Alpha- and Betacryptovirus within the family Partitiviridae cause no visible effects on their hosts and are only transmitted by cell division and through gametes. The bipartite dsRNA genome is encoding a RNA-dependent RNA polymerase (RdRp) and a coat protein (CP). Aside from sequence and structural analysis, the investigation of protein interactions is another step towards virus characterization. Therefore, ORFs of two type members White Clover Cryptic Virus 1 and 2 (WCCV-1 and WCCV-2), as well as the related viruses from Red Clover and Dill were introduced into a bimolecular fluorescence complementation assay. We showed CP-CP dimerization for all tested viruses with localization for alphacryptoviruses at the nuclear membrane and for betacryptoviruses close to cell walls within the cytoplasm. For CPs of WCCV-1 and WCCV-2, deletion mutants were created to determine internal interaction sites. Moreover, RdRp self-interaction was found for all viruses, whereas CP-RdRp interactions were only detectable for the alphacryptoviruses. An intra-genus test of CPs was successful in various virus combinations, whereas an inter-genus interaction of WCCV-1CP and WCCV-2CP was absent. This is the first report of in vivo protein interactions of members in the family Partitiviridae, indicating distinct features of the alpha- and betacryptoviruses.Plant-infecting viruses of the genera  Alpha- and Betacryptovirus of the family Partitiviridae [Partitivirus and Cryspovirus, which include viruses infecting fungi and protozoa, respectively [Partitiviridae frequently occur in various species, often in mixed infections with different cryptic viruses and other kinds of dsRNA viruses, such as endornaviruses [Southern tomato virus [Cryptic viruses, widespread in mono- and dicotyledonous plant species, are currently classified in the genera iviridae ,2. Additectively . The genectively ,5. Thereectively . Crypticectively . No visiectively ,7. Plantaviruses  and viruto virus ,10.Alphacryptovirus and Betacryptovirus was established. RdRp polymerase activity linked with virus particles was confirmed by enzyme assays [Beet cryptic virus 3 [White clover cryptic virus 1 (WCCV-1) was published by Boccardo in 2005 [White clover cryptic virus 2 (WCCV-2) was determined in 2013 [Alphacryptovirus in two clusters and a relationship between herbal and fungal viruses in the family Partitiviridae was shown [Lotus japonicus influenced the growth of the roots [First studies dealing with cryptic viruses were done in the early 1980’s, followed by the first description of their genome structure and particle sizes . Variouse assays . The fir virus 3 . The fir in 2005 , the fir in 2013 . Phylogeas shown . Severalas shown . In addihe roots . Howeverhe roots .A cryptic virus with a dsRNA genome, but also any other RNA containing virus using dsRNA as a replication intermediate, faces a problem during its replication cycle. Plants natural defense mechanisms generally recognize dsRNA, which is subsequently degraded. RNA viruses have evolved special proteins—suppressors of silencing—to protect themselves in various ways from RNA degradation . CrypticPenicillium stoloniferum virus F (PSV-F) a member of the genus Partitivirus, which is closely related to plant infecting alpha- and betacryptoviruses. The particle composition follows a T = 1 symmetry consisting of 120 subunits [Recent X-ray diffraction studies focused on the structural analyses of virus particles. A 3D model was established for subunits . Furtherin vitro methods [in vivo method to detect protein interactions. However, this system relies on the yeast nucleus under artificial conditions. Protein interactions requiring biologically relevant modifications or a specific subcellular localization are not detectable [After genetic studies concerning plant cryptic viruses ,14 ident methods , the yea methods  is the mtectable . Therefotectable ,25. The Partitiviridaein planta. The aim was to verify expected and hypothesized protein interactions. Firstly, we focused on the CP dimerization, which is the starting point of virus assembly. Sixty of these dimers are building the particle structure of Partitiviridae with a T = 1 symmetry, whereas no additional viral components are needed for this domain swapping. Furthermore, we hypothesized an interaction of CP and RdRp. This interaction is proposed for the last steps of the virus assembly to introduce the RdRp in the particle and to activate the transcription [versus proteins of two other virus members of the same genus (interspecies interactions). Moreover, an intergenus interaction with the CPs of WCCV-1 and WCCV-2 was performed. Additionally, we used deletion mutants to narrow down the part involved in the CP-CP interaction of the two type members of alphacryptovirus, WCCV-1 and betacryptovirus, WCCV-2, respectively.In this study, an optimized BiFC-system  was usedcription . AdditioDue to the formerly described infection cycle of the cryptic viruses in plants, different protein-protein and protein-RNA interaction could be expected. The primary domain shaping of CP proteins to dimers forms the basis of the final capsid structure. Multiple interaction sites were found by structural analyses ,27, so ain planta localization approach could be the first step to provide more hints to understand the “cryptic strategy”.Another important step in the virus life cycle is to ensure the passive transport of cryptic viruses during cell division, especially to the gametes. Due to the lack of movement proteins for active transport via plasmodesmata, the cryptic viruses had to develop mechanisms to establish in meristem cells, which enable them to withstand thermotherapy . An inteAlphacryptovirus and Betacryptovirus. For this purpose, the type members WCCV-1 and WCCV-2 from Trifolium repens [Trifolium pratense, namely Red clover cryptic virus 1 and Red clover cryptic virus 2 [Anethum graveolens of the family Apiaceae are also included in the study.An optimized BiFC system was used to elucidate protein interactions of six different cryptic viruses from the genera m repens  and two  virus 2  were use virus 2  from Anei.e., to verify protein-protein interactions detected by the BiFC system. The development of controls with proteins of cryptic viruses is limited, because these viruses encode only two proteins, which largely reduces the number of possible interaction partners. To circumvent this drawback, proteins of closely and distantly related cryptic viruses from two different genera were used in this study to broaden up the spectrum of potential interaction partners. To ensure the association of the monomeric red fluorescent protein (mRFP) fragments each CP and RdRp protein was fused to the N- as well as the C-terminal fragment. This allowed a screening of multiple combinations of fusion proteins for fluorescence complementation in all permutations  an interaction was found . This invice versa with the BiFC1/4 vectors .However, except for WCCV-1 a CP interaction was demonstrated in all permutations of the tested viruses, which indicates interacting domains or areas independent of free N- and C-termini of the CPs. The 40 N-terminal amino acids of Partitivirus CPs were not involved in the structure of the particle resolved by 3D structure analyses ,27. Probin situ hybridization. However, the distinctive location of primary virus assembly sites showed in this study may indicate that the viruses of the genera Alphacryptovirus and Betacryptovirus use different compartments to co-exist in one cell.A more precise localization in cell compartments could be reached with other techniques like immune labeling electron microscopy in the host plants or The putative interaction domain within the WCCV-1CP and WCCV-2CP was approximated by dividing the coding frame into three parts. The fragments vary from 150 to 273 amino acids, so that protein structures should be formed. However, possible secondary structures were not taken into consideration for the choice of the selected regions. Moreover, each potential interaction of different fragments was tested with only two fusion permutations, resulting in a limited degree of freedom for protein adjustments. The particle structure, as outlined above, implied multiple interactions within a single CP for dimer formation. In addition, protein regions are known that are probably not at the surface of viral particles ,27 and mTo narrow down the interacting domains in the CP of WCCV-1 and WCCV-2, six different deletion mutants were created . We obtaThe orientation of the fusion in respect to the reporter part seems to be critical for the dimerization. The association with the C-termini of the full-length CP resulted in five detected interactions with CP fragments, whereas the opposite direction did not. It is particularly interesting to note that only if both partners include the F3-part the fluorescence was located on the nuclear membrane. This might be an indication that the C-terminus is involved in the protein localization perhaps it provides its own signal peptide sequence for the protein targeting.A similar approach was performed for the Betacryptovirus WCCV-2  delivered a positive signal . Three iSo the localization, in case of WCCV-1 and RCCV-1, clearly differs from RdRp and CP self-interactions, where greater deposits in the cells were missing. A localization of CP-RdRp interaction at or near the outer nuclear membrane as described for the primary dimer fusion was found in one permutation only. This might be an indication for a later CP-RdRp interaction step within the framework of virus assembly in the cytoplasm.Betacryptovirus  combination as already described above, but not between WCCV-1 and RCCV-1. In addition, a dimerization for all RdRps of the betacryptoviruses was observed.In regard to the betacryptoviruses a similar localization for CP hetero-dimers were observed and overall fewer combinations showed a positive signal with a lower number of cells and fewer intensities of fluorescence, especially in combination with the more distantly related DCV-2. Interactions occurred in all tested virus combinations. In particular, it was noticed that interactions were not found with all permutations, compared to self-interactions, the fluorescence was weaker and the localization changed. This was also the case for the RdRp hetero dimerization detected within the genus N. benthamiana. Furthermore, an imperfect assembly of virus CPs might cause a malfunction in the further localization. In case of heterologous tests CP subunits of the same virus might preferentially interact, thereby not leading to a fluorescence signal, because of missing one reporter part. Heterologous protein interactions can also induce different localizations like the occurrence of deposits within the cytoplasm in case of the alphacryptovirus. Concerning this, further data are needed for the precise localization and description of steps involved in the virus replication cycle.However, in case of WCCV-1 only a few permutations were found to react positive, which could indicate that RCCV-1 and DCV-1 with much more interactions could be better analyzed in our BiFC system in The pCB:GOI-mRFPN (BiFC 1), pCB:GOI-mRFPC (BiFC 2), pCB : mRFPN-GOI (BiFC 3) and pCB:mRFPC-GOI (BiFC 4) expression plasmids were generated as described by Zilian and Maiss (2011) .BamHI or BglII and SalI or XhoI) for cloning into the BiFC vectors  approach [The coding sequence of the CP or RdRp, respectively of WCCV-1, WCCV-2, RCCV-1, RCCV-2, DCV-1, DCV-2 were RT-PCR-amplified using dsRNA preparations from White Clover, Red Clover and Dill with RevertAid Premium Reverse Transcriptase and Phusion Flash Master Mix (Thermo Scientific) as described previously . New seq vectors  Table A1approach . The seqThe open reading frame of each CP was divided into six fragments by PCR mutagenesis using Phusion Flash DNA polymerase (Thermo Scientific). The F1, F3, F1/2, F2/3 and F1/3 fragments of WCCV-1CP, encoding aa 1–150, 151–338 and 339–487, respectively were generated using the BiFC2:mRFPC-WCCV-1CP and BiFC3:WCCV-1CP-mRFPN vectors as templates. The same fragments of WCCV-2CP, using aa 1–200, 201–473 and 474–673 were created from the BiFC3: WCCV-2CP-mRFPN and BiFC4: WCCV-2CP-mRFPNC vectors.A. tumefaciens strain GV2260 [N. benthamiana plants 4 to 5 weeks old was performed by using A. tumefaciens mixtures containing the BiFC1-4 plasmids and pCB:p35TBSVp19 binary plasmid. All infiltrated plants were incubated at room temperature for 3 days. Discs of infiltrated N. benthamiana leaves were investigated with a Leica TCS SP2 confocal laser scanning microscope. The excitation at 543 nm of the mRFP domain was performed by using the green neon laser. The emitted light was captured at 600–610 nm, thus creating consistent-recording conditions. Visualization of the chlorophyll autofluorescence was made by excitation at 488 nm with the argon/crypton laser and subsequent fluorescence detection at 690–740 nm. Digital capture and processing of the images were performed by using the Leica confocal software.The BiFC plasmids and pCB:p35TBSVp19, encoding the TBSV p19 protein as a suppressor of gene silencing, were used for the electroporation into Fusarium poae virus 1 was analyzed by X-ray crystallography [Partitiviridae. We obtained expected CP–RdRp interactions only for the members of the genus Alphacryptovirus. The localization of the CP dimers were observed for WCCV-1, RCCV-1 and DCV-1 in the nuclear membrane, whereas the fluorescence signals for the WCCV-2, RCCV-2 and DCV-2 was located in inclusions within the cytoplasm of epidermis cells. CP mutants of WCCV-1 and WCCV-2 showed a different localization of interaction sites in the CP.Our results revealed various differences in protein interactions between alpha- and betacryptoviruses, which are not only caused by different protein and particle sizes. As already described, betacryptoviruses differ from alphacryptovirus in terms of the presence of prominent arches on the virus particle surface . For thelography ,27. As llography . NeverthAlphacryptovirus and Betacryptovirus in the same host. Together with the different localization of the CP–CP interactions and findings of the CP–RdRp interactions only in the alphacryptoviruses primary indications are given for striking differences in the molecular life cycle of these two virus genera. However, this study is the first protein interaction approach in planta for viruses of the family Partitiviridae so far and merely one further step to understand the biology of the viruses of the family Partitiviridae.From the perspective of the evolutionary relationship, it is interesting to verify protein interactions between viruses in one genus infecting distant host plants and to find no interaction between the type members of genus


In Puerto Rico, risk for transmission of B-virus from free-ranging rhesus monkeys to humans has become a serious challenge. An incident with an injured rhesus monkey, seropositive for B-virus, resulted in inappropriate administration of antiviral postexposure prophylaxis. This incident underscores the importance of education about risks associated with interactions between humans and nonhuman primates. Macaca mulatta) and patas monkeys (Erythrocebus patas) .Both species originated from the La Parguera Primate Facility, which was administered by the Caribbean Primate Research Center of the University of Puerto Rico’s Medical Sciences Campus from 1961 until 1982 . In 1974Macaca . First documented in 1932, this virus has received considerable attention because rhesus macaques are a species commonly used in research. Data indicate that 74%–100% of adult rhesus monkeys are seropositive for the virus  of seropositive monkeys will shed the virus at any given time  reported direct contact with the monkey’s blood, and none were bitten or scratched by the monkey. B-virus is not a bloodborne pathogen and is not transmitted by blood. However, the possibility of contact with other body fluids that represent a risk for B-virus transmission  could not be ruled out in four persons. All 25 persons were interviewed and evaluated by Puerto Rico’s Worker’s Compensation Agency and placed on prophylactic antiviral medication. All persons were employees from various municipal agencies responding to the accident. None wore latex gloves or other protective gear, despite the fact that most of the persons (84%) had access to personal protective equipment, including latex gloves, masks, and protective eyewear. Current protocols emphasize that the first few minutes after an injury or exposure are the most important . This finding is alarming, not only from a legal but also from a public health perspective. Trapping and confinement could potentially stress an animal enough to initiate reactivation of latent virus. Inexperienced handlers are at increased risk for a bite or scratch, and fear of legal implications might prevent a person from reporting or seeking medical attention for such injuries. Healthcare providers inexperienced with primates and associated zoonotic diseases might be largely unaware or unfamiliar with B-virus.2 island. The monkeys adapted well, and after nesting bird populations dropped radically on the island, government officials decided to remove the rhesus population (Cercopithecus aethiops sabaeus). This species was introduced >300 years ago with the slave trade. A study completed in 1994 indicated that despite trapping and removing more than 10,000 monkeys for 14 years, the population still increased by 4.5%; agricultural damage from the monkeys increased almost 30% during the same time span . In 1966, to study adaptation processes, a total of 57 rhesus macaques were released onto the remote 1.2-kmAlthough government agencies and other organizations continue to discuss interventions to address the free-ranging population of monkeys, the Puerto Rico Department of Health has begun the process of public education. Informational bulletins for government employees will stress the importance of using personal protective equipment when handling a nonhuman primate and what to do in case of an exposure to a rhesus monkey. In addition, every emergency room will receive a protocol for evaluation and management of exposed persons.The persons exposed in the incident in Bayamón suffered a low-risk exposure. While the macaque was seropositive for B-virus, most the persons involved were only aware of exposure to blood; no bites or scratches occurred. Exposure to a macaque’s blood does not constitute an exposure to B-virus because viremia is thought to be rare among infected macaques (


There are 219 virus species that are known to be able to infect humans. The first of these to be discovered was yellow fever virus in 1901, and three to four new species are still being found every year. Extrapolation of the discovery curve suggests that there is still a substantial pool of undiscovered human virus species, although an apparent slow-down in the rate of discovery of species from different families may indicate bounds to the potential range of diversity. More than two-thirds of human viruses can also infect non-human hosts, mainly mammals, and sometimes birds. Many specialist human viruses also have mammalian or avian origins. Indeed, a substantial proportion of mammalian viruses may be capable of crossing the species barrier into humans, although only around half of these are capable of being transmitted by humans and around half again of transmitting well enough to cause major outbreaks. A few possible predictors of species jumps can be identified, including the use of phylogenetically conserved cell receptors. It seems almost inevitable that new human viruses will continue to emerge, mainly from other mammals and birds, for the foreseeable future. For this reason, an effective global surveillance system for novel viruses is needed. First, we describe and analyse temporal, geographical and taxonomic patterns in the discovery of human viruses (§2). We then consider the processes by which new human viruses emerge §3). There are a number of definitions of ‘emergence’ . There a) show, tOf course, not all newly identified human virus species are ‘new’ in the sense that they have only recently started to infect humans; many of them have been present in humans for a considerable time but have only recently been recognized (see  for a mo2.(a)As a starting point for our survey, we used a previously published database (see ) obtaineThe year of discovery was taken to be the year of publication of the first report of human infection. The place of discovery was determined from the original report and recorded as the location of the diagnostic laboratory or, in the few instances where this was not clear, the address of the first author of the report. We did not attempt to locate the case itself, as this information was often lacking.We obtained a list of 219 ICTV-recognized virus species that have been reported to infect humans. 23 virus families were represented by species in this list.(b)a.The discovery curve is an ecological tool for estimating species diversity  comprisifigure 1As with all discovery curves, our curve reflects a number of different factors, including: (i) the technology available for detecting viruses ; (ii) thp < 0.05) upswings in the rate of virus discovery: in 1930  1927–1933) and in 1954 (1952–1955). Since 1954 the mean rate of discovery has been 3.37 species per year with variance 3.35, consistent with a Poisson process. However, there has been a slight but statistically significant downward trend in the rate of discovery (a linear regression of (count per year)0.5 against year has slope −0.010, 95% CIs −0.020 to 0.0, p = 0.049).Piecewise linear regression revealed two statistically significant ((c)a (ignoring four species for which the location of discovery could not be determined). That over 60 per cent of species were first discovered in North America or Europe almost certainly reflects considerable ascertainment bias [Numbers of species discovered by continent are shown in figure 2ent bias ,10. Rateb. The family containing the most human virus species is the Bunyaviridae with 40; six families contain just one human virus species. These numbers are too small for statistical analysis of rates of discovery: the most notable trend is that only a single new pox virus has been discovered since 1972 (compared with 10 up to that date). Nor are there any striking patterns using other classifications such as RNA viruses versus DNA viruses.Numbers of species by family are shown in figure 2(d)N virus species, each discovered in any given year with probability p. We considered fitting a distribution for values of p; however, provided that the individual p values are low, there was minimal improvement in model fit. The model was fitted to the data and evaluated using Markov chain Monte Carlo (MCMC) methods with flat prior information to calculate profile likelihood confidence intervals and the best fit parameters. The model defines the expected number of discovered viruses in year t, tλ, as binomially distributed so thatt = 1 corresponds to 1954.Following the approach described previously , we modeN, p) can be accurately approximated by a Poisson distribution with parameter Np for the range of values of N and p of interest. Thus, for a set of model parameters, the likelihood of observing data X = {ix}, the number of viruses discovered over years 1 to k, is given byHowever, the binomial distribution B, a lower 95% CI of 308 (89 remaining), an upper 90% CI > 2000 and an upper 95% CI that is undefined. Thus, although there is considerable uncertainty as to the size of the human virus species pool, this analysis suggests that there are at least dozens of new species to be discovered, and possibly a very much larger number.We compared the model with the observed data by calculating the mean, trend in the mean and variance for the number of virus species discovered per year . The model reproduces the observed data well: observed mean and variance 3.37 and 3.35, respectively; fitted mean and variance 3.36 and 3.41, respectively. Parameter estimates, however, are very uncertain owing to an unavoidable strong correlation between N was set at the 90% upper confidence interval. This gave a projected number of new virus species of 36 (95% CIs 20–57), corresponding to an average 2.4 species per year. This projection, of course, makes no allowance for any improvements in virus detection technology nor changes in discovery effort.To make shorter term projections, the model was extrapolated to year 2020, calculating 95% posterior prediction intervals using 2 million model simulations, taking into account parameter uncertainty and model stochasticity. An upper limit for (e)d looks likely to be met. Indeed, it would be unsurprising if it were exceeded, given the considerable recent interest in virus discovery and the advent of high throughput sequencing as a detection tool.From our systematic literature review, we identified at least 14 putative new species of human virus first reported during the 5 years 2005 to 2009 inclusive , though (f)b. Here, a family is included on the date of the first published report of human infection by a virus species from that family. There is too little data (n = 23) for detailed statistical analysis, but the figure does suggest a possible decrease in the rate of discovery, implying that the pool of undiscovered families may be relatively modest (see [The discovery curve for virus families is shown in figure 1est (see ).Strikingly, no new families have been added to the list since 1988, the longest such interval on record. However, several viruses  virus, TT mini virus and TT midi virus) newly reported since 1988 remain unassigned to a family.It should also be noted that there are three virus families that, although they do not contain any known human virus species, do contain species that infect other mammals: Arteriviridae ; Asfarviridae (African swine fever virus); Circoviridae . This suggests that the list of families containing human viruses may not yet be complete.3.(a)Homo sapiens (disregarding invertebrate vectors) [More than two-thirds of human virus species are zoonotic, i.e. they are capable of infecting vertebrate hosts other than vectors) ,26. By fThe remaining viruses, as far as we are aware, only naturally infect humans . Some o(b)A useful conceptual framework for thinking about the emergence of novel viruses is the pathogen pyramid ,31 figu. The pyrLevel 1 represents the exposure of humans to a novel pathogen; here, a virus. The source of viruses of interest is most likely to be other mammals or birds (see above) and ‘exposure’ implies any route by which a particular viral infection might be acquired, whether by contact with blood, saliva or faeces, contamination of food and water or via an arthropod vector. The rate of such exposure is determined by a combination of the distribution and ecology of the non-human host and human activities. It is likely that exposure to non-human viruses occurs commonly: a process referred to as ‘chatter’ .e) and the physiology of the exposed human .Level 2 represents the subset of viruses that are capable of infecting humans—that is, overcoming the ‘species barrier’. This is likely to reflect both the molecular biology of the virus . Again, this will mainly reflect the host–pathogen interaction, especially whether it is possible for the virus to access tissues from which it can exit the host, such as the upper respiratory tract, lower gut, urogenital tract, skin or (for some transmission routes) blood.R0 > 1 [Level 4 represents the subset of viruses that are sufficiently transmissible between humans to cause major outbreaks and/or become endemic in human populations without the requirement of a non-human reservoir. This equates with the epidemiological condition R0 > 1 , i.e. a a). We do not have a good estimate of the total species diversity of mammalian and avian viruses; however, we can get an indirect indication of the magnitude of the barrier between level 1 and level 2. It has been reported elsewhere  that of the virus species known to infect domestic animals —to which humans are presumably routinely exposed—roughly one-third are also capable of infecting humans. The species barrier exists: but it is clearly very leaky. Based on data from [R0 may exceed one (level 4). That a significant minority of  viruses should be capable of extensive spread within human populations  is consoming so ). The mo(c)Several reviews ,26,36 haOther ideas about drivers of emergence are even harder to test formally. One such is that we are currently living through a ‘perfect storm’ in which many potential drivers of emergence events  are acting in concert . Upward trends in many drivers can be quantified, but it is not entirely clear that the frequency of emergence events is increasing: one recent study suggested that it increased during the first decade of the HIV/AIDS pandemic, but has decreased thereafter .any change in the human environment  is likely to favour one pathogen or another, which responds by invading the newly accessible habitat. This idea would imply that emerging pathogens possess different life-history characteristics to established, long-term endemic pathogens. As noted earlier, the most striking difference identified so far is that the majority of recently emerging pathogens are viruses rather than bacteria, fungi, protozoa or helminths.A slightly different way of thinking about drivers of emergence is to draw an analogy between emerging pathogens and weeds . The idea here is that there is a sufficient diversity of pathogens available—each with their own biology and epidemiology—that (d)For viruses, one of the key steps in the emergence process is the jump between one host species and humans . . Moreover, for pathogens generally, host range seems to be phylogenetically labile, with even closely related species having very different host ranges . Clearly(e)One likely biological determinant of the ability of a virus to jump between species is whether or not they use a cell receptor that is highly conserved across different  hosts. We therefore predicted that viruses that use conserved receptors ought to be more likely to have a broad host range.To test this idea, we first carried out a comprehensive review of the peer reviewed literature and identified 88 human virus species for which at least one cell receptor has been identified. Although this is only 40 per cent of the species of interest, 21 (of 23) families were represented; so this set contains a good cross-section of relevant taxonomic diversity. Of these 88 species, 22 use non-protein receptors (e.g. heparin sulphate) and, of the remainder, two of the proteins were not entered in the UniProt database [On the basis of a previously published study of virus host ranges , we accop = 0.015).The result is shown in We conclude that the use of a conserved receptor is a necessary but not sufficient condition for a virus to have a broad host range encompassing different mammalian orders. It follows that a useful piece of knowledge about a novel mammalian virus, helping to predict whether or not it poses a risk to humans, would be to identify the cell receptor it uses. However, this may not always be practicable: at present, we do not know the cell receptor used by over half the viruses that infect humans, and this fraction is considerably smaller for those that infect other mammals.4.The lines of evidence described earlier combine to suggest the following tentative model of the emergence process for novel human viruses. First, humans are constantly exposed to a huge diversity of viruses, though those of others mammals (and perhaps birds) are of greatest importance. Moreover, these viruses are very genetically diverse and new genotypes, strains and species evolve rapidly (over periods of years or decades). A fraction of these viruses (both existing and newly evolved) are capable of infecting humans. It is not clear whether some of these human-infective viruses will already be capable of reaching higher levels of the pathogen pyramid—so-called ‘off-the-shelf’ pathogens—or whether subsequent evolution of their ability to infect and transmit from humans is usually required—‘tailor-made’ . The disWhichever of these two models is correct (perhaps both), there is a clear implication that the emergence of new human viruses is a long-standing and ongoing biological process. Whether this process will eventually slow down or stop (if the bulk of new virus species constitute extant diversity) or whether it will continue indefinitely (if a significant proportion of newly discovered virus species are newly evolved) remains unclear, although this makes little difference to immediate expectations. There is a hint, from the slower accumulation of new virus families found in humans, that virus diversity may be bounded, but that does not preclude there being a much larger number of virus species ‘out there’ than we are currently aware of. If anthropogenic drivers of this process are important then it is possible that we are in the midst of a period of particularly rapid virus emergence and, in any case, with the advent of new virus detection technologies, we are very likely to be entering a period of accelerated virus discovery. The unavoidable conclusion is that we must anticipate the emergence and/or discovery of more new human viruses in the coming years and decades. By no means all of these will pose a serious risk to public health but, if the recent past is a reliable guide to the immediate future, it is very likely that some will.The first line of defence against emerging viruses is effective surveillance. This topic has been widely discussed in recent years ,41, but Improving the situation will require both political will and considerable investment in infrastructure, human capacity and new tools ,41. Howe


Flaviviridae isolated in Cameroon in 1966.Presentation of pyrosequencing data and phylogenetic analysis for the full genome of Ntaya virus, type virus of the Ntaya virus group of the  The Flavivirus Ntaya virus (NTAV) was originally isolated from mosquitoes in Uganda in 1951 . Serosur2 tissue culture flasks  at 37 °C and 5 % CO2. At 90–100 % CPE (12 dpi), culture supernatants of infected cells were collected, and purification and RNA extraction were performed as described [A lyophilised 10 % suckling mouse brain suspension of NTAV isolate IPD/A of the CRORA collection at the Institute Pasteur Dakar collected in Cameroon in 1966 was passaged twice on Vero E6 cells in 175-cmescribed . In ordeescribed . The genThe NTAV polyprotein  shows the typical modular flavivirus structure of capsid protein C, polyprotein propeptide, precursor glycoprotein prM, glycoprotein E, non-structural proteins NS1, NS2A, NS2B, peptidase S7 and NS3 serine protease, P-loop-NTPase, non-structural proteins NS4A, NS4B, AdoMet-MTase and RNA-directed RNA-polymerase NS5 (Fig.


In contrast to most other cancer therapies, early research with oncolytic viruses (OV) tended to use direct or intratumoural routes of administration, initially motivated by concerns that the major limitation of intravenous delivery would be immune attack against the viruses, whether by complement, cytokine or most critically neutralising antibodies. Even in the absence of preformed circulating neutralising antibodies, antiviral immunity develops rapidly over the course of multiple viral administrations . PreclinGiven these perceived limitations, many have chosen direct administration to maximise intratumoural delivery: H101 (an oncolytic adenovirus with selectivity against p53-defective tumour cells) is approved in China and is given intratumourally for head and neck cancers. Several viruses have been administered into the tumour bed perioperatively for glioma. Measles has been administered intraperitoneally in ovarian cancer. One of the field-leading products is T-Vec (aka OncoVex), an engineered herpes virus. Its recent acquisition by Amgen was a fillip for those in the OV field, and was based on promising phase II data in melanoma where the virus was injected intratumourally into all accessible lesions ; a phaseWe previously recovered replication-competent reovirus from tumour following intravenous infusion in three patients with head and neck cancer,  a findinThese vital observations that two different viruses, administered intravenously, reach tumour tissues strongly support the further development of OV for systemic use in metastatic disease. However, neither of these studies completely addressed the issue of how a systemically administered virus might successfully run the gauntlet of anti-viral immunity.Reovirus is an endemic human pathogen and, therefore, neutralising anti-reovirus antibodies (NARA) are extremely prevalent in the population. In contrast vaccinia is not present in the environment and routine vaccination against smallpox ended in North America in the mid-1970s. In fact only one of the subjects treated with high-dose JX-594, in whom virus was detectable in the tumour, had pre-existing neutralising anti-vaccinia antibodies. In addition it is highly likely that successful virotherapy will require several doses of OV, which will induce high titres of neutralising antibodies in most patients.Against this background, we have recently confirmed successful intravenous delivery of reovirus in a population of pre-immune cancer patients . In a phex vivo-cultured carrier cells is an area that we and others have explored preclinically [in vivo following intravenous administration of neat virus. Further work to characterise and enhance the mechanism of viral carriage is underway, and will be critical to optimising systemic virotherapy.Importantly, all patients in the study had detectable neutralising antibodies before treatment and NARA titres peaked in all patients by the time of surgery. Accordingly, although reovirus was detected by PCR in the plasma of patients shortly after infusion, we were unable to recover replicating virus from plasma. This finding is compatible with NARA functionally neutralising the virus in the circulation. However we recovered replication-competent virus from the peripheral blood mononuclear cell, granulocyte and platelet fractions of the patients’ blood. These data indicate that virus hitchhikes on these circulating blood cells, circumventing the anti-viral immune response to access tumour. Hitchhiking virus onto inically ,8, but hIt is an exciting time to be involved in OV research. Preclinical developments continue to inform the clinical approach and provide novel targeted or armed agents for therapeutic testing . Clinica


To the Editor: The Chinese giant salamander (Andreas davidianus) is one of the world’s largest amphibian species and is often referred to as a living fossil. They primarily inhabit drainage basins of the Yangtze River, the Yellow River, and the Pearl River in the People’s Republic of China  . The sequences of the 3 PCR products from the virus-infected Chinese giant salamanders  showed >96% homology with the corresponding sequences of the 5 iridovirus strains. Additionally, neighbor-joining tree analysis showed that the virus was clustered in 1 lineage with frog virus 3, soft-shelled turle iridovirus, and tiger frog virus  was extracted from the isolated virus by using a commercial kit . PCR was performed by using 3 sets of primers targeting 681 bp, 568 bp, and 616 bp iridoviral fragments respectively, from the major capsid protein gene , the ATPase gene , and the thymidine kinase gene . Resulting PCR products from 10 salamanders were sequenced and compared with the corresponding sequences of the 5 known iridovirus strains by using a BLAST search (og virus . These rGopherus polyphemus) (Rana dybowskii) , People's Republic of China, 2010.Gross anatomic and histologic changes in sick Chinese giant salamanders (


Cobalamin and folate are essential micronutrients and are important in DNA and RNA synthesis, cell proliferation, growth, hematopoiesis, and cognitive function. However, data on cobalamin and folate status are lacking particularly from young children residing in low and middle income countries.To measure cobalamin and folate status and identifies their predictors among 6 to 35 months old children presenting with acute diarrhea.This was a cross-sectional study in 823 children presenting with acute diarrhea. We measured plasma cobalamin, folate, methylmalonic acid and total homocysteine who sought treatment for acute diarrhea between June 1998 and August 2000.p = <0.001). The prevalence of hyperhomocysteinemia (>10 µmol/L) and elevated methylmalonic acid (>0.28 µmol/L) were 73% and 52%, respectively. In the regression analyses, the plasma cobalamin concentration was positively associated with age, and introduction of animal or formula milk.The mean (SD) plasma concentrations of cobalamin, folate, total homocysteine and methylmalonic acid were 206 (124) pmol/L, 55 (32) nmol/L, 11.4 (5.6) µmol/L and 0.79 (1.2) µmol/L, respectively. The prevalence of low plasma cobalamin (<150 pmol/L) was 41% but less than 2% (15) children had low folate concentration (<10 nmol/L). Plasma homocysteine and methylmalonic acid concentrations were negatively associated with cobalamin concentration but not associated with folate status. The prevalence of cobalamin deficiency was higher in breastfed than non-breastfed children (44% vs 24%; Our study indicated that poor cobalamin status was common particularly among breastfed children. Folate deficiency was virtually none existent. Possible consequences of cobalamin deficiency in young children need to be explored. Cobalamin and folate are essential nutrients for young children and is important for DNA and RNA synthesis, cell proliferation growth, hematopoiesis and cognitive function. Recent studies suggest that deficiencies of these nutrients are common in young children The main source of vitamin B12 is animal-derived foods, which are expensive and for cultural and religious reasons often not eaten at all Deficiencies of both folate and cobalamin can result in an increase in the total homocysteine (tHcy) concentration In an antenatal clinic based study from Kathmandu valley, 61% had elevated serum MMA, and 49% had low serum cobalamin concentration The study was conducted in Bhaktapur municipality and its surrounding suburbs, which is located 15 km east of the capital Kathmandu. Bhaktapur is situated about 1400 meter above sea level and there is a cold and dry winter from November to March, the summer months are generally hot and humid, and the autumn is relatively warm. The municipality has approximately 84,000 inhabitants; Bhaktapur is the most densely populated district in Nepal This was a cross-sectional study and included children 6 to 35 months with acute diarrhea, participating in a double blind randomized clinical trial, aimed to measure the effect of zinc administration during an episode of diarrhea + hemoglobinometer  immediately after blood sampling according to the manufacture's instruction. The blood was then centrifuged at 760×g for 10 min in room temperature, and plasma was divided into polypropylene vials  and wrapped immediately by aluminum foil to prevent from light and stored at −40°C until transfer to Norway on dry ice for analysis. Then the specimens were stored at −70°C for 3–4 years and analyzed at the pharmacology laboratory, Oxford University, UK. After thawing, cobalamin and folate (n = 823) were analyzed by microbiological assays using a chloramphenicol-resistant strain of Lactobacillus casei and colistin sulfate-resistant strain of Lactobacillus leichmannii, respectively All baseline and morbidity information was recorded on the day of enrollment. The children were weighed using a scale to the nearest 100 g  and length or height was measured using a locally made length board to the nearest ± 0.1 cm. A study physician collected blood from each child's cubital vein into heparinized polypropylene tubes , mainly between 9 AM and 1 PM. The hemoglobin concentration was measured with a HemoCue® Hb 201We used the WHO definitions of cobalamin and folate deficiency which is <150 pmol/L of plasma cobalamin and <10 nmol/L of plasma folate P value of <0.05 was considered to reflect statistical significance. Simple and multiple regression analyses were used to examine the associations between the dependent variables  and different independent variables. The variables listed in P value <0.15 as described elsewhere (http://www.r-project.org) adjusting for the same variables as in the multiple linear regression models.All forms were checked manually by supervisors and physicians for completeness and consistency. The data was then double entered into Microsoft Visual FoxPro databases with computerized logic, range and consistency checks. Weight for age, length/height for age, and weight for length/height Z-scores were calculated using the most recent WHO growth charts The study had ethical clearances from the Nepal Health Research Council, Kathmandu. The implementation of all aspects of the project was in agreement with the International Ethical Guidelines for Research Involving Human Subjects as stated in the latest version of the Helsinki Declaration. Informed written consent was obtained from at least one of each child's parents.The baseline characteristics of the study population are presented in p = 0.003). In the same age group, the median (IQR) folate was higher in breastfed  than in non-breastfed  infants and it declined with increasing age. The median plasma MMA and tHcy concentrations were also higher among those who were breastfed than in those who were not  (The mean (SD) cobalamin, folate, total homocysteine and methylmalonic acid concentrations were 207 (125) pmol/L, 55 (32) nmol/L, 11.4 (5.6) µmol/L and 0.79 (1.2), µmol/L respectively. The median  cobalamin concentration was lower in breastfed  than in non-breastfed children  among 6–11 months old infants ( <0.001) Table 3.p = <0.001 and 1.0 vs 0.5 µmol/L, p = <0.001 respectively). None of the children had a plasma folate concentration <5 nmol/L and only 15 children (2%) had a folate concentration of <10 nmol/L. Most of the children (n  = 14) with low folate were more than one years old. Only one (0.3%) child had both a cobalamin concentration < 150 pmol/L and a folate concentration <10 nmol/L.Low cobalamin concentration (<150 pmol/L) was found in 40.7% (335) and 56% had plasma cobalamin <200 pmol/L. Forty four percent (299) of the breastfed and 24% (36) of the non-breastfed children had a cobalamin concentration <150 pmol/L. The mean tHcy and MMA concentrations were also higher in those with low cobalamin than in those with adequate plasma cobalamin concentration  and tHcy . The folate concentration was higher in those who did not consume animal or formula milk regularly [adjusted mean difference 8.6  nmol/L] and it was lower by −10.2  nmol/L in non-dehydrated children compared to those who were dehydrated. We also found a significantly higher plasma folate concentration during the spring season [adjusted mean difference 7.3  nmol/L] than in the summer. MMA concentration was negatively associated with age. Plasma tHcy was also negatively associated with age, however, only in breastfed children (p interaction <0.001) and it was lower in the spring than during the summer. Both cobalamin and folate and their metabolic markers were not significantly associated with anthropometric measurements or with clinical parameters listed in The results from the multiple linear regression analyses Table 5 et al. also reported that the cobalamin concentration in breast milk was positively correlated with maternal serum cobalamin concentrations which were lower in strict vegetarian women than in women on omnivorous diets We assessed cobalamin and folate status and identified their predictors in 6–35 months old Nepalese children with acute diarrhea. We found that poor cobalamin status was affecting almost one in every second child, which was more common in breastfed than in non-breast infants. Similar findings have been reported from studies conducted in India The median plasma folate concentration in this study was 48.7 nmol/L which is approximately twice as high as in a study of a similar age group children in New Delhi The main strength of the current study was its large sample size and that the data collection spanned over more than two years, including three hot and wet seasons. We also included measurements of the functional metabolic markers of cobalamin MMA and tHcy These limitations need to be kept in mind when drawing conclusions on the status of these vitamins in the general child population; however, we did not find any significant association of plasma cobalamin and folate with the duration and numbers of loose or watery stool. Nor did we observe any significant association with dysentery or vomiting episodes (data not shown). We did not obtain detailed information on dietary intake, exclusive breastfeeding pattern, the use of folic acid supplements by children and mother. Nor we were able to measure vitamin B6 which also can influence tHcy concentration Our findings are to a certain extent generalizable to young children in other low-income countries with similar socio-economic conditions, and where the food habits are comparable. The present study was clinic based and included children with acute diarrhea. Most of the people in this study were from the Newar ethnic group and were non-vegetarian although regular intake of meat and fish was not common. Therefore we are uncertain whether or not our findings of prevalence of cobalamin deficiency can be generalized to all children and other parts of Nepal where the dietary habits may differ Cobalamin deficiency was common in children with acute diarrhea in this community, suggesting that this may represent a health problem. Breastfed infants had the highest prevalence of cobalamin deficiency. Future studies including healthy children need to be undertaken in order to expand on these findings and to explore consequences of poor and sub-optimal cobalamin status. If these finding are confirmed in other parts of Nepal, it may be justified to consider including cobalamin supplementation in a national strategy to improve nutritional status in children.


Idiopathic Toe Walking (ITW) is present in children older than 3 years of age still walking on their toes without signs of neurological, orthopaedic or psychiatric diseases. ITW has been estimated to occur in 7% to 24% of the childhood population. To study associations between Idiopathic Toe Walking (ITW) and decrease in range of joint motion of the ankle joint. To study associations between ITW (with stiff ankles) and stiffness in other joints, muscle strength and bone density.In a cross-sectional study, 362 healthy children, adolescents and young adults (mean age (sd): 14.2 (3.9) years) participated. Range of joint motion (ROM), muscle strength, anthropometrics sport activities and bone density were measured.A prevalence of 12% of ITW was found. Nine percent had ITW and severely restricted ROM of the ankle joint. Children with ITW had three times higher chance of severe ROM restriction of the ankle joint. Participants with ITW and stiff ankle joints had a decreased ROM in other joints, whereas bone density and muscle strength were comparable.ITW and a decrease in ankle joint ROM might be due to local stiffness. Differential etiological diagnosis should be considered. Toe-walking is the inability to generate a heel strike during the initial contact phase of the gait cycle, the absence of full foot contact during the entire standing phase and is a common pattern seen in healthy developing children less than 3 years old . IdiopatToe-walking without a decrease in range of joint motion of the ankle joint is reported to be present in children with autism, in children with communication (language) disorders and learning disabilities, as well as in children with fine motor, visuomotor- and gross motor delays ,5., [No consensus is found in the literature between the presence of ITW and a decrease in local range of motion (ROM) (ankle joint). Shulman et al. reported that over 50% of the children who walked on their toes had normal or only mildly limited dorsal extension of the ankle joint, . Westber, .ITW with decreased range of joint motion in the ankle joint was present in a majority of children with musculoskeletal complaints and stiffness of other joints defined as symptomatic generalised joint hypomobility. Generalised joint stiffness which may lead to musculoskeletal complaints was possibly caused by an increased amount of stiffness of the connective tissue, as illustrated in skin biopsies (increased collagen and cross-linking contents) .In present study, we studied the association between a positive history or present occurrence of ITW and a decrease in ROM of the ankle joint at assessment.Secondly, we studied the association between ITW (with decreased ROM of the ankle joint) and decreased ROM in other joints.Thirdly, we studied muscle strength and qualitative measurements of bone in order to study if ITW is a local or generalized pattern and thereby provide more insight in the (patho)physiology.In a cross-sectional study, 362 children, adolescents and young adults (mean age (sd): 14.2 3.9) years, range: 8.0 - 19.9; 236 girls, 126 boys) participated. One hundred and seventeen out of 362 participants were recruited from from primary schools , whereas 167 out of 362 participants  were recruited from a secondary school in the city Zeist, the Netherlands. Another 78 participants were recruited from the University of Applied Sciences, Education of Physiotherapy, in Utrecht, the Netherlands . All participants served also in a age-matched reference group for research involving children with symptomatic generalized joint hypermobility and joint hypomobility. All participants were healthy without pathophysiological conditions involving the musculo-skeletal system [ years, rWe defined ITW as present when toe walking without heel strike was present for at least three months with the absence of past or present signs of any rheumatic, neurologic, skeletal, metabolic, or collagen disease.The Medical Ethics Committee of the Wilhelmina Children's Hospital  approved this study, while informed consent was obtained from all parents (when indicated due to age) and participants.We asked about ITW by questionnaire and encouraged participants to ask their parents if toe walking was present for a period of more than 3 months after three years of age. We also asked if any medical care was provided for this reason from birth onwards.The active range of joint motion of the shoulder (anteflexion), elbow (flexion and extension), wrist , hip (flexion and extension), knee (flexion and extension) and ankle  was measured bilaterally to the nearest 2 degrees with a standard 2-legged 360-degree goniometer, using the "anatomical landmark" method . In the The ROM of the lower extremity was assessed when the participant was positioned in supine.A severe decrease in active dorsal extension of the ankle joint, measured in supine position with full extension of the knee joint, was defined when active dorsal extension, was ≤ 0 degrees. Since the position of the knee joint affects the amount of dorsal extension of the ankle joint, 0 - degrees extension is the knee joint is indicated.Before start of the study, inter- and intrarater reliability was measured. Two assessors had to measure 25 joint excursions in 12 participants. All measurements were performed by the same trained physical therapists under supervision of the first author.The mean difference between two measurements of active ROM within one assessor (intra-rater) was 2.4 degrees (sd: 4.6). The mean difference between two measurements of active ROM within two assessors (inter-rater) was 5.0 degrees (sd: 4.7). These data were gathered in joints with large excursions (e.g. shoulder joint: flexion) as well as in joints with small excursions (e.g. elbow extension). The results of the reliability study indicate that 95% (= 2 standard deviations) of the difference between all measurements was less than 9.4 degrees which we found acceptable for measuring range of joint motion. When analyzing intrarater reliability of the ankle joint only, the mean (sd) difference was 1.9 (2.5) degrees indicating that 95% (= 2 standard deviations) was less than 5.0 degrees. The left and right-sided values of the ROM were averaged. We analysed range of joint motion of all excursions bilaterally. Only in the extension of the elbow, dorsal extension of the wrist and ankle, a significant difference existed (mean (sd) ROM elbow: left - right: 5.0 (6.1) - 4.4 (6.0); p-value: 0.004, mean (sd) ROM wrist: left - right: 75.8 (9.4) - 75.1 (9.1); p-value: 0.02, mean (sd) ROM ankle: left - right: 7.8 (6.2) - 8.3 (6.3); p-value: 0.01 respectively. To provide an indication of ROM in all joints, total range of joint motion was a summation of all measurements without that of the ankle joint. Measurements were performed by asking the participants to actively stretch or bend the joint maximally without interference by the investigator, and without support of the ipsilateral muscles by use of contralateral limbs.Maximum voluntary isometric muscle strength (expressed in Newtons) of the shoulder abductors, hip flexors, ankle dorsal flexors, and grip strength was measured bilaterally. The 'break' technique was used to test muscle strength. This technique is defined as 'the examiner applying force to a participant's limb until the child's capacity to hold is exceeded and the limb gives way". This methode has been shown to be reliable . MeasureWeight (in kilograms) was measured using an electronic weight scale. Height (in meters) was measured using a wall-assembled stadiometer. Body Mass Index (BMI) was calculated with the formula weight/height .We asked the participants to estimate the mean amount of hours spent on sport activities during last month, we asked about the occurrence of musculoskeletal complaints after sport activities and the presence of injuries.Quantitative ultrasound (QUS) measurements of the right os calcis were performed, according to the protocol of the manufacturer. A non-invasive method for assessing bone quantity and bone stiffness measurements was used  and broad band ultrasound attenuation  and speed of sound  were measured ,14. AcouData were analyzed using SPSS 16.0 .Normally distributed data are described with descriptive statistics .ITW (yes-no) and restricted joint motion of the ankle joint as outcome (yes-no) with a 95% confidence interval (95% CI).The Odds ratio was calculated for associations between dichotomous variables  was performed to compare clinical characteristics in participants with ITW and restricted ROM of the ankle joint versus controls. Results are expressed as a regression coefficient with a 95% confidence interval (CI). In multivariate regression analysis adjustments were made for confounding variables as age, gender, body height and weight. For all tests p-values <0.05 (two-tailed) were considered statistically significant.Smoothed centile curves were computed Figure  by usingThree hundred forty eight of 362 participants provided detailed information about the presence of ITW, so 14 participants were excluded.Thirty-one of 348 (9%) participants reported ITW and had a decreased ROM of the ankle joint (9 of 122 boys (7%) and 22 of 226 girls (10%). In 14 of 348 participants (4%), decreased ROM of the ankle joint was found without a history of ITW. In 9 of 348 (3%) ITW was present without a decreased ROM of the ankle joint. Two-hundred ninety three of 348 (84.4%) did not report ITW and no decrease in ROM of the ankle joint was present. Participants with ITW had a 3.2 times greater chance of having a decrease in ROM of the ankle joint .Data from participants with ITW and decreased ROM of the ankle joint n = 31) compared to participants without ITW and without decrease in ROM is are presented in Table  comparedChildren with ITW and restricted ROM of the ankle joint were significantly taller and weighted more than the control group. Total range of joint motion and quantitative bone density were significantly decreased, whereas total muscle strength was significantly increased. After adjustment for age, gender, body height and weight, only a significant decrease in total range of joint motion remained (regression coefficient (95% CI): 21.9 (2.1 - 41.7) : 2.8 degrees (1.8 - 3.9).Total range of joint motion was significantly negatively associated with body weight (regression coefficient (95% CI): -0.5; (-0.9 - -0.2) and gender . These observations did not change after multivariate analysis.In a general population of children, adolescents and young adults in the Netherlands we found a prevalence of 12% for ITW. Nine percent had a history of ITW and severely restricted ROM of the ankle joint. Participants with ITW had a three times higher chance of a severe restriction in their active dorsal extension of the ankle joint. After adjustment for confounders, participants with ITW and decreased ROM of the ankle joints had a significantly decreased range of joint motion in other joints, whereas quantitative ultrasound measurements and muscle strength were comparable.Several intrinsic limitations of this study might have influenced the findings and should be taken into account. The nature of the present study is cross-sectional and information on the presence of ITW in the past was obtained retrospectively from the participants. The children and adolescents might not have recalled the presence of ITW leading to an underestimation of the prevalence, although they were encouraged to ask their parents. However, we feel reassured that the prevalence of ITW in our study is comparable with data in the literature where ITW has been estimated to occur in 7% to 24% of the childhood population .Another intrinsic limitation was that we asked the participants to estimate the mean amount of hours spent on sport activities during last month. In future studies physical activity could be quantified by using a physical activity questionnaire.The "diagnosis" ITW was based on retrospective data gathered by a questionnaire. Data form medical records  were lacking. Although these observations were not the primary goal of our study, these data might be very important to look into the different history of treatment and outcome.The amount of stiffness was measured as a result from ROM. In future studies, it would have been interesting to use a dynamometer to quantify precisely the amount of stiffness.Localized stiffness as a possible cause of ITW fits 9% of our study sample. However, 6.3% of your sample did not fit this theory . A comparatively large group did not fit in our theory.ITW without stiffness was assessed in 3%. This might be explained by the fact that toe walking is often observed in children as a transient developmental expression .Stiff joints without the presence of ITW might be explained that although children have a severe decrease in the ROM of their ankle joint, this is not always translated into toe walking, but more in compensatory valgus of the hindfoot with an exotorsion of the upper and lower leg. Compensatory strategies are frequently observed, especially in older children in which weight increases. Therefore the compensation in the walking pattern might not be recognised and reported as ITW.Toe walking is often observed in children as a transient developmental expression and might eventually resolve spontaneously in the majority of children . On the The study populations were examined as a control group for children with symptomatic generalised hypermobility and hypomobility. Therefore only a history of toe-walking was available. As a consequence of the design of the study, no observational gait analysis could be performed regarding their current walking pattern.In our study, we assessed active ROM because we are interested in the functional ability of the children's joint status. Passive ROM provides information of the maximum ROM of a joint but the standardization of the extended external force to reach passive ROM is discutable.The cut-off point for defining decrease in range of joint motion is arbitrary. We defined a severe decrease in ROM of the ankle joint when the ankle could not actively dorsally extend more than 0 degrees with full extension of the knee joint. The position of the knee joint could have influenced our findings. However, no slight lack of full extension or slight hyperextension was observed.We chose the above mentioned cut-off point because interpretation in measuring the ankle joint ROM by goniometry is easy to perform. Moreover, the 0 degree position of the ankle-foot was clearly defined with the foot is positioned perpendicular to the table. This method is also reported in other studies ,16.In the literature, the amount of toe-walking is primarily focussing on gait analysis and kinematics . Little The combination of ITW and decreased ROM of the ankle joint has been presented by Alvarez et al. who studied gait analyses in 133 children with toe-walking (mean age (yrs): 9, range: 4-16) and classified toe-walking in three severity groups, based on range of joint motion, sagittal joint powers, knee kinematics and EMG data [Reference data on ankle ROM in children are scarce. Engelbert et al. reported in a group of 284 children a mean  active ROM of 9 degrees  [An advantage of our study was that we performed an intrarater and interrater reliability study concerning range of joint motion. We reported a mean difference between two measurements of 2.4 degrees (sd: 4.6) respectively 5.0 (sd 4.7) indicating that 95% (=2 standard deviations) was less than 9.2, respectively 9.4 degrees. This applies for all joints, also joints with a large range of possible joint motion, e.g. the shoulder, hip and knee joint. When analyzing reliability only of the ankle joint, the mean (sd) difference was 1.9 (2.5) degrees indicating that 95% (=2 standard deviations) was less than 5.0 degrees.In this study, participants with ITW and decreased ROM of the ankle joint were as active as controls as reported by the same amount of hours spend on sport activities. Our study population did not report the occurrence of pain during sport activities. This is in contrast with the study by Engelbert et al. They described toe-walking with a decrease in ROM of the ankle joints in combination with musculoskeletal complaints (pain during sport activities). The authors also reported a decrease in ROM in other joints who were possibly due to increased collagen and cross-linking contents in skin biopsies .In that study, the decrease in joint mobility in other than the ankle joint was more pronounced.Children with ITW and decreased ROM of the ankle joint had a decrease in total ROM between 2 and 42 degrees (95% CI) in other joints than the ankle joint. Although significant, the clinical relevance is probably limited (22 from 1400 degrees = 2%).Since we found no differences in bone quantitative measurements, muscle strength and clinically relevant differences in total ROM we think that ITW and decreased ROM of the ankle joints are rather a local stiffness than a local manifestation of a systemic problem. The findings of the study may have implications for treatment.The amount of total ROM is gender related. In our study, total ROM in female is increased compared to male. In general, ROM decreases when growing older. Although the design of the study was cross-sectional, we observed in older participants a decrease in ROM in the proximal joints (shoulder- and hip joint) whereas in the distal joints ROM remained the same or slightly increased (data not illustrated).In cases where decreased range of ankle-foot joint motion in ITW is present, treatment strategies to improve local ROM are not always successful. Stretching by a physiotherapist and casting protocols alone have not been shown to be successful with incomplete correction and high recurrence rates -20. AlsoA group of young children where found to have good results with Botulinum Toxin A with the majority of children having a heel toe walking pattern 12 months following the injections . This apIn cross- sectional studies on ITW, we do advise to use disorder specific instruments. Recently, Williams et al developed the Toe Walking Tool, a valid and reliable method of assessing children who present to the general allied health clinician with toe walking. This tool can assist with the decision of when to refer a child for further specialist investigation of their toe walking ,26.Differential diagnostics is indicated to study the aetiology of ITW. Different diagnosis related to ITW in the literature are presented in Table In a general population of children, adolescents and young adults in the Netherlands. we found a prevalence of 12% for ITW. Nine percent had a history of ITW and a severe restricted range of joint motion of the ankle joint. Children who had been walking on their toes had an three times higher chance of a severe decrease in their active dorsal extension of the ankle joint in their adolescence compared to children without a history of idiopathic toe walking. Since we found no differences in bone qualitative measurements, muscle strength and clinical relevant differences in total range of joint motion we think that ITW and stiff ankle joints might be an expression of a local problem rather than generalized stiffness or systemic aetiology. Since the underlying aetiology of ITW is unknown, differential diagnostics regarding possible etiological factors should be considered.ITW: Idiopathic Toe Walking; ROM: Range of joint motion; BMI: Body Mass Index; BUA: Broad band ultrasound attenuation; SOS: Speed of sound; 95% CI: 95% confidence interval.The authors declare that they have no competing interests.RE, CU, EP and PH designed the study. RE, CU and EP performed data acquisition.RE, CU, EP and JWG analysed and interpreted the data, RE wrote the first draft of the manuscript, which was reviewed by all authors. JWG revised the manuscript. All authors read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/61/prepub


Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1. Aedes aegypti) waned, particularly in Central America and the Amazon region. Not only has the number of DF cases on the continent (Dengue fever (DF) is a common viral disease of the tropics. Only a few countries in the Americas, including Chile, have not reported cases of this disease. Distribution of dengue virus in the Americas has increased since 1970, when efforts to eradicate the vector , myalgias, arthralgias, headache, and a maculopapular rash for 7 days. Laboratory analysis of a blood sample indicated low leukocyte and platelet counts. While still febrile, she traveled to Santiago, the capital of Chile, and was admitted to a private hospital; DF was suspected. On March 13, 2002, DF was confirmed by an in-house dengue immunoglobulin (Ig) M enzyme-linked immunosorbent assay (ELISA) in our laboratory. This case of DF was the first acquired in Chile.The Ministry of Health organized an outbreak investigation team. As part of the study and with the goal of recovering and identifying the virus, blood samples were taken from 16 febrile patients who were assessed and satisfied the clinical definition of suspected dengue case made by the Ministry of Health. The samples were brought to our laboratory, and plasma was used for viral culture and for reverse transcription–polymerase chain reaction (RT-PCR). Serologic testing was not performed on these samples.Viral culture was attempted from 15 acute-phase plasmas. Plastic flasks (T-25) seeded with Vero cells were injected with 200 μL of plasma diluted 1:5 with medium 199, 2% fetal bovine serum, gentamycin 50 μg/mL. After 1 hour of absorption at 37°C, cultures were incubated 10 days at the same temperature and observed once a day for cytopathic effect (CPE). Cells were harvested for indirect immunofluorescence antibody testing (IFAT) after CPE was first observed . Initially, IFAT was performed with polyclonal antisera reactive with all serotypes (D1–D4); then samples with positive results were stained with monoclonal antibodies specific for each subtype to identify dengue serotypes.Taq polymerase (Gibco) in a single reaction vessel with 50 μL final volume. The thermocycler was programmed to incubate for 1 h at 42°C and then 35 cycles at 94°C, 55°C, and 72°C. The second step used 10 μL of diluted 1:100 dengue cDNA from the first reaction and contained primers to amplify the four dengue serotypes . The results had bands of different sizes, depending on the serotype  after 20 cycles at the same temperatures as the first reaction.A nested RT-PCR developed by Lanciotti  visiting Easter Island came from Brazil. A lower proportion came from the Pacific Islands, where the same virus serotype was circulating at the time the outbreak started. Knowing the serotype is important to keep a strict surveillance of febrile patients and mosquitoes to determine if a different dengue virus serotype is introduced and to determine if cases of DHF are appearing on the island.Laboratory tests, like serology (IgM and IgG ELISA), and RT-PCR for dengue virus, were already available at our laboratory, whereas viral culture with IFAT for virus identification was quickly developed when the DF outbreak was identified. The further genotyping of the isolated dengue virus will allow us to compare with other DENV-1 viruses circulating in other parts of the world and determine the origin of Easter Island DENV-1.Because of diagnosis of the first indigenous case of DF in a country where tropical infections are unusual, being able to make a differential diagnosis and having laboratory resources for a variety of emerging infectious diseases are important, particularly for an immunologically naive community, such was the case of Easter Island and the Chilean population.


Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized.To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition.A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses. The influenza virus particle is spherical, about 100 nm in diameter, and encapsulated by a lipid membrane derived from the host cell. Two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), encoded by the virus genome are localized to the viral envelope. HA binds specifically to sialic acids, which serve as receptors for virus attachment Influenza virus RdRp composed of three virus-coded subunits, PB1, PB2 and PA, and the RdRp in viral particle catalyzes transcription et al. et al. et al. et al. The nuclear membrane is disassembled during S/G2/M-phase, prior to cell division, and subsequently reassembled after cell separation. Furthermore, the cell shape alters dynamically during the cell cycle In this study, using optical tweezers we show that the influenza virus attaches preferentially to cells in the G1-phase rather than those in the S/G2/M- phase. To our knowledge, this is the first successful application of optical tweezers for virus infection into individual cell.In addition, our results indicate that the sialic acid content of the membrane was higher on cells in G1- than in S/G2/M-phase. In concert the difference in sialic acid content of the membrane, we also succeeded to measure the difference in membrane stiffness between G1- and S/G2/M-phase cells using optical tweezers. The chemical and physical changes in membrane were associated with high-level expression of globotriaosylceramide (Gb3), globotetraosylceramide (Gb4), and glycosylceramide (GlcCer) in S/G2/M- than G1-phase cells. Taken together we propose a molecular basis of the cell membrane influencing influenza virus susceptibility.To determine whether influenza virus binds preferentially to cells at a particular phase of the cell cycle, purified influenza virus particles were labeled with a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). First, the binding of the fluorescence-labeled virus to host cells was confirmed by HA titration using chicken erythrocytes see . The DiIInfluenza virus attached cells were easily detected by the fluorescent probe Dil, while cells in S/G2/M-phase were detected by the GFP expressed from pFucci-S/G2/M Green vector. Among 50 Dil-labaled influenza virus attached cells that the pFucci-S/G2/M Green vector were transfected, 48 cells were not observed GFP expression, but only 2 cells were observed GFP expression. It was examined whether the different fluorescence-labeled influenza virus could attach to the specific cell phase. For this purpose the influenza virus particles were labeled with DiO or Syto21 respectively. DiO intercalates into lipid bilayer, whereas Syto21 binds to RNA. As shown in Previously, we developed a thermo-reversible hydrogel and optical tweezers to infect a cell with DiI-labeled influenza virus in a microchip  using glass beads, and subjected to centrifugation at 700×g for 10 min in a TOMY MX-301 centrifuge. The pelleted fraction was then treated with 100 μl of extraction buffer  and centrifuged at 186,000×g in a Beckman Optima ultracentrifuge . Ten microliters of the supernatant clarified by further centrifugation at 186,000×g was subjected to SDS-PAGE and transferred onto a PVDF membrane. To detect sialic acid, the membrane was probed with digoxigenin-conjugated lectins, which was then detected with an alkaline phosphatase-conjugated anti-digoxigenin antibody .G1- and S/G2/M-phase cells , and fixed with 10% formalin in PBST for 30 min. The binding of virus particles to glycopolymers was detected by incubation with an antibody against influenza virus A/PR/8/34 for 60 min at 37°C.Receptor specificity was determined using a solid-phase binding assay as described previously by Guo et al 6 cells in 20 volumes of 2∶1 chloroform:methanol. The extracts were separated by TLC in chloroform:methanol:water (40∶12∶1 v/v/v) and visualized by spraying with orcinol in 70% sulfuric acid.Cellular lipids were extracted from 3×10Type I collagen was coated onto carboxylate particles (2 μm in diameter)  via the cross-linker 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), using the method of Suzuki Figure S1Observation of binding activity of DiI-labeled virus on the cell treated with sialidase. A. DiI-labeled influenza virus was added to sialidase-treated (a) and –untreated (b) H292 cells respectively, and incubated for 15 min. The unbound viruses were removed and cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature and then observed under a Nikon Ti E confocal microscope fitted with a 100× objective lens. Red colored particles indicated by arrow head in white represent virus particle. B. Chicken erythrocyte aggregation by DiI-labeled influenza virus was assayed. Fifty μl of DiI-labeled influenza virus was added in the 50 μl chicken sialidase-treated and -untreated erythrocyte each. After 1h incubation at room temperature, the aggregation of chicken erythrocyte was observed. wells 1, 2, 3 and 4, influenza virus (+); well 5, influenza virus (−); wells 1 and 2, DiI-unlabeled virus; wells 3 and 4, DiI-labeled virus; wells 1 and 3, sialidase-untreated chicken erythrocyte; wells 2 and 4, sialidase-treated chicken erythrocyte.(EPS)Click here for additional data file.Figure S2Observation of various fluorescence-labeled influenza viruses on cell membrane. The various fluorescence-labeled influenza viruses were added on the H292 cells that were transfected with pFucci-S/G2/M Green vector and incubated for 15 min. The unbound influenza viruses were removed and cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and then microscopic observation was carried out under the ×100 objective lens. A. DiI-labeled influenza virus binding onto H292 cells. B. DiO-labeled influenza virus binding onto H292 cells, C. Syto21-labeled influenza virus binding onto H292 cells. Red colored particles indicated by arrow head in white represent virus particle, green color represents the expressed GFP.(EPS)Click here for additional data file.Figure S3Suction of a single cell using a capillary. Cells in the chamber were washed with 0.04% EDTA in PBS and suctioned with a glass capillary. The cell indicated by the arrowhead was manipulated. Left, before suction; right, after suction; below, manipulated cell.(EPS)Click here for additional data file.Movie S1Real-time observation of influenza virus on the cell. Movie showing the movement of a DiI-labeled influenza virus particle on an H292 cell following manipulation with optical tweezers.(MOV)Click here for additional data file.


To the Editor: Highly pathogenic avian influenza (HPAI) virus (H5N1) has been responsible for 603 confirmed human cases worldwide, including 356 that resulted in death, and for >7,000 epizootic outbreaks (All experiments were conducted by using HPAI virus (H5N1) strain A/chicken/Cambodia/LC1AL/2007 (GenBank accession nos. HQ200574–HQ200581). All animal experiments were conducted in the biosafety level 3 laboratory of Institut Pasteur in Cambodia (IPC), in compliance with the European Community 86/609/CEE directive and approved by the Animal Ethics Committee of IPC (permit: AEC/IPC/003/2010). Specific pathogen–free (SPF) chickens were provided by the National Veterinary Research Institute of Cambodia.7.8 50% egg infective dose of HPAI virus (H5N1) particles. The contaminated soil was then sprinkled on the bottom of an isolator (surface area 0.2 m2) in which the chickens were housed. Oropharyngeal and cloacal swab samples and feathers were collected daily from the chickens and underwent quantitative reverse transcription PCR (qRT-PCR) testing targeting the H5 hemagglutinin gene (We used 3 types of soil: 1) sandy topsoil collected from around rice fields in Phnom Penh Province, Cambodia; 2) building sand purchased from a local building company; and 3) soil-based compost purchased from a local tree nursery. Physicochemical and microbiologic parameters were measured for water extracts obtained for each type of soil , and lowNo clinical symptoms, deaths, or seroconversion for HPAI virus (H5N1) were observed in chickens exposed to contaminated sandy topsoil, regardless of the dose protocol used . HoweverSoil-based compost and building sand, although existing in natural settings, are not the most common substrates found in places where free-ranging poultry are raised in Cambodia. Therefore, despite the high mortality rate observed in our study after exposure to highly contaminated soils, the role of these soil types in transmission of HPAI virus (H5N1) infection to poultry or other species, including humans, appears limited when replaced in actual epizootic or enzootic field conditions. Our results also show that exposure of chickens to moderately contaminated soil may result in a protective immune response.Sandy topsoil, on the other hand, did not allow any transmission of HPAI virus (H5N1) from the environment to chickens. This type of soil, which covers ≈40% of the rice-growing areas of Cambodia .In Cambodia, as in several other countries affected by HPAI virus (H5N1), decontamination of the environment after an outbreak is recommended by authorities; for example, disinfectants such as cresols are sprayed over environmental surfaces. However, because of resource limitations, only limited areas can be treated. Our results provide evidence that, even when abundantly contaminated, some soil types are unlikely to allow transmission of the virus to poultry and, consequently, probably not to other animals or to humans. These results suggest that limited resources could be better concentrated in high-risk areas, where the nature of the soils would be more likely to lead to poultry infection after natural contamination. These data may aid in the design of more cost-effective and solid-based decontamination measures for preventing transmission of HPAI virus (H5N1) to humans and animals.Physico-chemical and bacteriological parameters measured in soils tested, and the low- and high-dose contamination procotols used for testing.


AbstractPropelma Trjapitzin 1963 is removed from synonymy under Eupelmus Dalman 1820  and treated as a valid genus within Neanastatinae Kalina 1984 based on examination of the holotype female of Propelma rohdendorfi Trjapitzin. Propelma rohdendorfi is redescribed, illustrated by photomacrographs, and compared to other described extant and extinct genera of Neanastatinae. Taxonomic, morphological and geological diversity of Neanastatinae relative to Eupelminae and Calosotinae is also discussed relative to potential age of the subfamily.The extinct Eocene Baltic amber genus  Propelma based on Propelma rohdendorfi (Hymenoptera: Eupelmidae), which he described from a single female in Eocene Baltic amber. Propelma under EupelmusEupelminae Walker 1833. The synonymy was based primarily on the lateral habitus drawing of Propelma rohdendorfi given by PageBreakPropelma he stated that it was similar to MetapelmaMetapelma is one of seven genera currently classified in NeanastatinaeCalosotinae Bouček 1958 and Eupelminae comprise the three recognized subfamilies of Eupelmidae. Of the seven neanastatine genera, four are extant, including EopelmaLambdobregmaMetapelma and Neanastatus Girault 1913. The other three genera, Aspidopleura, Brevivula and Neanaperiallus, were all described by Gibson (1999) from Baltic amber inclusions and are extinct. Description of the three extinct genera greatly expanded morphological limits of Neanastatinae and the new knowledge led me to re-examine Propelma rohdendorfi and question the validity of synonymizing Propelma under Eupelmus. Resulting study of the amber holotype of Propelma rohdendorfi subsequently showed that Propelma with Metapelma relative to subfamilial affinities, and that Propelma represents a fourth, extinct, valid genus of Neanastatinae from Baltic amber. The purpose of this paper is to correct my erroneous synonymy and redescribe and illustrate Propelma rohdendorfi so that its classification is better established in Eupelmidae.When Trjapitzin described Propelma rohdendorfi . The holotype is in a mostly dark orange-colored block of amber ; tgl = tegula. Measurements of the antennomeres, fore wing venation, and metasomal tergites were all taken at the same magnification; measurements between square brackets are repeated from the original description.Terminology follows Propelma Trjapitzin, stat. rev.Propelma Trjapitzin, 1963: 89–91. Type species: Propelma rhodendorfi Trjapitzin, by original designation and monotypy. Synonymy under Eupelmus Dalman by Trjapitzinresurrected combinationhttp://species-id.net/wiki/Propelma_rhodendorfiPropelma rhodendorfiPropelma rohdendorphi, Trjapitsyn 1963, Holotypus”. Trjapitzin, 1963: 91–94. Holotype: female in Baltic amber. Label data: “Eupelmidae 364/360, Eupelmus rhodendorfiPropelma under Eupelmus. Trjapitzin; Gibson, 1995. New combination by inference through synonymy of Female . Length PageBreakgion and frontovertex minutely coriaceous-granular with minute setiferous punctures; lower parascrobal region and gena more vertically coriaceous-alutaceous with short white setae similar to upper parascrobal region, frontovertex and interantennal region. Head in lateral view , which accept dorsolateral angles of pronotum when this rotated horizontally in same plane as mesoscutum); finely coriaceous-granular to coriaceous-alutaceous with short black setae except for a line of longer setae along posterior margin. Mesoscutum slightly wider than long, with ridge-like medial elevation extending between exposed transverse anterior portion and transscutal articulation (2h) over metascutellum (3), apparently uniformly setose; axillula with dorsal margin carinate. Tegula (2), triangular, 1.4× as long as high basally, with dorsal and ventral margins convergent to narrowly rounded posterior angle; finely coriaceous, bare. Acropleuron (3) and metanotal lateral panels  using the key to genera of Neanastatinae in PageBreakuniformly setose  and four extant genera, of which one (Lambdobregma) is restricted to the New World, one (Eopelma) is restricted to the Oriental region, one (Neanastatus) is Old World in distribution, and one (Metapelma) is more widely distributed throughout both the Old and New World. Metapelma is also the only extant genus with a described extinct species, Metapelma archetyponEupelmidae from Grassy Lake Canadian Cretaceous and Charentese French Cretaceous amber, respectively. I have been unable to obtain these inclusions for examination, but the earliest known verifiable eupelmids are all Neanastatinae from Baltic amber. Extant Neanastatinae is far less diverse, measured either by number of described species (82) or genera (4), than either Calosotinae (152/8) or Eupelminae (729/33)  of Baltic amber , including one taxon identified as the extant genus Zaischnopsis Ashmead , and at least some members of Lambdobregma could be egg parasitoids based on a single putative rearing of Lambdobregma schwarzii (Ashmead) from cricket eggs (Orthoptera: Grylloidea)  or as a paraphyletic assemblage relative to Cynipencyrtidae and Tanaostigmatidae .The recognition of (729/33) . It is uic amber  or becau Ashmead , but theiologies , though chnopsis , are parlloidea) . Such a lloidea)  compared


It has been confirmed that respiratory virus infections can induce abberant cytokine production in the host. These cytokines may be associated with both elimination of the virus and complications in the host, such as virus-induced asthma. Representative host defense mechanisms against pathogens, including bacteria and viruses, are mediated by the innate immune system. Cells of the innate immune system express essential molecules, namely pattern recognition receptors (PRRs), such as Toll-like receptors, nucleotide-binding oligomerization domain-like receptors, and retinoic acid-inducible gene-I-like receptors. These PRRs can recognize components of pathogens such as bacterial lipopolysaccharide, viral antigens, and their genomes (DNA and RNA). Furthermore, PRRs activate various signaling pathways resulting in cytokine production against pathogen infection. However, the exact mechanisms remain unknown. In this review, we mainly focus on the representative mechanisms of cytokine production through PRRs and signaling pathways due to virus infections, including respiratory virus infections. In addition, we describe the relationships between respiratory infections and virus-induced asthma. Coordination between innate and adaptive immunity against pathogens is indispensable in higher organisms including humans . In partin vivo. Indeed, we use recombinant IFNs to treat chronic viral infections such as hepatitis C  are the most common diseases in humans. Accumulating evidence suggests that around 80% of the causative agents of ARI may be respiratory viruses . The proTo better understand host defense mechanisms against viruses, it is important to clarify these molecular mechanisms. In this review, we focus on cytokine production and signaling pathways during viral infection. We also discuss the relationships between cytokine profiles and virus-induced asthma under the main theme “virus-induced asthma.”Figure 1.Host defense mechanisms against microbial infections constitute the main purpose of innate immunity  are localized in the cytosol and may be able to bind to some ssRNA viruses such as RSV, influenza virus, dengue fever viruses (DFV), and hepatitis C virus, leading to the production of type I IFN (IFN-α/β) in fibroblasts . In partInflammasome as a PRR is a concept of the inflammatory reaction-associated protein complex . It is sFigure 1; Figure 4. Briefly, a previous report showed that RSV infection in human fetal lung fibroblasts (MRC-5 cells) induces various cytokines through the activation (phosphorylation) of Akt , p38MAPK (mitogen activated protein kinase), ERK1/2 , and IκB-α  virus]-infected human airway epithelial cells produces significant amounts of IL-1, IL-6, and IL-8 (The summarized data of the cytokine production profiles in some respiratory virus-infected cells are shown in  in vivo . The finand IL-8 . Productand IL-8 . HRV-infand IL-8 . RSV-infand IL-8 . HPIV-3 and IL-8 .in vitro studies have demonstrated that elevated IL-6, IL-8, and RANTES are found in sputum and serum in influenza virus infection (Previous nfection . IL-6 annfection . Systeminfection . These rnfection .Viral infections clearly induce inflammation at infected sites. A variety of complicated pathophysiological events occur at these sites. In broad terms, these events may constitute converged cell death and regeneration . The proSince the discovery of PRRs, remarkable progress has been made toward understanding the role of innate immunity against pathogens. However, the precise roles of PRRs, the mechanisms of intrinsic signaling pathways, and cytokine production with regard to PRRs are not fully understood. In addition, recent studies suggest that PRRs may be associated with various inflammatory diseases such as gout, rheumatoid arthritis, and atherosclerosis. It would be beneficial to clarify the functional relevancy of infectious diseases and other inflammatory diseases in the near future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.