. hirsutum and G. barbadense) roots were sequenced and compared with those from mock-treated roots. This investigation identified a number of conserved miRNAs, including miR2118, and 14 new cotton miRNAs, which were up- or down-regulated upon V. dahliae infection Cotton is the most important textile fiber crop in the world. The cotton genus  small RNA datasets so are not further analysed.To determine whether these h, and i . No homoi genome ; howeverG. raimondii, and are just 164 bp apart from each other. The G. raimondii genomic sequence containing these two miRNAs is predicted to form two hairpins. Gra-miR482h/miR482h.2 and gra-miR482e.2 are located within each of these two hairpins  database (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=cotton), we found a tentative consensus sequence TC257237 (1446 bp in length), which was assembled from a few G. raimondii expressed sequence tags (ESTs) and is fully reverse complementary to the G. raimondii genomic sequence containing gra-miR482h/miR482h.2 and gra-miR482e.2. This finding suggests that TC257237 is likely to be the primary transcript for gra-miR482h/miR482h.2 and gra-miR482e.2 and that its orientation is probably mis-annotated in the DFCI database  and Dt (G. hirsutum) genomes  sequence contains three single nucleotide polymorphisms (SNPs) in miRCan1, which would abolish the base pairing between miRCan1 and miRCan1* and change the hairpin structure of the A genome sequence  of these 300 NBS-LRR genes were potentially targeted by various numbers of the gra-miR482 (or ghr-miR482) family members. Of these 36 candidate NBS-LRR targets, 16 were uniquely targeted by only one member of the gra-miR482 family and 20 were targeted by at least two members  of the miR482 family (The P-loop sequence of the  a codon . These c2 family .V. dahliae-infected and mock-treated roots of G. hirsutum and G. barbadense) onto the G. raimondii genome and analysed the distribution of small RNAs across all the predicted targets of ghr-miR482/miR482.2 using the method previously described NBS-LRR targets, the majority had small RNAs mapped to, and nine showed a phased distribution of those small RNAs (phase score >2) spreading downstream from the primary target site of ghr-miR482/miR482.2 (Gorai.011G075600 is shown in th nucleotide counting from the 5' end) of ghr-miR482d rather than that of ghr-miR482d.2, suggesting that production of the phased small RNAs was triggered by ghr-miR482d-mediated cleavage. 21-nt small RNAs corresponding to the first phased register of ghr-miR482 were also observed in Gorai.007G320300 and Gorai.007G320500 although no phase signal was bioinformatically detected for these two genes probably because the number of phased small RNAs were below the threshold. For Gorai.007G320500, ghr-miR482-mediated cleavage was also confirmed by cleavage analysis . Expression of ghr-miR482a/miR482e.2 was detected in leaves collected at 1 dpi from plants infected with V. dahliae by root dipping, but no V. dahliae-induced down-regulation of ghr-miR482a/miR482e.2 was observed  have a corresponding ghr-miR482.2. In addition, ghr-miR482e.2 but not its corresponding ghr-miR482e is found in the G. hirsutum small RNA datasets we analysed. By blasting the G. raimondii genome sequence, we found that ghr-miR482a has no homolog and thus could be a newly evolved ghr-miR482 member in G. hirsutum. Although ghr-miR482b and ghr-miR482c each have a single match in the G. raimondii genome, ghr-miR482d has five matches , suggesting that ghr-miR482d could be generated from multiple loci in the Dt genome of G. hirsutum. All seven members of the ghr-miR482 family , but not with CMV (cucumber mosaic virus), TMV (tobacco mosaic virus) or Sclerotinia sclerotinorium, a necrotrophic fungal pathogen. Bra-miR1885 has been shown to cleave a TIR-NBS-LRR (TNL) type disease resistance gene miRNAs have been shown to play an important role in response to biotic and abiotic stresses in plants NBS-LRR target genes were induced two to three-folds N. benthamiana mRNAs encoding NBS-LRR proteins were found to be silenced by tomato miR482, the authors have proposed that plants are able to exploit virus- and bacterium-derived suppressors of RNA silencing to induce expression of defense-related genes to achieve non-race-specific resistance against viral and bacterial pathogens NBS-LRR regulatory loop as a counter mechanism to minimize the cost of over-expression of NBS-LRR genes in the absence of a pathogen, and to ensure rapid induction of disease resistance proteins upon pathogen attack.Expression of miR482 was found to be suppressed in tomato plants infected with viruses or bacteria, and consequently some of its disease resistant NBS-LRR genes triggers production of phased siRNAs  of miR482 is one of the conserved motifs in the NBS-LRR proteins. The miR482 family is thus expected to regulate the expression level of a number of NBS-LRR genes. We found that ∼12% of NBS-LRR genes in the G. raimondii genome are potential targets of the miR482 family, and that 10 of the 11 analysed could be induced upon V. dahliae infection. On the other hand, the number of NBS-LRR genes regulated by miR482 in each species could be evolutionally determined by the balance between minimizing the disadvantageous effect of over-expression of NBS-LRR genes in the absence of a pathogen and maximizing the induction of NBS-LRR genes in the presence of a pathogen. This is supported by the observation that both the miR482 sequences and the predominant amino acid sequences of the P-loops of NBS-LRR proteins in cotton are different from those in tomato, soybean and M. truncatulaNBS-LRR targets in each species. Gain and loss of regulation of NBS-LRR genes by miR482 could be an on-going evolutionary event because for each type of P-loop the nucleotide variation was always found at the synonymous 3rd position of a codon , respectively, as probe. In view of the expression level of ghr-miR482b and its significant reduction in the d leaves , this asd leaves . Therefod leaves .NBS-LRR genes targeted by ghr-miR482/miR482.2, we demonstrated that V. dahliae infection represses certain members of the miR482 family and induces expression of specific disease resistance NBS-LRR genes in cotton. This suggests that the miR482-NBS-LRR regulatory loop is part of the immune responses induced not only by viral and bacterial pathogens but also by fungal pathogens.In conclusion, on the basis of characterization of the miR482 family and identification of G. hirsutum  and G. arboreum  plants were raised from seeds and grown in glasshouse at 28±2°C with 16 hrs day and 8 hrs night regime. One-true-leaf whole seedlings (including roots) of TM-1 and YZ were used in DNA isolation. The same stage seedlings of Sicot71 were used in V. dahliae inoculation by root dipping. This was done by submerging the roots of cotton plants into a suspension of V. dahliae conidia for 5 minutes. The inoculated plants were then planted in soil in 8-cm pots. The V. dahliae inoculum was prepared by growing V. dahliae in potato dextrose broth  on a shaker (25°C) for 9 days. The conidial suspension was diluted to ∼108 spores/ml with full strength PDB before inoculation. Leaf and root samples were separately collected from V. dahliae infected and mock-treated (dipping in water) plants at one day-post-inoculation (dpi).http://www.ncbi.nlm.nih.gov/geo/and used in identification of miR482 and miR482.2. Of these datasets, GSM699074–699077 were aligned to the G. raimondii genome sequence NBS-LRR genes generating phased secondary small RNAs, which was performed according to the approach described previously with a phase score >2 Previously published small RNA datasets, including GSM699074–699077 G. raimondii short sequences with 0–1 mismatch in comparison to their closest member of the miR482 family of G. hirsutum were first identified using each ghr-miR482 or ghr-miR482.2 as a query. Two-hundred base pairs of G. raimondii sequence flanking each identified short sequence were then retrieved and subjected to hairpin structure prediction using RNA-fold. A MIRNA-like hairpin contains a ghr-miR482, ghr-miR482.2 or their isoform was considered as MIR482 or MIR482.2 of G. raimondii.G. raimondii transcripts http://plantgrn.noble.org/psRNATarget/) Putative targets of gra-miR482/miR482.2 were first predicted based on the annotated NBS-LRR genes was carried out using total RNA by 5′ RACE (rapid amplification of cDNA ends) as previously described Confirmation of ghr-miR482/miR482.2-mediated cleavage of NBS-LRR genes in V. dahliae infected and mock-treated cotton leaves and roots were analysed as previously described −ΔCt. Significance was analysed by t-test. Cotton Histone 3 (Accession no. AF024716) was used as the reference for normalization Approximately 25 µg of total RNA isolated using the hot borate approach Figure S1Precursors of the members of the gra-miR482 family.(TIF)Click here for additional data file.Figure S2Precursors of the isoforms of gra-miR482d and gra-miR482f-i .(TIF)Click here for additional data file.Figure S3Alignment of the precursor sequence of gra-miR482h/miR482h.2 and gra-miR482e.2 with the tentative consensus sequence TC257237 (1446 bp).(TIF)Click here for additional data file.Figure S4Hairpin structure of the region containing miRCan1 and miR482e.2 in different genomes.(TIF)Click here for additional data file.Figure S5NBS-LRR genes generating phased secondary small RNAs.(TIF)Click here for additional data file.Figure S6Comparison of cotton seedlings  inoculated with Verticillium dahliae (right pot) and mock treated (left pot).(TIF)Click here for additional data file.Table S1Predicted targets of miR482/miR482.2 and miRCan1 in G. raimondii.(XLSX)Click here for additional data file.Table S2Oligos used in this study.(DOC)Click here for additional data file.


The published affiliation of Dr Laurent Rénia is incorrect. The correct affiliation of Laurent Rénia is:3 Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore.


Successful replication within an infected host and successful transmission between hosts are key to the continued spread of most pathogens. Competing selection pressures exerted at these different scales can lead to evolutionary trade-offs between the determinants of fitness within and between hosts. Here, we examine such a trade-off in the context of influenza A viruses and the differential pressures exerted by temperature-dependent virus persistence. For a panel of avian influenza A virus strains, we find evidence for a trade-off between the persistence at high versus low temperatures. Combining a within-host model of influenza infection dynamics with a between-host transmission model, we study how such a trade-off affects virus fitness on the host population level. We show that conclusions regarding overall fitness are affected by the type of link assumed between the within- and between-host levels and the main route of transmission . The relative importance of virulence and immune response mediated virus clearance are also found to influence the fitness impacts of virus persistence at low versus high temperatures. Based on our results, we predict that if transmission occurs mainly directly and scales linearly with virus load, and virulence or immune responses are negligible, the evolutionary pressure for influenza viruses to evolve toward good persistence at high within-host temperatures dominates. For all other scenarios, influenza viruses with good environmental persistence at low temperatures seem to be favored. It has recently been suggested that for avian influenza viruses, prolonged persistence in the environment plays an important role in the transmission between birds. In such situations, influenza virus strains may face a trade-off: they need to persist well in the environment at low temperatures, but they also need to do well inside an infected bird at higher temperatures. Here, we analyze how potential trade-offs on these two scales interact to determine overall fitness of the virus. We find that the link between infection dynamics within a host and virus shedding and transmission is crucial in determining the relative advantage of good low-temperature versus high-temperature persistence. We also find that the role of virus-induced mortality, the immune response and the route of transmission affect the balance between optimal low-temperature and high-temperature persistence. Influenza A viruses infect both humans and animals, causing frequent outbreaks One useful approach for studying the whole infection and transmission process is through the use of multiscale studies, wich have seen increased general development and use in recent years .All statistical analyses and simulations were done in the R programming environment Both the within- and between-host models contain a term for virus decay, namely ts  and   and 18. In a recent study The analysis of this dataset can be taken as suggestion for the presence of a trade-off between stability at low and high temperatures – at least for the panel of strains we investigated here. Since this is a small sample of strains, we do not want to over-emphasize the finding. However it seemed real and interesting enough to ask the questio: “How would such a potential trade-off lead to interactions on the within-host and between-host levels and affect overall virus fitness?”. We address this question in the remainder of the paper.As a potentially interesting side question – not further considered in the remainder of this paper – we wondered whether there are systematic differences between strains belonging to different groups. Based on amino acid differences, strains with different HA types can be clustered into two groups, as indicated in Anas platyrhynchos) To simulate a within-host infection, we need to specify parameter values for the within-host model. While parameter estimates are available for influenza infections in humans and mice st model –3 to accFor each strain listed in smission , the abismission , fitnessThe two cases, environmental only and direct only transmission represent extremes in terms of potential trade-offs. For direct transmission alone, there is no trade-off; optimizing within-host fitness is always the best strategy. The environmental transmission only scenario represents the case where the importance of the environmental stage is as large as it can possibly be. Mixture of the two transmission routes leads to values with intermediate importance of environmental persistence. While it is certainly possible to consider the general case with both direct and environmental transmission and compute absolute and relative fitness values, this would require making rather arbitrary assumptions about values for some of the unknown parameters of proportionality. Since considering such a general mixed transmission scenario would not add much beyond the results for the two simpler extreme cases, we focus on these two extreme cases in the following.In smission , better Not surprisingly, for the environmental transmission scenario , the treSo far, we analyzed decay data for specific influenza strains and documented differences in their ability to persist well at low and high temperatures. We can go one step further and study the hypothetical fitness of strains that we did not measure. To do so, we can vary To determine between-host fitness for a generic strain with given west see . For envFor the scenario where shedding and infection rate are proportional to the logarithm of virus load Click here for additional data file.Figure S2Relative fitness for different strengths of the immune response. Left column shows direct transmission scenarios, right column shows environmental transmission scenarios. The rows show from top to bottom the different forms of linking within-host virus load to between-host transmission, i.e. (TIFF)Click here for additional data file.Figure S3Fitness as measured by  and   for direct transmission and environmental transmission, with immune response at . The dashed vertical lines indicate the levels of (TIFF)Click here for additional data file.Figure S4Relative fitness for the A) direct and B) environmental transmission scenario for different shedding definitions in the presence of virulence. Fitness for H6N4 in the environmental transmission scenario with link-function (TIFF)Click here for additional data file.Text S1Additional Results for a within-model including an immune response and a scenario including virulence.(PDF)Click here for additional data file.


In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors. These may involve its tissue or organ tropism, its ability to deal with the host's immune system and/or its efficacy of replication. In the literature the definition of a virulence factor or virulence determinant is not always clear-cut. Thus, many studies concluded that if a genetic mutation in a gene or a complete knock out of the gene function results in an attenuated phenotype, this particular gene or amino acid sequence is a virulence determinant. However, some proteins or protein domains are involved in basic replication processes, making them essential for virus reproduction. Hence, the terms "virulence factor" and "virulence determinant" should be used with care. Our definition of a virulence determinant is: a naturally occurring genetic difference between strains of the same species that is responsible for their difference in virulence. However, nowadays reverse genetics allows the genetic modification of viral genomes almost at will and as a consequence the effects of genetic modifications that have not been encountered in nature can also be studied. Indeed, these techniques have resulted in much more detailed information on the involvement of viral genes and proteins in the virus life cycle and consequently on their contribution to virulence. Therefore, it is arguable whether a particular "artificial" genetic difference still meets the above mentioned definition of a virulence determinant. Similar effects may occur in nature but may be countered by other factors or may be too subtle to be determined by the particular test used to measure differences in virulence.Newcastle disease (ND) is one of the most important infectious diseases of poultry. It is distributed worldwide and has the potential to cause large economic losses in the poultry industry ,2. Its cNDV occurs in the field as a variety of strains which differ extensively in the organ systems that they affect and in the severity of the symptoms that they produce in infected birds. Based on the severity of the disease in chickens, NDV has been classified into three pathotypes: lentogenic, mesogenic and velogenic. Lentogenic NDV strains cause subclinical infection with mild respiratory or enteric disease and are considered low-virulent. Mesogenic NDV strains are of intermediate virulence causing respiratory infection with moderate mortality < 10%), while velogenic NDV strains are highly virulent causing mortality rates up to 100% . Velogen0%, whileLow virulent NDV strains need the addition of exogenous trypsin to spread from cell to cell and to form syncytia in cell culture monolayers, whereas virulent strains do not ,9. ThereUseful in vivo tests for the assessment of virulence are the mean death time (MDT) in embryonated chicken eggs , the int3 plaque forming units (PFU) were used per chicken, which is probably much less than the amount of virus in the inoculum prescribed by the World Organisation for Animal Health (OIE) and EU guidelines that consists of a 1 to 10 dilution of allantoic fluid with an haemagglutination (HA) titre of at least 24 41]). Cur112G-R/K-Q-G-R↓L117, and are cleaved extracellularly by trypsin-like proteases found in the respiratory and intestinal tract. Mesogenic and velogenic strains have a multi-basic amino acid motif at the F cleavage site, 112R/G/K-R-Q/K-K/R-R↓F117 and can be cleaved intracellularly by ubiquitous furin-like proteases [Entry of many enveloped viruses, including NDV, into host cells often requires the activation of viral fusion glycoproteins through cleavage by intracellular or extracellular proteases. It has been shown that viral glycoprotein activation is often mediated by proteases recognizing either monobasic or multi-basic cleavage sites . In the roteases ,60,61. Troteases . It coulroteases ,61.Consistently, studies with recombinant NDVs generated by means of reverse genetics showed that the virulence increased significantly when the cleavage site of a lentogenic strain was converted into that of a velogenic strain ,62,63. ISeveral studies have shown that the consequences of an infection are not solely determined by the presence of a multi-basic cleavage site motif in the viral fusion protein. A recent study with an avirulent avian paramyxovirus serotype 2 (APMV-2) virus showed that this virus does not require exogenous protease supplementation for growth in cell culture. In addition, recombinant APMV-2 viruses in which the cleavage site was replaced by that of APMV serotypes 1 to 9 gained in cleavability, replication and syncytium formation in infected cells, but remained avirulent for chickens . Also, aThe HN protein is responsible for the attachment of virus particles to sialic acid-containing receptors on cell surfaces and for triggering the fusion activity of the F protein during entry of the virus into the host cell. In addition it acts as a neuraminidase, removing sialic acid from progeny virus particles to prevent viral self-aggregation . CompariL with its parent rBC and confirmed the suggestion that the HN protein determines tissue tropism [H, NDFLtag(HN)LH and NDFLtag(HN)HL , which lacks an intact V ORF [The interferon (IFN) system is the first line of host defence against virus infection. Interferons induce an antiviral state that can inhibit virus replication and control virus spread. Paramyxoviruses have evolved mechanisms to escape or prevent both IFN production and IFN responsive signal transduction to evade or antagonize the innate immune response of its host -84. Manyct V ORF . The NDVct V ORF ,86. The ct V ORF ,87,88, wct V ORF -84.A recent study showed that the V protein of the mesogenic Beaudette C strain exhibits a greater antagonistic effect on IFN induction in vitro than that of the lentogenic LaSota strain. This might correlate with their different virulence properties in vivo . PreviouIt has been shown that viruses with mutated V proteins, in contrast to their parent viruses, are unable to degrade the STAT1 protein , which iNo studies have been published in which the V genes have been exchanged between different NDV strains, as has for instance been done for the F, HN, or L genes. In an unpublished study, the V proteins of the low virulent LaSota virus and the highly virulent Herts virus were compared by expressing them in a V-negative Herts backbone with an intact P ORF. The V genes were inserted between the P and M ORFs. No differences in ICPI value between the two recombinants were observed . Altogether, the studies mentioned clearly indicate that the V protein is essential for survival and replication of the virus in its host. However, more studies, especially using the natural infection route, must be performed to examine whether the V protein is also involved in determining differences in virulence between NDV strains.A correlation between virulence and the efficiency of viral replication has been observed for many viruses. Although not fully understood, it is quite conceivable that higher levels of viral replication result in more virus production which may overwhelm the host immune response, hence causing enhanced pathogenesis. It has been reported for NDV that reduced levels of RNA synthesis are associated with reduced virulence . For sevThe involvement of the NP, P and L replication proteins in NDV virulence has been examined by generating chimeric viruses in which genes were exchanged between strains of different pathotypes ,112. In We studied the adaptation of a PPMV-1 strain to chickens during brain passage. After five passages 3 mutations became dominant in the virus population, two in the L protein (N1564S and V1694E) and one in the P protein (N37D) . These mThe viral matrix (M) protein has been identified as another regulator involved in viral replication. While the M protein is considered to be the central organizer of viral morphogenesis and budding , it has Untranslated regions (UTRs) in viruses have been shown to play a role in the regulation of viral transcription and translation. In measles virus and canine distemper virus, the long 3'UTR of M and 5'UTR of F play important roles in replication and virulence of the virus ,126. A 6Deletion of the entire 5'UTR of the HN gene was shown to affect transcription and translation of the HN mRNA and thereby virulence . FurtherThe sizes of the intergenic sequences (IGSs), i.e. the number of nucleotides between the transcription end-box and transcription start-box, of consecutive genes of NDV vary from a single nt for the first three gene boundaries to 31 nt for the F-HN and 47 nt for the HN-L boundaries, respectively. A study has shown that although NDV can tolerate an IGS length of at least 365 nt, the extended lengths of IGSs down-regulated the transcription of the downstream gene. Furthermore, all viruses that had extended or decreased IGS lengths were attenuated in embryonated chicken eggs, day-old chicks and 6-week-old chickens . While iMore and more studies illustrate that virulence is a complex trait that is determined by multiple genetic factors. For NDV, the multi-basic amino acid cleavage motif in the F protein is an absolute prerequisite for virulence. In addition, other factors are critically involved in determining virulence, as shown in this review and summarised in Figure Many questions still remain concerning the molecular mechanisms used by NDV to regulate tropism, transcription and translation, interference with the host defence machinery, and how they affect virulence. Nevertheless, the studies summarised in this review have contributed significantly to our understanding of the biology of NDV and can serve as an important guide for future research on the molecular principles that determine its virulence.The authors would like to thank MGJ Tacken, OS de Leeuw and LA Cornelissen for sharing their unpublished data.The authors declare that they have no competing interests.JD developed the structural design of the review and drafted the manuscript. GK and PR reviewed the manuscript. BP supervised the study, and reviewed the manuscript. All authors read and approved the final manuscript.