In part, this is because viruses so intimately associate with their hosts that decoupling host and virus biology is difficult, and virus-host interactions occur at multiple scales, from within cells to populations, each of which is intrinsically complex. As a consequence, ecologists, population biologists, evolutionary biologists, and researchers from quantitative fields, including mathematics, statistics, physics and computer science, make significant contributions to the field of virology. Our understanding of virus dynamics and evolution has substantially benefited from these multidisciplinary efforts. It is now common to see advanced phylogenetic reconstruction methods used to determine the origins of emergent viruses, to estimate the effect of natural selection on virus populations, and to assess virus population dynamics. Mathematical and statistical models that elucidate complex virus and host interactions in time and space at the molecular and population level are appearing more regularly in virology and biomedical journals. Massive quantities of data now available due to technological innovation in imaging, increased disease surveillance efforts, and novel approaches to determine social contact structure are changing approaches to study the dynamics and evolution of viral infections in heterogeneous environments. The next decade presents exciting new opportunities and challenges for the expanding field of researchers investigating dynamics of viral infections that will lead to innovation and new insight on virus interactions in both individual hosts and in populations. The compilation of articles in this 1.1.et al. [in vitro infection experiments. These investigators assess the dynamics of host innate defenses to two strains of Respiratory Syncytial Virus (RSV) that have distinct epidemiological patterns but can coinfect an individual. The authors used spatial statistical methods to evaluate how the resistance of naïve cells to a challenge infection with one of the RSV strains changed as a function of proximity to a cell infected with a different RSV strain and the time between the primary and challenge infection. In this experimental setting, every infected cell is a data point. An important result of their study is that even in cell lines, asynchrony in growth and replication in a culture can change the susceptibility of cells to infection, which is presumably due to an intrinsic property of replicating or differentiating cells and not an innate immune response to virus infection. However, despite the background heterogeneity in cell susceptibility to infection, the authors demonstrate that one RSV strain, but not the other, can limit the ability of the heterologous strain to establish infection; an effect that would be obscured if the authors had used aggregate instead of point process analysis in their study. Further development of spatial statistical models will be imperative as data on responses of individual cells in population of cells become more widely available.Tissue culture is a mainstay for virologists studying cellular response to virus infection and efficacy of therapeutic interventions and treatment; response variables are typically aggregate counts of infected cells or production of a soluble factor. However, averaging values over cells, which are in different growth phases or stages of viral infection, can obscure or confound important responses to virus infection. Simenov et al.  report oSpecial Issue on “Virus Dynamics and Evolution” reveal important features of virus-host interactions by integrating detailed knowledge of viral life history strategy with viral sequence evolution over the course of infection of the natural host organisms. Virus evolutionary changes are determined by mutation rate and viral generation time and by selective pressures imposed by the host environment. These metrics inform variation in virus replication under changing host conditions. However, there are many features of virus life history strategy that can confound estimating these standard evolutionary parameters. For example, the efficacy of viral strategies to modulate host immunity is not constant over time. As the host environment changes, so does viral replication and this can complicate rate estimates that are based on viral sequence data. Harrison et al. [Three manuscripts in this n et al.  explain et al. [et al. [et al. [Carpenter et al.  address  [et al.  discuss  [et al.  for HBV.et al. [Viral infections, although complex on their own, rarely occur in isolation. Polymicrobial infections can exacerbate clinical consequences to the host but can also attenuate disease. Feline immunodeficiency virus (FIV) infection of its domestic cat host is an exemplary system to explore the mechanisms by which an avirulent FIV strain (PLV) attenuates CD4 cell loss induced by a virulent FIV in the absence of measurable antibody or cellular defenses [et al.  compared1.2.et al. [in vivo infection, virus cycling among host species does not unambiguously constrain virus fitness in either host or lead to a fitness trade off if specialization occurs. On the contrary, fitness can increase in sequential passage of virus between insects and vertebrates and, of significance, virus fitness in some vertebrate or mosquito species may be less constrained than in related host species. Thus, specialization or enhanced fitness in one environment does not necessitate trade-offs. These experimental studies highlight important areas for future research. Much of the work in this field relied on consensus sequencing and therefore failed to assess the full extent of virus diversity. Advances in sequencing technology will allow thorough exploration of sequence diversity from in vitro evolution experiments. However, our understanding of the significance of diversity in viral populations is also limited . DetailWhereas virus life history strategy is a key factor driving virus dynamics and evolution within a host, host life history, behavior, and social structure have profound effects on the patterns of virus transmission and consequently the dynamics of viruses in populations. For example, the number and type of contacts an individual has affects virus transmission in a specified population. Network science provides an intriguing approach to study virus spread in a population of known contact structure . The rel1.3.et al. [Virus infections affect the population structure of host organisms and can have a profound influence on the interaction among individuals and their environment. Hence, the consequences of virus and other pathogen infection are central to the interests of ecologists. Vandegrift et al.  discuss et al.  in this At cellular to population scales, our paradigms in virology are largely based on virus infections that have a detrimental affect on the host. However many—and probably most—viruses are not pathogens and some are beneficial to their host. Not surprisingly, the ecology and biology of apathogenic viruses have not been extensively investigated. The article by Roossinck  provides2.Special Issue reveal new information about virus host interaction at each of these scales and demonstrate the unique perspective that virus evolution provides into the complex environment of the host. New tools will continue to capture virus dynamics and evolutionary change at finer spatial and temporal scales, which will lead to new challenges and opportunities for researchers trained in a variety of disciplines to contribute to data analysis and interpretation. Collectively, these efforts will advance our knowledge of viral pathogenesis, elucidate innovation in evolving protein structures, provide specific targets for therapeutic intervention, and inform models of infection spread in heterogeneous populations.Virus infection is inherently a dynamic process from the protein to the population level. The articles in this


To the Editor: St. Louis encephalitis virus (SLEV) is a flavivirus that can asymptomatically infect humans or cause clinically apparent disease that manifests with fever, headache, nausea, and vomiting . The woman’s recent travel was limited to a 70-km radius from this town. Because influenza was suspected, an oropharyngeal swab specimen was collected as part of an influenza-like illness surveillance project, which had been approved by the US Naval Medical Research Center Institutional Review Board and endorsed by the Peruvian Ministry of Health. No blood specimen was obtained because blood was not collected in this respiratory infection–focused protocol. The swab specimen was inoculated onto Madin-Darby canine kidney cells; no cytopathic effect was observed, and the culture was negative for influenza virus and for other respiratory viruses amenable to culture .Nearly 6 years later, as part of a retrospective study of previously negative respiratory specimens, the sample was reevaluated for arboviral infection. Universal transport medium , containing the swab specimen, was inoculated onto Vero 76 cells; cytopathic effects were revealed on day 7. The cells were harvested, and an indirect immunofluorescence assay was performed by using a panel of mouse polyclonal hyperimmune ascitic fluid specific to a variety of flaviviruses. The initial screening tests indicated reactivity to yellow fever virus, dengue virus, and SLEV. Subsequent immunofluorescence assay analyses using monoclonal antibodies against yellow fever virus and all 4 dengue virus serotypes were negative.Viral RNA was recovered from the Vero culture supernatant and amplified by conventional reverse transcription PCR/nested PCR with generic flavivirus primers against the nonstructural 5 coding region, which confirmed that the isolate was a flavivirus. Real-time reverse transcription PCR with specific SLEV primers confirmed SLEV.A total of 10,850 bp, almost the full genome sequence of the virus, were sequenced (GenBank accession no. KF589299), and 10,236 nt from these sequences were compared with other SLEV sequences in GenBank. The strain showed 98.4% similarity with a SLEV strain isolated from a bird in Brazil in 1973, 98.4% similarity with an SLEV strain isolated from mosquitoes in Peru in 1975, and 97.9% similarity with an SLEV strain isolated from mosquitoes in the United States in 2003. The US strain may have been carried by migratory birds from Latin America  and withCulex spp. mosquitoes, the main vectors of SLEV in Brazil and Argentina, have been shown to carry SLEV in Peru (,Our findings bolster previous serologic investigations, adding Peru to the South American countries reporting this virus in humans. This discovery is not surprising because Although SLEV disease in humans is usually confirmed by testing blood or cerebrospinal fluid, the isolate described here was recovered from an oropharyngeal swab specimen. The presence of this virus in human respiratory samples was previously suggested by a study in which nasal wash samples from SLEV-infected persons were injected intranasally into mice, which induced immunity to a subsequent intracerebral challenge with SLEV (Our findings have many implications. First, SLEV may cause human disease in a wider area of South America than was previously known. In addition, SLEV can cause influenza-like illness and may elude identification unless specific assays are used. Finally, the upper respiratory tract may offer a less invasive way of recovering SLEV isolates than by lumbar puncture or even by drawing blood, although further investigations are needed.


However, the advent of numerous immunological and molecular methods has facilitated the study of BRSV enormously. The knowledge gained from these studies has also provided the opportunity to develop safe, stable, attenuated virus vaccine candidates. Nonetheless, many aspects of the epidemiology, molecular epidemiology and evolution of the virus are still not fully understood. The natural course of infection is rather complex and further complicates diagnosis, treatment and the implementation of preventive measures aimed to control the disease. Therefore, understanding the mechanisms by which BRSV is able to establish infection is needed to prevent viral and disease spread. This review discusses important information regarding the epidemiology and molecular epidemiology of BRSV worldwide, and it highlights the importance of viral evolution in virus transmission.The bovine respiratory syncytial virus (BRSV) is an enveloped, negative sense, single-stranded RNA virus belonging to the pneumovirus genus within the family Paramyxoviridae. BRSV has been recognized as a major cause of respiratory disease in young calves since the early 1970s. The analysis of BRSV infection was originally hampered by its characteristic lability and poor growth  The bovine respiratory syncytial virus (BRSV) has been recognized as a pathogen in cattle responsible of an acute respiratory disease syndrome in beef and dairy calves since the early 1970s . The impPneumovirus genus within the subfamily Pneumovirinae, family Paramyxoviridae [BRSV is an enveloped, non-segmented, negative-stranded RNA virus belonging to the oviridae . The BRSoviridae .BRSV is closely related to human RSV (HRSV), and the epidemiology and pathogenesis of infection between these two viruses share some similarities and also many differences . The simUnderstanding of the global epidemiology and molecular epidemiology of BRSV has significantly improved over recent years. In this review, we discuss various aspects of the epidemiology and molecular epidemiology of BRSV as well as their relationship with viral evolution.BRSV infection is widely spread around the world, most likely as a direct result of the movement of cattle . RegardlBRSV outbreaks commonly occur during winter . Thus, cBRSV outbreaks can become epidemics affecting animals in all age groups. However, the age distribution of BRSV infection seems to be a function of exposure. In other words, herds that have been previously exposed to the virus tend to experience infections that are limited to younger, more susceptible animals. In consequence, morbidity is commonly high during the occurrence of outbreaks . ImportaThe mechanisms that are responsible for the survival of the virus within a given population are not fully understood. Controversial information has been reported about viral persistence. Nonetheless, chronicity has been proposed as a mechanism that might play role in disease spread . BRSV caThe prevalence of the disease varies greatly in North America. For instance, in the United States, original studies conducted during the early 1970s showed a frequency of infection of 67% among adult cattle . HoweverIn Canada, initial reports have shown that the overall frequency of BRSV infection might be nearly 36% ,17. HoweIn Europe, shortly after the virus’s discovery, it was reported to have widely circulated in different parts of the continent . In SwedIn Africa, Ethiopia and South Africa have also been shown to have high incidences of BRSV infection ,46,47,48Thus, more information is required to understand the mechanisms that allow for viral survival in a given geographical region. Monitoring of outbreaks among herds is likely to provide valuable information that might help us to understand in greater detail the epidemiology of BRSV infection.Limited information is available regarding the molecular epidemiology of BRSV infection. However, the rapidly changing field of DNA sequencing might help to unveil the molecular mechanisms exploited by BRSV to assure transmission. Additionally, the field of phylogenetics has contributed to identify the existence of diverse BRSV genotypes and their genetic relationship while helping understand the molecular basis of BRSV genetic history ,51. PhylThe occurrence of antigenic variation among BRSV isolates was first suggested by the lack of reaction between a polyclonal serum made against one particular viral strain, which failed to recognize a different isolate . FurtherThe study of the molecular epidemiology of BRSV began with the identification of the nucleotide sequences of the glycoprotein (G), fusion (F), nucleocapsid (N), matrix (M), phosphoprotein (P), small hydrophobic (SH) and M2 proteins during the early 1990s ,65,66,67More comprehensive studies, which analyzed the reaction patterns against anti-BRSV G protein and the genetic diversity occurring in a larger segment of the G protein gene from several isolates obtained from different geographical regions, showed that the intragroup genetic variation among BRSV strains ranged between 88% and 100% . The corA large-scale study addressing the global molecular epidemiology and evolution of BRSV included 54 European and North American isolates, in addition to previously reported sequences . The stu4 to 10.1x104 substitutions per nucleotide. These findings suggest that BRSV populations evolve as complex and dynamic mutant swarms, despite the virus’s apparent genetic stability.Importantly, BRSV intrahost population has been shown to exist as a complex mixture of viral variants, ambiguously referred as “quasispecies” . AnalyseMolecular epidemiology approaches have shown the circulation of identical viruses among animals within the same herds, especially during the occurrence of outbreaks . However–2011) occurring among 30 different herds [The global molecular epidemiology of BRSV has remained elusive. Only sparse reports addressing the distribution of BRSV groups are available in the literature. Consequently, the molecular characterization of BRSV isolates from representative regions of the world is still rather incomplete. A study conducted in Japan in the early 2000s reported the circulation of strains belonging to subgroup III. The isolates were further subdivided into two distinctive lineages . Unfortunt herds . The repnt herds . Thus, bIn summary, the study of the molecular epidemiology of BRSV has evolved considerably over the past two decades. The information generated by the study of the molecular characterization, phylogenetics and evolution of BRSV strains have broadly advanced our understanding of the molecular mechanisms controlling virus transmission and disease spread. However, this field requires extensive research to unveil the means exploited by the virus to attain persistence in a given population. The establishment of molecular surveillance of BRSV in different geographical regions will likely improve the identification of outbreaks, resulting in the implementation of preventive measures aimed to control the disease. The advent of next-generation sequencing platforms on the eve of the “DNA-sequencing era” could also provide a unique opportunity for the discovery of the underlying processes responsible for viral replication and survival in the host.BRSV has been recognized as an important cause of respiratory disease in cattle for nearly four decades. The characteristic heterogeneity of the viral genome and its low fidelity in replication are some of the most important features that the virus exploits to assure its survival and persistence within the host. The instability of the viral particle usually leads to unsuccessful attempts to isolate viral strains in the laboratory from clinical specimens. Consequently, molecular approaches are rapidly becoming the gold standard for the correct identification and characterization of BRSV in clinical cases. As result, the field of the molecular epidemiology of BRSV has gained significant strength and has further enhanced our knowledge about BRSV distribution and transmission patterns worldwide. The arrival of new and more sophisticated molecular methods, including next-generation sequencing, will most likely help to unveil the genetic makeup of the circulating viral population in different geographical regions, as well as the mechanisms on which the virus relies for survival, persistence and transmission.


Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the  Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus, and other medically important viruses, is a cause of encephalitis in humans and animals. SLEV is considered endemic in the Americas, and currently there is no vaccine or specific treatment available for controlling of preventing SLEV-induced encephalitis. In this study we describe the first isolation of SLEV from an adult male horse with neurologic disease, which was further characterized by molecular and serological methods. Phylogenetic analysis of a 903 base pairs amplified sequence from partial Envelope (E) gene region indicated that the isolate from the horse was within the cluster of the VB genotype. In addition, inoculation of the SLEV isolate intracranially in newborn mice resulted in circulatory and neurological changes. This is the first report of isolation of SLEV from a horse with neurological disease in Brazil.St. Louis encephalitis virus (SLEV), a member of the  St. Louis encephalitis virus (SLEV) is a mosquito-borne virus that causes human and animal encephalitis in the Western hemisphere. SLEV is considered endemic in the Americas, with encephalitis cases being diagnosed from Canada to Argentina Flavivirus genus in the Flaviviridae family, together with several important pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), Dengue virus (DENV), Yellow fever virus (YFV) and others SLEV is a single-stranded positive sense RNA virus, with approximately 50 nm in diameter and a genome of 11 kb. SLEV is a member of the Human infections with SLEV are mostly asymptomatic. Infected individuals can present mild malaise or flu-like symptoms, especially young or middle-aged patients SLEV has been detected in Brazil for over 40 years, isolated from arthropods Here we describe the first isolation of SLEV from a horse with neurological signs in Brazil. SLEV identity was confirmed by molecular and serological techniques, and by inoculation of newborn mice. Our findings highlight the importance of effective arboviral surveillance.Our animal study followed national guidelines , which governs the use of animals for experimental procedures. All experimental procedures were approved and complied with the University of Minas Gerais (UFMG) Committee for Ethics in Animal Experimentation (CETEA) regulations, under protocol number 163/2011.ad libitum, under specific pathogen-free conditions, in the animal warren at the Departamento de Clínica e Cirurgia Veterinárias of UFMG.Pregnant female mice were acquired from Centro de Bioterismo (CEBIO) of UFMG . Newborn Swiss mice (24 hours old) were used in animal model development experiments. All mice were kept under controlled temperature (23°C) with a strict 12 h light/dark cycle, food and water available RRYATGGGYGAGTATGGRACAG, SLE 2517 (−) CTCCTCCACAYTTYARTTCACG, and primers for the final amplification were SLE (+) 2002 TGGAYTGGACRCCGGTTGGAAG and SLE (−) 2257 CCAATRGATCCRAARTCCCACG.Brain tissue from horses that presented neurological symptoms before death were sent to Laboratório de Saúde Animal at Instituto Mineiro de Agropecuária (LSA/IMA) in Belo Horizonte, Brazil. Samples that were PCR negative for Rabies virus were sent to Laboratório de Patologia Molecular at UFMG and stored at −80°C. Tissue samples were processed for RNA extraction and screened by nested RT-PCR. There were no tissue samples available for histopathology. Reaction parameters and primers were used as described by Ré and colleagues SLEV RT-PCR amplicon band was purified from an agarose gel and sequenced in Megabace 1000 sequencer. Edited sequences were aligned by CLUSTAL/W, using the BioEdit program, version 5.09. The resulting sequence was deposited in GenBank (accession number KF718857).www.megasoftware.net), version 4 (MEGA 4) http://blast.ncbi.nlm.nih.gov/). Construction of the phylogenetic was based on 39 other SLEV sequences available at GenBank, which were distributed within the following genotypes: IA, IB, IIA, IIB, IIC, IID, IIE, IIF, IIG, III, IV, VA, VB, VI, VII, VIIIa; VIIIB and 3 out-groups: WNV, JEV, and DENV, as detailed in A phylogenetic tree was generated using the Molecular Evolutionary Genetics Analysis software  daily. Viral stocks were collected for up to five days post-infection and re-inoculated (500 µL of supernatant on a fresh culture) two times, for a total of three passages. The viral titer from supernatant of the third passage was determined by focus immunodetection assay (FIA) as previously described To characterize the isolated SLEV strain, 40 µL of SLEV stocks were inoculated in newborn mice by intracranial route . Homogenates of a pool of brain samples (10% in PBS) from the littermates were used for preparing the inoculum for the next passage. Infected newborn brains were collected at the onset of neurological disease or at day 7 post-infection, to produce new virus stocks or to process for histological analysis. Brain suspensions were passed seven times in newborn mouse brains and relevant experimental controls were maintained. Clinical alterations were assessed daily in newborns after each SLEV passage and representative pictures were taken.Tissue samples, including brain, kidney, liver, lung, heart, and fragments of the thoracic and pelvic limbs, were obtained from two newborn mice at each SLEV passage, immediately fixed in 4% buffered formaldehyde, processed and embedded in paraffin. Tissue sections (4 µm thick) were stained with hematoxylin and eosin (HE), and examined under light microscopy. Micrographs were taken using a Spot Insight color camera coupled to Olympus BX41 microscope.th SLEV passage in newborn mice, at a multiplicity of infection (MOI) of 1, for 1 h. At 24 hours post-infection, cells were fixed, permeabilized and stained with a mouse anti-SLEV monoclonal antibody  followed by Alexafluor 488-labelled secondary anti-mouse . Experiments were performed with control group with or without primary antibodies. Stained coverslips were mounted in Mowiol 4–88  and analyzed using an Olympus BX61WI microscope equipped with a FV300 confocal scanning unit. Images were analyzed with imageJ software.C6/36 cells were harvested and seeded in 24-well plates with gelatin-coated coverslips, and incubated at 28°C for at least 3 hours. Cells were infected with a virus stock derived from the 7Sarcocystis neurona.From a total of 170 brain samples from horses with neurological disease received and analyzed by PCR at the Laboratório de Patologia Molecular at UFMG, one sample was identified as positive for SLEV. This brain sample was obtained in March 2009 (late summer) from a 12 years-old male horse of undefined breed, which died 72 hours after presenting neurological signs. Those neurological signs were described as incoordination, depression, and flaccid paralysis of the hind limbs. The horse came from a farm in Abaeté, countryside of Minas Gerais State, 207 kilometers from the capital, Belo Horizonte. In the same farm there were two additional horses that remained clinically healthy. Importantly, this SLEV-positive brain sample was negative for other pathogens commonly associated with encephalitis in horses, including Rabies virus, Equine Herpesvirus-1, Equine Herpesvirus-4, West Nile virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan encephalitis virus, and The SLEV amplicon originated by the RT-PCR reaction was sequenced and deposited in GenBank (Submission ID #1663732), referring to SLEV strain MG150.http://blast.ncbi.nlm.nih.gov/). Among the foreign isolates with a similar level of nucleotide identity (i.e. 98%), one (F72M022), also genotype VB originated in Florida from opossum in 2006 Phylogenetic analysis of a 903 bp amplified sequence from partial Envelope (E) gene region 3 FFU/mL of supernatant.After confirmation of the virus identity by sequencing, we focused our efforts on isolating SLEV from the horse tissue. A brain fragment from the SLEV-positive horse was homogenized and inoculated in C6/36 mosquito cells, which is a cell lineage suitable for arbovirus propagation. All passages were tested for SLEV by RT-PCR, and were all positive beginning at the second passage. The virus was isolated after three passages, obtaining a SLEV stock of 1×10in vivo, we performed serial passages of SLEV by intracranial inoculation of newborn mice, which allowed us to gather some data on MG150 strain pathogenicity. Increase of clinical signs and circulatory changes were associated with increased mortality that reached 100% at the 7th passage  resulting from each passage in mice were submitted for RNA extraction for confirmation of viral detection by RT-PCR (data not shown). The same procedure was performed with organs that had gross changes. Pool of organs, including the liver, heart, kidney, and lung from the 4th SLEV passage, fixed and stained 24 hours post-infection. Infected cells were positively stained with the anti-SLEV antibody, which was not observed in mock-infected cells, confirming the identity of this SLEV strain in an antibody-based test (data not shown).To further confirm SLEV identity, we performed an immunofluorescence assay, using a commercial monoclonal antibody to detect SLEV proteins in cell culture. Monolayers of C6/36 mosquito cells were infected with the 7The first isolation of SLEV from a horse that died due to a neurological disease in Brazil is a significant event. Virus isolation from horse brain tissue, together with molecular and immunofluorescence data, confirms that SLEV was the agent that caused disease and, ultimately, the horse death in this case. To our knowledge, this is the first observation that SLEV can cause disease in wild or domestic animals, which indicate that some aspects of SLEV viral cycle and its ability to cause disease need further studies. Furthermore, a model of newborn mice infection for characterization of SLEV was thoroughly described.In terms of public health and epidemiology, the first identification and isolation of SLEV in the State of Minas Gerais adds to previous reports regarding SLEV detection in Brazil Despite the existence of eight lineages and fifteen subtypes of SLEV, namely IA, IB, IIA, IIB, IIC, IID, IIG, III, IV, VA, VB, VI, VII, VIIIA, and VIIIB, phylogenetic studies based on the E gene indicate that genotypes I and II are found predominantly in North America, whereas genotypes III to VIII have been isolated in South and Central Americas in vivo, to study its pathogenicity, we noticed the virus progressively adapted to serial passagens in newborn mice. The mouse has been previously used as a model for assessing SLEV virulence In our efforts to characterize SLEV strain MG150


Cryptococcus neoformans-gattii species complex, the etiologic agents of cryptococcosis, can be differentiated by biological, biochemical, serological and molecular typing techniques. Based on their differences in carbon and nitrogen utilization patterns, cost effective and very specific diagnostic tests using D-proline and canvanine-glycine-bromthymol blue (CGB) media have been formulated and are widely used for identification of the two species. However, these methods have yet to be tested for strains with confirmed molecular types to assess the degree of specificity for each molecular type in the two species. We collected global isolates of every major molecular type available and tested their patterns of nitrogen utilization. We confirmed specificity of the CGB test to be 100% regardless of molecular type while the D-proline test yielded 8–38% false negative results in three of the four C. gattii molecular types, VGI–VGIII. The utilization pattern of a new set of amino acids: D-alanine, L-tryptophan and L-phenylalanine, showed species specificity comparable to that of D-proline. We discovered that the transcription factor Gat1 (Are1) regulates the utilization of nitrogen differently between C. neoformans and C. gattii strains. Unlike in C. neoformans, expression of the genes encoding glycine decarboxylase complex in C. gatti was only partially suppressed by nitrogen catabolite repression in the presence of ammonium. GAT1 in C. neoformans controlled the induction of three of the four genes encoding the glycine decarboxylase complex when glycine was used as the sole nitrogen source while in C. gattii its regulation of these genes was less stringent. Moreover, while virulence of C. neoformans strains in mice was not affected by Gat1, the transcription factor positively influenced the virulence of C. gattii strain.Two members of the  Cryptococcus neoformans-gattii species complex (Csc) are basidiomycetous yeasts that causes cryptococcosis in humans and animals world-wide C. neoformans and C. gattii each composed of various subtypes are recognized within the complex Csc can be separated into 8 molecular types, VNI-IV and VGI-IV that correlate with four serotypes C. neoformans: VNI and VNII – serotype A, var. grubii; VNIII – serotype AD; VNIV – serotype D, var. neoformans and C. gattii: VGI-IV – serotype B or C, which was raised to species level in 2002 C. neoformans, VNB, was recently discovered as a unique cryptococcal population in Botswana C. neoformans mainly causes meningoencephalitis in HIV infected patients worldwide C. gattii causes diseases more commonly in non-HIV patients and has gained its importance recently as the causative agent of the cryptococcosis outbreak on Vancouver Island in Canada C. neoformans reported world-wide is usually associated with pigeon guano C. gattii, on the other hand, is known to be associated with a variety of trees, especially Eucalyptus trees C. gattii has been expanded to areas with temperate climates such as the Pacific Northwest region of USA and Canada Two members of C. neoformans but not in C. gattiiC. neoformans from C. gattiiC. gattii is known to utilize glycine and dicarboxylic acids as carbon sources more effectively than C. neoformansC. gattii strains utilized glycine, only 20% of C. neoformans strains were reported to utilize glycine as a carbon source C. neoformans and the 70 strains of C. gattii tested Previous studies have reported that the two species are significantly different in their patterns of nitrogen and carbon utilization. Utilization of creatinine as a nitrogen source was among the first such studies undertaken and found that the strains of serotype A utilize creatinine less efficiently than strains of the other serotypes GAT1 also contributes to the virulence of pathogenic fungi in both humans GAT1 significantly reduced the virulence of Candida albicansAspergillus fumigatusC. neoformans, the role of GAT1 was extensively characterized where a relationship was observed between the transcription factor and the genes that are associated with cryptococcal virulence GAT1 has been shown to regulate melanin synthesis, capsule production, mating in vitro and virulence in either invertebrate and vertebrate hosts C. neoformans and C. gattii in the utilization of several nitrogen sources, the role of GAT1 in regulation of nitrogen assimilation has not been studied.Utilization of limited nitrogen sources in the host environment is crucial for growth of any pathogenic organism. In order to efficiently regulate the use of nitrogen when abundant, nitrogen catabolite repression (NCR) or nitrogen metabolite repression is induced. NCR ensures the use of preferred nitrogen sources first by suppressing the degradative pathways for secondary nitrogen sources until the readily assimilable nitrogen sources have been exhausted C. neformans and relatively little attention has been paid to C. gattii. Comparative biological studies between the two species were mostly carried out prior to the advent of AIDS epidemics C. gattii on Vancouver Island Canada in 1999 C. gattii has gained prominence as a primary pathogen. Genetic characterizations of C. gattii have been conducted mostly to decipher the virulence traits of the species GAT1 regulation of the amino acid utilization and its effect on virulence.Although cryptococcosis caused by both species was discovered more than 100 years ago C. neoformans and C. gattii, respectively, since these two genome sequenced strains are typically used for molecular genetics and pathogenesis studies URA5-RFLP and then the strains were tested for utilization of the four amino acids as the source of nitrogen  C. gattii strains were false negative. False negatives were obtained for three of the four C. gattii molecular types (VGI–VGIII) with the highest frequency observed among strains of molecular type VGI (38%) followed by VGIII (18%) and VGII (8%). No false negative results were obtained with VGIV strains. As is the case with D-proline, no C. neoformans strains utilized D-alanine . Among the C. gatti strains tested, only 6% could utilize L-Phenylalanine while none could utilize L-Tryptophan as a nitrogen source  and VNIII, the serotype A/D hybrid  molecular type strains. Most VNIV strains (82%) poorly utilized L-Phenylalanine or L-Tryptophan as the sole nitrogen source  gat1Δ strains of H99 and R265, as representatives of the two species, differed significantly in their ability to utilize various nitrogen sources. Growth of R265gat1Δ and H99gat1Δ strains was markedly reduced in all nitrogen sources tested except for proline and arginine where only a slight reduction was observed for both species. Unlike C. neoformans, only a slight reduction of alanine utilization was observed in C. gattii. Growth of R265gat1Δ on L-leucine, L-lysine, and L-aspartate was significantly reduced compared to H99gat1Δ. On the other hand, GAT1 disruption caused only slight reduction in the growth of R265 on glycine and creatinine while the GAT1 disruption in H99 totally abolished its ability to utilize the same nitrogen source . In H99, expression of all four genes was significantly up-regulated when glycine was used as the sole nitrogen source compared to ammonium sulfate  yet to be identified also regulates their induction when glycine is used as the sole nitrogen source. In R265gat1Δ, on the other hand, the expression levels of these four genes were close to wild type levels. These data suggest that the role of GAT1 is less prominent in R265 than in H99 with respect to expressison of the four glycine decarboxylase genes when glycine is the sole nitrogen source.We also examined the roles of GAT1 in H99 altered its virulence GAT1 deletion in H99 and R265 on the expression of virulence factors in vitro including melanin and capsule production, ability to grow at 37°C and cell wall integrity in the presence of caffeine. Expression of the LAC1 gene which encodes laccase responsible for melanin production gat1Δ strains of both H99 and R265 exhibited an increase in melanin production  and the expression of LAC1 was found to be upregulated    . When cactively) . Howeverild type . Howeverild type , a cell Csc also has been effectively carried out by both nitrogen and carbon utilization tests C. gattii while we found a much lower specificity; only 80% of C. gattii strains yielded positive results in contrast to the 100% that were negative for C. neoformans. The CGB media, on the other hand, showed 100% specificity between the two species regardless of molecular type. Remarkably, the false negatives observed for D-proline utilization were found mainly among strains of the VGI and VGIII (5 negatives among 6 strains) molecular types. According to the phylogenetic analysis of global Csc strains based on multi gene sequence analyses, the VGI and VGIII molecular types formed a separate cluster with high bootstrap supports C. gattii.The ability to utilize specific nitrogen and carbon sources is among the standard methods used for the identification of yeast species C. neoformans from C. gattii strains with reasonably high specificity. In fact, a previous study with much fewer numbers of strains also showed the discriminating power of phenylalanine and tryptophan utilization between the two species C. gattii strains that produce questionable CGB results can be tested for growth on tryptophan media in order to correct identify the questionable strains. Although molecular techniques enable confirmation of species identity, use of the one-step diagnostic media employing a species specific nitrogen source could be a cost effective option for developing countries with limited resources.Upon screening for utilization of various amino acids as sole nitrogen sources, we discovered a new set of amino acids, phenylalanine, tryptophan and D-alanine, that could discriminate C. neoformans was studied based on the role of the GATA-type transcriptional activator Gat1 C. gattii was not as stringent as in C. neoformans while it was opposite in the utilization of aspartate. These results were supported by transcription analysis of the four genes encoding the glycine cleavage enzyme complex where the induction of these genes was positively controlled by Gat1 in C. neoformans but to a less extent in C. gattii. In addition, the NCR of glycine utilization in the presence of ammonium was clear-cut in C. neoformans but not in C. gattii. The difference in NCR of glycine utilization between the two species is reminiscent of creatinine metabolism where creatinine deiminase was inhibited by the accumulation of ammonium only in C. neoformans and not in C. gattii. As a result, growth of C. neoformans on creatinine media is considerably slower compared to C. gattii and creatinine bromthymol blue agar was the first one-step diagnostic media formulated to differentiate between the two species S. cerevisiae, glycine can be synthesized from or converted to serine and threonine by serine hydroxymethyltransferase (Shm1 and Shm2) and threonine aldolase (Gly1). However, both species of Csc do not utilize threonine as effectively as other amino acids . The nitrogen catabolic pathway, therefore, appears to be complex and further studies are needed to elucidate the pathways in Csc.The nitrogen regulation by NCR in no acids  suggestiAspergillus fumigatus and C. albicansgat1Δ is similar to the findings of Lee et al. gat1Δ behaved differently between the two species in controlling the expression of some virulence factors in vitro and virulence in animals. It was surprising to observe that several virulence factors including capsule production, melanin synthesis and cell wall integrity, were negatively controlled by Gat1 in C. gattii while the virulence of R265gat1Δ was decreased in mice. In the host environment, the impact of impaired nitrogen utilization due to the lack of Gat1 function may be compensated by the increase in the virulence factors. The different impact of Gat1 in virulence between the two species might stem from the differences in their natural habitat and hence their pathobiology. It has been speculated that the virulence factors of the Cryptococcal species have evolved to combat their environmental predators such as soil amoeba and nematodes C. neoformans, is high in ammonium concentration and Gat1 may be constantly down-regulated while the primary natural habitat of C. gattii is detritus of trees which should be much lower in ammonium content than pigeon guano. This would have had a different impact on evolution of the role of Gat1 in regulation of virulence factors. However, whether the slight impact of Gat1 on the regulation of virulence factors observed in this study benefits survival of Csc in nature is unclear.The ability to survive in a limited nutrient environment in the host is crucial for the growth of pathogenic organisms. For example, Gat1 influences virulence of several pathogenic fungi including, C. neoformans and C. gattii, two closely related etiologic agents of cryptococcosis. The range of nitrogen sources that can be used to differentiate between the two species has been expanded from previous studies to include a few more amino acids. The difference in nitrogen metabolism was confirmed by differences in nitrogen regulation by Gat1 and ammonium NCR which has a distinct effect on virulence between the two species. The difference in nitrogen utilization and its regulation by Gat1 shown in this study provides a compelling reason for characterization of the genes and metabolic pathways responsible for the differences. Global screening techniques namely transcriptome comparisons via cDNA microarrays and construction of Agrobacterium tumefaciens mediated insertional libraries Csc.In conclusion, our study points out the different aspects of nitrogen metabolism between The animal experiments were carried out with the approval and oversight of the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, United States National Institutes of Health, Bethesda MD.C. neoformans and 36 C. gattii strains were revived from −80°C freezers of Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, MD, USA and Molecular Mycology Research Laboratory, Westmead Hospital, Westmead, NSW, Australia. Molecular types of each strain were confirmed by URA5-RFLP as previously described (data not shown) A global collection of 8 molecular types consisting of 31 S. cerevisiae genome project (http://www.yeastgenome.org/) were used to perform the BlastP in C. neoformans H99 genome project (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) and C. gattii R265 genome project (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans_b/MultiHome.html). Genes were identified as follows (C. neoformans/C. gattii): GAT1– CNAG_00193/CNBG_0368, GCV1 – CNAG_02818/CNBG_3472, GCV2 – CNAG_01594/CNBG_3846, GCV3 – CNAG_06316/CNBG_5135, LPD1 – CNAG_02851/CNBG_5871.Protein sequences of each gene from GAT1 were created using overlapping PCR to link the 5′ and 3′ flanking region to nourseothricin antibiotic cassette GAT1 gene into gat1Δ using biolistic transformation. As none of gat1Δ was able to utilize L-serine as the sole nitrogen source, transformants grown on serine media (2% glucose YNB with 10 mM L-serine) were analyzed for the homologous complementations by PCR and confirmed by southern hybridization.  and re-suspended (1 OD600) in 2% glucose YNB supplemented with 10 mM of the specified nitrogen source. Cells were then grown at 30°C for 2 hrs and harvested for RNA extraction. Cell pellets were frozen and lyophilized. RNAs were extracted using RNA by Trizol® reagent  and cDNAs were generated by the Supercript III first-strand synthesis system  according to the manufacturer's protocol. Quantitative real-time PCR were performed using SYBR greener supermix  in Applied Biosystems 7500 Real-Time PCR System. Experiments were performed in triplicates using a relative standard curve method. .Quantification of laccase activity for melanin synthesis was performed in triplicate according to the described method 3, pH 7.3 and grown to a cell density of 106–107 cells/ml at 37°C with 5% CO2 for 72 hr Amount of capsule produced was quantified by comparing the size ratio between cell with the capsules vs naked yeast  as has been reported previously Strains were grown on YPD with or without 1 mg/ml caffeine, a known cell wall perturbing agent 6 cells/ml. Mice were anesthetized by isoflurane and partially hung by placing their incisors on a string while their lower back lie to a support patch. Tongues of the mice were gently held in full extension with padded forceps while a 20 µl suspension was pipetted onto the base of the tongue. Mice were allowed to breathe in the solution for 10 seconds. Tongues were then released and mice were placed in their cages to recover. Survival was monitored for 50 days.BALB/c mice were inoculated with 50,000 yeast cells via intrapharyngeal inhalation as described All statistical analyses specified were performed in SPSS 17.0 .Figure S1Examples of GAT1/AREA complementation which restored nitrogen utilization to the wild type level. Wild type, gat1Δ and complemented strains were grown on 2% glucose YNB with 10 mM of each nitrogen source for 5–7 days at 30°C. . 5 µl of cells at OD600nm of 10, 0.1 and 0.001 were spotted on the media.(TIF)Click here for additional data file.Table S1Primers details.(DOCX)Click here for additional data file.


IGF2-H19 locus, resulting in insulin-like growth factor 2 (IGF2) transcription from both paternally and maternally inherited chromosomes, and overall IGF2 overexpression. As the DLK1-GTL2 locus is structurally similar to the IGF2-H19 locus, the status of parental imprinting of the DLK1-GTL2 locus was studied in RMS. We observed that while both embryonal and alveolar rhabdomyosarcomas  show LOI of the DMR at the IGF2-H19 locus, imprinting of the DMR at the DLK1-GTL2 locus varies in association with the histological subtype of RMS. We found that, while ERMS tumors consistently show LOI of the DMR at the DLK1-GTL2 locus, ARMS tumors have erasure of imprinting (EOI) at this locus. These changes in imprinting status of the DLK1-GTL2 locus result in a higher GTL2/DLK1 mRNA ratio in ARMS as compared to ERMS. This difference in imprinting elucidates a novel genetic difference between these two RMS subtypes and may provide a potential diagnostic tool to distinguish between these subtypes.Parental imprinting of differentially methylated regions (DMRs) contributes to appropriate expression of several developmentally important genes from paternally or maternally derived chromosomes. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is associated with altered expression of certain parentally imprinted genes. As previously reported, RMS cells display loss of imprinting (LOI) of the DMR at the  DLK1) and gene trap locus 2  gene cluster is located on human chromosome 14. The importance of the DLK1-GTL2 locus in skeletal muscle development is supported by the fact that a single-nucleotide polymorphism at this locus deregulates its expression and leads to DLK1-mediated skeletal muscle hypertrophy, as seen in callipyge sheep (The delta-like homolog 1 (Rhabdomyosarcoma (RMS), a family of cancers related to the skeletal muscle lineage, is the most common soft-tissue sarcoma in children. From a histological and clinical perspective, there are two major histological subtypes of RMS: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) . ClinicaIGF2 and DLK1) are methylated within DMRs on paternally derived chromosomes.Evidence has accumulated that imprinted genes play a role in RMS pathogenesis ,5. In thAccording to the parent-offspring conflict theory, while paternally expressed imprinted genes enhance embryo growth and size of the offspring, maternally expressed genes inhibit cell proliferation and negatively affect cell size –9. BasedIGF2-H19 and DLK1-GTL2 tandem loci reveals significant similarities. The molecular structure of these imprinted loci is very similar. The DMRs for these tandem genes are both located between the two genes within each locus  (IGF2 encodes insulin-like growth factor-2 (IGF-2) and DLK1 encodes transmembrane delta-like 1 protein, which contains six epidermal growth factor (EGF) repeat motifs and is involved in cell differentiation in a juxtacrine/paracrine manner. Both DLK1 and IGF2 are transcribed from paternal chromosomes and are involved in skeletal muscle development as stimulators of skeletal muscle growth (Comparison of the ctively) ,11, and e growth ,13. By cIGF2-H19 locus located at the 11p15.5 chromosome region is observed both in ERMS and ARMS. Furthermore, this LOI have been reported in both sporadic tumors and in RMS originating in Beckwith-Wiedemann syndrome patients  in this regulatory region. Despite EOI of the IG-DMR within the DLK1-GTL2 locus, ARMS cells highly express DLK1 mRNA. Therefore, regulation of DLK1 seems to be more complicated in RMS cells than initially thought. These findings have biological implications and may also have diagnostic utility.Loss of imprinting (LOI) of the IG-DMR at the For 35 of the samples analyzed, frozen tumor samples were provided by the Pediatric Division of the Cooperative Human Tissue Network without patient identifiers. The only information provided was histologic diagnosis and fraction of tumor cells in a frozen section from the block from which the sample was taken. In the samples, the fraction of tumor cells varied from 65 to 90%. In 17 of the samples, the histologic subtype was embryonal rhabdomyosarcoma and in 18 of the samples the histologic subtype was alveolar rhabdomyosarcoma. In the latter 18 cases, the presence of the characteristic gene fusions was ascertained by RT-PCR revealing 14 cases with a PAX3-FOXO1 fusion and 4 cases with a PAX7-FOXO1 fusion  or the EZ DNA Methylation Kit  according to the manufacturer’s instructions. IG-DMR regions were amplified by nested PCR  using bisulfite-treated gDNA as template and specific primers . The DNA methylation status of IG-DMRs at the  primers . For DLKescribed ,17. The ®2.1-TOPO® vector and transformed into TOP10 bacteria using a TOPO TA Cloning Kit . The plasmids were prepared using a QIAprep Spin Miniprep Kit (Qiagen Inc.) and sequenced with M13 forward and reverse primers. The methylation pattern in DMRs was analyzed using CpG viewer software . Eluted amplicons were ligated into the pCRsoftware .Methylation-specific PCR was performed on the bisulfite-treated genomic DNA with two different sets of primers recognizing either methylated or unmethylated alleles . PCR wasTotal RNA from RMS patient samples was isolated using RNA STAT-60  in accordance with manufacturer’s instructions. RNA was screened for expression of DLK1 and GTL2 using premade qRT-PCR assays from Applied Biosystems . All qRT-PCR assays were run on 384-well plates in the ABI Prism 7900 Sequence Detection System. The control 18S RNA was assayed in separate wells of the same run using a premade qRT-PCR assay (Applied Biosystems).Statistical analysis of the data was done using the Fisher’s exact test with p<0.05 as the criterion for significance.DLK1-GTL2 locus exhibits structural and molecular similarity to the IGF2-H19 locus, we investigated the imprinting status of the IG-DMR at the DLK1-GTL2 locus, which is located between the two genes and controls coordinated expression of both genes at this locus (BstUI) to determine whether the fragment has retained or lost the corresponding CpG-containing restriction enzyme recognition site. The digested DNA was subsequently separated and visualized by agarose gel electrophoresis. As shown in BstU1 site was cut in all five ERMS samples and thus this region was hypermethylated in these cases. As there is often no evidence of a residual uncut of hypomethylated allele, this finding is indicative of LOI in the ERMS cases. By contrast, all five analyzed ARMS patient samples have lost the BstU1 recognition site and thus exhibited hypomethylation of the IG-DMR at the DLK1-GTL2 locus. On the other hand since there is often no evidence of a residual cut of hypermethylated allele, this finding is consistent with elimination of imprinting (EOI) in these ARMS cases.Since the DLK1-GTL2 locus in RMS, we employed bisulfite modification of DNA followed by sequencing of selected ERMS (patient E3) and ARMS (patient A3) samples. The PCR-amplified bisulfite-treated DNA fragments were subcloned and multiple subclones were individually sequenced. These sequencing results further supported our COBRA results, as all the copies of the IG-DMR at the DLK1-GTL2 locus were hypermethylated in the ERMS case (consistent with LOI), and were hypomethylated in the ARMS case (consistent with EOI) (To further confirm these imprinting changes at the ith EOI) . As expeith EOI) .After these initial studies, to further confirm our observation we analyzed an additional 17 ERMS and 18 ARMS patient samples by the COBRA assay. Combined densitometric analysis of all analyzed patient samples showed a highly relevant difference in methylation pattern between ARMS and ERMS. The average methylation of ARMS samples was ∼10%, whereas for ERMS samples, we observed 80% methylation . TherefoDLK1-GTL2 locus and thus can be potentially employed in RMS diagnostics  known as DLK1-DIO3 , MEG8 (RIAN), and the antisense gene RTL1. Moreover, in addition to two long non-coding intergenic RNAs (GTL2 and MEG8), this region contains one of the largest miRNA clusters in the human genome, encoding a total of 54 miRNAs, which play an important role in tissue homeostasis and, when aberrantly expressed, are involved in tumorigenesis , which develop spontaneously or as a part of Beckwith-Wiedemann syndrome  in ERMS and hypomethylated (EOI) in ARMS cells. Therefore, in contrast to the LOI that is consistently observed within the IGF2-H19 locus in both ERMS and ARMS samples, methylation of the IG-DMR at the DLK1-GTL2 locus is RMS-subtype specific. This different pattern of methylation could have some diagnostic value. By employing a specific pair of primers that recognize methylated DNA sequences, we have demonstrated that it is possible to quickly phenotype both subtypes of RMS by PCR.In our studies, performed on DNA samples from 23 ERMS and 23 ARMS tumors, we observed that the IG-DMR at the DLK1-GTL2 locus correspond to changes in mRNA expression we analyzed by employing RQ-PCR the GTL2/DLK1 ratio in 10 fusion-negative and 16 PAX3-FOXO1 fusion-positive ARMS samples. Our mRNA expression data correlated with imprinting status of the DLK1-GTL2 locus and demonstrated a significantly higher GTL2/DLK1 ratio in ARMS tumor samples compared to ERMS tumors.To address if these changes in imprinting at DLK1-GTL2 locus. However, we are aware that in addition to epigenetic regulation some other mechanisms could be involved. In support of this, an insulator protein located 18 kb upstream of the DLK1-GTL2 locus, which plays an important role in regulating imprinting of this locus, has been identified in human acute leukemia cells (In conclusion our data indicate that DLK1/GTL2 locus is differently regulated by imprinting in ARMS vs. ERMS cells. We developed an easy PCR-based assay to phenotype ERMS and ARMS, based on the methylation status of the IG-DMR at the ia cells . Thus, r


In a stable of eight horses in Northern Iceland, six horses presented with clinical signs, such as ataxia and reduced appetite, leading to euthanasia of one severely affected horse. Serological investigations revealed no evidence of active equine herpes virus type 1 infection, a common source of central nervous system disease in horses, nor equine arteritis virus and West Nile virus. Another neurotropic virus, Borna disease virus, was therefore included in the differential diagnosis list.Serological investigations revealed antibodies against Borna disease virus in four of five horses with neurological signs in the affected stable. One horse without clinical signs was seronegative. Four clinically healthy horses in the stable that arrived and were sampled one year after the outbreak were found seronegative, whereas one of four investigated healthy horses in an unaffected stable was seropositive.This report contains the first evidence of antibodies to Borna disease virus in Iceland. Whether Borna disease virus was the cause of the neurological signs could however not be confirmed by pathology or molecular detection of the virus. As Iceland has very restricted legislation regarding animal imports, the questions of how this virus has entered the country and to what extent markers of Bornavirus infection can be found in humans and animals in Iceland remain to be answered. Borna disease virus (BDV) is a negative-stranded RNA-virus infecting and causing neurological disease in several warm-blooded animals ,2. The cHere, we describe the first evidence of antibodies to BDV in Iceland, detected in an outbreak of neurological disease in horses.In a geographically isolated fishing village in northern Iceland there is a cluster of twelve stables that house about 70–80 horses during the winter. In February 2011, one of the stables (stable A) with 8 horses reported neurological signs in horses  were taken for serological analyses 9–10 days after they presented with neurological signs and three weeks after onset of signs in the index case (day 23). No antibodies towards equine herpes virus (EHV) types 1 and 4 were found by complement fixation. Presence of IgG-antibodies against EHV-1 detected by ELISA  in one horse (no. 5) was not regarded to indicate an active EHV-1 infection and none of the other investigated horses in the outbreak stable had this finding in the observation period (from day 23 to 440). Antibodies to equine arteritis virus (EAV) and West Nile virus (WNV) were not detected by serum neutralization test and ELISA , respectively. Infection with an alphavirus was considered unlikely based on the clinical signs and epidemiology. We therefore decided to include BDV as a differential diagnosis.An indirect immunofluorescence assay (IFA) was employed as previously described , except Around 10 days after onset of clinical signs, all horses showing clinical signs (horses no. 2–5) were positive in the first IFA, and two of the horses (horses no. 2 and 5) were positive by the second IFA, and thus defined seropositive Table . Blood aMolecular diagnostics was performed using previously described real-time and conventional RT-PCR assays targeting three genes of BDV  ,9,10. ToThe euthanized horses were not sent for necropsy; thus, there are no pathology data confirming inflammatory changes of the central nervous system indicating virus infection. Nevertheless, this report shows the first evidence of antibodies to BDV in Iceland, a country with extremely strict animal import regulations, indicating that these horses have been exposed to BDV or a closely related virus. In 1998, we conducted a study of 17 Icelandic horses with pyrexia of unknown reason, which found all to be seronegative by BDV-ELISA . How the virus has entered Iceland remains to be determined. One possible route of transmission is by migratory birds, because wild birds have been indicated as potential reservoirs of BDV . A uniquIn conclusion, we present the first evidence of antibodies to Bornavirus in Iceland, found in horses with and without neurological signs. The aetiology of the neurological signs has not been confirmed, and other causes, such as toxins or other viruses, cannot be excluded. Acquired equine polyneuropathy has recently been described in Swedish, Norwegian and Finnish horses, but this syndrome does not give rise to ataxia . Hence, ABV: Avian Bornavirus; BD: Borna disease; BDV: Borna disease virus; EAV: Equine arteritis virus; EHV: Equine herpes virus; ELISA: Enzyme-linked immunosorbent assay; FITC: Fluorescein isothiocyanate; IFA: Immunofluorescence assay; IgG: Immunoglobulin G; RT-PCR: Reverse transcription polymerase chain reaction; WNV: West Nile virus.The authors declare that they have no competing interests.SB conceived and designed the study, performed sampling, clinical evaluation, interpreted the data and helped to draft the manuscript. EA performed sampling, clinical examinations and treatment. ALB performed molecular diagnostics and helped to draft the manuscript. ILÖ performed and analysed serological assays. LTB participated in the conception and design of the study, and helped to draft the manuscript. VS conceived and designed the study, interpreted the data, and helped to draft the manuscript. JJW conceived, designed and coordinated the study, performed and analysed serological assays, interpreted the data and drafted the manuscript. All authors approved the final manuscript.


Musculoskeletal diseases are becoming increasingly important due to population aging. However, studies on the economic burden of musculoskeletal disease in Korea are scarce. Therefore, we conducted a population-based study to measure the economic burden of musculoskeletal disease in Korea using nationally representative data.This study used a variety of data sources such as national health insurance statistics, the Korea Health Panel study and cause of death reports generated by the Korea National Statistical Office to estimate the economic burden of musculoskeletal disease. The total cost of musculoskeletal disease was estimated as the sum of direct medical care costs, direct non-medical care costs, and indirect costs. Direct medical care costs are composed of the costs paid by the insurer and patients, over the counter drugs costs, and other costs such as medical equipment costs. Direct non-medical costs are composed of transportation and caregiver costs. Indirect costs are the sum of the costs associated with premature death and the costs due to productivity loss. Age, sex, and disease specific costs were estimated.Among the musculoskeletal diseases, the highest costs are associated with other dorsopathies, followed by disc disorder and arthrosis. The direct medical and direct non-medical costs of all musculoskeletal diseases were $4.18 billion and $338 million in 2008, respectively. Among the indirect costs, those due to productivity loss were $2.28 billion and costs due to premature death were $79 million. The proportions of the total costs incurred by male and female patients were 33.8% and 66.2%, respectively, and the cost due to the female adult aged 20-64 years old was highest. The total economic cost of musculoskeletal disease was $6.89 billion, which represents 0.7% of the Korean gross domestic product.The economic burden of musculoskeletal disease in Korea is substantial. As the Korean population continues to age, the economic burden of musculoskeletal disease will continue to increase. Policy measures aimed at controlling the cost of musculoskeletal disease are therefore required. Musculoskeletal disease includes osteoarthritis, rheumatoid arthritis, osteoporosis, and low back pain. These diseases are public health concerns and have a heavy burden on society owing to their chronic characteristics and the associated severe pain. In addition, they also affect the psychological health of patient families and caregivers . The burth in DALYs for males, while rheumatoid arthritis ranks fifth and osteoarthritis ranks eighth for females [Korea is not an exception to this trend. In Korea, musculoskeletal disease follows cancer, cardiovascular disease, digestive disease, respiratory disease, and neuropsychiatric disease to be ranked seventh in total DALYs for major disease groups in 2002. Additionally, among musculoskeletal diseases, rheumatoid arthritis ranks 14 females .Musculoskeletal disease is also a significant economic burden. The economic cost of musculoskeletal disease in Canada accounted for 3.4% of the GDP in 1994, and it is estimated that the cost of musculoskeletal disease was 7.7% of the GDP in the USA, which reached $849 billion dollars in 2004 . The selThe prevalence of most musculoskeletal diseases increase with age and are affected by lifestyle and habits such as physical activity and obesity . SignifiSocial changes in Korea such as population aging, increasing rates of obesity will soon make musculoskeletal disease a central public concern ,15. AdeqIn this study, the importance of musculoskeletal disease in terms of economic burden was determined using a number of data sources such as NHIC statistics, the Korea Health Panel study, and cause of death reports generated by the Korea National Statistical Office (NSO).This study measures the economic burden of musculoskeletal disease using the prevalence approach, which estimates the economic cost of all cases in a given period . The cosIn this study, economic burden was measured using the methodology outlined in previous studies ,20. DireThis study limited musculoskeletal disease to "M00-M99" as defined by the International Classification of Diseases (ICD) 10 . TherefoBecause Korea has a social health insurance system and the NHIC is the exclusive insurer, NHIC claims data are nationally representative of medical care costs covered by the Korean insurance program ,24. HoweThe Korea Health Panel study was conducted in 2007 through collaboration between the Korea Institute for Health and Social Affairs and the NHIC to determine the health service use behaviour of patients and medical expenditures . The surIn our study, the data sources were utilized as follows. First, the first round of the Korea Health Panel data was used to determine the prevalence of musculoskeletal disease. Musculoskeletal disease patients were defined as those who responded that they had a chronic musculoskeletal disease that had been diagnosed by a doctor. In the Korea Health panel, diseases are coded according to the Korean classification of 298 diseases. Patients diagnosed with any of the 11 musculoskeletal diseases in this classification were defined as musculoskeletal patients (inflammatory polyarthropathies (M05-M14), arthrosis (M15-M19), acquired deformities of the limb(s) (M20-M21), other arthropathies , systemic connective tissue disorders (M30-M36), cervical or other intervertebral disorders (M50-M51), other dorsopathies , soft tissue disorders (M60-M79), disorders of bone density and structure disorders (M80-M85), osteomyelitis (M86), and other disorders associated with musculoskeletal disease (M87-M99)). Some subjects in the survey had multiple chronic diseases. Subjects were diagnosed as musculoskeletal patients if they had at least one musculoskeletal disease. The prevalence rate was measured as the proportion of musculoskeletal patients among the participants of the Korea Health Panel according to age group . The population of Korea was multiplied by the prevalence rate to estimate the numbers of patients with musculoskeletal diseases.Among the economic cost, medical care costs paid by insurer were estimated using NHIC statistics for the year 2008 . All meaInpatient, outpatient, and emergency service due to musculoskeletal disease according Korea Health Panel data were analysed to estimate the payment by patients. Some patients had multiple reasons for using these services, but if at least one of the patient diagnoses was musculoskeletal disease, these visits were considered in this study. The costs paid by patients were adjusted by the number of chronic diseases reported. However, the cost of each musculoskeletal disease was classified according to the major diagnosis. The study period for the first round of the Korea Health Panel was 192.1 days and was adjusted to annual cost. The total cost to the Korean population was estimated as the total population compared to the number of subjects surveyed in the Korea Health Panel. Also, the cost of OTC drugs and other costs were determined from the Korea Health Panel data. Other costs included the cost of medical equipment, herbal medicine expenditures, health supplement food, and sanitary aid associated with musculoskeletal disease. OTC drugs and other costs were surveyed by household in the Korea Health Panel. Musculoskeletal disease patients were identified, and household OTC costs and other costs were analysed from this data. Finally, costs were adjusted according to the number of persons in each household and the number of chronic diseases in the musculoskeletal patients. Cost paid by the patient = (inpatient costs + outpatient costs + emergency service costs)*(365/192.1)*(the population of Korea in 2008/the number of subjects surveyed).OTC drugs costs =  * (365/192.1) * (the population of Korea in 2008/the number of subjects surveyed).Other costs = *(365/192.1)*(the population of Korea in 2008/the number of subjects surveyed).In the analysis of direct costs, transportation costs were estimated using data from the Korea Health Panel study. The frequencies of inpatient and outpatient service use due to musculoskeletal disease were determined, and the average cost of transportation for musculoskeletal disease patients was estimated. The average cost of using medical services was estimated at $0.58 per one way trip for outpatient service and $8.68 per one way trip for inpatient service.Transportation Costs = inpatient transportation costs + outpatient transportation costs =  *(365/192.1) *(the population of Korea in 2008/the number of subjects surveyed).In the case of caregiver costs, the cost and percentage of patients who used a caregiver, including an informal caregiver such as a relative or friend, were estimated from data from the Korea Health Panel study, and the admission day was estimated using NHIC statistics. The average cost per day of paid caregivers for patients with musculoskeletal disease was $45, and the percentage of patients that used a caregiver was 74%.Caregiver costs = )* .Indirect costs were divided into productivity loss costs and premature death costs. Costs due to loss of productivity were calculated from productivity loss due to outpatient and inpatient visits for patients between 20 and 64 years of age. Average wage per day according to age group was estimated from Ministry of Labor statistics . For inpProductivity loss = productivity loss of inpatients and outpatients = days of hospitalization * average wage per day + frequency of outpatient visits * average wage per day*(1/3).In order to estimate the cost of premature death, mortality due to musculoskeletal disease was determined for each age and sex group using cause of death reports generated by the National Statistical Office (NSO) . Lost fuPremature death costs = present value of future income according to age & sex * frequency of mortality according to age &sex group.This study used public data from the NHIC, the Korea Health Panel and the NSO which do not have personal identifiers, and data publishing was approved by each organization's ethics committee. This survey conformed to the Declaration of Helsinki as well as to local legislation. The data were collected and analysed using Microsoft Excel and SAS 9.2 .Table The five most costly musculoskeletal diseases are noted in Table Direct medical care costs, direct non-medical care costs, and indirect costs are shown in Table Indirect costs were composed of productivity loss costs and costs due to premature death. The cost due to productivity loss was estimated to be 2.28 billion dollars. The total number of deaths due to musculoskeletal disease was 1,699 according to NSO statistics . The leading cause of death was disorders of bone density and structures including osteoporosis, which affected 676 patients. Mortality rates were higher in females 65 years and older (565 people). The cost due to premature death was 79 million dollars, and systemic connective tissue disorders accounted for the highest proportion of these costs (40 million dollars).When costs were compared according to age group and sex, costs for female adults aged 20-64 were the highest (3.21 billion). However, when compared with the population, the burden of musculoskeletal disease was highest for aged females ($430 per person), followed by aged males ($291), female adults ($205), and male adults ($103). The proportions of costs for male and female patients were 33.8% and 66.2%, respectively, and those of direct medical care costs, direct non-medical care costs, and indirect costs were 60.8%, 4.9%, and 34.3%, respectively. Total costs, which included direct costs and indirect costs, were 6.89 billion dollars . Males accounted for only one-third of the total costs.The prevalence of musculoskeletal disease in this study is relatively low compared to those in previous studies in Korea ,12. One Differences in case definition and the methodology used to estimate prevalence could also result in differences in the estimation of disease prevalence. Hur et al.  defined Musculoskeletal disease prevalence varies in other studies. For example, one study in the United Kingdom reported that 14.5% of adults had symptoms of musculoskeletal disease in 2003 . AnotherFurthermore, variation in estimates of the economic burden of musculoskeletal disease is due to differences in the types of diseases included as well as the cost calculation methods used. For example, in Canada, direct and indirect costs associated with musculoskeletal disease were estimated to be 5.6 billion dollars and 13.5 billion dollars, respectively. Total economic costs in 1994 were estimated to be 19.2 billion, about 3.4% of the Canadian gross domestic product (GDP) . In the In this study, direct costs were 65.7% of total costs and the total cost of musculoskeletal disease was about 0.7% of the Korean GDP . These rWhen compared with other Korean studies using a similar study design, the total cost of musculoskeletal disease in 2002 ($6.89 billion) was lower than that of cancer ($9.4 billion) . In compThough the prevalence and economic burden of musculoskeletal disease vary, the fact that the prevalence of musculoskeletal disease was found to be higher in the aged and in females in our study sample is consistent with the results of most other studies ,12,29,34In this study, other dorsopathies  is the most costly disease entity. This includes the low back pain category  of the Global Burden of Disease Study and also includes deforming dorsopathies and dorsalgia [In this study, the classification of disease was based on Korean standards. For example, though the definition of arthrosis was consistent with that used in the Global Burden of Disease Study , apart fSeveral additional limitations should be noted in this study. First, the Korea Health Panel which was used to estimate components of cost and prevalence does not include weighting factors to estimate national statistics, therefore the accuracy of the cost components using Korean Health Panel could be limited. Second, this study did not consider the cost of disability due to disease or other intangible costs. Some musculoskeletal diseases such as osteoarthritis could result in disability or sequelae, so could result in reduced quality of life. Hence, if these costs were considered, the economic burden of musculoskeletal disease would be greater . AdditioIn conclusion, this study estimated the economic burden of musculoskeletal disease including non-covered care costs, OTC drugs costs, and other costs such as medical equipment expenditures, which were not sufficiently considered in previous studies. Furthermore, this study is the first to estimate the economic burden of all musculoskeletal diseases in Korea. In this study, the estimated population prevalence of musculoskeletal disease was 11212.7 persons per 100,000, and the economic burden was 2.33 billion in males and 4.56 billion in females, totalling 6.89 billion dollars and 0.7% of the Korean GDP. The average economic burden of musculoskeletal disease per person was highest in aged females. As the Korean population ages, the economic burden of musculoskeletal disease will continue to increase. Therefore, adequate policy measures that minimize the cost of musculoskeletal disease will be required in the near future.The authors declare that they have no competing interests.SJY planned and supervised all aspects of this study. IHO interpreted the data and prepared a draft manuscript. EJK participated in the statistical analysis, and YAK and SHK contributed to the study and manuscript revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/157/prepub


To the Editor:Salmonella enterica is among the leading causes of foodborne diseases worldwide. Multidrug-resistant S. enterica serovar Typhimurium definitive phage type (DT) 104 emerged during the early 1990s in the United Kingdom and spread worldwide thereafter . The repeat unit sequence was CTCAGAA/TTCTGAG, spanning 1952121–1952274 on the reference genome or 22 repeats of 7 nt.The design of an MLVA system relies on the analyzed genome sequences. In this study, we found and evaluated a variable-number tandem-repeat region, or locus, designated DT104o. The locus is specific to S. enterica serovar Typhimurium collected during 1981–2012; 103 were from human samples and 163 from non-human sources. Bacteriophage typing was performed according to Anderson’s method and scheme (We used 266 apparently independent isolates of D) identified in each locus are summarized in the D value of 0.974; use of MLVA5 plus the DT104o locus (MLVA6) identified 83 types with a D value of 0.984. These results indicate that the DT104o locus is highly specific and therefore useful as an additional molecular epidemiologic marker for analyzing S. enterica ser. Typhimurium DT104.For all 266 isolates tested, the number of alleles and the Simpson’s index of diversity score (Because DT104o was highly variable, 5 DT104 strains were tested for the frequency of variants at each locus after 5 serial passages by using liquid culture: cultures were diluted 1:1,000 at each passage. Sixteen colonies of each strain were tested by using MLVA6 . No variWe also found that DT104o could provide more discriminatory power to MLVA5 in some settings . We compThe DT104o locus is located at the proximal region of fragment 180 comprised of a prophage structure, which was proven to be DT104-specific in a previous study (S. enterica ser. Typhimurium DT104. The MLVA system could be improved by adding loci based on the genome sequence of such pandemic strains. In this study, we showed that the newly identified DT104o locus could be useful in identification and subtyping of S. enterica ser. Typhimurium DT104.In conclusion, development of an MLVA system is dependent upon the genome sequences available, and the system is usually used for molecular subtyping of a certain serotype in a particular organism. However, a specific group of strains could cause a pandemic and become a target of public health concern, as was Salmonellaenterica serovar Typhimurium definitive phage type 104 isolated from nonhuman samples.Variation of locus and repeat copy number in settings identical in MLVA5 but not in MLVA6 in