Leporipoxviruses, a genus of Chordopoxvirinae, double stranded DNA viruses, whose members infect leporids and squirrels, inducing cutaneous fibromas from which virus is mechanically transmitted by biting arthropods. However, in the European rabbit (Oryctolagus cuniculus), MYXV causes the lethal disease myxomatosis. The release of MYXV as a biological control for the wild European rabbit population in Australia, initiated one of the great experiments in evolution. The subsequent coevolution of MYXV and rabbits is a classic example of natural selection acting on virulence as a pathogen adapts to a novel host species. Slightly attenuated mutants of the progenitor virus were more readily transmitted by the mosquito vector because the infected rabbit survived longer, while highly attenuated viruses could be controlled by the rabbit immune response. As a consequence, moderately attenuated viruses came to dominate. This evolution of the virus was accompanied by selection for genetic resistance in the wild rabbit population, which may have created an ongoing co-evolutionary dynamic between resistance and virulence for efficient transmission. This natural experiment was repeated on a continental scale with the release of a separate strain of MYXV in France and its subsequent spread throughout Europe. The selection of attenuated strains of virus and resistant rabbits mirrored the experience in Australia in a very different environment, albeit with somewhat different rates. Genome sequencing of the progenitor virus and the early radiation, as well as those from the 1990s in Australia and Europe, has shown that although MYXV evolved at high rates there was no conserved route to attenuation or back to virulence. In contrast, it seems that these relatively large viral genomes have the flexibility for multiple pathways that converge on a similar phenotype.Myxoma virus (MYXV) is the type species of the  Oryctolagus cuniculus) population of Australia is the classic example of host-pathogen coevolution following a species jump. In its natural host, Sylvilagus brasiliensis, the South American tapeti or forest rabbit, infection with MYXV causes an innocuous cutaneous fibroma, which persists for some weeks. Virus is passively transmitted from the fibroma on the mouthparts of biting arthropods. This benign host-pathogen coexistence has been presented as a text book example of an evolutionary climax in which the pathogen causes little harm to the host and both host and pathogen survive [The introduction of myxoma virus (MYXV) into the European rabbit . The virus naturally infects the South American tapeti, causing a cutaneous fibroma at the inoculation site. However, in the European rabbit, which is exotic to the Americas, MYXV causes myxomatosis, a generalized, lethal disease characterized by swollen head, eyelids and ears, raised cutaneous lesions over the body, ears and legs, ano-genital oedema, blepharoconjunctivitis and mucopurulent ocular and nasal discharge. This disease was first described following an outbreak in laboratory rabbits at the Institute of Hygiene in Montevideo, Uruguay in 1896 [MYXV is the type species of the Sylvilagus floridanus (eastern cottontail), which is native to the eastern parts of North America and extending south into Central America and in S. bachmani (brush rabbit) found on the west coast of North American and into Mexico. However, infections with these viruses cause very different outcomes in European rabbits. Rabbit fibroma virus (RFV), found in S. floridanus, induces a cutaneous fibroma at the inoculation site in immunocompetent European rabbits and is used as a heterologous vaccine against myxomatosis [S. bachmani have extreme virulence for European rabbits, often killing the rabbit before the classic signs of myxomatosis can develop [Lepus europaeus) in Europe  from which it can be transmitted by mosquitoes. In brush rabbits infected experimentally, most of the fibromas scabbed within four weeks, and thus became unsuitable for mosquito transmission, but some persisted for much longer providing a source of infection for 2–3 months [S. bachmani, which has a limited distribution along the west coast of North America, and S. brasiliensis or S. floridanus.Cal MYXV induces a cutaneous fibroma in 3 months ,24. It w3 months ,25,26. H3 months ,27,28. M3 months ,30. ThisS. floridanus (eastern cottontails) [S. floridanus has also been introduced into Europe for hunting but whether RFV was also inadvertently introduced is not known [RFV, also known as Shope fibroma virus, is the best studied of the leporipoxviruses in its natural host and provides a model for their biology. RFV normally infects ontails) ,32; and ontails) ,34,35. Fontails) . Infectiontails) . A fasciontails) ,39. S. fot known ,41.Lepus europaeus) [Lepus californicus (black tailed jack rabbit) [Lepus capensis) from Kenya [Hare fibroma virus is the only leporipoxvirus naturally found outside the Americas. It induces relatively large (1–3 cm diameter), protuberant cutaneous fibromas in European hares (ropaeus) . The oriropaeus) ,43,44. I rabbit)  and smal rabbit)  but it iom Kenya , but virSciurus carolinensis) in North America. Generalized disease can occur in suckling squirrels with proliferative lesions over the body and in the lungs, liver, lymph nodes and kidney [Marmoto monax) are also susceptible to infection and can transmit virus via mosquito feeding [Sciurus niger) and porcupines (Erethizon dorsatum) from the eastern USA [Squirrel fibroma virus induces cutaneous fibromas and proliferative epidermal lesions in eastern gray squirrels  in California. Classification as a leporipoxvirus was based on serological cross-reaction with Californian MYXV, although it did not cross-react with South American MYXV and electron microscopy showed particles with surface striations that were not typical of other leporipoxviruses [A putative leporipoxvirus has been described from skin lesions on western gray squirrels . HoweveInterestingly, while RFV can replicate to high titres in the fibroma on European rabbits, it could only rarely be transmitted by mosquitoes, probably because the immune response controls the fibroma before high titres of virus are reached in the overlying epidermis, which appears critical for transmission by mosquitoes ,56. DespS. audubonii and S. nuttalli, were susceptible to experimental infection with MYXV and efficient sources of virus for mosquito transmission [S. audubonii overlaps with S. bachmani but this species does not support transmission of Cal MYXV with virus infection inducing a rapid and intense T cell response at the inoculation site [Lepus timidus) can occasionally be infected, naturally or experimentally, with MYXV but are generally refractory [Two species of North American cottontails, smission . The dision site ,58. In afractory ,44,52.The establishment of MYXV in European rabbits demonstrates that if a susceptible host is available then species jumps can be successful. However, on many occasions such infections must simply burn out without establishing an ongoing transmission cycle because the host does not support efficient transmission, the population size is too limited, the infection is too virulent or vectors are inefficient. This seems to have been the fate of the early attempts to introduce MYXV in Europe and Australia and of infections that occurred in domestic and laboratory European rabbits in South America ,7,8.The Lausanne (Lu) strain of MYXV (Brazil/Campinas 1949) has a genome of 161,777 bp of double stranded DNA with closed single strand hairpin termini. It encodes 158 unique open reading frames (ORFs), 12 of which are duplicated in the 11,577 bp terminal inverted repeats (TIRs) ,60. By cin vivo comes from studies in European rabbits.Genes encoding proteins involved in replication and structure are relatively conserved with other poxviruses and tend to be in the central part of the genome ,63, whilS009 gene, which means most of this ORF and all of the S008.2 ORF, which is the equivalent of the M156R gene in Lu, are duplicated, but leaving the RH copy of S009 as a truncated pseudogene. Apart from the expansion of the TIRs, the gene order is conserved between Lu and RFV. Gene loss appears to have proceeded by fragmentation and gradual loss of the ORFs. At least six genes implicated in virulence or with potential immunomodulatory functions in Lu are missing from RFV, which may explain why this virus is unable to disseminate and replicate at distal sites in immunocompetent European rabbits. Immune suppressed or very young European rabbits are however susceptible to more generalized disease following inoculation of RFV [The Kasza strain of RFV has been completely sequenced . The genn of RFV ,65,66,67M156R, M154R, M153R, M152R, M151R ORFs and part of the M150R ORF from the RH end of the genome duplicated and inserted in the LH TIR with deletion of a large part of the M009L ORF [The MSW strain of Cal MYXV has a genome of 164,600 bp. This increase in size, compared to the other sequenced leporipoxviruses, is predominantly due to an expansion of the TIRs to 15,464 bp with the 009L ORF . MSW is M008.1L/R and M152L/R genes, which have been implicated in virulence for European rabbits in Lu [M000.5L/R, M008.1L/R and M152R lost in both viruses .Surprisingly, given the extreme virulence of MSW for European rabbits ,17, therts in Lu ,105,111.ts in Lu , the oriet al. (1999) [Based on these three genomes, the South American Lu strain appears to have a full complement of genes suggesting that it resembles a putative ancestral leporipoxvirus, although from the RFV genome, Willer . (1999)  have pro. (1999) ,61.In contrast to MYXV, both MSW and RFV have multiple gene disruptions suggesting that they may have undergone host switches and adaptation with loss of genes, perhaps to decrease virulence and in turn increase transmissibility. Alternatively, the genes that were lost may not be required for replication in the new host. This appears to have happened more recently in MSW than in RFV, based on the fragmentation and loss of the genes in RFV compared to multiple indels in MSW.8 pfu/g) with profound depletion of lymphocytes from lymph nodes and, depending on the strain, spleen. Very high titres of virus occur in secondary cutaneous lesions, the swollen eyelids and particularly the very swollen tissues at the base of the ears, which are probably important for insect transmission. Virus is also found in tissues such as lung and liver but generally at lower titres [Pasteurella multocida and Bordetella bronchiseptica is common, but early studies reported that internal organs were usually free of bacteria [In European rabbits, virulent MYXV inoculated intradermally replicates in MHC-II + cells at the dermal/epidermal interface and then spreads to the draining lymph node, where it is found initially in the cells of the subcapsular sinus and then in lymphocytes in the T cell zones of the node. From here, the virus disseminates to distal tissues carried in lymphocytes and possibly monocytes; there is little or no free virus in the bloodstream ,113,114.r titres ,113,115.bacteria . Bacteribacteria ,118.Sylvilagus species to suppress the local immune response sufficiently to allow virus to persist at the cutaneous site, but in European rabbits these proteins profoundly suppress the immune system, which results in fatal disease. The actual cause of death is obscure. Mims (1964) [et al. (1983) [The ability of the virus to replicate and disseminate in lymphocytes and establish and replicate to high titres at distal sites is critical for virulence in European rabbits . Its (1964)  could nos (1964) , but this (1964)  and rabbs (1964) ,17. Ducl. (1983)  suggest . (1983) ).The European rabbit likely evolved in the Iberian Peninsula and southern France from where it was distributed around Europe often as semi-domesticated stock, managed for meat, fur, and hunting, that later reverted to the wild. The current domestic breeds were developed from these rabbits ,122,123.Domestic breeds of European rabbits were introduced to Australia from the time of the earliest European settlement in 1788, and although some local spread occurred it was the introduction of a small group of wild rabbits from Britain in 1859 that led to the continent-wide invasion and establishment of rabbits as Australia’s major vertebrate pest ,125. MyxStarting in December 1950, MYXV spread from one or more trial sites in the Murray Valley across vast swathes of south eastern Australia during the summer of 1950/51. This was facilitated by extensive inland flooding, allowing mosquito breeding in normally dry country, and the presence of huge numbers of completely susceptible rabbits at high population densities. It was also likely deliberately spread by landholders transporting infected rabbits, and there were other deliberate introductions outside the main epizootic but these seem not to have spread ,4,8.By the autumn, in March/April of 1951, the main epizootic was over both because mosquito activity declined with cooler weather and the profound loss of susceptible rabbits; however, the virus trickled on in isolated populations, usually ones not affected during the summer, and re-emerged in epizootic form in the following spring from September 1951 . Naturalet al. (1952) [The number of rabbits in Australia pre-1950 is not known but it was probably in the hundreds of millions and may have been an order of magnitude higher, as numbers had risen significantly in the immediate post-war period. Similarly, the reduction in population size during the first epizootic of 1950–1951 could not be measured, and only part of the continent was affected, but Ratcliffe . (1952)  describe. (1952) .The strain of MYXV released in Australia was termed the Standard laboratory strain (SLS), also called the Moses strain. The original inoculum was produced from Martin’s strain B (Martin 1936), which was obtained from the Rockefeller Institute in the USA and was originally derived from an infected European rabbit at the Institute Oswaldo Cruz in Brazil around 1910 . The virOnce ecological studies were underway it became apparent that field strains of MYXV were emerging that were slightly attenuated compared to the progenitor SLS, and that they tended to outcompete SLS when both viruses were present ,132,133.While SLS was not a cloned virus in the sense of having been pock-purified on the chorioallantoic membrane of fertile eggs or cloned by limit dilution in rabbits, Fenner and Marshall (1957)  stated tThe virulence of field isolates of MYXV was systematically analysed by infecting small groups of laboratory rabbits, usually five or six, and at least four months old, with low doses of each virus. Virulence was classified into five broad grades based on the CFR, average survival time (AST) and clinical signs ,16. The Grade 1 viruses such as SLS quickly became relatively rare in the field and the predominant viruses were of grade 3 virulence with smaller numbers of grade 4 viruses . Where v7 rabbit ID50/g) for long periods whereas grade 1 viruses, although rapidly reaching transmissible titres, killed the rabbits quite early. Grade 5 virus was poorly transmitted probably because the rabbit immune system was able to quickly control virus replication. The grade 5 virus, neuromyxoma, used in this study may not be representative of field strains as it reaches very low titres in the epidermis; other grade 5 viruses have higher titres but are still relatively quickly controlled in the epidermis [MYXV can be transmitted by direct contact; virus is shed from eroded cutaneous lesions and mucosal sites such as conjunctivae and nasal passages and can be inoculated into the upper respiratory tract or conjunctivae during social interactions or cutaneously by fighting. However, for epizootic spread, mosquitoes or other mobile, biting arthropods are the most effective means of transmission. Mosquito transmission studies using laboratory rabbits infected with viruses of different virulence grades demonstrated that Grade 4 viruses were the most transmissible  Figure . The titpidermis ,113.Despite the transmission advantage of grade 4 viruses in laboratory rabbits, grade 3 viruses predominated in the field. The most likely explanation for this is that rabbits were undergoing intense selection for resistance to myxomatosis. The emergence of attenuated viruses allowed some survival of infected rabbits, although even in the first epizootic some rabbits recovered from infection ,140. OveIn much of Australia, rabbits have an annual breeding season, during which does may produce 4–5 litters; the generation interval is approximately 12 months. Systematic studies using rabbits from a field site at Lake Urana in southern New South Wales demonstrated that over a seven year period, corresponding to seven annual epizootics of myxomatosis and approximately seven generations of selection, CFRs dropped from 90% to 26%. This was measured by infection of non-immune rabbits from each generation with the KM13 strain of MYXV under laboratory conditions ,144.Resistant rabbits have reduced disease severity and higher survival rates, rather than resistance to infection, and resistance can be overcome by viruses of higher virulence such as Cal MYXV or by suppressing the Th1 immune response ,145,146.Although only limited studies were done, resistance does not seem to have emerged uniformly across Australia. It developed more rapidly in the hot dry areas, such as the Mallee in Victoria, than in cool moist climates ,144,151 Modeling simulations suggest one MYXV strain should predominate: grade 4 in populations with no resistance but grade 3B in more resistant populations, which generally fits the observed field data ,154. HowAn important nexus between virulence and transmissibility was further demonstrated by experiments with flea transmission of MYXV in wild rabbits. Rabbits that survived infection, regardless of the actual virus virulence, were relatively poor sources of virus based on the proportion of fleas that were able to transmit infection from the infected rabbit. The best transmission was from those rabbits that survived for several weeks with active myxomatosis but ultimately died . These wTransmission is obviously a central component of virus fitness. The drivers of arthropod transmission are the duration of high titres of virus in the epidermis, whether the infected animal survives and the survival time. Importantly, the same virus will have a different disease phenotype (and transmission outcome) in resistant wild rabbits compared to unselected domestic rabbits, but even within wild rabbit populations individual levels of resistance will vary. In addition, biotic and abiotic factors that suppress the rabbit immune response, such as, age of the rabbit, nutritional stress, cold, parasites or intercurrent disease may lead to a more virulent phenotype and hence alter transmission potential ,162,163.In June 1952, the owner of an estate at Maillebois in north western France inoculated two wild rabbits with MYXV in an attempt to control rabbits on his property. The virus (Brazil/Campinas 1949) was obtained from the Virus Culture Collection in Lausanne, Switzerland and has since been known as the Lausanne strain (Lu). Unlike SLS, it had undergone very few passages in European rabbits since its isolation and infected rabbits had different lesion morphology to SLS with large purplish protuberant cutaneous lesions. However, the CFR and AST were indistinguishable from SLS in unselected rabbits ,16. As iSpilopsyllus cuniculi), which is an efficient vector of MYXV, and other fleas, with more limited geographic distribution, such as the Spanish rabbit flea (Xenopsylla cunicularis), were also efficient vectors. These fleas were not originally present in Australia but were later introduced to aid in the dissemination of MYXV [Rabbits in Europe carried the European rabbit flea , an indel in the M083L gene and an indel in the 3' end of the M152R gene leading to read-through and an altered sequence at the C-terminus of the M152 protein. Whether these mutations were present in the original isolate or arose during rabbit passage cannot be determined. As already noted, while both SLS and Lu are of grade 1 virulence when tested in laboratory rabbits, Lu is actually more virulent than SLS when tested in resistant wild rabbits [The progenitor Australian strain of MYXV, SLS, was isolated in Brazil nearly 40 years earlier than Lu and had been maintained by rabbit passage for 40 years. It has 72 nt differences from Lu including indels but only counting one TIR . These i rabbits . In addi rabbits  suggestiM014L, M130R and M153R ORFs causing premature termination of translation. The M153 protein has a significant role in virulence by downregulating MHC-I from the surface of infected cells [A rapid divergence occurred in Australia following the spread of MYXV in the period 1950–1953. Only two virus isolates from the first epizootic in the summer/autumn of 1950/51 were tested for virulence and both were grade 1 . Genome ed cells ,107,108.ed cells , althouged cells . Interesed cells ,195. Alted cells . UnfortuM014L found in Glenfield, suggesting that this mutation was relatively widespread. Ur has an additional indel in M005L/R at the same location as the original SLS indel plus an indel in the 3' end of the M134R gene, this is also present in KM13 and Glenfield but where these viruses have 3A inserted in M134, Ur only has 2A inserted leading to a predicted premature termination of translation. Both viruses have only low numbers of other substitutions but interestingly four amino acid substitutions in KM13 are present in all recent Australian isolates sequenced [Genome sequences have been completed for two viruses isolated during the third epizootic, the KM13 strain (Corowa 12-52/2), the prototype grade 3 virus, isolated in December 1952 ,132, andequenced .There are no Australian sequences for the period between 1953 and 1990. However, genome sequences for 21 viruses from 1991 to 1999 have been completed. Virulence classifications are known for six of these, with three viruses classified as grade 1 virulence and three of grade 4 or 5 virulence . MoleculM014L ORF is disrupted in all three viruses sequenced from the early radiation. However, more recent sequences do not have the M014L ORF disrupted and intriguingly all of the modern viruses sequenced have an intact M083L ORF, despite this being disrupted in SLS and the early viruses. All but one of the recent viruses have the M009L ORF disrupted by an indel. Both the M009L indel and the M083L mutations occur on the long branch of the tree separating the 1951–1953 viruses from the 1990 viruses. Some virus isolates have further disruptions to the M009L ORF, or mutations forming stop codons, suggesting that it is becoming a pseudogene , which may indicate an earlier duplication event during leporipoxvirus evolution. Fragmentation or duplication of genes has the potential to allow evolution of new functions, for example, the CPXV12 gene in cowpox virus, is a gene fragment which has evolved a novel role in MHC-I downregulation and hence avoidance of T cell recognition [Another group of viruses have deleted 923 nucleotides of the ognition .M009L has also been lost by an expansion of the TIR boundaries and duplication of sequences from the RH end of the genome in the Cal MYXV MSW strain and, despite the disruption of at least two virulence genes, this virus is highly virulent in European rabbits.Interestingly, M036L ORF; this is also present in Nottingham, which has additional indels in the M150R gene and in the M134R gene. It seems likely that the multiple indels are responsible for attenuation of Nottingham, but whether the disruption to M036L is responsible for the attenuation of Sussex is not clear as a virulent Australian virus also has this gene disrupted [The first reported outbreak of myxomatosis in Britain was in October 1953; sequencing of three viruses from the early radiation: Cornwall, April 1954 (grade 1); Sussex, September 1954 (grade 3); Nottingham, April 1955 (grade 5), showed that the virulent Cornwall had eight nucleotide differences from the Lu progenitor, seven of which were in ORFs and five were non-synonymous ,195. Howisrupted . Sussex M009L, M036L, M135R and M148R, the latter two have demonstrated roles in virulence [M036L is independent of the mutation in Nottingham and the indel in M009L is different to that in the Australian isolates. Outside of these indels there were 67 nucleotide substitutions between the grade 5 6918 strain and the Lu progenitor over the 43 years of evolution in European rabbits. Viruses with the same mutation in the M135R gene as 6918 have been isolated in the Netherlands and so may be widely distributed in Europe [M135 gene orthologue [M009L and M036L as well as M002L/R and M017L [M009L disrupted [The only other complete genome is that of the highly attenuated Spanish 6918 strain of MYXV isolated in 1995 . Four geirulence , providin Europe  but, in n Europe . Interesthologue . In partnd M017L  and someisrupted .The poxvirus early, intermediate and late promoter sequences appear to be conserved across the leporipoxviruses sequenced . HoweverOverall, the conclusion from the genome studies is that in these large, complex DNA viruses there is no common pathway to attenuation or virulence, but a convergence for phenotype that is compatible with multiple different genotypes and possibly involving complex epistatic interactions. Variants would undergo local selection but, as already noted, it is likely that a broad range of genotypes is compatible with successful spread depending on the local ecological conditions and rabbit population dynamics, including the level of resistance in the population. However, it is clear that from very early in the adaptation of MYXV to European rabbits, these local variants outcompeted virulent viruses released into the same populations ,133,155.The sequence data for RFV and Cal MYXV suggest that in some evolutionary pathways loss of genes is compatible with successful transmission, but whether these genes were actively selected against to enhance transmission by reducing virulence cannot be determined. Similar reductive evolution potentially associated with adaptation to new environments or selection for attenuation has been described in the orthopoxviruses ,207. TheGeographically, multiple lineages of MYXV can coexist over quite small distances with particular variants dominant on sites in different years and multiple variants isolated on single study sites during a season ,190,195 The emergence and gradual domination of attenuated strains of MYXV in Australia occurred in the face of ongoing widespread releases of the virulent SLS and, in NSW and later Victoria, the SLS-derived Glenfield strain. Following its spread in Europe, the Lu strain was released experimentally in Australia in 1954  but was Sequence studies provide the basis for understanding the molecular changes that led to attenuation, although it is difficult to determine the roles of individual mutations and gene disruptions. Similarly, it is difficult to know whether virulence was maintained by circulating virulent viruses or re-evolved in attenuated viruses. That is, do particular virulence lineages maintain stable circulation or is it relatively easy to flip back and forth between virulent and attenuated viruses with local selection determining which phenotype is successful? Further important questions include whether there is ongoing selection pressure on the virus driven by increasing resistance in the rabbit population, what capacity the rabbit has for further evolution, and what the cost of resistance is to the rabbit? This is further complicated by ongoing selection pressure on the European rabbit imposed by the virulent rabbit haemorrhagic disease virus (RHDV), a calicivirus, first described in China in 1984, that has spread in Europe and, more recently, Australia causing high mortality in rabbit populations ,209.S. brasiliensis or S. bachmani.Writing 50 years ago, Fenner and Ratcliffe (1965)  suggesteSuch an outcome is dependent on the rabbit being able to evolve very strong resistance that prevents dissemination and generalized disease, limiting virus replication to the inoculation site. This would require the virus to maintain sufficient immune suppression capacity to prevent clearance at the inoculation site but not overwhelm the rabbit. Obviously, the previously characterized grade 5 viruses would not be selected as they would be too readily controlled by the rabbit immune system. Viruses selected in these conditions of high resistance could be of very high lethality for non-resistant rabbits. The alternative is that the virus mutates to lose the ability to disseminate within the host while retaining its resistance to immune clearance from the inoculation site (perhaps by recombination with RFV). However, it is questionable how well such a virus would compete with field strains that cause generalized disease, because multiple virus-rich tissues would provide more opportunities for insect transmission than a single fibroma unless it had very prolonged persistence.The second possibility was that selection for genetic resistance in the rabbit population reaches a plateau because further selection for virus virulence is not possible.In this scenario, a relatively stable situation occurs whereby virus strains with sufficient virulence to cause the generalized lethal disease optimal for transmission continue to predominate in the field, causing appreciable mortality in the population, but not driving further selection. Rabbit resistance may also plateau because the costs of resistance are too high or there is insufficient genetic polymorphism for further selection in the absence of mutation in the rabbit genome. Depending on the eventual level of resistance in the rabbit population, this could see the emergence of viruses with hypervirulent phenotypes in unselected rabbits.A third option not considered by Fenner and Ratcliffe (1965)  is that After 65 years of evolution, there is very little evidence for the emergence of a fibroma type disease and some evidence that grade 1 and 2 viruses are more common in the field in Europe and Australia, suggesting that genetic resistance may still be driving virus evolution. Clearly, further systematic studies of viruses and rabbits are needed to understand how this complex evolutionary story will play out and to understand the biological and molecular mechanisms of this evolution. A single model may not prevail with different environments favouring different evolutionary solutions.


As we reported previously, Getah virus infection occurred in horses at the Miho training center of the Japan Racing Association in 2014. This was the first outbreak after a 31-year absence in Japan. Here, we report a recurrent outbreak of Getah virus infection in 2015, sequential to the 2014 one at the same site, and we summarize its epizootiological aspects to estimate the risk of further outbreaks in upcoming years.P < 0.01). Therefore, the horses newly introduced from other areas at this age were susceptible, whereas most horses aged 3 years or older, which had experienced the previous outbreak in 2014, were resistant. Among the 2-year-olds, the prevalence in horses that had been vaccinated once (10/45 [22.2 %]) was significantly higher than that in horses vaccinated twice or more . Horse anti-sera raised against an isolate in 2014 neutralized both the homologous strain and a 2015 isolate at almost the same titers (256 to 512), suggesting that these viruses were antigenically similar. Among horses entering the training center from private surrounding farms in 2015, the seropositivity rate to Getah virus increased gradually . Thus, increased virus exposure due to the regional epizootic probably allowed the virus to spread in the center, similarly to the outbreak in 2014.The outbreak occurred from mid-August to late October 2015, affecting 30 racehorses with a prevalence of 1.5 % of the whole population (1992 horses). Twenty-seven (90.0 %) of the 30 affected horses were 2-year-olds, and the prevalence in 2-year-olds (27/613 [4.4 %]) was significantly higher than that in horses aged 3 years or older (3/1379 [0.2 %], The 2015 outbreak was caused by a virus which was antigenically close to the 2014 isolate, affecting mostly 2-year-old susceptible horses under epizootiological circumstances similar to those in 2014. The existence of 2-year-olds introduced from regions free from Getah virus could continue to pose a potential risk of additional outbreaks in upcoming years. Vaccination on private farms and breeding farms would help to minimize the risk of outbreaks. Alphavirus in the family Togaviridae ) was significantly higher than that in horses aged 3 years or older . All of the samples analyzed had completely identical nucleic acid sequences, and the sequences were also identical to that of the 14-I-605 strain  isolated during the 2014 outbreak [We tried to isolate Getah virus from blood samples that were positive on RT-PCR. In testing on 23 blood samples, primary cocultivation resulted in the isolation of two strains confirmed as Getah virus by RT-PCR. We analyzed the sequences of the n = 2) raised against MI-110 neutralized the homologous virus at titers of 512 and neutralized the 14-I-605 strain at almost the same titers (256) [n = 2) raised against the 14-I-605 strain—which were also used in the previous study [To assess whether the current Getah virus vaccine was effective against the circulating virus in 2015, we performed cross-neutralization tests between the vaccine strain (MI-110) and the strains isolated in 2014 (14-I-605) and 2015 (15-I-752). The results of the cross-neutralization tests are summarized in Table rs (256) . In the us study —neutraliDuring the 2014 outbreak, we found that epizootic Getah virus infection occurred not only at the training center but also on private farms surrounding the center . To asseThe epizootic Getah virus infection in 2015 seems to have started earlier than that in 2014, which started in mid-September , whereasThe significantly higher prevalence in the 2-year-olds (4.4 %) than that in the 3-year-olds or older population (0.2 %) was one of the most characteristic aspects of the current outbreak, because such significant difference was not observed in the 2014 outbreak . In thisMore than 30 private farms are located in Ibaraki Prefecture, where the Miho training center stands, and in neighboring Chiba Prefecture, and horses are repeatedly transferred between the farms and the center for training and rest. Unlike in the training centers, where Getah virus vaccination is mandatory, on the farms vaccination is not common. In addition, this area is one of the biggest producers of pigs. Although the exact prevalence of Getah virus in pigs and its association with the recent outbreaks in horses have not been studied, the horses in this area are considered to be at high risk of infection. As in the 2014 outbreak, in 2015 our results indicated that there was a high prevalence of Getah virus infection among horses on the private farms (Fig. Aedes vexans nipponii and Culex tritaeniorhynchus, and these two species were considered to be involved in the circulation of Getah virus in Japan [Alphaviruses to which Getah virus belongs [As described above, the 2014 and 2015 outbreaks of Getah virus infection occurred after an absence of detectable outbreaks for more than three decades. The sudden outbreaks in the two sequential years at the same site suggest the occurrence of direct factors such as mutations that might alter the features of the virus. Although we were initially concerned about antigenic mismatch between the circulating virus and the vaccine strain, our previous and current results suggest that the 2014 and 2015 isolates are antigenically similar to the vaccine strain (Table in Japan . In our in Japan . The car belongs , and als belongs . In thisIn conclusion, an outbreak of Getah virus infection occurred at the Miho training center in 30 horses from mid-August to late October 2015. It was sequential to the 2014 outbreak at the same site. The 2015 outbreak was caused by a virus closely related to the 2014 isolate and affected mostly 2-year-old susceptible horses under epizootiological circumstances similar to those in 2014. The existence of 2-year-olds introduced from non-epizootic regions could continue to pose a risk of additional outbreaks in upcoming years. Vaccination on private farms and breeding farms would help to minimize the risk of outbreaks. Continuous surveillance at the training center, as well on the farms surrounding the center, will be required.MEM, minimum essential medium; nsP3, non-structural protein 3; VN, virus-neutralizing


During the past 20 years liver transplantation has become the definitive treatment for most severe types of liver failure and hepatocellular carcinoma, in both children and adults. In the U.S., roughly 16,000 individuals are on the liver transplant waiting list. Only 38% of them will receive a transplant due to the organ shortage. This paper explores another option: bioengineering an autologous liver graft. We developed a 20-year model projecting future demand for liver transplants, along with costs based on current technology. We compared these cost projections against projected costs to bioengineer autologous liver grafts. The model was divided into: 1) the epidemiology model forecasting the number of wait-listed patients, operated patients and postoperative patients; and 2) the treatment model forecasting costs -related costs; and 10-year post-transplant-related costs) during the simulation period. The patient population was categorized using the Model for End-Stage Liver Disease score. The number of patients on the waiting list was projected to increase 23% over 20 years while the weighted average treatment costs in the pre-liver transplantation phase were forecast to increase 83% in Year 20. Projected demand for livers will increase 10% in 10 years and 23% in 20 years. Total costs of liver transplantation are forecast to increase 33% in 10 years and 81% in 20 years. By comparison, the projected cost to bioengineer autologous liver grafts is $9.7M based on current catalog prices for iPS-derived liver cells. The model projects a persistent increase in need and cost of donor livers over the next 20 years that’s constrained by a limited supply of donor livers. The number of patients who die while on the waiting list will reflect this ever-growing disparity. Currently, bioengineering autologous liver grafts is cost prohibitive. However, costs will decline rapidly with the introduction of new manufacturing strategies and economies of scale. Approximately 30 million people in the U.S. have a liver disorder. About 40,000 of them will progress to end-stage liver disease, which is responsible for approximately 30,000 deaths annually in the U.S. , 2. MediIn many ways, liver transplantation has been a victim of its own success. As transplantation science evolved, indications for this therapeutic modality expanded to include many causes of acute and chronic liver failure, cirrhosis, inherited metabolic diseases and some cases of cancers . Yet theNumerous unconventional strategies have been explored to increase the number of livers available for transplantation. These include: the use of marginal donors, an ill-defined group comprised of donors over the age of 60; donors with greater than 30% hypernatremia or macrosteatosis; donors with positive serologies for the hepatitis C or hepatitis B virus; donors with a cold ischemia time of greater than 12 hours; non-heart-beating donors; and grafts from split-livers or living-related donors  and the Because the liver has the ability to regenerate completely after severe liver failure, it benefits from methods of temporary hepatic support and hepatocyte transplantation . AuxiliaCreating a universally available liver graft from autologous tissue and cells would increase the number of organs available for transplantation and eliminate the need for a life-long regimen of immune suppression drugs and their complications. Clearly there is a compelling social need to perform liver transplantation more widely. Now that many of the scientific elements are falling into place, it will likely be possible to bioengineer a liver from autologous cells.The transplant community faces major challenges treating end-stage liver disease—social, political and monetary. In this paper we attempt to define the size of the problem by creating an economic model for liver transplantation over the next 20 years. We projected the population in need plus the costs of treatment with conventional allogeneic liver transplantation. Then we looked at the prospective costs of transplanting livers bioengineered from autologous induced pluripotent stem cells (iPSCs).Our model is divided into two parts: The Epidemiology Model and the Treatment Costs Model. The Epidemiology Model forecasts the population dynamics of liver transplantation: wait-listed patients; operated patients, and postoperative patients out to 10 years. A simulation period of 20 years is considered for the cohort of wait-listed patients. (Patients prior to 2014 or after 2033 are excluded).The Treatment Costs Model forecasts costs generated during the simulation period. Costs are divided into pre-transplant-related costs, transplant (admission)-related costs and 10-year post-transplant-related costs. The patient population has been categorized using the Model for End-Stage Liver Disease (MELD) score. The pre-transplant-related costs are allocated per MELD score . They account for the costs generated one year prior to transplant (for severe illness) or while patients are on the waiting list (for moderate illness with waiting time exceeding one year). In this way we were able to capture the costs of treatment prior to transplantation for severely ill patients—who will be on the waiting list for days to a couple of months—compared to patients who are less ill and could be on the waiting list for up to five years. Due to the lack of published data, the admission-related costs and post-transplant-related costs are shown for all patients without regard to the MELD score. All costs are expressed in 2014 U.S. dollars (USD) and, where appropriate, were adjusted to 2014 USD using the Gross Domestic Product (GDP) deflator-based calculator . The 20-The outcomes of the Epidemiology Model are the number of new patients listed each year and their distributions based on MELD score and age at listing. Additionally, the following metrics are forecast: (Pre-liver transplantation) number of patients who will die or suffer severe illness on the waiting list; (Pre-liver transplantation) number of patients who will not receive a liver transplantation; (Pre-liver transplantation) number of patients who will receive a liver transplant; (Admission) number of patients surviving surgery; and (Post-liver transplantation) number of patients surviving Year 1 through Year 10 after liver transplantation.Outcomes of the Treatment Cost Model are: 1) the discounted treatment costs during the pre-liver transplantation phase, per to-be transplanted patients and per MELD score; 2) the discounted total resources used from the day of transplantation to discharge, per transplant-operated patient; and 3) the discounted total costs of treatment 10 years post-liver transplantation per patients surviving for 10 years. All metrics are shown over the 20-year period of simulation and at Years 1, 10 and 20.In numerical modeling, an uncertainty analysis employs different techniques for determining the reliability of model predictions and accounts for various sources of uncertainty in model input and design. In our model an uncertainty analysis is performed to generate plausible value ranges of the costs metrics at Year 20 (2033), and to assess the certainty at which the base case can be achieved. For this purpose, parameters are varied according to a realistic distribution and 30,000 Monte Carlo iterations are run using Oracle Crystal-Ball software. We then performed a sensitivity analysis, which determines how different values of an independent variable will impact the financial output metrics.The epidemiology and costs data are collected from various literature sources and are summarized in http://www.cellulardynamics.com). Factored into the per-vial pricing are the manufacturing costs, royalty payments for IP rights to commercialize, and a profit margin for the vendor.For the purposes of the manufactured liver cost model, we have made several key assumptions. The initial assumption is that hepatocytes, endothelial cells, and fibroblasts will be sufficient to build an entire liver from scratch. The second assumption is that there will be no proliferation of cells within the bioengineered organ—therefore, every cell will be provided in the transplanted model—which is unlikely, but ensures a conservative model. We have also made the assumption that the cells comprising the bioengineered liver will be purchased as individual cryovials at current catalog list pricing. In this case study, Cellular Dynamics International (CDI) is used as the vendor  will be patients scoring 10–19, whereas in 2033 the 20–29 MELD score category will be the largest at 39%. This study predicts that patients' age at listing will increase from an average of 57.7 years in 2014 to an average of 62 years in 2033, a 7% increase.Based on the OPTN database, this model projects that 65% of the simulated patients over a 20-year period would receive a liver transplant, 18% would not undergo the transplant operation . Additionally, the model outcomes predict that 6% of the patients who are operated on would die during hospitalization for transplantation and 63% of transplanted patients would survive the 10-year period following transplantation. The second set of analyses of forecasting the financial metrics show that the treatment of a patient in the pre-liver transplantation phase will cost between $49,407 (MELD score <9) and $613,020 (MELD score >40) on Year 1. It will increase, respectively, to $71,621 and $867,564 in Year 20. The weighted average treatment costs in the pre-liver transplantation phase are predicted to be $168,386 in Year 1 and will increase by 83% to $307,610 by Year 20. The treatment costs for a patient receiving a liver transplantation (admission phase) and those 10 years post-transplantation will increase by 42% between Year 1 and Year 20 from, respectively, $588,580 to $836,788 and from $670,839 to $949,391 . The totcertainty to achieve the cost projections (at or above), at Year 20, is 36%-39% for the pre-transplant-related costs, 37% for the hospital admission-related costs and 36% for the 10-year post-transplant-related costs  and the annual cost discount rate (sensitivity index -17% to -28%) . Pre-traThe manufacture and universal availability of a liver graft from autologous tissue and cells would change the paradigm of organ transplantation by increasing the number of organs available for transplantation, and eliminate the need for life-long immune suppression and its complications. Several strategies are being developed for construction of whole organs using the decellularized whole organ matrix, and subsequent re-seeding with relevant cell types, in physiologically appropriate bioreactors –30. EngiIn The simple truth is that significantly more livers are needed for transplantation. Transplantation is the definitive treatment for end-stage liver disease, yet one in ten people on the transplant waiting list die because of the dearth of organs. Additionally, the number of individuals on the waiting list gauges only a fraction of the true need. According to clinicians, thousands more could benefit from a transplant but are precluded from the waiting lists because they have not yet reached critical status. The shortfall of organs is predicted to rise in the coming decades given the increasing prevalence of liver-damaging viruses, a surge in fatty liver disease, and a buildup of environmental toxins.The social need for greater availability of livers for transplant is bolstered by an economic argument. The epidemiology and treatment costs models forecast demand for liver transplantation will increase by 10% in 10 years and by 23% in 20 years. Total weighted treatment costs of a liver transplantation—including pre-operative, admission and post-operative phases—will increase from $1,427,805 per patient in Year 1 to $2,093,789 in Year 20 .This study is subject to certain limitations with the major challenge being access to databases. To build and run the model, data was collected from different sources and databases available online  which maMoreover, our model excludes several new developments that are likely to modify the course of liver transplantation. Currently, the top four causes of chronic liver disease in patients on the U.S. liver transplant waitlist are chronic hepatitis C virus (HCV) infection, alcoholic liver disease (ALD), nonalcoholic steatohepatitis (NASH), and a combination of chronic HCV infection and ALD (HCV/ALD). However, these rankings may change. New direct-acting antiviral agents are transforming the treatment of chronic hepatitis C. A recently approved single-tablet regimen of the hepatitis C virus (HCV) NS5A inhibitor ledipasvir and the NS5B inhibitor sofosbuvir  has been reported highly effective –38. HoweAdditionally, several novel cellular therapies to induce tolerance in solid-organ transplant patients have entered early-phase clinical trials . AlthougHowever, despite these limitations the financial outcomes forecasted in Year 1 in our model are in similar ranges as those reported by Buchanan  and co-wFrom the results of Buchanan et al  one shouLiver allocation in the U.S. is donation-based with the sickest patients prioritized using the Model for End-Stage Liver Disease (MELD) score. Following implementation of the MELD system, the number of patients who died while on the waiting list fell, but the health status of listed patients deteriorated. Care of wait-listed patients is expensive. The cost of treating severely ill patients, reflected by a high MELD score, requires longer hospital admissions, more frequent and longer use of Intensive Care Units, and more laboratory tests and medications .eligible donor organs into actual donor organs. Schnitzler et al (2005) [Further complicating the problem, organ availability is limited not only by the number of donors, but also by the inability to convert l (2005)  have sugl (2005) , and orgl (2005) . Still, Social policy may ameliorate the shortage in the short term. Adopting an opt-out system, for example, will make all citizens automatic donors unless they choose to opt out and will certainly increase the availability of organs. This will put the eligible-donor/actual-donor conversion rate at 100% and presumably will not fall below the current rate. Additionally, creating a marketplace for organs has proponents, but this proposed solution raises numerous ethical concerns, generating considerable opposition. Neither an opt-out system nor establishing a marketplace for organs is a likely probable solution.The shortage may be overcome by the manufacture of bioengineered livers from autologous stem cells. Elements of the technology, although not yet validated by a reproducible protocol, are in place. Critical aspects of the developmental biology of the liver remain unknown but the manufacture of replacement organs is no longer science fiction. Relatively soon anyone needing a liver could have one bioengineered from one of their own skin cells. Because there will be no immune reaction, there will be no need for immunosuppressive drugs, an added clinical and financial benefit over using a cadaveric organ.How far are we from a bioengineered organ? The factors that drive the self-organization of the cells comprising the liver are not yet fully detailed. The question becomes, do we need to recapitulate all the evolutionary programming that drives cells to form metazoan structures? Perhaps there are shortcuts. That’s the promise of decellularized scaffolds . Will dein vitro or in vivo bioreactor, or by means of bioprinting, these hepatocyte precursor cells will provide the building blocks for a customized liver that originates from patient-specific somatic cells.Blood or skin cells from a person with a high MELD score are already the sources of iPSCs from which hepatocyte precursor cells are made. In an Certainly the current tremendous cost ($9.7M) to bioengineer just 35% of a liver—a tissue mass that may be sufficient to maintain critical metabolic function—is not a viable option. However, these costs will decrease dramatically as this therapeutic approach gains traction. Why? Because the bulk of the manufacturing cost is associated with cell production and these costs will fall with economies of scale.The time required for reprogramming and selecting a clone for production can be reduced by banking personal iPSCs , which places the focus on reducing the costs associated with multiple growth and differentiation media used in the manufacturing process.Reagents comprised of growth factors and small molecules are used to activate and/or repress signaling pathways at key checkpoints that determine cellular fate. Pulling these molecular levers at the precise moments necessary to drive well-defined tissue specification across the appropriate germ layers of endoderm, ectoderm and mesoderm requires knowledge of the signaling mechanisms affecting gene regulation and protein signaling cascades.Technicians begin the cell-production process upstream of differentiation with the initial culture of the stem cells. Stem-cell culture is typically effected in mTeSR or the more cost efficient and defined E8 media, both of which are composed of growth factors and signaling molecules. These media provide stable stem cell cultures in the absence of a feeder layer, minimizing variability and maintaining the cells in a state of readiness to enter differentiation.There are multiple hepatocyte differentiation protocols. All follow the same approximate timeline of 25–30 days and employ similar induction factors –51 , or recellularized organ tissue scaffolds that could be maintained in vivo, ex vivo or as a de novo engineered scaffold used in combination with a bioreactor device. Any of these approaches could lead to improvements in production scale and concomitant cost reductions.In addition to the inevitable reduction in production costs of reagents, there will be corresponding efficiencies in cell production that will increase batch sizes and reduce labor costs depending on the choice of bioreactor (defined as any production mechanism outside of flask culture used to differentiate and expand the cells). Bioreactors could be envisioned as The cost of sequencing the first human genome was approximately $300 million , 53. , necessitating iPSC differentiation for these cell types. Additionally, differentiated cells derived from iPSCs may still be immature, functionally resembling fetal or neonatal phenotype. Cell maturity may prove to be critical for diseases requiring functional differentiated cells. So although proof-of-principle for whole-organ assembly and transplantation has been shown for three of the solid organs , numerous obstacles must be overcome before the cells generated can be used widely in preclinical studies. Perhaps other innovative technologies already on the horizon (e.g. organ reprogramming) may change the future of liver transplantation –57.S1 Economic Model(XLSX)Click here for additional data file.


The mortality rate of gallbladder cancer (GBC) is considerably high in India and world over. The Adenomatous Polyposis Coli (APC) gene is widely reported for its role in cancer. APC is known to have two promoter regions, 1A and 1B, that show differential role in various cancers. However, role of these promoters and their exons in the molecular pathogenesis of GBC is obscure. Our aim was to study the epigenetic control of APC promoters in GBC and to evaluate their utility as prognostic/diagnostic biomarker of GBC.We carried out methylation specific PCR of the modified genomic DNA from GBC and GSD tissues, followed by semi-quantitative and quantitative PCRs (qPCR). We compared the transcript levels of the two exons of APC in GBC and GSD as compared to their adjacent control tissues. We also performed Immunohistochemistry (IHC) on tissue micro array (TMA) of GBC and GSD. The scoring of different tissue cores was done by the expert pathologist and student’s t-test was performed to check the significance.The APC 1A promoter was found significantly methylated in both GBC  and GSD , but 1B was not. The down-regulation of APC exon 1 was observed in both GBC and GSD, whereas exon 2 appeared normally expressed. Immunohistochemistry of APC protein on Tissue Microarray (TMA) revealed down regulation with negative score of 34.48% in GBC (p=0.057), 1+ in 24.14% GBC (p=0.005), 2+ in 25.85% in early stage or GSD (p=0.091).We infer that APC is epigenetically down regulated in advance cases of GBC and might be a key step in the tumorigenesis of gallbladder. In future, APC may be considered for diagnostic, prognostic and therapeutic purposes in GBC.


The objective of this biomechanical study was to evaluate the stability provided by a newly developed shape memory alloy hook (SMAH) in a cadaveric transforaminal lumbar interbody fusion (TLIF) model.in vitro flexibility experiment by applying pure moments of ±8 Nm in flexion/extension, left/right lateral bending, and left/right axial rotation. After intact testing, a TLIF was performed at L4-5. Each specimen was tested for the following constructs: unilateral SMAH (USMAH); bilateral SMAH (BSMAH); unilateral pedicle screws and rods (UPS); and bilateral pedicle screws and rods (BPS). The L3–L4, L4–L5, and L5-S1 range of motion (ROM) were recorded by a Motion Analysis System.Six human cadaveric spines (L1-S2) were tested in an Compared to the other constructs, the BPS provided the most stability. The UPS significantly reduced the ROM in extension/flexion and lateral bending; the BSMAH significantly reduced the ROM in extension/flexion, lateral bending, and axial rotation; and the USMAH significantly reduced the ROM in flexion and left lateral bending compared with the intact spine (p<0.05). The USMAH slightly reduced the ROM in extension, right lateral bending and axial rotation (p>0.05). Stability provided by the USMAH compared with the UPS was not significantly different. ROMs of adjacent segments increased in all fixed constructs (p>0.05).in vitro. Further studies are required to determine whether the SMAH can achieve fusion in vivo and alleviate adjacent segment degeneration.Bilateral SMAH fixation can achieve immediate stability after L4–5 TLIF  Over the last 100 years, lumbar spinal fusion has evolved as the conventional treatment for infection, tuberculosis, fracture, malformation, arthritis, and degenerative disorders. Traditional posterior lateral fusion has been replaced by interbody fusion gradually, which provides solid fixation of spinal segments while maintaining a physical load-bearing capacity and proper disc height Transforaminal interbody fusion (TLIF) was developed by Harms TLIF involves fixation of adjacent vertebral bodies with implants to achieve fusion. Pedicle screw and rod fixation was popular, but widespread application is limited by postoperative complications and high costs in vitro flexibility experiment. We hypothesized that the semi-rigid fixation effect of the SMA construct would decrease the range of motion (ROM) of fixed segments compared to intact spines, and the ROM of adjacent segments, compared to fixation with pedicle screws and rods.Since Buehler found nickel-titanium alloy had a shape memory effect in 1963 The cadavers were donated to the Department of Anatomy of Guilin Medical College for the purpose of teaching and preclinical research. Written informed consents from the donors or the next of kin were obtained before donation. Permission from the ethics committee of Guilin Medical College was obtained before the study. And the study was conducted according to the principles outlined in the Declaration of Helsinki.2 ranging from 0.91 to 1.38 g/cm2. All specimens had normal bones without osteoporosis. Specimens were thawed at 4°C overnight. Subsequently, the paraspinal muscles were carefully removed while preserving the intervertebral ligaments, capsules, joints, and discs intact. Specimens were implanted with steel nails at L1 and S2, and embedded at L1 and S1–S2 vertically with polymethylmethacrylate (PMMA), finally wrapped with physiological saline gauze, sealed, and stored at −20°C until the flexibility test.Six male fresh-frozen human cadaver lumbosacral spines were used in this study . Anterior-posterior and lateral radiographs of the spines confirmed the absence of any neoplastic disease, significant degeneration, fractures, or deformities The Flexibility Test System was composed of cables, pulleys, suspended counterweights, a loading disc, and a basement. The lower side of the PMMA block was placed on the basement, the upper side of the PMMA block was fixed to the loading disc. The cables connected to the loading disc and suspended counterweight transferred equal but opposing forces on to the top of the specimen . Thus, tSpecimens were initially tested in the intact state. Subsequently, TLIF was performed; each specimen underwent a right-sided L4-5 facetectomy and discectomy. Facetectomy was performed 1 cm away from the facet surface. The superior edge of the L4 lamina, the inferior edge of the L5 lamina, and the part of the lamina connected to the vertebral pedicle were intact. Therefore, force applied to the lamina was delivered to the vertebral body under physiological conditions, and hooks instrumented on the superior edge of the L4 lamina and the inferior edge of the L5 lamina could achieve stability and resistance to failure. Each specimen was then tested for the following surgical constructs: USMAH, BSMAH, UPS, and BPS. A maximum moment of ±8 Nm was applied on all specimens as previously described For the USMAH, an appropriately sized TLIF spacer was inserted into the middle third of the L4-5 disc space. The length between the superior edge of the L4 lamina and inferior edge of the L5 lamina was measured to allow selection of a suitable SMAH. Different size SMAHs ranging from 39 mm to 59 mm in length were designed to meet individual needs. Every 2 mm is an interval. Therefore, if the shortest length between the superior edge of the L4 lamina and inferior edge of the L5 lamina was 45 mm, a 43 mm or 41 mm SMAH was chosen. The smaller size hook provided a persistent compressive force to stabilize the lumbar segments. After an SMAH was instrumented, stability was checked by shaking the hook. The hook was unfolded in an ice-water mixture using a stretcher, and instrumented on the right side superior edge of the L4 lamina and inferior edge of the L5 lamina using a holder . Gauze sDuring the testing progress, physiological saline was sprayed on the specimen every 5 minutes to keep it moist and fresh. Testing was conducted at room temperature. X-rays were taken after each test . All opeStatistical analyses were performed using SPSS 19.0 software. Comparison of ROM was performed using one-way analysis of variance (ANOVA) and post hoc analysis by Tukey for multiple comparison procedures. p<0.05 was considered significant.In flexion, all instrumented constructs significantly decreased ROM compared with the intact spine (p<0.05). BPS and BSMAH decreased ROM by 71% and 60%, respectively. UPS and USMAH decreased ROM by 41% each. There was no significant difference between the stability offered by USMAH, BSMAH, UPS and BPS.In extension, BSMAH, UPS, and BPS significantly decreased ROM by 51%, 46%, and 70%, respectively, compared with the intact spine (p<0.05). USMAH decreased ROM by 26%; however, there was no significant difference compared with the intact spine (p>0.05). There was significant difference between the stability offered by USMAH and BPS (p<0.05).In left lateral bending, all instrumented constructs significantly decreased ROM compared with the intact spine (p<0.05). BSMAH and BPS decreased ROM by 68% and 72%, respectively; USMAH and UPS decreased ROM by 33% and 35%, respectively. There was no significant difference between the stability offered by USMAH and UPS, or BSMAH and BPS. Bilateral fixation constructs had lower ROM compared with unilateral fixation constructs (p<0.05).In right lateral bending, BSMAH, UPS, and BPS significantly decreased ROM compared with the intact spine (p<0.05). BSMAH and BPS decreased ROM by 52% and 71%, respectively. USMAH and UPS decreased ROM by 29% and 31%, respectively. There was no significant difference between the stability offered by USMAH and UPS, or BSMAH and BPS.In axial rotation, BSMAH and BPS significantly decreased ROM by 51% and 54%, respectively, compared with the intact spine, (p<0.05). There was no significant difference between the stability offered by USMAH and UPS compared with the intact spine .At L3-4 and L5-S1, there was no significant difference in ROM between the constructs compared with the intact spine (p>0.05). Mean ROM of BSMAH and BPS appeared higher compared with the intact spine and USMAH and UPS, but the differences were not significant , 6.in vitro spinal reconstruction and biomechanical testing. In 1998, Harms and Jeszenszky introduced TLIF as an alternative technique to PLIF in an attempt to overcome risk for damage to the conusmedullaris and dural sac In vitro biomechanical tests have demonstrated that TLIF can achieve immediate stability following surgery with unilateral or bilateral pedicle screw and rod fixation In vivo analysis has revealed that 30% of the vertebral endplate surface area is the minimum required to achieve rigid interbody fusion In this study, TLIF was chosen as the model for Ni-Ti SMA instruments have a combination of good mechanical strength, specific properties such as the shape memory effect and superelasticity, and good biocompatibility The Ni-Ti alloy can be bent when cooled and then retake its original shape when heated. This property, known as shape-memory, has resulted in the application of shape-memory alloy wire implantations for gradual correction of scoliosis We predicted that the SMAH would be an ideal implant if it could provide enough stability after TLIF. In accordance with previous reports, our study demonstrated that the ROM of an operated segment was decreased after TLIF using unilateral or bilateral pedicle screw and rod fixation In right lateral bending and axial rotation, USMAH did not significantly decrease ROM compared to the intact spine; this may have been due to a small sample size. The degree of stability required to attain lumbar fusion is unknown, but it is generally believed that the ROM of an operated segment should not be larger than the intact spine. Although UPS cause a non-significant decrease in ROM in axial rotation, clinical studies have shown that TLIF with unilateral pedicle screw and rod fixation can achieve the same fusion rate and satisfaction as bilateral instrumentation Adjacent segment degeneration (ASD) has been recognized as a potential long-term complication of rigidly instrumented fusion in vivo requires further experiments and supportive clinical data. Third, we could not make a clear conclusion about the effect on the adjacent segment. These studies should be extended to include measurement of intravertebral facet-joint pressure and the use of a finite element analysis model to simulate the ROM, the intravertebral and facet-joint, and the strain distribution of the whole unit, to enable more definitive conclusions to be made. Fourth, because stability and resistance to failure of SMAH implants are dependent upon the integrity of the lamina, we performed facetectomy within 1 cm from the facet surface to ensure the lamina remained intact and was strong enough to instrument with an SMAH. However, in clinical practice, for the purpose of decompression, wide resection of lamina may be needed. Under these conditions, application of an SMAH may not be the optimal choice. Fifth, some patients have severe lumbar spinal degeneration with thickened lamina and spinal canal stenosis, which will make SMAH implantation difficult. Even if implantation is successful, epidural compression is possible. We recommend that surgeons use pre-operative CT scans to guide their decision-making, and that SMAH should be used for patients with mild osteo-degenerated lumbar diseases that require TLIF . We propose that the SMAH represents another option for spinal surgeons treating these diseases. Sixth, we did not test the stability of the SMAH construct when the placement of the spacer in the disc space was varied. Therefore, we plan further studies using cadaveric experiments and Finite Element Analysis.There are several limitations in our study: First, the sample size was necessarily small as we conducted our investigations using a cadaveric model. Second, our results were only indicative of immediate stabilization. Lack of experimental equipment limited our ability to apply axial pressure to simulate the physical load, and perform tests for fatigability and pull out strength. The biomechanical properties of implants after TLIF in vivo are complex. The ability of this newly developed SMAH to provide stabilization and achieve fusion In conclusion, bilateral SMAH fixation can achieve immediate stability at L4-5 after TLIF. The newly developed SMAH is an ideal implant for lumbar fusion, with the possibility to reduce operative time and healthcare costs and improve patient outcomes. Further studies are warranted to identify the stress distribution of the whole unit and whether this fixation method could achieve an acceptable fusion rate in other segments and in vivo.


The genome of a virus isolated from an outbreak of foot-and-mouth disease (FMD) in Morocco in 2015 is described here. This virus is classified as lineage Ind-2001d within serotype O, topotype ME-SA (Middle East-South Asia). This lineage is endemic on the Indian subcontinent but has caused outbreaks in the Middle East and North Africa since 2013. Aphthovirus (family Picornaviridae), an etiological agent for an economically important vesicular disease that spreads rapidly among susceptible cloven-hoofed animals. Outbreaks of FMD can lead to major economic losses due to reduction in livestock production and disruption in national and international trade. The virus is classified into seven immunologically distinct serotypes, each containing numerous variants that are often restricted to specific geographical locations (topotypes) and lineages (Foot-and-mouth disease (FMD) virus is a single-stranded positive-sense RNA virus of the genus lineages ; due to During November 2015, the FAO World Reference Laboratory for FMD (WRLFMD) received samples from cattle displaying clinical signs of FMD in the Sidi Bennour Province, Morocco. Subsequently, FMD virus (FMDV) serotype O was confirmed to be the causative agent. This was the first detection of FMDV in Morocco since 1999 . VirusesHere, we report the complete genome sequence of an isolate (O/MOR/1/2015) from the O/ME-SA/Ind-2001d lineage causing outbreaks in Morocco in October 2015. This virus was isolated from a clinical sample by a single passage in primary bovine thyroid (BTy) cells. The genome was sequenced using MiSeq technology , as previously described . SequencThe genome sequence of O/MOR/1/2015 was 8,115 nucleotides (nt) in length [including 15 nt of a poly(C) tract of unknown length located within the 5′ untranslated region (UTR)]. The first 6 nt of the genome were not determined. A single large open reading frame of 6,999 nt was predicted to encode a polyprotein of 2,333 amino acids (aa) containing four structural  and 10 nonstructural proteins . A comparison to the genome sequence of O/LIB/2/2013 (Libya) belonging to the same lineage  showed 9After the initial emergence of the Ind-2001d lineage from the Indian subcontinent to the Middle East and North Africa in 2013, the virus became established in North Africa. The emergence and continued spread of the Ind-2001d lineage reinforce the need to monitor and report outbreaks of FMD not only to understand the global epidemiology of the disease but also to develop tailored control strategies.KU291242.The nucleotide sequence of FMDV O/MOR/1/2015 has been deposited in GenBank under the accession no.


Porcine epidemic diarrhea virus (PEDV) has been found throughout Europe and Asia, and has emerged in North and South America. A whole genome sequence was obtained from a paraffin-embedded tissue sample from the Instituto Colombiano Agropecuario (ICA), Colombia through Next Generation Sequencing techniques to further understand the evolution of PEDV. Porcine epidemic diarrhea virus (PEDV) is a single stranded, positive sense RNA virus that causes dramatic mortality in neonatal piglets. The pathogen was discovered in the United Kingdom in 1971, and was later found throughout Europe and Asia . In 2010On 2 May 2014, the Veterinary Diagnostic Laboratory at the University of Minnesota received paraffin-embedded tissue from a 1-week old piglet that had diarrhea submitted by the Instituto Colombiano Agropecuario (ICA) of Colombia. The sample was strongly positive for PEDV by immunohistochemistry. To determine the phylogenetic relationship of the Colombian PEDV strain to global PEDV strains, the total RNA was extracted using MagMAX FFPE total nucleic acid extraction and sequenced using Next Generation Sequencing technology , 6. The Through continued sequencing of PEDV from diverse geographical areas, we hope to keep monitoring and learning about the evolution of PEDV in the Americas. Phylogenetic analysis will help to prevent or mitigate outbreaks of PEDV in the future.KU569509.The COL/Cundinamarca/2014 PEDV sequence has been deposited into GenBank under the accession number


Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of  Annually, up to 10% of the U.S. population is affected by symptomatic influenza infection. It had been reported that more than 220,000 persons are hospitalized, of which 24,000 die due to influenza-associated illness each year Influenza is a serious public health problem that causes severe illnesses and deaths for higher risk populations. Influenza A viruses are responsible for seasonal epidemics and have caused three pandemics in the 20Influenza virus, which is an enveloped, negative-strand RNA virus with a segmented RNA genome, is characterized by frequent mutations - antigenic drifts  and antigenic shifts . To combat the virus, a number of treatments are currently available. Vaccines, such as cell-based whole-virion inactivated vaccines and dose-sparing adjuvants Cryptoporus volvatus belongs to Eumycota, Cryptoporusth century a.d. when the record of Cryptoporus volvatus appeared in “Materia Medica of Yunnan” Cryptoporus volvatus reveals that it contains many physiological activators, such as polysaccharose, amino acid, volatile oil, and cryptoporic acids etc. Cryptoporus volvatus has been reported to have anti-tumor, anti-allergy, anti-inflammation, and immunomodulatory activities Natural products can be candidates to be identified as new generations of antivirals Cryptoporus volvatus has potential antiviral effects against porcine reproductive and respiratory syndrome virus (PRRSV) infection in vivo and in vitroCryptoporus volvatus has the ability to inhibit influenza virus infection. We first examined its potential to inhibit different influenza virus strain replication in vitro, and then determined if the extract could protect mice from lethal challenge with 2009 pandemic H1N1 influenza virus. Our results showed that the extract from Cryptoporus volvatus inhibited influenza virus infections in vitro through targeting an early stage in the replication cycle, very likely the virus entry into host cells, and the release of the virus from cells. More importantly, Cryptoporus volvatus efficiently inhibited 2009 pandemic H1N1 influenza virus in vivo. These results implicate that the aqueous extract from the fruiting body of Cryptoporus volvatus has the potential to be an antiviral therapeutics against influenza virus infection.We previously showed that the aqueous extract from the fruiting body of All animal research was approved by the Beijing Association for Science and Technology  2007–0023) and complied with the guidelines of Beijing Laboratory Animal Welfare and Ethics of the Beijing Administration Committee of Laboratory Animals.Madin-Darby Canine Kidney (MDCK) cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin.50).The pandemic H1N1/2009 influenza virus, A/Beijing/7/2009(H1N1/09), was isolated from a young patient with an influenza-like illness in December 2009 Cells were fixed with cold methanol-acetone (1∶1) for 10 min at 4°C, washed with phosphate-buffered saline (PBS), and then blocked with 5% normal goat serum for 30 min at room temperature. After blocking, cells were stained with mouse monoclonal antibody AA5H  against influenza A virus NP. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgG (H+L)  for 60 min at 37°C. After three washes in PBS, cells were counter-stained with DAPI and examined by fluorescence microscopy.Cryptoporus volvatus was purchased from Yunnan Province, China. The dry fruiting body of Cryptoporus volvatus was crushed by grinder and soaked in distilled water (1 g dry fruiting body in 20 ml H2O) overnight at 4°C, and then centrifuged at 8000–10,000 g for 30 min. The supernatant was harvested and freeze-dried, and then stored at −80°C until use. When used, the freeze-dried powder was re-dissolved with normal saline or culture medium and filtered with 0.22 mm filters. The final concentration was determined by the weight of the dry fruiting body and the volume of solvent finally used,The Cryptoporus volvatus extract on cell viability. MDCK cells in 96-well plates were treated with sequential dilutions of the extract or normal saline in a total of 100 µl growth medium for 48 h. And then, 20 µl of freshly made 5 mg/ml MTT solution was added to each well, and the cells were incubated at 37°C for another 5 h before the medium was replaced with 200 µl DMSO to dissolve the crystals. The plates were further incubated at 37°C for 5 min to dissolve any air bubbles before the MTT signal was measured at an absorbance of 550 nm. The 50% cytotoxic concentrations (CC50) were analyzed by GraphPad Prism .The MTT  assay was used to examine the effect of the Cryptoporus volvatus extract and 2 mg/ml TPCK-treated trypsin. Twenty-four hours later, the supernatant was collected for virus titration and cells were fixed for indirect immunofluorescence assay. The 50% effective concentration (EC50) was determined using a 4 parameter, nonlinear regression of dose response inhibition by plotting log (inhibitor(concentration) vs. viral titer (variable slope) using GraphPad Prism .Confluent MDCK cell monolayers in 96-well plate were inoculated with the different influenza virus (multiplicity of infection [MOI]  = 0.1) in the presence of different concentrations of the Confluent MDCK cell monolayers in 96-well plate were inoculated with influenza virus A/WSN at an MOI of 1 at 4°C for 2 h and then shifted to 37°C (this time point was set up as 0 h). And the extract was added at −1 h, 0 h, 1 h, 2 h, 4 h, 6 h or 8 h p.i.. At 9 h p.i., the supernatants were collected for virus titration.5 TCID50 was incubated with different concentrations of the Cryptoporus volvatus extract for 1 h or 3 h at 37°C. Following the treatment, viral infectivity was determined on MDCK cells.Influenza virus A/WSN of 10Cryptoporus volvatus extract and A/WSN (MOI of 2) at 4°C for 2 h. After cells were washed 6 times with cold PBS, cell lysates were prepared by rapid freeze-thaw. Virus titer was determined as described above.MDCK cells were incubated with different concentrations of the 5′-AGCAAAAGCAGG-3′). A (GAPDH)-specific primer (5′- GAAGATGGTGATGGGATTTC-3′) was also included in the RT reaction mixture. The quantitative real-time PCR was carried out with a 20 µl reaction mixture containing primers specific for influenza virus M1 gene (5′-ACAGATTGCTGACTCCCAGC-3′ and 5′-TCTCATCGCCTGCACCATTT-3′) or for GAPDH RNA (5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′) by using SYBR green DNA dye (TAKARA) following the manufacturer's introductions.MDCK cells in 6-well plates were infected with influenza virus at an MOI of 10 at 4°C for 1 h. The inoculum was aspirated, and the cell monolayer was washed three times with cold PBS, replaced with fresh DMEM medium containing different concentration of the extract, and switched to 37°C. At 1 h post switching to 37°C, cells were washed twice with acidic PBS-HCl (pH1.3) to remove any un-internalized viral particles on cell surface, followed by washing twice with PBS. Intracellular virus was analyzed using quantitative RT-PCR. Total cellular RNAs were extracted using Trizol Reagent (Invitrogen) and reverse transcription (RT) was conducted using oligonucleotides specific for vRNA (Cryptoporus volvatus extract or BFA (1 mg/ml). At 0.5 h and 1 h following medium replacement, supernatants were collected and cells were lysed by rapid freeze-thaw on dry ice-ethanol and a 37°C water bath. Influenza virus RNA copies in the supernatants (extracellular virus) and cell lysates (intracellular virus) were then quantified using quantitative real-time PCR as described above.MDCK cells were infected with A/WSN (MOI = 0.1). At 10 h p.i., cells were washed 3 times with PBS and replaced with fresh medium containing different concentrations of the 3.5 pfu BJ09 H1N1 virus and then treated with the Cryptoporus volvatus extract . Treatment was given twice daily for 8 days post virus inoculation (dpi) with half of the dosage via intragastric administration and the other half via thigh muscle injection. One group of mice was administered with the extract (50 µg extract/g body weight) without virus inoculation. One group of mice inoculated with BJ09 H1N1 virus was provided twice daily with normal saline, and one group of mice was just provided with normal saline. At 1, 4, and 7 dpi, three mice in BJ09/normal saline, or BJ09/50 µg/g treatment groups were euthanized and the lungs were collected for virus detection and titration. Also, at 4 dpi, lung sections from three mice in BJ09/normal saline, BJ09/16.5 µg/g, or BJ09/50 µg/g treatment group were collected for immunohistological analysis. Five mice in each group were monitored daily for 14 days for clinical signs post inoculation. Any mouse that lost >25% of its pre-inoculation body weight was euthanized.Six-week-old female BALB/c mice  were used in five treatment groups, and mice were anaesthetized with Zoletil  before treatment. Two groups of mice were intranasally inoculated with 10+SD .The lung sections were fixed in 10% (wt/vol) buffered formalin. Formalin-fixed tissues were then embedded in paraffin wax, sectioned into 5-mm slices, and mounted on glass slides. Tissues were stained with hematoxylin and eosin (H&E) for light microscopy, or stained for viral NP by immunohistochemistry (IHC) with a mouse monoclonal antibody as previously described One-way ANOVA except for the animal experiment, which was analyzed using Student's t test. Differences were considered to be statistically significant if the P value is less than 0.05. *P<0.05; **P<0.01.Results were analyzed using Cryptoporus volvatus extract, we first investigated whether the extract could inhibit influenza virus replication in vitro. Madin-Daby canine kidney (MDCK) cells were inoculated with A/Beijing/7/2009 H1N1 (H1N1/09) influenza virus and then treated with the extract at different concentrations. Twenty-four hours post infection, infected cells were directly observed under microscope following immunofluorescence staining of the virus . To further verify its anti-influenza virus activity, we examined whether the Cryptoporus volvatus extract could inhibit different influenza virus strain replication in MDCK cells. As illustrated in Cryptoporus volvatus extract also inhibited seasonal influenza virus A/Jiangxi/262/05(H3N2) and laboratory-adapted A/WSN/33 H1N1 (A/WSN) infections. Virus titer was decreased about 60-fold for H3N2 when the extract was at 5 mg/ml. For A/WSN, the suppression even reached to 104 fold with the extract at the concentration of 5 mg/ml. The extract inhibited influenza virus infection with 50% effective concentration (EC50) values of 0.45 mg/ml for H1N1/09 strain, 1.21 mg/ml for H3N2 strain, and 0.37 mg/ml for A/WSN strain.To evaluate the therapeutic potential of the he virus  and the he virus . Our resCryptoporus volvatus extract using the MTT assay (Cryptoporus volvatus retained approximately relative viability of 100% compared with control. And the relative viability of MDCK cells was reduced to less than 10% when the Cryptoporus volvatus extract concentration in medium was 400 mg/ml. The 50% cytotoxic concentration (CC50) of the Cryptoporus volvatus extract for MDCK cells was 148 mg/ml, which greatly exceeded its EC50. The therapeutic index (CC50/EC50) in MDCK cells was 328 for H1N1/09 virus strain,68 for H3N2 virus strain, and 400 for A/WSN virus strain.To exclude the possibility that nonspecific toxicity induced by the extract could affect influenza virus replication, we evaluated MDCK cell viability under various concentrations of the TT assay . Forty-eCryptoporus volvatus extract could inhibit influenza virus infection in vitro.These results suggested that the Cryptoporus volvatus extract, we examined the time course of their inhibitory effects. MDCK cells were infected with A/WSN virus at an MOI of 1 and, then treated with the extract at various time points post infection from -1 h to 8 h p.i.. We then measured the titer of infectious viral particles released into the supernatant at 9 h p.i. Cryptoporus volvatus extract was added at −1 h p.i., virus production was strongly blocked by a factor of about 3000 folds, while the extract displayed partial antiviral effect when added at 1 h p.i. or later to 8 h p.i., suggesting that the Cryptoporus volvatus extract is able to inhibit earlier and late stages in the virus replication cycle. However, for ribavirin which inhibits virus RNA synthesis, virus production was continuously inhibited between 1 h and 8 h p.i. as a function of compound addition. To verify whether the Cryptoporus volvatus extract can directly inactivate the virus infectivity, we incubated A/WSN (H1N1) virus with the Cryptoporus volvatus extract at 37°C for 1 h or 3 h. As shown in Cryptoporus volvatus extract had no effect on viral infectivity, suggesting that the inhibitory effect of the Cryptoporus volvatus extract is not due to its direct inactivation of H1N1 virion particles.To characterize the specific step(s) of the influenza virus life cycle that is inhibited by the Cryptoporus volvatus extract could block virus attachment or entry into host cells.Early events of influenza virus infection cycle include virus attachment and cell entry. Thus, we investigated whether the Cryptoporus volvatus extract in virus attachment. As shown in Cryptoporus volvatus extract did not affect the quantity of infectious virus particles that can attach to host membranes. To test whether the Cryptoporus volvatus extract acts at the internalization stage of infection, we used an assay described before Cryptoporus volvatus extract, and the cells were transferred to 37°C. Cell-attached viruses started to enter cells through endocytosis when the temperature was shifted from 4°C to 37°C. At 1 h post switching to 37°C, cells were washed with acidic PBS-HCl (pH = 1.3) to remove any un-internalized viral particles on the cell surface, and intracellular virus was analyzed using Real-Time PCR for the M1 gene. As illustrated in Cryptoporus volvatus extract, and only about 20% virus entry into cells compared of control was detected when the extract was at 5 mg/ml.We first determined the role of the Cryptoporus volvatus extract inhibits virus release, we used an assay described previously To investigate whether the Cryptoporus volvatus extract to inhibit H1N1/09 virus replication in mice, we collected the lungs of three mice in two of the three treatment groups to quantify H1N1/09 virus at day 1, 4, and 7 post infection . Similar results were also observed at 7 days post infection. These findings indicated that the Cryptoporus volvatus extract treatment was capable of suppressing H1N1/09 virus replication in mice. We further investigated influenza virus distribution in lungs by IHC . However, mice still showed obvious clinical signs, including decreased activity, huddling, hunched posture, and ruffled fur. Strikingly, high-dosage of the Cryptoporus volvatus extract treatment prevented all 5 mice from death. Mice in this group did not show obvious clinical signs except for slight weight loss  infection in vivo and in vitro. In this study, we found that the aqueous extract from the fruiting body of Cryptoporus volvatus also had broad and robust activity against influenza virus infection. Our data demonstrated that the extract could inhibit different influenza virus strain infection. And most importantly, we showed that the extract completely protected mice from lethal challenge with H1N1/09 influenza virus in a mouse model when we treated the mice with a high dose of the extract.Chinese herbal medicines are a unique source of medical complexity and diversity, and they have been exploited extensively in pursuit of new antiviral agents Cryptoporus volvatus extract to inhibit influenza virus infection in vitro, and its toxicity on cells. We showed that the CC50 of the Cryptoporus volvatus extract for MDCK cells was 148 mg/ml, which greatly exceeded its EC50 . The therapeutic index (CC50/EC50) in MDCK cells was 328 for H1N1/09 influenza virus, 68 for H3N2 virus, and 400 for A/WSN virus strain.We first investigated the potential of the Cryptoporus volvatus extract is not due to its direct inactivation of H1N1 virion particles, as its incubation with A/WSN (H1N1) virus at 37°C for 1 h or 3 h had no effect on viral infectivity (Cryptoporus volvatus extract blocks PRRSV entry into cells. Both viruses use similar entry routes and so it is conceivable that the extract maybe target cellular factor(s) which is essential for virus entry. However, as the extract is a mixture of many chemical compounds, we could not exclude the possibility of targeting viral proteins that mediate virus entry into cells, e.g. the receptor binding protein haemagglutinin (HA). Further studies are needed to illustrate the mechanism by which the extract inhibits virus entry.Then we investigated on which step(s) of influenza virus life cycle the extract exerted its effect to inhibit virus infection. The inhibitory effect of the ectivity . In the Cryptoporus volvatus extract has the potential to be used to treat H1N1 influenza virus infected mice. However, we could not exclude the fact that the H1N1 influenza virus infected mice treated with high dose of the extract still lost some weight compared to saline/no virus controls, even though these mice survived. Even though we showed that Cryptoporus volvatus could significantly inhibit H1N1 influenza virus replication in lungs of mice, the reduction of virus titre was minimal. We propose that there might be other mechanisms existed for Cryptoporus volvatus to protect mice from H1N1 influenza virus lethal challenge, e.g. suppressing inflammation. Indeed, Cryptoporus volvatus is used to treat bronchitis Cryptoporus volvatus is a crude extract, which includes many components. The antiviral effects of the extract might result from a mixture of active compounds rather than from a single chemical entity. The efficacy of Traditional Chinese Medicine (TCM) is a characteristic of a complex mixture of chemical compounds present in the various herbs. The concept of combinatorial medicines has been exemplified by the drug cocktail used in the treatment of acquired immunodeficiency syndrome Cryptoporus volvatus. Obviously, more work remains for us to do to identify molecules in the Cryptoporus volvatus extract.More importantly, in the animal study, we found that the extract could completely protected mice from lethal challenge with H1N1/2009 influenza virus. To make sure that the dose we used in mouse study did not cause much direct damage to the mice, the highest dose we used was 50 µg/g body weight. Indeed, the extract alone at this dose did not cause obvious weight loss and lung lesions compared to the saline/no virus control. To our surprise, the extract at high dose (50 µg/g body weight) not only reduced virus loads in lungs of mice, but also protected mice from lethal challenge with H1N1 influenza virus. These results suggest that the Cryptoporus volvatus exhibits antiviral activity against influenza A virus in vitro and in vivo, and has the potential to be developed into a new antiviral agent. Further studies are in progress to identify the molecules that are responsible for the inhibitions of virus replication.In conclusion, our findings reveal that the aqueous extract of