A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch’s postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions. A number of factors have influenced the development of interest in these viruses. Many early studies dealt with reptiles as hosts for arboviruses that also infect humans as well as other mammals and birds, such as flaviruses and togaviruses. Interest in these viruses in reptiles has increased since the emergence of West Nile Virus in the western hemisphere at the end of the 20th century. A number of studies have shown that various arboviruses can infect a number of different reptile species and that temperature affects the development of viremia in these animals. Several studies have demonstrated that some arboviruses can persist in reptiles over winter, a factor that may play a role in the epidemiology of these viruses. Other studies have focused on the role of viruses as pathogens in reptiles. In many cases, however, Koch’s postulates have not been fulfilled for viral diseases of reptiles, so that the connection between virus and disease is postulated based on clinical, pathological and histological observations. Viruses that have been shown to be important pathogens in reptiles include ranaviruses and herpesviruses in chelonians, and paramyxoviruses in snakes. Other viruses, such as reoviruses, have not or have inconsistently induced disease in transmission studies, so that their role as primary pathogens has been questioned. However, a number of these viruses do appear to play a role in the health of reptiles, and may be part of factorial disease processes influenced by the viruses themselves, other infectious agents, and environmental factors. A third factor influencing interest in reptilian viruses is their taxonomic position and evolution. Analysis of the genomes of some reptilian viruses has placed a number of these in separate genera from viruses of other vertebrates, leading to proposals for new genera and, in some cases, a better understanding of coevolution of viruses and their hosts.The detection and study of viruses of reptiles relies on a wide range of tools, including classical virological methods such as cell culture, as well as molecular methods, including PCR and sequencing as well as metagenomics. The combination of these methods has been successfully used to increase our understanding of viruses in this group of animals, although much remains to be learned, both about the viruses themselves and about their effect on the animals they infect.The viruses that have been most commonly detected in reptiles include herpesviruses, especially in chelonians, adenoviruses, especially in lizards and snakes, reoviruses, especially in lizards and snakes, paramyxoviruses, especially in snakes, picornaviruses in tortoises, and iridoviruses, with ranaviruses detected predominantly in chelonians and invertebrate iridoviruses detected in lizards. Of these, the herpesviruses and adenoviruses have long been detected histologically based on the inclusion bodies they cause in tissues of infected animals, herpesviruses of tortoises, picornaviruses of tortoises, reoviruses, and iridoviruses can all be isolated in cell culture, and sensitive PCRs are available for the detection of herpesviruses, adenoviruses, paramyxoviruses, and iridoviruses.This review aims to provide an overview over the viruses detected in reptiles to date as well as some information on the diseases associated with these viruses. The taxonomic positions of the reptilian viruses relative to viruses of other vertebrates are provided if information on this is available. The study of these viruses has grown beyond the point at which a full overview over every aspect of this topic could be provided in such a review, so that specific information on some aspects of reptile virology, particularly specifics on diagnostic testing and samples as well as serological aspects of infection, are only briefly mentioned for each virus family. The viruses in the review are presented according to taxonomic position, with DNA viruses  presented first, followed by RNA viruses .2.Chordopoxvirinae. These genera include viruses of mammals and birds [Chordopoxvirinae with Nile crocodilepox virus as the type species of the genus [Poxviruses are large double-stranded DNA viruses. Their morphology is somewhat pleomorphic, ranging from brick-shaped to ovoid with a lipoprotein surface membrane. Vertebrate poxviruses are currently grouped into eight different genera in the subfamily nd birds . Recentlhe genus .Caiman crocodilus) in the USA [Poxviruses have been repeatedly detected in crocodilians with skin lesions around the world. The first report of poxvirus-associated disease in a reptile was in captive caimans , poxvirus infections have been associated with brownish wart-like skin lesions that can occur over the entire body. Infection is associated with high morbidity but low mortality [et al. [Chordopoxvirinae. An atypical form of crocodile pox has also been observed in Nile crocodiles. This virus was associated with deeply penetrating skin lesions in farmed crocodiles in Africa. A PCR targeting a unique region of CRV, ORF 019, was developed and used to amplify a portion of the genome of the virus associated with these lesions. Analysis of the sequence of the PCR product (533 bp) showed that this virus was related to, but not identical with CRV [In Nile crocodiles  were found to contain pox-like viruses [Chamaeleo dilepis) in Tanzania was found to have two different types of intracytoplasmic inclusions in circulating monocytes. The inclusions were found to be caused by a Chlamydia-like organism and a pox-like virus [Poxvirus infections have also been detected in individual cases in other reptiles by electron microscopy. Papular skin lesions around the eyes of a Hermann’s tortoise  in lizards; and erythrocytic necrosis viruses in lizards, snakes, and turtles.The iridoviruses belong to the Nucleo-Cytoplasmic Large DNA viruses (NCLDV), a group of DNA viruses that infect very diverse hosts. The NCLDV also include mimiviruses, phycodnaviruses, African swine fever virus, and poxviruses. Although the genome size of these viruses varies greatly (between 100 kb and 1.2 Mb), they appear to form a monophyletic group based on a subset of about 30 conserved genes . Among tytivirus . Until r3.2.Testudo horsfieldii), eastern box turtles (Terrapene c. carolina) [Trionyx sinensis) [Testudo hermanni) [Trachemys scripta elegans), Burmese star tortoises (Geochelone platynota), gopher tortoises (Gopherus polyphemus), and Florida box turtles (Terrapene carolina bauri) [Terrapene carolina carolina) [Testudo kleinmanni) [Geochelone pardalis) [Testudo marginata), and spur-thighed tortoises (Testudo graeca) [Terrapene ornata ornata) and red-eared sliders, i.m. injection of a ranavirus isolated from a Burmese star tortoise led to disease, including lethargy, anorexia, ocular discharge, conjunctivitis, and oral plaques, and death of the animals in some cases [Chondropython viridis) in Australia [Uroplatus fimbriatus) in Germany [Lacerta monticola) in Portugal [Ranaviruses have been increasingly shown to be important pathogens of ectothermic animals. They have been regularly isolated from reptiles since the late 1990’s. They have been mostly described in chelonian species worldwide, including Russian tortoises (arolina) ,16, Chininensis) , Hermannermanni) , red-earinmanni) , a leopaardalis) , margina graeca) . In thesustralia . The sna Germany  and a moPortugal . In the Ranavirus. The complete sequence of the soft-shelled turtle virus has been determined [The origin of infection in reptiles has not been documented. Characterization of the detected viruses has generally relied on sequencing of a portion of the highly conserved MCP gene. These analyses have shown the detected viruses to be closely related to frog virus 3 (FV3), the type species of the genus termined . The gentermined .Elseya latisternum and Emydura krefftii) [Spread of ranavirus infection within a mixed-species group of tortoises  as well refftii) .3.3.Iridovirus had only been described in invertebrates. These viruses are of particular interest because of their potential use for controlling important agricultural pests and vector insect species, in which they induce lethal infections [Gryllus campestris and Acheta domesticus [Gryllus bimaculatus [Gryllus campestris and Acheta domesticus from a Dutch breeder [Gryllus bimaculatus [Until recently, viruses of the genus fections . At the mesticus  and Grylaculatus . In both breeder . The infaculatus .Iridovirus, on the basis of genome sequences and host range. No differences were detected between these two viruses in transmission studies with various insect species. Different gene loci including the MCP gene were analyzed, compared and led to the conclusion that CrIV and IIV-6 are not different species within the Iridovirus genus and that CrIV must be considered to be a variant and/or a novel strain of IIV-6 [Gryllus bimaculatus iridovirus (GbIV) was sequenced. The obtained sequence was 97% identical to the corresponding sequence of IIV-6 [CrIV was compared with IIV-6, the type species of the genus of IIV-6 . In the of IIV-6 .Iridovirus have only recently been described in reptilian hosts. In 2001 a German group reported the isolation of IIV-like viruses from the lung, liver, kidney, and intestine of two bearded dragons (Pogona vitticeps) and a chameleon (Chamaeleo quadricornis) and from the skin of a frilled lizard (Chlamydosaurus kingii) on viper heart cells (VH2) at 28 °C. The frilled lizard showed pox-like skin lesions and one of the bearded dragons had pneumonia. The other lizards had died with non-specific symptoms. A 500 bp portion of the MCP gene of the isolates was sequenced and had 97% identity to the nucleotide sequence of IIV-6 and 100% identity to the nucleotide sequence of GbIV. A host-switch of this virus from prey insects to the predator lizards was postulated [IIVs belonging to the genus stulated .Chamaeleo hoehnelii). A 500 bp portion of the MCP gene was identical to the corresponding sequence of GbIV. The pathogenicity of this isolate for crickets of the species Gryllus bimaculatus was tested, resulting in mortality rates between 20 and 40%. Virus was reisolated from several fat body samples. In some fat bodies of infected crickets massive arrays of viruses could be detected by electron microscopy. These findings support the hypothesis that IIV from insects are able to infect reptiles [An IIV was isolated from several tissues of a high-casqued chameleon (reptiles . IIV-likreptiles .3.4.Toddia and Pirhemocyton sp.) [Lacerta monticola and Lacerta schreiberi showed that infection with these agents can, in some cases, become systemic and may lead to death [Iridoviridae. Approximately 60% of the erythrocytes examined in this snake were severely hypochromic and exhibited anisocytosis and polychromasia. Sequence analysis of a 628 bp portion of the DNA dependent DNA polymerase gene showed that this virus was distinct from other known iridoviral genera and species and that it may represent a novel genus and species in the family Iridoviridae. The virus was given the name Thamnophis sauritus erythrocytic virus (TsEV) [Lacerta monticola from Serra da Estrela, Portugal was also partially characterized based on a 596 bp length portion of the same gene. Analysis of the obtained sequence showed that it clustered together with the TsEV, with 65.2/69.4% nt/aa% homology with that sequence, although ultrastructural differences between the viruses were detected by electron microscopy [Iridoviridae. Their final classification and understanding of their relationship to erythrocytic necrosis viruses of amphibians and fish require further study.Viral erythrocytic infections associated with irido-like viruses have been described in fish and amphibians as well as lizards, snakes, and turtles . These vton sp.) ,36. Pathto death . Recentls (TsEV) . A lizarcroscopy . This st4.4.1.Herpesviridae is a member of the order Herpesvirales, which also contains the family Alloherpesviridae, which incorporates fish and frog viruses and the family Malacoherpesviridae, which currently contains a bivalve virus. The family Herpesviridae contains mammal, bird, and reptile viruses. It is divided into three subfamilies, the Alpha-, Beta-, and Gammaherpesvirinae. All of the reptilian HVs described so far belong to the family Herpesviridae, but none have yet been assigned to any of the existing genera [Herpesviruses (HVs) are large, enveloped viruses with a double-stranded DNA genome and an icosahedral capsid. The family g genera . It is cg genera .Herpesviridae. A detailed analysis of available sequence information from two HVs from sea turtles, the green turtle HV (GTHV) and lung eye and trachea disease-associated HV (LETV) showed that these both originated from the lineage leading to the recognized members of the Alphaherpesvirinae and concluded that these viruses belong to a private clade in that subfamily. Analysis of the limited sequence data available from lizard HVs ) did not yield a clear classification of these viruses, concluding that additional sequence data is necessary to clarify the relationships between these viruses and other HVs and to understand the evolution of these viruses [Alphaherpesvirinae of the family Herpesviridae [Herpesviridae, and that these viruses be classified together in a new genus with the suggested name “Chelonivirus” [In reptiles, HVs have been detected in lizards –48, snak viruses . Of the sviridae ,62,66–69nivirus” .4.2.A number of different HVs have been detected in lizards, while relatively few have been described in snakes. In snakes, HVs have been detected in venom glands of various species, in some cases associated with decreased venom production , and in Lacerta viridis) [Alphaherpesvirinae, but does not cluster with the chelonid HVs [In lizards, a number of different HVs have been detected. HV-like particles, as well as particles resembling papova- and reoviruses were detected by electron microscopy in papillomas of green lizards (viridis) . Herpesvviridis) . Based oonid HVs .Varanus prasinus) with proliferative stomatitis [Gerrhosaurus major) and a black-lined plated lizard (Gerrhosaurus nigrolineatus) with stomatitis. Analysis of a portion of the DNA polymerase gene of these viruses indicated that they may belong to the subfamily Alphaherpesvirinae, but that they appear to be only distantly related to other known HVs [A number of cases have been documented in which lizard HVs were associated with oral lesions in infected animals. The varanid HV 1 was detected in the oral mucosa and brain of green tree monitor lizards (omatitis . Three dnown HVs .Agama agama) in the USA [Sauromalus varius). Partial sequence analysis of the DNA polymerase gene of this virus showed that it also appeared to belong to the Alphaherpesvirinae, but that an exact taxonomic classification was not possible based on the limited data available [A few reports are available of HVs in lizards associated with lesions in the liver. A HV was detected by electron microscopy in hepatocytes of red-headed agamas ( the USA . Iguanidvailable .Iguana iguana). Transmission of the isolate to other lizards did not lead to the development of clinical signs [Iguanid HV 1 is the only lizard HV that has been isolated in cell culture. This virus was isolated from cell cultures derived from an infected green iguana  in Australia with a crust on the abdominal skin [Alligator mississippiensis) with lesions in the cloaca [HVs have been reported in crocodylians in two cases. The first was the detection of HV-like particles in the skin of saltwater crocodiles , painted turtles (Chrysemys picta), and map turtles (Graptemys spp.). Clinical signs reported in affected animals include lethargy, anorexia, and subcutaneous edema. Characteristic necropsy findings include hepatomegaly and pulmonary edema. Areas of hepatic necrosis with the presence of intranuclear inclusion bodies in hepatocytes were reported. Inclusions have also been demonstrated in the spleen, lungs, kidneys, and pancreas [Of the reptilian HVs, the chelonian HVs are most common and have been best characterized so far. A number of reports are available on HV infection in water turtles. These have been described in Pacific pond turtles (pancreas –60. NoneChelonia mydas). Aquaculture reared, 2- to 3-month-old turtles appear to be most commonly affected. The virus was described by electron microscopy, and no further data are available on the virus involved [Gray patch disease was one of the first HVs to be described in chelonians. It infects green sea turtles  has also been described in green sea turtles. Clinical signs associated with infection are gasping, harsh respiratory sounds, buoyancy abnormalities, inability to dive properly, and the presence of caseated material on the eyes, around the glottis and within the trachea. Some of the infected turtles died after several weeks, while others became chronically ill. A HV (LETV) was isolated from diseased turtles in green sea turtle kidney cells and has since been further characterized ,66,71.Caretta caretta), Hawksbill (Eretmochelys imbricata), and olive ridley (Lepidochelys olivacea) sea turtles around the world. Infected turtles develop fibropapillomas and individual or multiple tumors can occur externally all over the body. Internal tumors are also possible. The viral etiology has been tested by tumor transmission using cell-free tumor extracts [In sea turtles, fibropapillomatosis has been associated with HV infection and has been described in many different species of marine turtles including green, loggerhead (extracts . The fibextracts . In anotextracts .Caretta caretta); loggerhead genital-respiratory HV (LGRV), and loggerhead orocutaneous HV (LOCV). LGRV was associated with ulcers in the trachea, around the cloaca and on the base of the phallus, while LOCV was associated with ulcers in the oral cavity and cutaneous plaques which were covered with exudate and had an erythematous border as well as with pneumonia [Two HV-associated disease syndromes have been described in wild-caught loggerhead sea turtles (neumonia .HV infections have been reported in many different species of tortoises (Testudinidae). Clinical signs commonly associated with infections include rhinitis, conjunctivitis, stomatitis and glossitis, which frequently develop into a diphtheroid-necrotizing stomatitis and glossitis, with diphtheroid membranes covering parts of the oral cavity and extending down into the trachea and esophagus. Edema of the neck is a common sign. Affected animals are generally anorexic and lethargic. Animals that survive acute HV infection may develop central nervous system disorders including paralysis or incoordination . In a trHistologically, HV infections in tortoises may be associated with eosinophilic or amphophilic intranuclear inclusions in infected tissues, most frequently in epithelial cells of the tongue, oral mucosa, and upper respiratory tract as well as in the gastrointestinal tract. Occasionally, inclusions can also be found in epithelial cells of the urinary tract, in the brain, liver, and spleen. .Herpesviridae. However, many of the viruses listed there were only described by electron microscopy, and no further characterization of the viruses is possible. Their relationship to one another and to newly described reptilian HVs can no longer be elucidated. A revised method of naming newly described and characterized chelonian HVs, particularly HVs of tortoises, has therefore been used in recent publications. The first tortoise HV from which sequence data from a small portion of the DNA polymerase gene became available, allowing a preliminary taxonomic analysis of the isolate, was from Russian and pancake tortoises  in Japan [Gopherus agassizii) in the USA [Chersina angulata) in a zoo in the USA [et al. [T. graeca), marginated tortoises (T. marginata), Hermann’s tortoises (T. hermanni), and Russian tortoises (T. horsfieldii). It is interesting that these species show different susceptibilities to HV-associated disease. TeHV3 infections in Hermann’s and Russian tortoises are generally associated with high morbiditiy and mortality, while spur-thighed tortoises appear to be relatively resistant to disease. This may be a reflection of the evolutionary history of the virus, and it has been speculated that this may be a virus of spur-thighed tortoises, and that infections of other tortoises represent host switches and are therefore associated with higher mortality rates. Antibodies against TeHV3 have also been detected in wild-caught spur-thighed tortoises in Turkey [The ICTV lists a number of chelonid HVs as unassigned viruses in the family in Japan . Studiesin Japan . Another the USA . A fourt the USA . Naming  [et al.  therefor [et al. ,75. TeHVn Turkey . However5.5.1.Adenoviridae contains non-enveloped viruses with a genome consisting of linear, double-stranded DNA 26–45 kbp in size [Adenoviridae suggests a coevolutionary lineage of the viruses with their hosts, and additional host switches [Mastadenovirus, for birds the genus Aviadenovirus, for reptiles the genus Atadenovirus, for amphibians the genus Siadenovirus and for fish the proposed genus “Ichtadenovirus”. However, recent findings on AdVs of chelonians and frogs shows that more work is necessary to fully understand the evolutionary relationships between these viruses and to fully appreciate their ability to switch hosts [Atadenovirus [Atadenovirus also contains viruses from ducks, geese, chickens, possums, and ruminants [Adenoviruses (AdVs) are the viruses most commonly identified in many species of lizards, particularly bearded dragons. The family  in size . They ha in size . AdVs oc in size . Currentswitches . Accordich hosts –82. All enovirus ,84. The uminants . The comuminants .5.2.Although they are most commonly detected in various lizard species, particularly agamids, AdV infections have also been detected in many other reptile species including various species of snakes, chelonians, and crocodiles. AdVs appear to occur worldwide in captive populations and antibodies to AdVs have been detected in wild Boa constrictors from Costa Rica  and in wSiadenovirus genus [Manouria impressa) and a Burmese star tortoise (Geochelone platynota) [Terrapene ornata ornata) with degeneration of liver cells, pronounced vacuolization of the cytoplasm, pyknosis of nuclei, and inclusion bodies in some hepatocytes. The virus detected in this animal differed distinctly from all previously described AdVs from reptiles [AdVs have only relatively recently been detected in several species of chelonians. In Sulawesi tortoises, infection was associated with severe systemic disease and a very high mortality rate 82%). Pathological findings in infected tortoises were multifocal hepatic necrosis, amphophilic to basophilic intranuclear inclusions and diffuse hepatic lipidosis, myeloid necrosis in bone marrow and severe necrotizing enterocolitis. The virus detected in these tortoises differed distinctly from the AdVs characterized from squamates so far and was determined to belong in the us genus . The samatynota) . A singlatynota) . In Hung%. Patholet al. [et al. [Boa constrictor) while Ahne and his co-workers isolated an AdV strain from a royal python (Python regius) [Pantherophis guttatus) showing clinical signs of pneumonia [A number of AdVs from reptiles have been isolated in cell culture, which has facilitated further characterization of individual viruses. However, many of the reptilian AdVs have not yet been successfully grown in cell culture. Snake AdVs have been isolated in several cases. Jacobson et al.  and Mars [et al.  each obt regius)  and fromneumonia . This coneumonia ,102 and neumonia  are idenneumonia . Althougneumonia .Heloderma suspectum) in both the USA and Europe, and the related but distinct Helodermatid AdV-2 found in beaded lizards (Heloderma horridum) [Pogona minor minor) in Australia [It has been hypothesized that atadenoviruses have co-evolved with reptiles and that atadenovirus infections in non-reptilian hosts represent host switches. This theory has also been supported by the finding that atadenoviruses of non-reptilian hosts have a biased genome with a relatively high AT content, which led to the naming of this genus. Another observation in many squamate AdVs is their relative species specificity: specific lizard AdVs are mostly found in a single host. However, there are a number of exceptions to this apparent rule: Eublepharid AdV-1 has been detected in two gecko genera in the subfamily Eublepharinae . SnAdV-1orridum) ,104. Howustralia .The AdV most commonly described in bearded dragons, Agamid AdV-1, has been shown to consist of a number of slightly different viruses. In one study, which compared bearded dragon isolates based on partial DNA polymerase sequences slight differences were detected between different cases from different countries (USA / Germany / Austria / Hungary) . A genot6.Papillomaviridae so far, all with mammalian or avian hosts [Lacerta viridis). The virus was identified by electron microscopy based on morphological characteristics. Herpes-like and reo-like viruses were also identified in the lesions [Platemys platycephala) [Testudo horsfieldii) [Caretta caretta) and a green turtle (Chelonia mydas) [Caretta caretta papillomavirus 1 (CcPV-1) and Chelonia mydas papillomavirus 1 (CmPV-1) [Papillomaviruses are non-enveloped viruses 55 nm in diameter. The genome of the viruses is a single molecule of circular dsDNA. The papillomaviruses are highly host specific and tissue-restricted. They generally cause benign tumors  in their natural host. Occasionally, they can also cause these in lesions in related species. Sixteen different genera have been defined in the family an hosts . The fir lesions . Since tcephala) , a Russifieldii) , a loggea mydas) . The Bola mydas) . The Rusa mydas) . Lesions(CmPV-1) . The com(CmPV-1) .7.Parvovirinae and Densovirinae. Viruses in the subfamily Densovirinae infect arthropods, while all vertebrate parvoviruses classified so far belong in the subfamily Parvovirinae. This subfamily has been suggested to be divided into at least seven different genera based on biological, genomic, phylogenetic, and serological features. These genera include the genus Dependovirus, which includes the only classified reptile parvovirus, serpentine adeno-associated virus, which is suggested to be a species within this genus [The parvoviruses are small (18–22 nm) non-enveloped round viruses with icosahedral symmetry and a single-stranded DNA genome. The family is divided into two subfamilies, is genus . DependoElaphe quatuorlineata) and of an Aesculapian snake (Elaphe longissima), both with gastrointestinal disease. Adeno-like viruses were also detected in the duodenums of both snakes. Herpes- and picorna-like viruses were also detected in the duodenum of the Aesculapian snake [Isospora sp. [Pantherophis guttatus) in iguana heart cells (IgH2), but was not further characterized [Python regius). Both parvoviruses were found to be identical and were given the name serpentine adeno-associated virus (SAAV). The genomic organization of this virus was determined to be most similar to that of described dependoviruses with two open reading frames, one encoding the putative non-structural (Rep1 and Rep2), and one encoding the capsid  proteins. It is unclear whether SAAV can replicate without a helper virus, as both isolates were obtained together with adenoviruses [Parias hageni) infected with an adenovirus [In reptiles, parvo-like viruses were first described by electron microscopy in the duodenum of a four-lined rat snake  with multifocal areas of necrosis in the spleen and liver. The virus was identified based on electron microscopy [Circoviruses are small, non-enveloped single-stranded DNA viruses with a circular genome. The family is currently divided into two genera, croscopy .9.Chelonia mydas) with fibropapillomatosis using metagenomics. This virus has a circular genome, with a hypervariable region and a conserved region. Numerous quasispecies were identified in both turtles. Most of the genome has no similarities to any known viruses. A single ORF (ORF2) has weak (25%) amino acid level similarity to the VP2 protein of chicken anemia virus. The virus was named sea turtle tornovirus 1 (STTV1). The authors of the study postulated that STTV1 might represent a new viral genus of the family Circoviridae or even a new viral family. Both of the infected turtles were severely afflicted with fibropapillomatosis with extensive external fibropapillomas and internal fibromas. STTV1 was detected in the fibropapillomas as well as in external swabs from the conjunctiva, oral cavity, cloaca, unaffected skin, and numerous internal tissues. The herpesvirus FPTHV was also detected in the fibropapillomas, but not in other tissues. STTV1 was also detected in leeches collected from one of the green sea turtles. STTV1 is not believed to be the cause of fibropapillomatosis, as it is not found in all fibropapillomatosis affected turtles, but both of the STTV1 positive turtles had severe fibropapillomatosis, and it was hypothesized that STTV1 might affect the immune system of infected sea turtles or that it might be an opportunistic pathogen [A novel single-stranded DNA virus with a circular genome approximately 1800 nucleotides long was detected in two green sea turtles  and members of the genus Betaretrovirus as A-type particles (immature capsids) in the cytoplasm. A-type particles then budded with either B- or D-type morphology [Vipera russelli) with a sarcoma [Gammaretrovirus, the type species of which is murine leukemia virus [Bothrops jararacussu that were apparently healthy [Python molurus bivittatus) with various neoplasms [Corallus caninus) with adenocarcinoma [Retroviridae [Gammaretrovirus, while CERV2 clustered distantly to all known retroviruses [Python molurus) that was kept together with IBD positive pythons. The viral genome was fully sequenced and found to be distantly related to both B and D types and the mammalian C type viruses, but the virus could not be classified. Further study indicated that this is a highly expressed endogenous virus of Burmese pythons that is not associated with IBD. A similar virus was also identified in blood pythons (Python curtus). These viruses were named python endogenous retrovirus (PyERV) [rphology . Retrovi sarcoma , named v sarcoma . VRV hasia virus . Two mor healthy . C-type eoplasms . A type-arcinoma . Retroviarcinoma ,127. A darcinoma . A studyoviridae . Furtheroviruses . Retrovioviruses ,132. No  (PyERV) .Bothriechis marchi) and an eastern king snake (Lampropeltis getulus) [Inclusion Body Disease (IBD) is a disease of snakes that has been described worldwide in captive snakes. The disease is characterized by the formation of intracytoplasmatic inclusions in neurons and in epithelial cells of various organs. The inclusions are made up of protein. IBD generally affects boid snakes, although it has also been reported in palm vipers (getulus) ,134. Cligetulus) ,132. Howgetulus) , making getulus) . The incgetulus) . The ori11.11.1.Reoviridae consists of non-enveloped viruses 60 to 80 nm in diameter with a segmented double-stranded RNA genome. The family contains 12 genera including the genera Orthoreovirus, Rotavirus, Orbivirus, Coltivirus, Seadornavirus, and Aquareovirus, which contain vertebrate viruses. All of the reoviruses detected in reptiles have been tentatively classified in the genus Orthoreovirus. These viruses infect only vertebrates and are spread by the respiratory or fecal-oral route. All members of the genus have ten RNA segments which can be divided into large (L), medium (M), and small (S) size-classes [Orthoreovirus species have been shown to cause significant and often fatal disease in birds, and primates. Five species have been classified in the genus Orthoreovirus: Mammalian orthoreovirus, Avian orthoreovirus, Nelson Bay orthoreovirus, Baboon orthoreovirus, and Reptilian orthoreovirus. All but the mammalian orthoreovirus isolates are fusogenic in cell culture [Orthoreovirus [Orthoreovirus. The reptilian viruses were monophyletic and formed three distinct clusters [Opheodrys aestivus) was found to be a novel strain distinct from the previously described reptilian orthoreoviruses at a level consistent with species difference based on the same partial sequence of the RNA-dependent RNA polymerase [The family -classes . While t culture . Sequencreovirus . Compariclusters ,139. A rlymerase .11.2.Lacerta viridis) [Iguana iguana) that died with no specific clinical signs in Germany [Uromastyx hardwickii) in the United Kingdom that died during a disease outbreak associated with changes in the lungs [Eublepharis macularius) in the USA [Reovirus infections have been relatively frequently described in reptiles. In lizards, reoviruses have been described by electron microscopy associated with papillomas in green lizards (viridis) . Isolati Germany  and fromhe lungs . An outb the USA . Serolog the USA ,145.Azemiops feyi) with enteritis, from a ball python (Python regius) [Corallus caninus) [Elaphe longissima) [Crotalus viridis) with signs of central nervous system (CNS) disease [Opheodrys aestivus) in the USA with a necrotizing hepatitis [Elaphe moellendorffi) and beauty snakes (Orthriophis taeniurus) with fatal respiratory disease. The virus isolated from that outbreak was inoculated intratracheally, orally and nasally into a black ratsnake (Elaphe obsoleta obsoleta). That snake died with pneumonia 26 days p.i. A reovirus was reisolated from the lungs of the dead snake [In snakes, reoviruses have been isolated from Chinese vipers ( regius) , an emercaninus) , an Aescgissima) , from th disease , and froepatitis . Rough gepatitis . Reoviruad snake . Reoviruad snake . A transad snake .Testudo graeca). The tortoise was cachectic and had a necrosis of the epithelium of the tongue [In tortoises, a reovirus has only been isolated in one case from a spur-thighed tortoise  in Australia. The virus was isolated in mice. Antibodies to this virus were also detected in a human [Ameiva ameiva ameiva in Brazil. Chaco virus was also isolated from another species of teiid lizard, Kentropyx calcaratus from the same area. All three viruses were isolated in suckling mice. Both Marco and Chaco viruses were also shown to multiply in the salivary glands of the mosquito Aedes aegypti and could be passaged serially in these mosquitoes [Dracaena guianesis) [In reptiles, rhabdovirus infections have been documented based on both serology and virus isolation. Little is known about the importance of rhabdoviruses in reptiles, but the rhabdoviruses detected in reptiles so far have been arboviruses that were also capable of infecting mammals. Antibodies against a vesicular stomatitis virus have been detected in wild-caught reptiles in Texas, USA . Neutral a human . Marco, squitoes . Marco, squitoes . A furthianesis) .13.13.1.Paramyxoviridae contains viruses with a negative sense single-stranded RNA genome. Virions are 150 nm or more in diameter, pleomorphic, and consist of a lipid envelope surrounding the nucleocapsid. The family is currently divided into the subfamily Paramyxovirinae with the genera Rubulavirus, Avulavirus, Respirovirus, Henipavirus and Morbillivirus and the subfamily Pneumovirinae with the genera Pneumovirus and Metapneumovirus. Members of the subfamily Paramyxovirinae encode 7 to 9 proteins. Virion proteins common to all genera include three nucleocapsid-associated proteins: an RNA-binding protein (N), a phosphoprotein (P), and a large polymerase protein (L); three membrane associated proteins: a matrix protein (M), a fusion protein (F) and an attachment protein . These genes are in the order 3′ N-P-M-F-HN-L 5′ in the genomes of members of this subfamily. There is, however, diversity in genome length, presence or absence of overlapping open reading frames (ORFs) within these common genes, genomic terminal sequences, and length and sequence of intergenic and untranslated regions. Among the genes common to all members of the Paramyxovirinae, the sequence relatedness between corresponding proteins is greater for N, P, M, and L than for F and HN, which are less conserved. Some, but not all members of this family have hemagglutinating and neuraminidase activity [The family activity .Paramyxovirinae within a new genus with the proposed name Ferlavirus [Paramyxovirinae subfamily to include the genus Ferlavirus, with FDLV as the type species [Paramyxoviruses (PMVs) have been isolated in cell culture from a number of different reptiles. The first PMV isolated from a snake, Fer-de-Lance virus (FDLV) is considered the type species of the reptilian PMVs. All related reptilian PMVs have hemagglutination and neuraminidase activity. The complete genome of FDLV has been sequenced and analyzed. It has a length of 15,378 nt. The genome encodes seven discrete, non-overlapping transcription units, one of which contains two overlapping ORFs. The genome organization is: 3′ N-U-P/V-M-F-HN-L 5′, with N, M, F, HN, and L defined as above. U encodes a protein of unknown function with no similarity to known sequences and P/V contains ORFs for paramyxoviral P and V genes. The analysis of the complete sequence showed that this virus should be classified in the subfamily rlavirus . It has  species .13.2.Ferlavirus [Crotalus unicolor). In that study, snakes were inoculated intratracheally with a PMV cell culture isolate from an Aruba Island rattlesnake. Several snakes developed pulmonary symptoms including blood in the lungs, trachea and oral cavity. The two animals that were not euthanized earlier died 19 and 22 days p.i. [PMV are an important cause of disease in snakes in both private and zoologic collections and have been described in many different parts of the world –167. Therlavirus . In snakrlavirus . In one ays p.i. . Lungs oays p.i. ,169.Draecena guianensis). These animals showed few clinical signs and were either found dead or were anorexic prior to death. Pathological and histological examination showed proliferative interstitial pneumonia [In lizards, PMV infections have been described much less frequently than in snakes. However, antibodies against PMV have been detected in both wild-caught and captive lizards ,145,170.neumonia .Testudo hermanni) with pneumonia. That virus was further characterized and found to be related to but distinct from the described ferlaviruses from snakes and lizards so far [Geochelone pardalis) with pneumonia. The detected viruses were each most closely related to ferlaviruses from snakes. However, it was unclear whether the virus was a causative agent, as no virus was detected in the lungs of the diseased animal [Descriptions of PMV infections in chelonians are also rare. These infections have been associated with dermatitis , and pnes so far . In anotd animal .Python reticularis) in the United Kingdom [Ferlavirus has also been described in squamatids [Comparisons of ferlaviruses have been carried out based on sequencing of various portions of the genome and on serological cross-reactivity. Hemagglutination inhibition (HI) assays were one of the first tools used to study the relationships among ferlaviruses and between ferlaviruses and other PMVs. In a study using antibodies raised in rabbits against three different snake PMV isolates (later identified to be members of subgroups A and B) and other PMVs to investigate antigenic relations, no cross reactivity was documented between the ferlaviruses and non-reptilian PMVs, but the three ferlaviruses did cross react . Another Kingdom . Studies Kingdom . Further Kingdom , 920 bp  Kingdom , and a 6 Kingdom  have all Kingdom . A thirduamatids .14.Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. The first four of these are capable of infecting vertebrates. Orthobunyaviruses, nairoviruses, and phleboviruses replicate alternately in invertebrates. They are generally cytolytic for their vertebrate hosts, but cause little or no cytopathogenicity in their invertebrate hosts. Different viruses have different arthropod vectors including mosquitoes, ticks, phlebotomine flies and others [Bunyaviruses are spherical or pleomorphic enveloped viruses 80–120 nm in diameter. The viral genome comprises 3 molecules of negative or ambisense single-stranded RNA, which are called L (large), M (medium), and S . The family is currently divided into five genera: d others .Trionyx spinifer emoryi) which was wild-caught in Texas, USA. The virus was isolated in suckling mice by intracerebral inoculation and caused 100% mortality in inoculated mice. The virus was not further characterized, but was found to serologically cross-react with Cache Valley and Tensaw viruses, both mosquito-borne viruses of the genus Orthobunyavirus [Bunyaviridae, has been isolated from both mosquitoes and a skink (Ablepharus boutonii virgatus) in Australia [A bunyavirus was isolated from the blood of a Texas soft-shelled turtle . Both snakes showed signs of gastrointestinal disease. An adeno-like virus was also found in the duodenum and spleen of the Boa constrictor, while parvo-, adeno-, and herpes-like viruses were detected in the duodenum of the Aesculapian snake [Testudo graeca), but are also found in marginated tortoises (T. marginata), Hermann’s tortoises (T. hermanni), leopard tortoises , and Egyptian tortoises (T. kleinmanni). They have been associated with various signs of disease in infected animals, including softening of the carapax in young animals, diphtheroid-necrotizing stomatitis, rhinitis, conjunctivitis, and ascites, but have also been isolated from clinically healthy animals [Mycoplasma spp. [et al. [Picornaviridae, but differs significantly from described picornaviruses. It has therefore been proposed that this virus belongs to a novel picornavirus genus [The family n module . The famd genera . None ofan snake . Picornaan snake . These v animals ,188. In sma spp. . Heuser  [et al.  have nowus genus . Sequenc16.Vesivirus is an exception, with a broad host range. Transmission is generally via contaminated food, water, fomites, and sometimes via aerosolization [Caliciviruses (CVs) are non-enveloped, 27–40 nm in diameter and have icosahedral symmetry. The genome consists of a positive sense single-stranded RNA. The family is divided into four different genera. CVs infect a broad range of animals, and most individual CV species exhibit a natural host restriction. However, the vesicular exanthema of swine virus (VESV) in the genus lization .Crotalus unicolor), a rock rattlesnake (C. lepidus), and an eyelash viper (Bothrops schlegeli)), as well as from Bell’s horned frogs (Ceratophrys orate) in a single collection in the United States [Crotalus viridis helleri) showed that these animals could be infected and virus could be reisolated (in one out of two snakes inoculated), but no pathognomonic disease was observed in the infected snake. Transmission of the virus to pigs led to seroconversion, but not disease. The isolated viruses were considered identical based on serological comparisons, but were serologically distinguishable from other CV serotypes. The isolated virus was therefore named reptilian calicivirus Crotalus type 1 (RCV Cro-1) [Vesivirus [In reptiles, CVs have been isolated from four different snake species  . SequencV Cro-1) . Reptileesivirus . A PCR fesivirus , but no 17.Flaviviridae is divided into the genera Flavivirus, Pestivirus, and Hepacivirus. All of the flaviviruses described in reptiles belong to the genus Flavivirus, which contains viruses that are transmitted by hematophagous arthropods (arboviruses). Some flaviviruses are able to infect and replicate in a wide variety of vertebrate hosts  [Flaviviruses are enveloped, single-stranded RNA viruses. The family eptiles) , and theGeochelone pardalis) with epistaxis, cloacal hemorrhage, biliverdinuria, and anemia. Herpes and adenoviruses were isolated from other animals in the same collection, and intraerythrocytic inclusions of unknown etiology were also described in these tortoises [Antibodies against various flaviviruses, including St. Louis encephalitis virus, Powassan virus, Japanese encephalitis virus, and WNV have been found in chelonians, snakes and crocodilians in various parts of the world –203. Japortoises ,206. TheIguana iguana), Florida garter snakes , red-eared sliders (Trachemys scripta elegans), and North American bullfrogs (Rana catesbeiana), the iguanas, snakes, and frogs became infected and the iguanas and frogs both developed low level viremias. No disease was reported in any of the infected animals [Thamnophis siralis) that were infected subcutaneously, 5 of 9 infected snakes became viremic, while five had persistently low levels of neutralizing antibodies. Four of the experimental animals died and high titers of virus were found in multiple organ samples [WNV is a flavivirus that primarily cycles between mosquitoes and birds. It was first documented in North America in 1999 . A numbe animals . In a tr samples . Another samples .Alligator mississippiensis) in a number of different states including Georgia [Culex quinquefasciatus), and high virus loads were also documented in the cloaca [WNV has been shown to be pathogenic for crocodilians and causes high titer viremias in these animals. Antibodies against WNV have been detected in crocodilians in many different countries, including in crocodiles at a commercial farm in Israel , in wild Georgia , Florida Georgia , and Lou Georgia . In Flor Georgia . In an o Georgia . WNV has Georgia . In a tre cloaca . West Nie cloaca .18.Togaviridae contains spherical virions with a lipid envelope and a linear, positive sense single-stranded RNA genome 9.7–11.8 kb in size. The family is divided into two genera: Alphavirus and Rubivirus [Alphavirus, which contains viruses that have the ability to replicate in and be transmitted horizontally by mosquitoes. Most alphaviruses can infect a wide range of vertebrates, mostly birds and mammals, but several have also been reported in reptiles. Studies on alphaviruses in reptiles have mostly focused on the possible role of these animals for the transmission of alphaviruses to humans and livestock. This has led to a focus on persistence of alphaviruses in reptiles, particularly viral persistence over winter in temperate regions in the absence of mosquito activity (reviewed in [Thamnophis) indicates that recurrence of viremia in these animals has a cyclical rhythm and appeared to be independent of temperature [Clemmys guttata) with EEE virus led to the development of viremia and neutralizing antibodies in infected turtles [Gopherus berlandieri) with WEE virus led to high titer viremia over an extended period of time. Viremia was longer at lower temperatures than at high environmental temperatures [The family ubivirus . The togiewed in ). Detectiewed in  for a reperature . Experimperature –221. A t turtles . Subcutaeratures . None of19.The factors involved in the development of viral disease in reptiles are, in many cases, unclear. Both host and viral factors are involved. Transmission studies have been carried out with a number of viruses in specific host systems, as described above. However, as our understanding of reptilian viral infections grows and as methods for the detection of these viruses become both more sensitive and specific, it becomes more and more clear that additional factors can also be involved. It has long been known that environmental factors, particularly temperature, influence the immune system of reptiles. Increasing evidence also suggests that other factors can play an important role in the outcome of viral infections in reptiles. For example, higher disease rates of fibropapillomatosis in sea turtles have been shown to occur particularly in watersheds with a high nitrogen footprint, which was hypothesized to affect the immune system, possibly through changes in nutrition .Opheodrys aestivus) with clinical wasting [Pantherophis guttatus) in Germany, co-infection with PMV, several different adenoviruses and a reovirus was documented [Concurrent infections with multiple infectious agents, including multiple viruses, have been shown to occur and the interactions and effects of these concurrent infections are not yet understood. Although fibropapillomatosis is believed to be caused by a herpesvirus, other viruses have been demonstrated in fiborpapillomatosis positive sea turtles, leading to hypotheses that these might affect the immune system of infected turtles and possibly the course of disease . Concurr wasting . Coinfec wasting . It is i wasting , while i wasting . In a cacumented . In tortcumented ,226, andcumented . These m


Latent virus reactivation might occur by superinfection of latently infected cells. Previous studies have suggested that latent viruses contribute to recombination We used both Jurkat and primary cell latency models. In Jurkat cells, wt and drug-resistant latent populations were superinfected with wt or drug-resistant viruses, including nef or tat mutants, and treated with various inhibitors. Viral reporter gene expression was measured by FACS. Sequence tags on each resistant virus permitted identification of recombinants. We also performed superinfection experiments in primary cells.2 = 0.98) with the extent of superinfection across a wide range of viral inocula. Latent virus reactivation required gene expression of the superinfecting virus, since latent viruses were reactivated in the presence of a protease inhibitor, but not a reverse transcriptase (RT) or integrase inhibitor. Latent virus reactivation occurred following superinfection with nef-deleted or tat-attenuated, but not tat-inactivated, viruses. These results suggest that neither gp120-induced CD4/CXCR4 signalling nor Nef-induced NFkB/NFAT modulation were required for latent virus reactivation, but that superinfecting virus Tat was required. Drug-resistant latent viruses (RT K103N) were reactivated following superinfection with additional drug resistant viruses (RT M184V). The resulting supernatants (containing heterozygous virions) were used to infect new cells, to which the RT inhibitors FTC and EFV were added. Sequencing and restriction digestion confirmed that recombination frequently occurred, leading to FTC/EFV-resistant virions encoding unique sequence tags derived from both parental viruses. We also established latency in unstimulated primary CD4T-cells and subjected them to superinfection. Results from nine individual donors indicate that latent viruses were reactivated by superinfection; these results were statistically significant (p=0.001).Latent viruses were reactivated by superinfection in both latency models. In Jurkat cells, the extent of latent virus reactivation was strongly correlated (rOur results suggest that superinfection of latently infected cells can reactivate latent viruses, which can then recombine with the superinfecting virus. Since all viral quasispecies including drug-resistant viruses can be latently archived, reactivation of latent viruses by superinfection or other means could contribute to the emergence of replicatively fit viruses in the face of strong selective pressures.


Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes  that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases. Closteroviridae, which consists of Closterovirus, Crinivirus, and Ampelovirus genera with mono-, bi-, or tripartite genomes, provide some of the better examples of combinations of conserved and unique genes. They all encode a mixture of conserved signature gene modules along with unique genes with no relationship found in other members of the family. The conserved gene products are involved primarily in replication and virion assembly. In fact, some domains and cis-acting elements involved in replication can be exchanged between different viruses. Additionally, members within a genus possess 1–5 unique genes. These gene products are thought to have evolved to interact exclusively with their specific hosts  has a 19.3-kb single-stranded positive-sense RNA genome -tagged CTV in peeled bark that exposes phloem cells. In all citrus hosts, long-distance movement appears to be limited to relatively few initial infection sites. In the more susceptible hosts, Closterovirdae or other phloem-limited viruses.In the more susceptible citrus species, CTV also has limited cell-to-cell movement that produces small clusters of infected cells. However, in less susceptible citrus species, it appears that little or no cell-to-cell movement occurs. The virus is able to exit sieve elements but cannot spread to adjacent cells, resulting in infection of isolated single cells. Thus, CTV provides a new pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature. Such a movement pattern has not been described previously. It is not known whether this pattern is characteristic of other members of the CTV generally has been moved long distances into new areas by transport of infected planting (or propagating) materials. Prior to the advent of rapid shipping in the nineteenth century, importation of citrus occurred only as seed, avoiding CTV spread as the virus is not transmissible by seed. However, as navigation improved, citrus was moved as plants or budwood, and so was CTV. Presently, the problem is that since even severe isolates are symptomless in some of their hosts, the virus often is spread by well-meaning individuals moving an infected but non-symptomatic plant or budwood from such a plant into a new area. Afterwards, local spread is by aphids, where transmission is in a semipersistent manner. This combination has effectively spread CTV  in a wider range of citrus species and relatives within the CTV host range and found that they are needed for systemic infection of some of the hosts  on a series of different plants from the host range suggested that CTV has the largest number of distinct phenotypes of any plant viruses  induced greatly increased SP, while other combinations (p13 or p13 plus p18) resulted in reduced SP  tended to have the most SP, which might suggest that the p13 gene product was involved in induction of stem pits. However, the triple deletion mutant, which did not have the p13 gene, induced severe SP, demonstrating that interpretation is not so simple. In contrast, increased SP generally was associated with deletion of the p33 ORF. Mutants with the absence of the p33 ORF  induced severe SP. Thus, mutants retaining the p33 gene  had the least amounts of SP. These results suggest that the presence of the p33 protein could be correlated with reduced SP (its absence increases it). However, the mutant with the deletion of the p18 ORF (p33 and p13 retained) induced moderate SP. Overall, the production of stem pits or no stem pits appears to be related more to a balance between expression of the p33 and p13 and possibly p18 genes  and a mild strain (T30). Remarkably the T30 strain does not induce decline. In comparing the two strains, it appears that T36 contains determinants that induce decline that T30 does not have. In an attempt to identify the decline determinants, we have made hybrids in T36 in which T36 sequences are removed and substituted by T30 sequences, similar to the mapping exercise to identify SY determinants. However, this project has lingered due to our inability to assay for decline in the greenhouse with small trees. Under these conditions, sweet orange on sour orange rootstocks grow normally. Apparently, the small trees replace phloem as fast as the virus causes damage to it. We now have a field test on which we await results.The potential control strategy is to use cross protection (superinfection exclusion: see Folimonova in this series) to protect trees on the sour orange rootstock. Since T36 and T30 are from different strains, T30 cannot be used to protect trees from T36 (Folimonova et al., Is the viral counter-attack of the host RNAi system a component of disease induction? It has been shown that ectopic expression of one of the CTV suppressors of RNAi, p23, induces virus-like symptoms (Ghorbel et al., * (Bazzini et al., In non-citrus species is has been shown that ectopic expression of viral suppressors of silencing alters mRNA expression levels and induces symptoms (Soitamo et al., C. macrophylla, apparently causing gene product ratios that induced abnormalities. In both cases, the symptoms resulted from an alteration of development. Interestingly, both of these disease symptoms are non-continuous. SY symptoms usually are transient. Plants respond only briefly and new growth does not exhibit the symptoms. SP is spatially sporadic. Some infected areas develop abnormally resulting in pits, but most other infected areas continue to develop normally.CTV non-conserved genes apparently evolved to allow systemic infection of its hosts. These are genes involved in cell-to-cell and long-distance movement and in counter surveillance. Some are not needed for all hosts. These non-conserved genes can also be involved in induction of disease symptoms. A specific region was mapped to be involved in the SY syndrome. In contrast, deletion of genes was involved in induction of SP in Viruses evolve to survive in hosts with which they are presented. This involves acquiring and modifying genes to interact precisely with their hosts. A range of potential host species creates a bewildering array of selective factors; each species will differ to some degree in physiology, gene expression, metabolism, and antiviral defenses, and an isolate at an adaptive peak in one host may be less fit in another. The process of adaption to one host may also create the potential to cause disease in another. In citrus for example, most isolates are mild to asymptomatic in pomelo, mandarin, and citron (Garnsey et al., However, the results described above come from simple systems—a pure culture—a single strain of virus from a cDNA clone. Yet, most virus infections in the field are complex populations of mixtures of different strains and defective RNAs. Little is known concerning how these populations equilibrate and which components of the population interact with the host to elicit or prevent disease symptoms. Do components of the population complement to induce disease symptoms? Do some components counteract other components? The next frontier in plant virology is developing an understanding of populations.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the considerable health impact of coeliac disease (CD), reliable estimates of the impact of diagnosis on health care use and costs are lacking.To quantify the volume, type and costs, in a United Kingdom primary care setting, of healthcare resources used by individuals diagnosed with CD up to ten years before and after diagnosis, and to estimate medical costs associated with CD.A cohort of 3,646 CD cases and a parallel cohort of 32,973 matched controls, extracted from the General Practice Research Database (GPRD) over the period 1987–2005 were used i) to evaluate the impact of diagnosis on the average resource use and costs of cases; ii) to assess direct healthcare costs due to CD by comparing average resource use and costs incurred by cases vs. controls.Average annual healthcare costs per patient increased by £310  after diagnosis. CD cases experienced higher healthcare costs than controls both before diagnosis  and after diagnosis . These differences were driven mainly by higher test and referral costs before diagnosis, and by increased prescription costs after diagnosis.This study shows significant additional primary care costs associated with coeliac disease. It provides novel evidence that will assist researchers evaluating interventions in this area, and will challenge policymakers, clinicians, researchers and the public to develop strategies that maximise the health benefits of the resources associated with this disease. Coeliac disease (CD) is an inherited chronic autoimmune disorder that can potentially affect many organ systems beyond the gastrointestinal tract Although the health impact of CD is considerable due to increasing prevalence rates, associated non-specific morbidity and long-term complications The main objective is to quantify the volume, type and cost of healthcare resources used by individuals diagnosed with CD up to ten years before and after diagnosis, with particular reference to consultations, tests, referrals and prescriptions in the primary care setting where non-acute care is mainly managed, and to compare these with the costs incurred by a matched control-cohort, using the General Practice Research Database (GPRD). Unlike previous published studies and uniquely in the area of coeliac disease research, we also analyse in detail the distribution of prescriptions costs across the 15 British National Formulary (BNF) categories The UK GPRD is one of the largest computerised databases of anonymised longitudinal medical records from primary care. There was, therefore, no need of patients’ consent. GPRD contains data from 1987 onwards and currently covers a population of over 5 million patients throughout the UK. It includes medical diagnostic codes for consultations, tests, referrals and details of prescriptions issued. Data are routinely subject to quality checks in order to guarantee they are ‘up-to-standard’ We used information supplied by GPRD staff over the period June 1987 to October 2005. Our case-cohort comprised all patients within GPRD with a recorded diagnosis of CD. Following the methods of a previous published study A diagnosis date, defined as the date of the first record of CD, was attributed to each patient with CD. Controls were assigned a ‘pseudo-diagnosis’ date coinciding with that of their matched case. To ensure maximum reliability of data we distinguished between ‘incident cases’ – those patients with a date of diagnosis of CD at least 12 months after their up-to-standard record on GPRD began – and ‘prevalent cases’, including all other subjects with CD. Primary analyses made use only of incident cases.Details of demographic and diagnostic information were extracted or constructed from the GPRD data for the CD and the control cohorts. They included patient age, gender, smoking status, BMI, Charlson comorbidity score, which, by accounting for both number and severity of comorbidity, classifies patients according to their disease burden Information relating to resource use for each patient over the study period was extracted from the GPRD data. It included number of primary care consultations, tests, referrals to out-patient hospital care For each patient, healthcare costs stratified by subcategory of interest  were computed by multiplying units of resource use by their unit costs. These were then summed over all resource use categories to obtain an annual cost and total cost for each patient. Details on the specific unit cost sources We analysed the 10-year period prior to and following diagnosis of CD for cases, and pseudo-diagnosis for the matched control-cohort. We considered the full 20-year period even if data on healthcare utilisation and costs for a case or control subject were available for less than 20 years, provided that data were available on at least some of the matched sets of cases and controls within the study population. Consequently, resource use and costs were calculated on a number of cases/controls that was variable each year over the period of study. Average annual amounts of healthcare resource use and associated costs were calculated and then compared for the identified CD cohort in the 10 years prior to and following diagnosis. The aim was to evaluate the impact of diagnosis on the average resource use and costs incurred by each patient within a primary care setting. Similar analyses were performed for the control-cohort. Analyses were stratified by resource use and cost categories of interest, and by age group at diagnosis .The direct healthcare costs due to CD were estimated by comparing the average annual amounts of healthcare resources use and costs incurred by the CD cohort with the analogous amounts incurred by the matched control-cohort before and after diagnosis/pseudo-diagnosis (for controls) of CD. These analyses were also stratified by resource use and cost category of interest, and by age group at diagnosis/pseudo-diagnosis . Similar analyses were conducted for costs of prescriptions by BNF category.We also investigated whether the widespread introduction of serological testing, approximately between 1993 and 2000 P-values less than 0.05.Consultations, referrals, tests and prescriptions were treated as continuous variables and reported as number of events per patient per year. Resource use rates and mean costs before and after diagnosis were reported together with their standard errors. Rates and mean differences in observed resource use and costs between the two time periods were reported alongside 95% confidence intervals (CI). Statistical differences in mean estimates were evaluated using Student’s two-sided t-test or dermatitis herpetiformis or a non-specific reference to coeliac disease or a prescription for gluten free products between 1st June 1987 and 31st October 2005 (wider definition of CD). Of these, 561 patients were excluded because of ascertained inconsistencies between the diagnosis date and the patient’s date of registration with the practice. No data on the variable ‘practice-up-to-standard’ were available for further 109 patients, who were excluded. Our decision to focus only on incident cases led to the exclusion of a further 5,420 prevalent cases, reducing the sample to 5,889 patients. A further 2,243 cases were removed because they did not satisfy the more restricted definition of CD. Results are, therefore, based on 3,646 incident cases and the corresponding controls matched on a 1∶10 ratio. A further refinement of the control-cohort was the exclusion of 3,487 individuals as ‘no consulters’ on the grounds that they did not consult over the 12 months before and after the pseudo-diagnosis date.The coeliac cohort had a lower rate of consultations (5.8 vs 6.9), tests (1.9 vs 3) and prescriptions (14.3 vs 40.7) in the 10 year prior to diagnosis than following diagnosis . Rate diTotal annual healthcare costs increased by £310  in the case-cohort, following CD diagnosis. This 91% increase was mainly driven by the trebling of prescription costs . The modest, but statistically significant increase in consultation costs  and test costs  was partially offset by the reduction in referral costs . Stratification of total annual healthcare costs by age group at diagnosis File S1,  show thaOver the 10-year period preceding diagnosis, the CD cohort experienced significantly higher total costs of £91 compared with the control-cohort  . This diA closer look at the prescription costs  reveals To the best of our knowledge, this is the first detailed evaluation of the resource use and costs before and after diagnosis of CD in the primary care setting of the UK healthcare system.Average per-patient annual healthcare costs in primary care significantly increased by 91% for CD patients after they had been diagnosed with the disease. The trend of increased expenditure was stronger in the immediate pre- and post-diagnosis period, which is consistent with clinical practice . DiagnosThis first result seems, at first glance, in clear contrast with the sparse previously published empirical evidence. Previous studies Similarly to the US results, coeliac patients in our study underwent a post-diagnosis reduction in referral costs. This partially counterbalanced the slightly higher level of costs for consultations and tests (but not prescriptions) in the post-diagnosis period. Increased consultations and tests may occur because CD patients have to be checked at regular intervals by a healthcare team, including a GP and a dietician, in order to monitor adherence to a gluten-free diet and to reinforce the importance of such compliance Differently from the US studies, our analyses included costs of prescriptions, which were found to be the major driver of the increase in post-diagnosis CD costs. Noticeably, prescriptions costs for BNF category 9 ‘Nutrition and blood’, which includes gluten-free products, underwent a 25-fold increase from an average annual pre-diagnosis cost of £10 to an average of £246 post-diagnosis per coeliac patient, 89% of the latter cost represented by prescriptions for food supplements .Increased post-diagnosis prescription costs for CD patients were also identified in other BNF categories . This may represent further evidence on the cumulative morbidity experienced by CD patients compared with the general population The stratification of healthcare costs by calendar year of diagnosis indicated that, on average, cases cost 43% more than controls before diagnosis up to 1999, compared with 34% more in the post-1999 period. After diagnosis, the difference remained almost unchanged. This is compatible with the hypothesis that improved methods for detecting CD – notably wide adoption of serological testing – and a higher awareness of the disease and its symptoms among health professionals In evaluating the healthcare costs attributable to CD prior to diagnosis, we found that the average cost per patient with undetected and untreated CD was 37% higher than the corresponding cost for a patient in the control-cohort. Cost comparisons between cases and controls up to 10 years after diagnosis showed that diagnosis and treatment of CD did not reduce direct healthcare cost in aggregate, but did reduce costs of tests (by 21%) and referrals (by 37%).Our estimates of the costs of undiagnosed and diagnosed CD will help inform clinical and public health policy, facilitate the economic evaluation of diagnostic and treatment interventions, complement the existing NICE guidelines on the management of CD Other strengths of this study include the large sample size, the time frame of 20 years, which was considerably longer than those adopted in previous published studies Several limitations of our study should be noted. Firstly, we used a fixed unit cost for each resource category during the whole 20-year period of our analyses. Although this is a standard practice in cost studies and was dictated by the lack of a long time series of unit costs, it might result in our total costs being slightly overestimated. Secondly, although cases and controls were identified on a ratio of 1∶10 and matched by age, sex and practice, we did not match by registration year. We also discarded single individuals if they had missing values in some of the relevant variables. Analyses were therefore conducted on a variable number of cases and controls over the duration of the study and did not strictly follow a matched case-control design. However, we consider it unlikely that this will have had a significant impact on our results, given the sample sizes involved. Thirdly, whilst GPRD is an accurate source of resource use data in the primary care context, the capture of data for secondary care resource use is, by the intrinsic nature of the dataset, less precise, especially with respect to inpatient episodes and follow-up outpatient visits. We have consequently not attempted to include these, and only include initial outpatient referrals from the GP to secondary care. GPRD now permits linkage of patient records to the Hospital Episodes Statistics database, and this will permit future research to more fully capture the primary-secondary care interface and reliably quantify hospital use in the coeliac and non-coeliac population. Fourthly, we focused only on the evaluation of direct costs incurred or initiated by NHS primary care services and did not include indirect costs associated with reduced productivity caused by impaired ability to work and absence from work, out-of-pocket costs such as those incurred in following a special and expensive diet, and other costs to those with coeliac disease and their families.In addition to economic costs, CD also imposes a significant impact on quality of life In conclusion, this study provides a uniquely detailed description of healthcare costs before and after diagnosis of CD in the UK healthcare system. By identifying and quantifying the relative contribution of different types of resource use to the primary care costs of each CD patient, and comparing these to carefully matched controls, this study may provide insights into the potential cost impact and cost-effectiveness of new ways of detecting and treating CD.Table S1Unit costs.(PDF)Click here for additional data file.Table S2Total costs per patient by age group in CD versus non-CD cohorts (for a maximum of 10 years before and after diagnosis).(PDF)Click here for additional data file.Text S1Specific composition of each resource use category and assumptions made for their count.(PDF)Click here for additional data file.


In a clinical setting, where motor-driven systems are not readily available, the major difficulty in the assessment of the stretch reflex lies in the control of passive limb displacement velocity. A potential approach to this problem arises from the use of manual sinusoidal movements  paced by an external stimulus. Unfortunately, there are conditions in which sinusoidal movements induce interfering phenomena such as the shortening reaction or postactivation depression. In the present paper, a novel manual method to control the velocity of passive linear movements is described and the results obtained from both healthy subjects and spastic patients are reported. This method is based on the synchronisation of movements with tones played by a metronome at different speeds. In a first set of experiments performed in healthy subjects, we demonstrated consistent control of velocity during passive limb movements using this method. Four joints usually examined during muscle tone assessment were tested: wrist, elbow, knee and ankle joints. Following this, we conducted a longitudinal assessment of the stretch reflex amplitude in wrist flexor muscles in patients with spasticity treated with botulinum toxin type A. The evaluators were not only able to vary the movement velocity based on the metronome speed, but also could reproduce the respective speeds two weeks later, despite the changing degree of hypertonia. This method is easy to perform in a clinical setting and hardware requirements are minimal, making it an attractive and robust procedure for the widespread clinical assessment of reflex hypertonia. Studies utilizing motor-driven systems that are capable of producing passive limb displacements at predetermined velocities have clearly shown that increased excitability of the stretch reflex (SR) is a primary effector of spasticity Movements executed by motor-driven devices to elicit the SR can be either sinusoidal or “ramp and hold” displacements (herein referred as “linear movements”). Sinusoidal displacements consist of rhythmic and consecutive flexion and extension movements, while linear movements involve displacement of a limb from one position to another and stopping without a consequent movement.In a clinical setting where motor-driven systems are not readily available, hypertonia is commonly evaluated by means of subjective clinical scales incapable of differentiating between the reflex and non-reflex components Without the aid of motor-driven systems, the major difficulty in SR assessment lies in the control of passive limb displacement velocity. A potential approach to this problem arises from the use of manual sinusoidal movements paced by an external stimulus. The evaluator can take advantage of these sinusoidal movements to displace the joint at consistent and accurate velocities, indicating a possible clinical strategy for SR evaluation th World Congress of Neurorehabilitation In the present paper, a novel manual method to control the velocity of passive linear movements is described and the results obtained from both healthy subjects and spastic patients are reported. This method is based on the synchronisation of the movements with tones played by a metronome at different speeds. In a first set of experiments performed in healthy subjects, we demonstrated consistent control of velocity during passive limb movements using this method. Four joints usually examined during muscle tone assessment were tested: wrist, elbow, knee and ankle joints. In a second set of experiments performed in patients with spasticity, the validity of this method was demonstrated in a longitudinal SR assessment performed in wrist flexor muscles before and after the injection of botulinum toxin type A (BoNT-A). Part of these findings have been presented at the 6The first set of experiments was performed on 6 right-handed healthy subjects .http://www.agenziafarmaco.gov.it/sites/default/files/det_20marzo2008.pdf). Demographic and clinical details of the 12 patients are reported in The second set of experiments was performed on 12 patients  with post-stroke hemiparesis who were selected according to the following criteria: (1) clinical presentation of a hemispheric stroke leading to unilateral motor deficit at least 9 months prior to participation; (2) CT or MRI documenting a single vascular lesion in the middle cerebral artery territory; (3) a stable clinical picture in the last three months; (4) presence of spasticity in the wrist flexor muscles with a Modified Ashworth Scale (MAS) score ranging from 1 to 3 ; (5) no previous treatment with BoNT-A. All subjects gave informed consent according to the Declaration of Helsinki. The participants provided their written informed consent to participate in this study, which was notified to the local ethical committee “Comitato Etico IRCCS Azienda Ospedaliera Universitaria San Martino – IST”, according to the “Determinazione AIFA 20 Marzo 2008” ; age: 33±10 years (mean±SD), range: 23–50 years], one for each tested subject.Passive movements on the 12 stroke patients were performed by two evaluators , herein referred to as evaluator A and evaluator B. Both evaluators tested all 12 patients. Therefore, each patient was tested twice.To ensure a standardized level of training, all evaluators were properly instructed in a 15–20 minute training session.For the kinematic recording of the passive movements and the assessment of the passive range of movement (ROM), we used two independent methods: the optoelectronic system “ELITE”  and the Biopac MP100 data acquisition system connected to a TSD130B twin-axis electronic goniometer .The ELITE system consists of six infrared cameras (100 Hz sampling rate) that track the motion of passive reflective markers in 3D coordinates. Synchronised acquisition and data processing was performed using Analyser software (BTS S.p.a.). Three reflective markers (15 mm in diameter) were positioned for each investigated joint: the first on the proximal segment, the second near the joint pivot and the third on the distal segment.The goniometer was placed across the investigated joint in order to optimally record the angle during the joint displacements; a sampling rate of 2 KHz was used.EMG activity was recorded by surface electrodes  placed over the muscle belly. The signal was acquired by a MP100 unit  with a 2 KHz sampling rate and underwent a Blackman −61dB 80–300 Hz band-pass filter for offline processing . A constant-current stimulator  was used to stimulate the median nerve.Movement timing was paced with a software emulated metronome.The method consisted of 4 phases. During all of the phases, the subject was instructed to stay relaxed and to avoid resisting or facilitating the joint movements applied by the evaluator. Upper limb joints were assessed with the subject in a supine position while lower limb joints were evaluated with the subject in a prone position, with the feet off the surface of the examination table.An audio tone frequency was set on a metronome. For instance, the frequency of 60 beats per minute (BPM) was set. This means that the interval between two consecutive tones  corresponded to 1000 ms. Only the evaluator could perceive the tones through earphones.While trying to cover the entire ROM, the evaluator applied continuous manual sinusoidal joint displacements, moving the distal segment of the joint smoothly. These movements were done at a constant pace so that the distal segment of joint would arrive at the extreme flexed and extended positions in synchrony with consecutive tones. In this way, the evaluator related the motion with the rhythm of the metronome.When the evaluator had comfortably synchronized the movement with the tones, the sinusoidal movement was stopped at one extreme position (flexed or extended). At this point, the evaluator continued to conceptualize the previously executed movement using first perspective motor imagery After at least 15 seconds of motor imagery, when the evaluator felt prepared to follow the pace of the metronome accurately, he moved the distal segment of the joint so that it arrived at the opposite position in synchrony with the following tone; then he stopped again .Through this procedure, linear movement was derived from sinusoidal movement while retaining the feature of velocity control. This was made possible by the utilization of both an external audio cue as well as first person motor imagery. This procedure was repeated several times, in order to collect several flexion and extension linear movements.The method was applied to wrist, elbow, knee and ankle joints all examined on the right side. Before the application of the method, the passive ROM of the tested joint was evaluated. In the assessment of wrist, elbow and knee joints, the metronome was set at the following BPM values: 40BPM (TI = 1500 ms), 60BPM (TI = 1000 ms), 120BPM (TI = 500 ms) and 180BPM (TI = 330 ms). Since the ankle is the joint with the smallest ROM, we used the BPM values of 60BPM, 120BPM, 180BPM and 240BPM (TI = 250 ms) in order to achieve joint velocities comparable with the other three joints. At each BPM value, 15 flexion and 15 extension linear movements were collected. Flexion was considered the movement reducing the angle between skeletal members.flexor carpi radialis muscle (FCR). The metronome was set at the following BPM values: 40 BPM (TI = 1500 ms) and 60 BPM (TI = 1000 ms). At each BPM value, 15 extension and 15 flexion linear movements were collected. Only the extension movements were analysed. As in healthy subjects, just before the application of the method, passive ROM of the wrist joint was evaluated. During the application of the method, EMG activity was recorded from FCR, which was stretched by the passive extension linear movements.The method was applied to the wrist joint of the affected side before (baseline condition) and 15 days after (test condition) the injection of BoNT-A in the The M-wave was recorded in the FCR through supramaximal electrical stimulation of the median nerve in the cubital fossa.Wrist flexor muscle hypertonus was clinically rated at baseline condition and test condition according to the MAS. BoNT-A was dosed from 30 to 75 Xeomin® Units, according to the clinically appropriate dose .Passive ROM evaluated before the application of the method was assessed as the difference between joint angles at the maximal flexed and extended positions.For each linear movement obtained in phase 4, onset and termination times were calculated. With the ELITE system, these times were identified on the velocity profile, considering the 1% of the peak velocity amplitude as threshold. With the electronic goniometer, they were visually detected, using a display gain of 20°/1cm and a temporal window of 340 ms/1cm.dynamic-ROM calculated as the difference between joint angles at the starting and ending positions; 2) movement mean velocity (mean-V), calculated as dynamic-ROM/movement duration (difference between onset and termination times). Dynamic-ROM is specified in order to differentiate it from the ROM measured before the application of the method in a static condition.Onset and termination times were used to calculate the following two parameters: 1) Since a preliminary analysis showed no difference between the kinematic parameters acquired using the optoelectronic system and the electronic goniometer, only the latter are shown in the results.The SR amplitude was measured as the mean amplitude of the rectified EMG during the passive displacements. The SR amplitude was divided by the amplitude of the M-wave elicited at baseline condition to obtain SR/M ratio.dynamic-ROM was performed using a factorial ANOVA, while the 4 BPM have been compared using repeated measures ANOVA for each joint separately and with BPM as within-subjects factor. Linear regression analysis was performed to determine the correlation between BPM and mean-V.In the normal subject group, the comparison between ROM and dynamic-ROM, mean-V and SR/M have been compared before and after the BoNT-A treatment using a repeated-measures ANOVA, with the variable “condition” (baseline and test) as within-subjects factor and the variables “BPM” (40BPM and 60BPM) and “evaluator” (A and B) as between-subjects factors. Baseline and test ROM were compared using a paired T-test.In the patients groups All analyses were considered significant for p<0.05. All the measures of variability are expressed as standard deviation.dynamic-ROM values for all the tested joints. In each joint, dynamic-ROM was lower than ROM  = 213, p<0.0001; elbow: F = 80, p<0.0001; knee: F = 38, p<0.0001; ankle: F = 134, p<0.0001), without any difference among BPM  = 0.72, p = 0.55; elbow: F = 0.57, p = 0.64; knee: F = 0.48, p = 0.70; ankle: F = 2.0, p = 0.15). The value of dynamic-ROM, expressed as a percentage of ROM, was 82±6% for the wrist, 96±6% for the elbow, 86±13% for the knee and 79±9% for the ankle. No significant difference was found between baseline and test ROM (p = 0.2).Mean-V has a direct linear relationship with BPM for all the tested joints . The fitdynamic-ROM values for the wrist joint obtained by evaluator A and B, at baseline and test conditions. The value of dynamic-ROM, expressed as a percentage of ROM, was 90±21% during baseline and 87±15% during test conditions. Dynamic-ROM was lower than ROM both at baseline  and test conditions . Dynamic-ROM was similar between baseline and test conditions . No significant difference was found between BPM  nor was it found between evaluators . No significant interactions CONDITION X BPM  = 0.04, p = 0.9) and CONDITION X EVALUATORS  = 0.008, p = 0.9) were found.In all the patients, passive wrist extension movements evoked a SR on the FCR. No EMG activity was recorded while the muscle was in the shortened position before the passive extension. SR/M ratios were higher during baseline condition compared to the test condition . No difference was found between evaluators  and between BPM  . No signdynamic-ROM was smaller than ROM. It should be considered that during ROM assessment, evaluators had sufficient time to reach the extreme joint positions, minimizing the risk of damaging the joint. However, during the application of the method when dynamic-ROM was assessed, the evaluators were likely aware that the kinetic energy applied to the distal segment could have damaged the joint. The evaluators’ consideration to preserve the joint probably limited the passive movement, thus explaining the reduction of dynamic-ROM in comparison to ROM.For all the joints, the dynamic-ROM and ROM was not a factor in this linear relationship as the difference was consistent across the tested BPM frequencies.The most important finding was that the mean-V increased linearly with progressive BPM values for all the tested joints. Furthermore, the gap between the The range of BPM was large enough to produce mean-V values from about 30 to 400°/s, covering all the angular velocities used in a clinical setting These results prompted us to investigate this method’s efficacy in assessing the SR in patients with spasticity.Dynamic-ROM was smaller than ROM without any difference among evaluators, BPM values, and between conditions. Theoretically, the SR in wrist flexor muscles could have played a major role in this discrepancy. During ROM assessment, the evaluators stretched the wrist flexor muscles at speeds below the SR threshold, whereas when the dynamic-ROM was measured a SR was evoked in these muscles. The presence of the SR may have limited extension movements, thereby reducing dynamic-ROM. However, we assert that this is not the case for the following reasons. First, a similar difference between the dynamic-ROM and ROM was also found in healthy subjects, in whom a SR was not evoked. Second, the difference did not change after BoNT-A injection, when the SR amplitude was decreased. Therefore, we believe that the most likely explanation for the discrepancy between dynamic-ROM and ROM is that the evaluators were concerned for the integrity of the joint, as was suggested for the healthy subjects. However another possible explanation can also be proposed. When the examiner performs the linear movements, he is very concentrated in keeping them synchronized with the metronome while he is less concerned about reaching the actual extreme joint position. Also in normal subjects, just before reaching the extreme position when moving a joint a mild resistance related to the fibro-elastic properties of the joint and muscles con be perceived. In this sort of “dual task” condition, the role of the mild resistance near the end of the movement might disguise the examiner in considering to have reached the extreme position when he starts perceiving this mild resistance, resulting in an overall reduced dynROM.In both the baseline and test conditions, the mean-V obtained at 60 BPM was higher than that obtained at 40 BPM. Furthermore, there was no difference in mean-V values between the two evaluators as well as between the two conditions. Therefore, the passive movements obtained with our method demonstrated all the features necessary for a longitudinal SR assessment.dynamic-ROM, the mean-V obtained at test condition would have been higher than that obtained at baseline condition. This was not the case in the present set of experiments, as the treatment did not increase the dynamic-ROM. This can be attributed to the fact that we only enrolled patients who did not have important ROM limitations of the wrist joint before BoNT-A injection.In the case that the treatment with BoNT-A had induced an increase of the Even if the treatment had resulted in an increased ROM, two strategies would have been used to achieve comparable velocities between baseline and test conditions. The first strategy would have been to predetermine and limit the joint displacement instead of moving throughout the entire ROM. The second strategy would have consisted of decreasing the BPM value during the test condition. Further studies performed in patients with severe spasticity and ROM limitations are needed to test these procedures.A SR was evoked in each patient during the baseline condition, reflecting the presence of reflex hypertonia in all 12 tested subjects. The SR amplitude was highly variable among the 12 patients as has been reported in the literature To reduce the inter-subject variability, we divided the SR amplitude by the M-wave amplitude obtaining the SR/M ratio SR/M ratios did not differ between the two evaluators. Furthermore, SR/M ratios did not differ between the two BPM values. Based on the findings in healthy subjects, we decided to use 40 and 60 BPM values to test the accuracy of our method in producing passive displacements at different velocities. We can now say that a gap of 500 ms between the two tested TIs  yielded two appreciably distinct movement velocities. As the difference between the two velocities was small (31°/s), we did not find any difference between the SR/M ratios obtained at the two BPM values. This is consistent with previous results obtained from motor-driven systems For both the evaluators, the SR/M ratios decreased after BoNT-A treatment. In each patient, the SR/M ratio at test condition was less the 50% of the ratio at baseline condition. Again, these results are corroborated by previous findings obtained with motor-driven systems The method was consistent at achieving discrete velocities during linear passive movements in both healthy subjects and stroke patients. In the latter, the method was useful in detecting the reflex component of hypertonia as well as the effects induced by BoNT-A treatment.The method is easy to perform in a clinical setting and the hardware requirements are minimal, making it an attractive and robust procedure for widespread clinical assessment of reflex hypertonia.