The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by experiments which only tangentially and indirectly examine the process, producing results that are difficult to interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as better purification protocols and improved methods of analysis have been brought to the study. Results have been obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two models and make the case for an alternative pathway of entry by alphaviruses. Alphaviruses are transmitted to vertebrates by hematophagic insects such as mosquitoes and ticks . These ialphavirus in the family Togaviridae is a group IV membrane containing virus with a positive sense RNA genome [Sindbis Virus (SINV), the prototype virus of genus A genome . It is wA genome ,11 and hAlphaviruses are small 70 nm viruses that have 240 copies each of three structural proteins, E1, E2 and capsid (C) assembled in a 1:1:1 stoichiometry. These three proteins create two nested T = 4 icosahedral shells that sandwich a host derived lipid bilayer  , chikungunya glycoproteins  and their fit into the SINV and/or Semliki Forest Virus cryo-reconstruction . These structures were produced by expressing the ectodomains of the E1 and E2 glycoproteins connected together by a linker (58). This fusion protein was constructed due to the inability of expressing E2 in E. coli. While it is common practice to remove troublesome domains from proteins to allow for crystallization to occur, how is it determined when there has been too much manipulation of the sequence? These structures have the same problems as E1*. The assumption that the transmembrane domains, which have been removed in the c DNA clone, and integration into the ER membrane play no role in the correct folding and assembly of the spike structure is an unproven assumption. For virus entry of the macromolecular SINV particle, structure is critical. The overall alphavirus icosahedral structure is unique in that it is a membrane containing virus that does not have the membrane exposed. The membrane itself is not the form determining factor as with influenza, and because it is beneath the outer protein shell is not readily available to fuse with host membranes. The essential role of the membrane may be to provide the scaffold upon which the virus is assembled. To fully explore the mechanism of virus penetration and entry other methods of analysis other than interpretations of structural fit can provide the missing events required for SINV infection.A second caveat which seriously affects the quality of the data is that the entity to be studied not be distorted by the ability of proteins to be manipulated and expressed in some form in The process of virus attachment/absorption to the host cell is probably a multistep event. It is not a new proposal that virus infection begins with scanning of the cell surface that begins with a general "sticking" to the cell via one or more proteins (or the cell membrane itself) followed by "rolling" on the cell surface as it locates the proper receptor to initiate the penetration step of virus entry. This type of interaction has been identified recently using single particle fluorescence resonance energy transfer (FRET). SINV saturated with a membrane intercalated fluorescent self-quenching dye can be visualized when the dye is excited with a specific wavelength light . Fusion As arboviruses, alphaviruses infect insect and vertebrate hosts. Since alphaviruses need to infect cells which provide widely divergent biochemical and genetic environments, it is likely that they either use a ubiquitous receptor, or are able to use multiple proteins as a receptor. The receptor/s has not been identified. Many proteins and polysaccharides have been implicated as being part of the receptor complex. The list includes, heparin sulfate -40, the The concept that the receptor is, at least in part, proteinaceous comes from a study which showed that protease treatment of cells prior to adsorption decreased the number of infected cells. Phospholipase and neuraminidase treatment did not have an effect on infection . Using cThe use of anti-idiotypic antibodies as receptor locators has been used as well to determine the receptor for alphaviruses. This approach is responsible for the discovery of the 63 kDa chicken protein  and evenThere are numerous techniques that can be used to examine the interactions between the virus and the cell. These techniques can generally fall into two categories: direct and indirect observation. Direct observation uses familiar techniques such as thin-section microscopy, cryo-electron microscopy, tomography, and other less familiar techniques like freeze-fracture immunolabeling to examine virus-cell interactions. When properly prepared and processed, the interactions between the virus and cell seen in the microscope are the actual interactions that occurred at the time of fixation. By examining different times during the infection process, the possible mechanism and pathway of virus entry can be elucidated. Unfortunately, thin-sections and other direct observation techniques cannot distinguish between a successful infection process, and that of an unproductive interaction. This makes microscopy very subjective and is the primary problem when observing virus-cell interactions, as even with a relatively low particle/pfu ratio of 1:10, most observations are of virus-cell interactions that do not lead to a successful infection. For SINV, the SVHR strain can be purified to a particle/pfu ratio of 1:1, (28) and this virus was used in EM studies of virus infection that led to the direct cell penetration hypothesis of virus entry . Many ofWhen direct observation is not possible, or cannot answer the question being asked, techniques that take advantage of indirect or secondary reporters have been used. Some indirect reporters that have been used as a measure of successful virus entry include virus RNA production , virus pa priori reason to conclude that because a virus has a membrane, penetration must be by fusion. Discussed here are two suggested modes of entry for the alphavirus and the supporting data.Once the virus has bound to its receptor, the virus outer shell must compromise the plasma membrane barrier. The classical mechanism of membrane containing viruses to breach this barrier is by triggering fusion of the virus membrane with the host cell membrane, releasing its contents into the cytoplasm . This isThe most commonly accepted mechanism of entry for alphaviruses is via endocytosis and the subsequent acidification of the vesicle leading to membrane fusion. This is similar to the mechanism accepted for influenza. The studies in support of this mechanism are extensive, and rely on the observations that alphaviruses have membrane fusion capabilities, and that inhibition of the acidification of endosomes inhibits the entry of the virus. The original fusion assays were done with cultured cells, and it was shown that treating cells with adsorbed virus to a brief low pH environment, resulted in cell-cell fusion (fusion from without) . This suTreatment of cells with chemicals that inhibit the acidification of endosomes has been used to assay the role of acid environment in the infection of mammalian cells by alphaviruses ,70. The Using a SINV construct that contained a GFP reporter, a thorough study was done to assess the effect of Bafilomycin A1 (BafA1) on the entry of alphaviruses into mammalian and mosquito cells . The reset al. were able to show full and empty particles . In these experiments, high MOI of SVHR was adsorbed onto mosquito cells for half an hour after which the virus was cross-linked to cell surface proteins with gluteraldehyde. These samples were then processed via a freeze-fracture method to examine the plasma membrane and its components. The technique was a modification of the FRIL (freeze-replicate-immunolabel) technique developed by Fujimoto ,88. In bDirect observation of alphavirus-cell interaction by electron microscopy and through the use of chemical inhibitors and interaction with artificial membranes has created seemingly conflicting data. In many direct observations, virus can be seen in endosomes inside the cell . These dThe use of chemical inhibitors of endosome formation and acidification has relied on reporter events which occur many steps away from the process of entry. Many of the agents have secondary effect on events that occur after entry which have led to erroneous conclusions.. Likewise studies of virus interaction with artificial membranes have produced a set of requirements for the fusion event that are not related to those occurring with living cells. It appears that, although alphaviruses possess the ability to fuse membranes this is is an event that occurs under special laboratory conditions and may be unrelated to the true entry process.Arthropod borne viruses are significant sources of disease in man and domestic animals and some are potential agents of bioterrorism. Strategies to control infections by these agents include the development of compounds which will block critical steps in the entry pathway. An accurate image of this pathway and the identification of the cell receptor participating in the entry process is critical for the development of agents which can block infection.The authors declare that they have no competing interests.JK, RH and DB contributed to and edited the manuscript. All authors read and approved the final manuscript.


Bacteria, Eukarya, and Archaea. While those infecting domain Eukarya are nearly universally described as “Viruses”, those of domain Bacteria, to a substantial extent, instead are called “Bacteriophages,” or “Phages.” Should the viruses of domain Archaea therefore be dubbed “Archaeal phages,” “Archaeal viruses,” or some other construct? Here we provide documentation of published, general descriptors of the viruses of domain Archaea. Though at first the term “Phage” or equivalent was used almost exclusively in the archaeal virus literature, there has been a nearly 30-year trend away from this usage, with some persistence of “Phage” to describe “Head-and-tail” archaeal viruses, “Halophage” to describe viruses of halophilic Archaea, use of “Prophage” rather than “Provirus,” and so forth. We speculate on the root of the early 1980's transition from “Phage” to “Virus” to describe these infectious agents, consider the timing of introduction of “Archaeal virus” , identify numerous proposed alternatives to “Archaeal virus,” and also provide discussion of the general merits of the term, “Phage.” Altogether we identify in excess of one dozen variations on how the viruses of domain Archaea are described, and document the timing of both their introduction and use.Viruses infect members of domains  For historical reasons, bacteriophage is widely used to refer to viruses of bacteria (and sometimes even archaea). The problem with such nomenclature is that it artificially divides the virosphere into two camps, with viruses of bacteria and archaea on one hand and viruses of eukaryotes on the other. Viruses are infectious agents that alternate between autonomous, encapsidated states known as virions, which are “packages of genes” , and uneArchaea. We agree with what we observe to be a near consensus within the field that the use of the term “Virus” as well as the qualification “Archae-” or “Archaeal”—as in, for instance, “Archaeal virus”—is both logical and reasonable, echoing, for example, the fairly common usage of “Bacterial virus” as an alternative to “Bacteriophage” or “Phage.” For approximately half of the 40 or so years that these viruses have been studied, however, the explicit phrase “Archaeal virus” did not exist in the published literature. The absence of this phrase prior to the early 1990s reflects the replacement only in 1990 of “Archaebacteria” [Archaea” [Among the viruses of microorganisms are those that infect microscopic eukaryotes along with those that instead infect prokaryotes. While the viruses of eukaryotes nearly exclusively are described as just that, that is, as viruses, the viruses of prokaryotes have been burdened with a more complicated naming history. Here we look at the naming conventions that have been applied to the latter, with some emphasis on considering the relative merits of the term “Phage” as a descriptor particularly of the viruses of domain Archaea”  as a deshttp://dx.doi.org/10.1155/2013/251245) as well as Table 2 .Here we consider the history of the general naming of archaeal viruses as found within the published literature. In addition to exploring the timing of the introduction of the term “Archaeal virus,” we also consider the transition from “Phage” to “Virus” as seen approximately a decade earlier as well as the use of various related terms including “Archaebacteria,” “Halophage,” and “Prophage.” A summary of some of the terms that have been used to describe archaeal viruses, along with what we have been able to ascertain are their dates of introduction into the literature, is presented in While schemes of organism classification have existed for millennia, prokaryotes were explicitly introduced into such systems only in 1938, by Copeland . This “nMonera were, rightly or wrongly [Bacteria and Archaea versus the eukaryotes, with the latter dubbed domain Eucarya (or Eukarya) [Archaea in 1990 [archaebacteria” by Woese and Fox [archaebacteria.The apparent antiquity of the methanogenic phenotype plus the fact that it seems well suited to the type of environment presumed to exist on earth 3-4 billion years ago lead us tentatively to name this urkingdom the As is well known especially to microbiologists, the five-kingdom system has given way to the three-domain system . There t wrongly , 43, furEukarya) . Note th in 1990  was firsArchaea should retain the “Phage” designation or instead assume the more general term of “Virus.” Here we consider the history of this issue especially in terms of published usage, focusing particularly on the relatively long transition from “Bacteriophage” to “Archaeal virus.” We additionally consider overall usage as determined by examination of individual publications.In terms of the viruses associated with each of these domains, an obvious question then was whether those infecting members of domain Myriad approaches were used to identify archaeal virus-associated publications. Where possible, publications were obtained, in various forms, and if not already searchable as PDFs (Portable Document Format) then digitally scanned and/or subject to optical character recognition (OCR). Articles were then computer searched using the Adobe Acrobat search function. This was initially done in bulk but ultimately individually, for various terms, where the context therefore could be observed. Terms found within reference lists were ignored and terms such as “Phage” that did not appear to be associated with considerations of archaeal viruses were also not considered . Note that generally we equate “Virus” with “Viruses” as well as “Viral” but not with “Virion” and also consider the use of “Phage” as well as “Virus” both alone and as suffixes. We have also disregarded trivial misspellings such as “Archeal virus” as used in the same publication as “Archaeal virus.”Acidianus bottle-shaped virus” [Methanobacterium phage ΨM1”, since technically this could be described instead as, for example, “Methanobacterium archaeal virus ΨM1” or simply “Methanobacterium virus ΨM1”. The result of this bias is what we consider to be a conservative underestimation of the use of “Virus” along with an overestimation of use of “Phage.” In terms of consideration of use of “Phage” within the literature, we feel that this approach is reasonable particularly since a publication will still be described as containing “Virus” if it also uses “Virus” to describe archaeal viruses outside of the names of specific viruses. The example at the beginning of this paragraph, on the other hand, was designated as not containing “Virus” since the quoted text was the sole use of “Virus” to describe an archaeal virus that we were able to locate in the nonreferences portion of that publication. In any case, “Virus” as well as “Phage” must have been used to describe one or more archaeal viruses to be counted towards these tallies.We avoid describing a publication as containing the term “Virus” if that term was limited to within the name of a virus, for example, “d virus” . We haveOur working assumption is that our reference list  is less than fully complete. Explicitly missed are references that were not published in English as too patents. We also have likely missed various chapters along with whole books, especially those on subjects tangential to that of archaeal viruses. This is true also for materials that we were unable to obtain and therefore examine as well as any references that were not yet indexed nor easily identified prior to approximately January 1, 2013. Altogether, 694 references were identified, examined, and otherwise recorded as referring in some manner to archaeal viruses. All of these are listed in the reference section of the supplementary materials.Just as human-generated indices can miss sought information, so too are digital searches fallible. It is possible also that relevant context was missed in the course of examining individual publications, though ultimately the decision as to whether a term such as “Phage” or “Virus” was used in a publication to refer to an archaeal virus, or not, was based on the experience of the lead author (S. T. Abedon). As with any “Experiment,” the resulting output—given limitations in technology, retrieval, and subjective judgment as well as simply “operator error”—should be viewed as representative, in this case of the archaeal virus literature, rather than either a complete or error-free sampling. Archaea described them as phages or bacteriophages. This usage was appropriate, especially at the point of initial discovery, as the classification of their hosts as distinct from bacteria was not yet appreciated; that is, and as noted above, the term “Archaebacteria” dates to 1977 [Nature, was titled “Bacteriophage of Halobacterium salinarum.”The earliest references to viruses of domain  to 1977 . That paNature and was titled “Salt-dependent bacteriophage infecting Halobacterium cutirubrum and H. halobium.” In each case neither “Virus,” “Viral,” nor the root “Arch…” appear in the article. With some irony, however, the article immediately following Torsvik and Dundas describes bacteriophage PM2—presumably, though not specified, of Pseudoalteromonas of domain Bacteria—as both a “Virus” and “Viral” [The Torsvik and Dundas paper was followed by a publication during the next year, 1975, by Wais et al. . This wa “Viral” . Equival “Viral”  and 1980 “Viral”  as well  “Viral” . Pauling “Viral” , 51.Halobacterium halobium” . Of interest, for their Table 2, Stube et al. cite Post, 1975, in an article titled “Life in the Great Salt Lake” [Halobacterium—Halococcus” (page 44).Somewhat ambiguously, and in a report rather than an otherwise formal publication, Stube et al.  provide lt Lake” . In thatHalobacterium halobium phage ϕH,” the word “Virus” to our knowledge makes it first unambiguous as well as mainstream appearance in what would become the archaeal virus literature. This is found in the first sentence of the abstract: “Phage ϕH, a novel virus of the archaebacterium Halobacterium halobium, resembles in size and morphology two other Halobacterium phages.” In addition, under keywords is found “archaebacteria/virus,” though otherwise “Phage” is used far more often than “Virus” in this publication. Furthermore, in two locations the term “Archaebacterial phage” can be found for the first time. This latter usage seems to reflect the citing of Fox et al. [Halobacterium belongs to a group of organisms now known as archaebacteria which differ in many respects from both eucaryotes and eubacteria … This paper presents the first analysis of the DNA of an archaebacterial phage.The genus In 1982, in an article by Schnabel et al.  titled “x et al.  as foundBy contrast, and notably, this publication by Fox et al. is not cited in Pauling's article of the same year , along wThe Schnabel et al.  article Science in 1980, and thus widely disseminated well prior to 1982, it can be argued  that “This is because Zillig was firmly of the view that archaea have viruses not bacteriophages. Also, because of the influence of Carl Woese, as the first conference on Archaebacteria was held in Germany, around this time . …Munich (that Carl attended)… at the very institute that Zillig and Schnabel worked.” As relayed to us by Stedman , “the 1981 meeting was at the Max Planck Institute for Biochemistry,  where both Wolfram Zillig  and Heinke Schnabel  worked as Group leaders.” The concept of archaebacteria thus appears to have been very much a part of the conversation among those individuals who then introduced the concept of “Virus” to what would become the archaeal virus literature.The simultaneous timing of the introduction of the terms “Virus” and “Archaebacteria,” and variants, into what up to that point had been nearly indistinguishable from the otherwise “Bacteriophage” literature was not necessarily coincidental. In addition to Fox et al.  having bHalobacterium halobium: virion DNAs and proteins,” the term “Viruses” is used . Also used is “Archaebacteria” . This article does not cite Fox et al. [These trends in usage continued in the following year, 1983. In an article by Rohrmann et al. , titled x et al.  but doesx et al. .Thermoproteus tenax.” The publication is interesting, etymologically, for at least two reasons.Halobacterium spp. did not serve as hosts. Related to that point, these are the first viruses to be described of kingdom Crenarchaeota, versus the kingdom Euryarchaeota [Halobacterium is a member of the latter.This is the first archaeal virus paper for which rchaeota , where gϕH which resembles phages of eubacteria in many respects.”There is an indication (page 45) of “particles also resembling viruses of eukaryotes rather than “normal” bacteriophages.” In particular, “These viruses are unlike bacteriophages known to date, including halobacteriophage Further though not yet complete trending away from use of “Phage” as a descriptor of these viruses is seen in an article published by Janekovic et al. , also inArchaea as “Phages” to instead describing them primarily as “Viruses.” In particular, the observation of virions that were not phage-like was suggestive of a kinship between the viruses of what would come to be known as domain Archaea and viruses that otherwise are described simply as “Viruses,” that is, eukaryotic viruses.We thus have a new host genus and kingdom as well as a new paradigm for the nature of archaeal viruses which, in at least some cases, are somewhat divergent from what is seen among the viruses of bacteria. It is possible that this combined novelty provided some basis for a change in perspective, that is, from describing these infectious agents of domain Although viruses infecting archaea are known since the early 1970s , they haConsistent with this perspective, though placing the date of the transition approximately ten years later than as indicated here, is this 2012 sentiment from Felisberto-Rodrigues et al. :AlthoughArchaea would continue at a rate of at least one reference per year to the present. At this point, consideration of these publications is less relevant to the transition to “Archaeal viruses” except for the sake of documenting ongoing use. We thus present these publications primarily in graphical as well as tabular form  or Haloarcula hispanica pleomorphic virus 1 (HHPV1), for example, as summarized by Krupovic et al. [In  viruses . Indeed, viruses . We specc et al. .As there is no centralized authority governing of the naming of archaeal viruses nor central control over whether they are described in names as viruses versus phages, the use of “Virus” in these names not only may help to drive the increasing use of “Virus” in the archaeal virus literature but also can represent a consequence of that trend. Particularly, the “rules” for governing virus naming range from proposals for formal guidelines  to the wA number of descriptors exist that are synonymous with the term “Archaeal virus” as summarized in part in Archaea” and therefore “Archaeal” came into being, with the former not invented until 1990 [As we have considered above, there first was a transition from use of “Phage” to use of “Virus” for what now are known, at least in part, as archaeal viruses. This transition occurred in the early 1980s and it appeared to have coincided—as discussed above—with the introduction of the term “Archaebacteria” into the literature considering their viruses. The transition to “Archaeal virus” by necessity, however, could not occur until the term “til 1990 . When, ttil 1990 . Highligtil 1990  .The term “Phage” appears to have first been applied to bacteriophages to describe a macroscopic phenomenon that is not necessarily always of viral origin, that is, the lysing of bacterial cultures such that, in particular, they are to clear from a turbid state. This transition can be legitimately described as an “eating” or “devouring” as is the Greek origin of “Phage,” or alternatively something that “develops at the expense of something else” . This “enot specifically within the context of the destruction of a cell culture [H. salinarium, phage particles were observed in some crudely purified flagellar preparations. Phage-containing preparations did not give rise to plaques when plated with exponentially growing H. salinarium cells. Attempts at ultraviolet induction of H. salinarium cultures resulted in normal death curves without any concomitant phage production. Maintenance of batch cultures, started from isolated H. salinarium colonies, in the logarithmic phase of growth, by means of serial transfer of cultures in late log phase to fresh media, resulted in eventual lysis of the cultures. Lysis may occur after the first transfer or be delayed for more than six transfers.During an investigation of flagella from Contrasting bacterial phages, the first archaeal virus was discovered, at least initially, Archaea had yet to be defined, such that H. salinarium was considered to be a bacterium. Is it legitimate therefore for researchers who study these viruses nevertheless to not feel beholden to the concept of phages, but instead to explicitly tie these viruses to the larger phenomenon of viruses? Our answer to that question is that it is not that the use of the term “Archaeal virus” should be questioned by phage researches but instead that “Phage” as a descriptor should be tolerated and perhaps even appreciated by virologists due to its historical roots and ubiquity of use. There should be no requirement, in other words, for emulation of use of the term “Phage” by other virologists, whether those individuals choose to study the viruses of eukaryotes, archaeans, or even of bacteria. Indeed, it is quite clear that those who study the viruses of Archaea prefer “Virus” to “Phage” as a descriptor of those infectious agents  but also from use of “Phage” in describing these viruses, a shift that began in earnest especially during the early 1980s , failure to adequately distinguish between the concepts of “Phage” and “Virus” as applied to archaeal viruses, use of the term “Phage” to generally describe head-and-tail viruses, use of “Phage” within the context of “Halophage” , and the use of “Phage” within the context of “Prophage” . In addition, perhaps a special case is the use of “Phage” to describe specific archaeal virus isolates, such as “atabases .Bacteria, Archaea, and Eukarya, respectively.Alternatives to “Archaeal virus” versus “Bacterial virus” versus “Eukaryotic virus” have been proposed by a number of authors. These include archaeovirus, bacteriovirus, and eukaryovirus as suggested by Raoult and Forterre  as well Archaea and “Bacterial virus” for viruses of domain Bacteria, or alternatively the various “Virus-” based proposed alternatives as listed immediately above. We suggest this merely for the sake of limiting ambiguity within the archaeal virus literature, with a clear distinction therefore maintained between archaeal viruses on the one hand and bacterial viruses on the other. By contrast, within the bacterial virus literature, or when referring to specific phages , it is neither likely nor necessarily desirable to abandon or reduce use of “Phage” or “Bacteriophage” as a general descriptor of the viruses of domain Bacteria.Within publications in which archaeal viruses are discussed, we nonetheless feel that the use of “Phage” should be limited in favor of “Archaeal virus” for viruses of domain Provided are annotated summaries of the archaeal virus literature, including 694 references as available before January 1 of 2013. Highlighted per reference is the use of either “Phage” or “Virus” within the context of archaeal viruses. Shown also is a tabular time line of the use and introduction of additional terms employed as synonyms for “Archaeal virus”.Click here for additional data file.


Marek's disease virus (MDV) is an economically important oncogenic herpesvirus of poultry. Since the 1960s, increasingly virulent strains have caused continued poultry industry production losses worldwide. To understand the mechanisms of this virulence evolution and to evaluate the epidemiological consequences of putative control strategies, it is imperative to understand how virulence is defined and how this correlates with host mortality and infectiousness during MDV infection. We present a mathematical approach to quantify key epidemiological parameters. Host lifespan, virus latent periods and host viral shedding rates were estimated for unvaccinated and vaccinated birds, infected with one of three MDV strains. The strains had previously been pathotyped to assign virulence scores according to pathogenicity of strains in hosts.Our analyses show that strains of higher virulence have a higher viral shedding rate, and more rapidly kill hosts. Vaccination enhances host life expectancy but does not significantly reduce the shedding rate of the virus. While the primary latent period of the virus does not vary with challenge strain nor vaccine treatment of host, the time until the maximum viral shedding rate is increased with vaccination.Our approach provides the tools necessary for a formal analysis of the evolution of virulence in MDV, and potentially simpler and cheaper approaches to comparing the virulence of MDV strains. Marek's Disease Virus (MDV) is an oncogenic poultry herpesvirus of considerable economic importance to the poultry industry. Virus strains have become increasingly virulent since the 1960s . The cauMDV is an airborne pathogen with infection occurring via inhalation . Virus sAlmost all industrialised countries have experienced MD losses in their poultry industry and a crude estimate of the cost of Marek's disease is said to be in the range of US $1-2 billion annually . ControlDefinitions of virulence are numerous and varied . MicrobiThere have been various attempts to define virulence in the context of MDV . For exaControl strategies for MDV require an understanding of the epidemiology of the disease, in particular how virulence relates to key parameters such as viral shedding rates and duration of infectious period. There have been efforts to find correlates with virulence of MDV isolates, most notably with viral load (virus within a bird tissue). This has been achieved by cell culture techniques  and intrHere we present methods for parameter estimation for MDV isolates, allowing a formal quantification of infected host lifespan, viral shedding rates and viral latent periods. The parameters determined from the analysis allow comparison between isolates of different virulence in both unvaccinated and vaccinated birds. It is anticipated that the parameter estimates will be used for a better understanding of the pathology and aetiology of the disease itself and as a platform for investigation into the causes of virulence evolution.In the first section, we used survival analysis with a Weibull mortality function to build a statistical model for the lifespan of birds infected with MDV. In the second section, we developed a dynamic model to simulate the shedding of virus by a group of birds and used Bayesian techniques to estimate the four key infectiousness parameters: primary and secondary latent periods and viral shedding rates.p < 0.01). The resulting graphs showing the data and Weibull model fit are displayed in Figure An additive covariate Weibull regression model was fitted to the bird survival data, since the interaction terms were not significant. The model estimates are given in Table The dynamic model output records the density of virus /mg dust), which fitted to the data from each isolator independently. The model fits can be compared directly in Figures α, is set to 15. Assuming α ∈ , the results obtained in the McMC method did not change, whatever value of α is chosen.For each isolator, the median of the posterior distribution for the four parameters in the model  are given in Tables p = 0.41), showing that the primary latent period did not differ among virus strains and nor was it altered by vaccine treatment. For the secondary latent period, neither virulence score nor HVT vaccine affected the duration of the latent period, however, Bivalent vaccine increased the duration of the second latent period significantly and to a greater extent for smaller virulence scores  nor virulence score (p = 0.055). There was, however, a strong positive association between the virulence score of a strain and the secondary viral shedding rate (p = 0.00076). There was no association between the secondary viral shedding rate and either HVT or Bivalent vaccine treatment . We used survival analysis with a Weibull mortality function to build a statistical model for the lifespan of birds infected with MDV. We were able to compare the relative impact of virulence score and vaccine covariates by calculating credible intervals via Bayesian methods. We developed a dynamic model to simulate the shedding of virus by a group of birds and used Bayesian techniques to estimate the four key infectiousness parameters: primary and secondary latent periods and viral shedding rates.We applied these methods to the data for three MDV strains varying moderately in virulence. From the survival analysis, we found that increasing the virulence score of a virus strain decreases the lifespan of the host Figure . VaccinaThe results described here are qualitatively in line with current thinking on MDV pathogenesis, but the new methods presented allow a formal comparison of strains in an epidemiological meaningful way. It is anticipated that this analysis will allow a more quantitative understanding of the aetiology of MDV and the mechanisms governing a functional rise in virulence for a strain. With this analysis it is hoped that a more epidemiologically significant classification of MDV strains will emerge which will eventually provide not only insight into the reasons for evolution to highly virulent strains but also a metric to evaluate future risks and the efficacy of possible control strategies.We look at a single experiment  with twoThe two variables recorded were the time to death of a bird (measured in days) and the density of virus within each isolator every week (measured in logVCN per mg of dust in the environment). The virus titres in the dust were determined via quantitative real-time PCR of dust collected from the isolator exhaust ducts, the methods of which have been discussed in separate studies . The datWe assumed that each infected bird starts shedding virus after an initial delay, then after a further delay, the rate at which it sheds virus rises to a long-term stable value. We estimated five key epidemiological parameters: host lifespan, primary latent period (the time until an infected bird becomes infectious), secondary latent period (the delay between an infectious bird first shedding virus and when it sheds virus at its secondary long term rate) and viral shedding rates . A survival analysis was undertaken for the mortality data because multiple covariates were needed to find the host lifespan. These covariates were vaccine treatment and virus virulence score. Latent period and rate of viral shedding could not be estimated directly from the data since the response variable (measured weekly in VCN per mg dust) could vary with the number of birds housed together, which changed as birds died. Therefore a dynamic model capturing this variability was used to estimate the two infectiousness parameters.10 throughout the paper.For ease of notation, log is written to represent logT, such that T ~ W where r, λ > 0 are the shape and scale parameters respectively. The associated probability density function isThe biology of Marek's disease suggests that the probability of death by MDV infection changes through time, since the virus undergoes lytic, latent stage , late lytic (where infection is extended to other organs of the host), and transformative (tumour-induction) stages. These stages are pathologically distinct and may lead to a different mortality rate. Therefore, a Weibull distribution was chosen as a candidate distribution for modelling survivorship curves, which is often used for time to death data since it is flexible and can mimic other distributions but only has two parameters in its non-location form. We therefore assumed that time to death can be modelled as a random variable, r = 1, the distribution collapses to an exponential distribution. When r > 1 or r < 1 there is an increasing or decreasing chance of death over time respectively. Coefficients, β, are such that λ = exp(β · x), where β =  are the covariate coefficients and x = T are the covariates. In this analysis, there were three covariates (one continuous: the transformation of the virulence score of an isolate (x1) and two binary: the presence or absence of HVT (x2) and Bivalent (x3) vaccine). Therefore there were 9 combinations on the set of possible covariates, thus j λ= βxj ·where j ∈  The likelihood function can therefore be writtenA Weibull model was fitted to the survival data in the study. In the case when i δis zero when the ith observation is censored and unity elsewhere. This function was maximized via the Newton-Raphson algorithm such that v, of an isolate is a percentage measure and to be used as an explanatory variable in a regression analysis, it should be transformed such that β, however further Bayesian analysis was required to estimate the associated credible intervals when the covariate covariance matrix does not approximate the identity matrix. An McMC framework was set up in WinBUGS [where  WinBUGS  to calcuThe prior distributions of each parameter were assumed to be uninformative and were thus taken as uniform, to ensure equivalence to maximum likelihood estimation. The burn-in was set to 22,000 iterations with every 10th sample taken from the subsequent 100,000 iterations ,38.The dynamic model tracked the density of MDV over time in each isolator. In this model it is assumed:1) After MDV infection, there is a delay before virus is first shed . This is2) After this primary latent period, virus is shed at a constant rate  for a set period of time, termed the secondary latent period.3) Once this secondary latent period is over, virus is shed at a constant rate  until the termination of the experiment.Additionally, the density of virus (VCN per mg dust) was calculated at the end of each day and any removal of birds was assumed to occur at the start of the day. While all parameters are assumed to be constant across all the birds in a single isolator, they can vary between isolators.T1 and T2 respectively. Birds shed virus at a rate, a1 (VCN per mg dust) from T1+1 to T1+T2 days post infection; and at a rate a2 (VCN per mg dust) from T2+1 until the end of the experiment (at 8 weeks post infection). The density of MDV in the dynamic model could be compared to the amount of virus recorded in the data (sampled every 7 days). We therefore estimated v ∈ {16.5, 36, 46} and each vaccine status j ∈ {Sham, HVT, Bivalent}.There are therefore four parameters estimated per isolator: the primary/secondary latent periods (in days) and the primary/secondary rates of viral shedding (VCN/mg dust). The primary and secondary latent periods are denoted by α). The daily quantity of dust shed per bird was calculated by fit ting a cubic spline to dust MDV data provided from the same experiment [The model accounts for the removal of birds given in the mortality data and the number of complete hourly air changes  and that the distribution has a mean of zero (p = 0.40). The resulting standard deviation of the difference between log-transformed data was estimated to be 0.33 logVCN/mg dust.To find the time point after which the data is assumed to have plateaued, four sets of points were examined: weeks 5, 6, 7 and 8. The differences between the logged data of weeks 7-8 provided the highest probability of both being drawn from a normal distribution (σ2 = 0.332/2. The burn-in time was set to 30,000 iterations and the posterior distribution was taken as every 10th sample from the following 90,000 iterations [Posterior distributions were found by means of McMC realisations by calculating the likelihood of the data given the parameter values, with lognormal errors of erations ,38.The authors declare that they have no competing interests.KEA performed the statistical analysis and drafted the manuscript. AFR was involved in the drafting and revision of the manuscript for intellectual content. NJS was involved in the Bayesian statistical analysis. SWB and KGR were involved in supplying data and associated technical advice. SWB was involved in the revision of the manuscript for intellectual content. MEJW was involved in design of the statistical analysis and the drafting and revision of the manuscript for intellectual content. All authors approved the final manuscript.


African swine fever virus p72 genotype IX, associated with outbreaks in eastern Africa, is cocirculating in the Republic of the Congo with West African genotype I. Data suggest that viruses from eastern Africa are moving into western Africa, increasing the threat of outbreaks caused by novel viruses in this region. Asfaviridae  is a serious disease of domestic pigs caused by a DNA arbovirus (African swine fever virus [ASFV]) belonging to the family The Republic of the Congo, located in western-central sub-Saharan Africa, shares borders with the Cabinda enclave of Angola, the Democratic Republic of the Congo, Central African Republic, Cameroon, and Gabon. The last ASF outbreaks in Congo were reported to the World Organization for Animal Health (OIE) during 2003. Since then, the disease has been officially declared endemic but without quantitative data. Sampling and characterization of currently circulating field strains from this region of western-central Africa are needed to fully understand virus spread and maintenance. Such data will have implications for regional control in western Africa.During August 2009, a United Nations Food and Agriculture Organization mission was undertaken with local counterparts in Congo to support development of an action plan to control ASF. A key factor in selecting the sites sampled was inclusion of the main pig-producing, marketing, and consuming areas, with a particular focus where suspected ASF outbreaks had been recently reported. From the survey conducted, 86 samples comprising serum (35 samples), whole blood (44 samples), and tissues (7 samples) were collected from 80 domestic pigs in the departments of Brazzaville, Pointe Noire, Kouilou, Bouenza, Niari, and Cuvette . These departments contain 80%–90% of the country’s pig population. Samples also were collected from Pool in southeastern Congo, where a recent hemorrhagic disease outbreak, characterized by case-fatality rates of ≈80%, had been reported. Clinical material was sent to the European Union Reference Laboratory for African Swine Fever  for confirmatory diagnosis and characterization of the ASFV strain(s) responsible for the outbreak(s).Specific ASF antibodies were detected by using the OIE-prescribed assays  of the p54 gene amplification, amplicons of 600–650 bp were generated from the isolates from eastern Congo, whereas the estimated size of the amplicons from western Congo was 500 bp (data not shown). Analysis of the tandem amino acid repeat sequences within the CVR demonstrated 3 different variants cocirculating in the country, 2 within the p72 type I virus genotype. The type of CVR sequence (AABNABTDBNAAAA) identified in the isolate from Pointe Noire (Con09/PN003) was identical to CVR subgroup XIII (,,Under the same thermal cycling conditions used for full We confirmed ASF in 5 of the 6 departments in which surveillance was conducted during August 2009 in Congo. Genotyping of 5 ASFV isolates from Congo resulted in identification of genetically distinct viruses circulating simultaneously in the country. In eastern districts of Congo, viruses were most genetically similar to those recovered from the outbreaks in Kenya and Uganda during 2006 and 2007. Therefore, genotype IX, associated with ASF outbreaks in eastern Africa during the previous 10 years, also occurs in western Africa. By contrast, viruses in western Congo were similar to genotype I viruses that historically circulated in western Africa. Analyses of the CVR identified 2 discrete variants within genotype I. The Pointe Noire ASFV was identical to viruses isolated in neighboring Angola during the 1970s, indicating a prolonged persistence of this virus type in the region. By contrast, the ASFV isolated from Niari was related to variants circulating during the 1980s in Burundi. These results are consistent with recent transfer and dissemination of ASFV genotypes from eastern to western Africa. Broadening of our initial findings requires extending analyses to western-central Africa, particularly to countries such as Cameroon and the Democratic Republic of the Congo, and providing more in-depth analysis in Congo.The apparent recent movement of a virus genotype previously associated with virulent ASF in eastern Africa to western Africa, where the viruses have hitherto always been classified in p72 genotype I, indicates the possibility of future outbreaks of disease caused by novel viruses in western Africa. The reality of this scenario recently was demonstrated in the Caucasus region. Our study confirms the continuing spread of ASFV.


Exclusive breastfeeding (EBF) for 6 months is the recommended form of infant feeding. Support of mothers through individual peer counselling has been proved to be effective in increasing exclusive breastfeeding prevalence. We present a costing study of an individual peer support intervention in Uganda, whose objective was to raise exclusive breastfeeding rates at 3 months of age.We costed the peer support intervention, which was offered to 406 breastfeeding mothers in Uganda. The average number of counselling visits was about 6 per woman. Annual financial and economic costs were collected in 2005-2008. Estimates were made of total project costs, average costs per mother counselled and average costs per peer counselling visit. Alternative intervention packages were explored in the sensitivity analysis. We also estimated the resources required to fund the scale up to district level, of a breastfeeding intervention programme within a public health sector model.Annual project costs were estimated to be US$56,308. The largest cost component was peer supporter supervision, which accounted for over 50% of total project costs. The cost per mother counselled was US$139 and the cost per visit was US$26. The cost per week of EBF was estimated to be US$15 at 12 weeks post partum. We estimated that implementing an alternative package modelled on routine public health sector programmes can potentially reduce costs by over 60%. Based on the calculated average costs and annual births, scaling up modelled costs to district level would cost the public sector an additional US$1,813,000.Exclusive breastfeeding promotion in sub-Saharan Africa is feasible and can be implemented at a sustainable cost. The results of this study can be incorporated in cost effectiveness analyses of exclusive breastfeeding promotion programmes in sub-Saharan Africa. Sub-Saharan Africa has the poorest child health record, accounting for over half of all deaths of children worldwide ,2. The mEBF promotion has been identified as one of the interventions with the highest life-saving potential globally, and if all children were optimally breastfed, this could potentially save 13% of child deaths worldwide . It is tEBF promotion programmes are also hampered by many other factors, including cultural aspects at the family level and scarcity of resources at the national level -19. Infohttp://clinicaltrials.gov/ct2/show/NCT00397150) conducted in four sub-Saharan African countries, namely Burkina Faso, South Africa, Uganda and Zambia. This paper presents the annual costs of the PROMISE EBF intervention in Uganda, and provides estimates of the resources required to fund the scale up to district level. The costing study was not done in Burkina Faso at the same time, due to the language insufficiency of the researchers, and both this and the South African report will be made at a later stage, albeit with slightly different focus. The Zambian study was not analysed due to disruptions caused by flooding at the time of the intervention.We set out to measure the costs of an individual peer counselling intervention, designed to increase EBF prevalence at 3 months postpartum among infants in sub-Saharan Africa . PROMISEMbale district is situated in Eastern Uganda with a population of about 700,000 and a population density of 535 per square kilometre . The stuIn the district, 24 communities (clusters) were selected and stratified based on similarities in terms of location, urban-rural set ups and socio-economic status. In each stratum, half of the clusters were randomized to the control and intervention groups, respectively. The intervention of peer counselling for exclusive breastfeeding was therefore set up in twelve clusters, each with an estimated population of about 1000 inhabitants, expected to generate 35 babies in a year given a birth rate of 3.5%. Each rural cluster consisted of one to three villages combined, depending on the village population size. All pregnant women in the geographical clusters were eligible for participation. The women were identified in the clusters, introduced to the project and recruited upon consent, according to eligibility criteria. The inclusion criteria were that the woman resided in the selected cluster, was 7 months pregnant and had no intention to move out of the cluster. Women with severe psychological and somatic illness, those having given birth more than 1 week ago, and those planning to replacement feed were not included in the study.In the intervention clusters, mothers were visited by a peer supporter at least five times, with the first visit occurring when a mother was about seven months pregnant. The remaining visits were scheduled at 1, 4, 7 and 10 weeks after delivery. Mothers with breastfeeding problems were given extra visits. Extra visits were also given if a mother called the peer counsellor for additional assistance outside the scheduled time or if the peer counsellor deemed it necessary. The peer counsellors chose the time most convenient to the mothers for meetings during the scheduled weeks. The peer counselling intervention was conducted in a period of about one and a half years. Women in the control clusters did not receive this counselling, but were encouraged to attend regular antenatal and postnatal clinics, which are available at all health facilities in Uganda. Antenatal attendance is very high in Uganda, with over 94% of pregnant women making regular visits .Sensitization workshops and meetings aimed at introducing the project to community leaders were held prior to commencement of the trial. The community leaders facilitated the mobilisation of local women to work as peer counsellors on the project. Twelve women were selected as peer supporters, one in each cluster table .At the beginning of the programme, all peer supporters were given six days training based on the WHO breastfeeding counselling course . During A team consisting of two peer supporter supervisors and the study coordinator were responsible for overall peer supporter supervision. The role of the peer supporter supervisor was to provide advice and support to the peer counsellors. Supervision was done through on-site observation of counselling sessions, which were conducted every fortnight. Monthly meetings for all peer supporters were also held at the office. These meetings provided an opportunity to share field experiences and sort out any problems encountered by the counsellors.PROMISE EBF composed of the following employees: a research coordinator, who was the overseer of the entire project, a data manager, in charge of the database, a data collectors' supervisor, whose job was to manage the data collectors, peer supporter supervisors who were responsible for the peer supporters, peer supporters, recruiters and a driver. During the PROMISE-EBF intervention, there was no clear distinction between the intervention and research component for workers such as the driver and research coordinator, as they worked on both activities. In the costing study, a distinction was made between project evaluation and the intervention. Only costs of the intervention are included in this paper. The intervention was basically the peer support program, and project evaluation consisted of the data collection process. Since the cost analysis was done in retrospect, there was no way to measure the time spent on different activities by workers who belonged both to the intervention and evaluation. The project personnel were, therefore, divided between project evaluation and intervention using percent effort, which was determined through interviews with project staff:Project evaluation: Research coordinator (60%), data manager (100%), data collectors' supervisor (100%), data collectors (100%), recruiters (50%), driver (60%).Intervention: Research coordinator (40%), peer supporter supervisors (100%), peer supporters (100%), recruiters (50%), driver (40%).The research coordinator, peer supporter supervisors and driver were permanent staff on the project, and were therefore offered competitive salary packages. Peer supporters and recruiters were not permanent members of staff. They were offered a token US$20 every month for their participation table , a figurCosting was undertaken from a local provider's perspective and involved the identification of all project costs relating to the intervention in the project's books of accounts and administrative records. Costs to the family and society at large are not included in this analysis. Costing was done across 5 major categories which were identified as the main activities of the peer support intervention. These were start-up, overheads, training, peer support and peer support supervision. The start-up category included all preparatory activity costs such as manual adaptation, training and workshops. The initial training of peer supporters for the intervention was included as a capital cost in start-up costs. All post-start up training of peer supporters was included as a recurrent cost in the category we refer to as Training. This includes all re-training of counsellors, and other trainings which may or may not have been related to the intervention. Overheads included items such as rentals, telephone and internet. Peer support included all items related to peer counselling, such as personnel and travel; and peer supervision included personnel, transport, equipment and costs of all activities related to overall project supervision.Data collection was based on the Costing Guidelines for HIV Prevention Strategies . ResourcCapital costs were annuitized, a process that is used to reflect their annual value . Start uWe conducted a time use assessment among peer supporters to enable us to understand how time was spent on project activities. We specifically sought to find out how much time was spent per visit. A daily log sheet was administered to peer supporters, where they were asked to record the time spent on each counselling visit and the time it took to travel to each counselling session. We did not have a defined sample of counselling sessions, but decided to collect data for at least 500 visits. Total time spent on all activities was divided by the total number of activities, to calculate the average time spent on each activity. Time data were also used to calculate total and average walking distance covered by peer supporters for every visit, based on an estimated average walking speed of 5 kilometres (km) per hour (h).The impact of the intervention was measured using the number of women counselled (which was taken as the number of women reached by the intervention) and exclusive breastfeeding up to 6 months post partum. The two main outputs of the peer support component from the costing perspective were total number of mothers counselled and total number of counselling sessions or visits. These were combined with total costs to calculate average costs per visit and per mother counselled. We used these to approximate the cost per weeks of exclusive breastfeeding (WEBF). WEBF were the sum of the duration, in weeks, that a child was exclusively breastfed. The cost per WEBF was expressed as the total cost divided by total WEBF at a given period.A univariate sensitivity analysis was undertaken to estimate the impact of a number of assumptions made in the analysis. The discount rate was varied by replacing the bond rate (20%) with 3% and 6%, which are both commonly used in health economic evaluations. We varied personnel costs up and down by 20%. We also estimated the impact of increasing or reducing the number of visits per mother (+/- 20%) and allocating staff time between project implementation and evaluation (+/-20%), for the project coordinator and driver, who were involved in both activities. In addition, we varied the percentage allocation of other costs shared between project implementation and evaluation (+/-20%). The +/-20% range was used because we did not have any reference studies or procedures, and we felt this range would capture as much variation as possible.PROMISE EBF was held in a trial setting, and as such, its design was oriented towards research. A number of activities and costs might not have been incurred in a programme setting, and total project costs might therefore be lower. We tested this assertion by modelling the costs of undertaking an alternative exclusive breastfeeding support programme at the scale of PROMISE EBF. We assumed that the community programme was supervised by the Ministry of Health (MOH), and undertaken through its network of community health workers such as traditional birth attendants and other voluntary workers, already established in most communities in Uganda. The aim was to assess the changes in costs and average costs of undertaking similar EBF support programmes with varying scenarios. The design of the alternative programme was based on a community randomized trial assessing the effects of community based promotion of exclusive breast feeding . In the We estimated the cost of scaling up the alternative scenario from the village level to district level, based on the calculated average costs and the expected number of annual births. The number of births was calculated based on an expected 35 babies born per thousand populations per year. We assumed that the cost structures observed in the sample were similar to those obtaining at district level.The results of the PROMISE EBF project show that the intervention was successful in increasing EBF prevalence. At 12 weeks of age, based on a 24 hour recall, the results in the intervention and control arms were: 81.6% and 43.9% . Similarly, at 24 weeks of age, the results were 58.6% and 15.5% . The 7 day recall prevalence ratios were similar to those obtained in the 24 hour recall. The full analysis and discussion of the intervention methods and results are presented elsewhere .Table Counselling and travelling time data were collected for a sample of 1,192 visits. The total project time recorded was 184,786 minutes. Over 60%  of this time was spent travelling. The mean travel time in a day was 101 minutes, ranging from a low of 3 minutes to a high of 335 minutes. A total of 64,213 minutes were recorded for counselling sessions, with an average of about 54 minutes  per counselling session. The mean distance walked per day per peer supporter was 8 km .A total number of 406 women were recruited into the PROMISE EBF intervention arm /1000] babies born per year in the district. We established that the average cost per mother-baby pair of the community intervention was US$74. Multiplying the average cost to the estimated number of babies yields an annual cost of US$1,813,000 . This is the estimated annual cost of implementing a breastfeeding intervention programme as an additional cost to routine public sector programmes. The total cost of scaling up the programme to a population of 1 million inhabitants was US$2,590,000.We have analysed the costs of implementing an individual peer counselling intervention, which was successful in increasing exclusive breastfeeding prevalence at 3 months post partum . A literPROMISE EBF was a moderately intensive study, but with very close supervision, and as such, personnel costs accounted for the largest share of total costs. Personnel costs were also driven up because permanent project staff were offered competitive salaries, equivalent to those prevailing in the private sector. Transport costs, and mostly fuel, also accounted for quite a large proportion of total costs. This was probably a result of the large distances that had to be covered during each visit, as evidenced by the results of the time use analysis, which indicated an average walking distance of 8 kilometres a day per peer supporter.At US$139 per mother, PROMISE EBF was quite an expensive undertaking, whose design would be difficult to implement in Uganda given the scarcity of resources. However, our sensitivity analysis indicates that it is possible to reduce these costs by over 70%, through variation of certain component costs. Since PROMISE EBF was vertically conducted in a trial setting, the costs incurred might not be reflective of actual costs that a similar project would attract in a real life setting. We explored this assumption by assuming a horizontal project undertaken under the auspices of the Ministry of Health, with its network of community health workers that are well established in most communities in Uganda. This analysis revealed that it was possible to reduce annual implementation costs by over 60%. This reduction is possible for a number of reasons, most important that supervision is not as rigorous in real life as it is under trial settings. Trial supervision accounted for the highest cost in PROMISE EBF. Costs such as personnel emoluments in a trial setting are usually pegged at competitive market prices, but in a real setting, and under a government programme, these costs are substantially reduced as government workers are often paid a much lower rate. Sustainability of the project in the long term by the community, particularly through government support might therefore be feasible, with government personnel taking on a supervisory role.Reducing costs as such, may be fairly easy. The real challenge lies in simultaneously maintaining the quality of intervention outcomes. The effect of cost reductions on the quality of outcomes has not been analysed in this paper, but it is necessary for future research to look into this issue.Costs of a peer counselling for the promotion of exclusive breastfeeding in Zambia; Unpublished], and found that while average costs were lower in Uganda, the scale up costs were much higher. The scale up costs at district level were estimated at about US$700,000, obviously a result of population differentials, with Mbale district in Uganda having a higher population. The cost per mother of US$139 in Uganda, compared to US$233 in Zambia indicated higher cost structures in the latter country, highlighting the need for policy makers to take the economic environment into consideration when planning such programmes. Both the Zambian and Ugandan costs, however, were comparatively lower than similar costs estimated for implementing such a programme in Kwazulu Natal, South Africa [The benefits of exclusive breastfeeding have been documented ,5, and th Africa . Horton Whether our estimated costs of scaling up the programme are attainable, or not, by the governments in our study areas is debatable, but it is important to acknowledge the availability of external funding sources, and that implementing such a programme on a large scale will require concerted efforts by the government and its partners in the donor community, private sector and non-governmental organisations. All parties involved in the implementation of such an intervention, should however, be aware of the challenges of implementing it both as a vertical or integrated programme. Implementing such an intervention as part of a government programme for example, could have a negative impact on the performance of an already overstretched workforce. Such project effects should be known before hand and solutions made to mitigate them.Investments do not depend on cost decisions alone, and it is prudent to weigh costs against the benefits of an intervention before making an investment. In the case of PROMISE EBF or similar breastfeeding support programmes, policy makers may need to reflect on the potential long term health and economic benefits that could arise from the promotion and maintenance of exclusive breastfeeding through peer support. Evidence confirms that it is possible to reduce costs of illness due to diseases such as diarrhoea and pneumonia, by increasing the level of exclusive breastfeeding, thereby accruing savings from reduced service provision -37. WhilFuture research should also look into the interaction of costs and time spent on the project. We did not analyse this relationship, though we conducted a time use survey among some project staff. It can be envisaged though, that the more time spent, the higher would be the costs. How this would affect the quality of counselling is debateable, but it can be argued that the effect would most likely be negative, as peer supporters might concentrate on increasing the number of visits in an effort to increase their monetary gain, rather than spending more time on each visit to ensure high quality support.The authors declare that they have no competing interests.All authors participated in the design of the study. LC, LN and BR contributed to data analysis, writing and editing of the manuscript. CK, JN, JT and TT contributed to the writing and editing of the manuscript. LC made the first draft of the manuscript. All authors read and approved the final manuscript.


More than 50 researchers and administrators from over a dozen countries attended a symposium on the emergence and control of zoonotic viral encephilitis. Held April 6–8, 2003, in a convivial setting at Les Pensières, Veyrier du Lac, near Annecy in the French Alps, this meeting was one of a series on the emergence and control of infectious diseases, sponsored and organized by the Mérieux Foundation. The general objectives were to review the biology of viral encephalitis, the virulence and genetic evolution of encephalitis viruses, and the factors involved in emergence of these diseases.Emergence or reemergence of viruses may be due to virus evolution, to the impact and influence of human populations on previously undisturbed ecosystems, or to better recognition. Clearly, we must understand the basic mechanisms by which these viruses emerge or reemerge and cause illnesses. Methods for detecting infections caused by neurotropic viruses and for detecting viruses or their genome sequences are available and improving. Methods for detecting antibodies also have improved.Paramyxoviridae, genus Henipavirus), both of which are neurotropic, and Australian bat lyssavirus , also a neurotrope. All three viruses, and others related to them, have been shown to have fruit bats (Pteropus spp.) as their natural hosts. Progress is being made in understanding transcription regulation and cell fusion by henipaviruses. In addition, basic epidemiologic procedures and classical prevention strategies have been put into place to prevent infections caused by these viruses.Examples of recently recognized viruses causing encephalitis in humans, livestock, or wildlife include Hendra and Nipah viruses (henipaviruses; family Since 1988, a worldwide effort has been under way to eradicate the nonzoonotic but encephalitogenic poliomyelitis viruses. The number of cases has been reduced by 99%, and the natural occurrence of these viruses now is limited to seven countries. The system established to conduct surveillance and response may provide a model for use in tracking and controlling other viruses causing encephalitis. Long-term studies of ecologic parameters, seasonality, and changing virus and vector prevalence rates are being used to determine risk factors in various arbovirus infections, including Japanese encephalitis virus in Thailand, and are being applied for prevention and control.In Russia, where West Nile virus has long been recognized but has not caused any major diseases, recent detection of various virus genotypes suggests a mélange of genotypes circulating in various areas and transported between areas by birds. Generation and maintenance of continuous genetic variation may lead to partial protection and escape mutants, which could provide a “pump” that generates more variants and “new” viruses. When these genotypes adapt to naïve populations of birds, horses, and humans, in the presence of competent arthropod vectors, epidemics may arise and new opportunities for these viruses and virus variants may occur, perhaps including West Nile virus into the New World in 1999. Evidence presented suggests that little genomic variation in New World West Nile virus has occurred since its 1999 recognition there; this situation is likely to change.Continuing to make the classical epidemiologic observations that have characterized disease investigations in the previous half-century is important. However, to understand the overall effects of virus outbreaks, denominators are needed. Numerous presentations demonstrated that we are beginning to understand the molecular mechanisms leading to pathogenetic events. Further studies may provide information useful for the development of antiviral compounds and candidate vaccines.Attendees were provided with an overview of various transmission cycles of arboviruses, which are concomitantly diverse in regards to their hosts and vectors. Viral neuroinvasiveness appears to depend on the uniqueness of phylogenetically diverse hosts, their ages, genetic predispositions, immune status, virus origin, passage level, dose, and other factors—a complex situation to investigate and comprehend. Critical factors impacting neuroinvasiveness and neuronotropism must be coupled to cause encephalitis. Viral mutations may affect the ability of the virus to replicate in cells, altering viral virulence; however, specific genomic and polyprotein sequence changes may account for the high viremias and replication in neurons that are central to emergence. The extent of the roles of various proteins in virus infections, neuronal involvement, and apoptosis are being recognized. Now we are beginning to understand complex signaling mechanisms, antibody-producing cell types, cytokines, and the cellular responses and pathways leading to both disease and protection from disease.Considerable progress has been made in understanding the relationships between genetic and functional diversities, neuronal receptors, transport, and cellular protein-virus interactions. Such understanding is critical to further insights to neurotropism, pathogenesis, pathogenetic mechanisms, and immunogenicity.Phylogenetic trees were used to describe the evolution of encephalitic flaviviruses, geographic exclusion, virus persistence, and flaviviral recombination as a mechanism of flaviviral evolution. In addition, data were presented that illustrated the persistence of, and immune modulation by, alphaviruses, which, in concert, allow the virus to replicate while preventing the host from responding to its benefit.Other than the classical techniques of preventing infection, little was mentioned about disease control during this symposium. Control must be based on rapid recognition of early cases, subsequent immunization of persons or animals at risk, or immunization of persons or animals with the potential to be at risk, such as travelers, laboratory personnel, and attending clinicians. Attendees learned about diverse methods being used to develop vaccines. Representatives from the World Health Organization explained that organization’s plans for responding to disease emergence and for preventing zoonotic diseases from reaching human populations.New paradigms for field studies of zoonotic diseases are necessary. These approaches must include longitudinal and in-depth investigations of agent, host, habitat, and environment if we are to predict risk and respond in an appropriate manner. At this time, zoonotic disease control comprises prevention and public education and not much more. Progress is being made in rapid diagnosis, production of sophisticated vaccines, and understanding of the molecular mechanisms by which zoonotic viruses persist and cause disease.


Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses—etiological agents of smallpox , monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)—as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.On May 8, 1980, the World Health Assembly at its 33 Orthopoxvirus of the family Poxviridae comprises the species variola  virus (VARV), with human as its only sensitive host; zoonotic species monkeypox virus (MPXV), cowpox virus (CPXV), vaccinia virus (VACV), and camelpox virus (CMLV); and several others. These orthopoxviruses are immunologically cross-reactive and cross-protective, so that infection with any member of this genus provides protection against infection with any other member of the genus Orthopoxvirus genus have been named primarily according to the host animal from which they were isolated and identified based on a range of biological characteristics The genus Orthopoxvirus, and this genome contains all the genes found in the other species of this genus Cowpox virus was proposed as the closest of all the modern species to the progenitor virus for the genus Orthopoxvirus, while the remaining species, Variola virus included, had appeared as a result of multistage reductive evolution With accumulation of the data on complete genome nucleotide sequences for various strains of orthopoxvirus species, it has been found that an interesting feature of the orthopoxvirus genomes is the presence of genes that are intact in one species but fragmented or deleted in another VARV, the most pathogenic species for humans, has the smallest genome of all the orthopoxviruses The conducted analysis of the available archive data on smallpox and the history of ancient civilizations as well as the newest data on the evolutionary relationships of orthopoxviruses has allowed me to suggest the hypothesis that smallpox could have repeatedly emerged in the past via evolutionary changes of a zoonotic progenitor virus Because of the cessation of the vaccination against smallpox after its eradication 35 years ago, a tremendous part of the world human population currently has no immunity not only against smallpox, but also against any other zoonotic orthopoxvirus infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, should alter several established concepts on the ecology and range of sensitive hosts for various orthopoxvirus species.variolae vaccinae, originates from zoonotic CPXV, introduced to immunization practice by Jenner as early as 1796 th century was it found out that the orthopoxvirus strains used for smallpox vaccination significantly differ in their properties from both the natural CPXV isolates recovered from cows and the other orthopoxvirus species examined by that time Vaccinia virusThe most intricate case is the origin of VACV. For many decades, VACV has been used for vaccinating humans against smallpox, and it was considered that this virus, th century. The vaccine lymph from the horse gave the most satisfactory results in inducing an anti-smallpox immunity as well as less side reactions Vaccinia virusThe issue of VACV origin was somewhat clarified after sequencing the complete genome of horsepox virus (HSPV) Since the 1960s, VACVs have been repeatedly isolated in Brazil Vaccinia virus, genus OrthopoxvirusIn the past, the outbreaks of buffalopox had occurred frequently in various states of India as well as in Pakistan, Bangladesh, Indonesia, Egypt, and other countries Vaccinia virus have a wide host range, are zoonotic, are currently spread over large areas in Eurasia and South America, and that their natural carriers are several rodents.Thus, yet incomplete data on the modern ecology of VACV and BPXV allow for speculation that the orthopoxviruses belonging to the species Cowpox virus–like and Vaccinia virus–like clades CPXV has relatively low pathogenicity for humans but has a wide range of sensitive animal hosts MPXV is a zoonotic virus causing a human infection similar to smallpox in its clinical manifestations with a lethality rate of 1–8% In its biological properties and according to the data of phylogenetic analysis of the complete virus genomic sequence, CMLV is closest to VARV, the causative agent of smallpox, as compared with the other orthopoxvirus species It is known that most of the emerging human pathogens originate from zoonotic pathogens There are no fundamental prohibitions for the possible reemergence of smallpox or a similar human disease in the future as a result of natural evolution of the currently existing zoonotic orthopoxviruses. An ever-increasing sensitivity of the human population to zoonotic orthopoxviruses, resulting from cessation of the mass smallpox vaccination, elevates the probability for new variants of these viruses, potentially dangerous for humans, to emerge. However, the current situation is radically different from the ancient one, since many outbreaks of orthopoxvirus infections among domestic animals and humans are recorded and studied.Recently, the efforts of scientists under WHO control are directed to the development of state-of-the-art methods for VARV rapid identification as well as design of new generation safe smallpox vaccines and drugs against VARV and other orthopoxviruses In the areas of high incidence of zoonotic orthopoxviral infections, it would be purposeful to vaccinate domestic and zoo animals as well as the persons closely associated with them using state-of-the-art safe vaccines based on VACV, which has a wide range of sensitive hosts. This would considerably decrease the likelihood for such infections to spread from wildlife into the human environment.In the African region endemic for monkeypox, which also displays a high rate of HIV infection, the population could be vaccinated with the VACV strain MVA, which has been recently demonstrated to be safe even for HIV-infected persons Taking into account the above mentioned increased incidence of outbreaks of animal and human orthopoxvirus infections and their potential danger, it is important to accelerate organization of the international Smallpox Laboratory Network, discussed by the WHO Advisory Committee on Variola Virus Research The international system for clinical sampling and identification of infectious agents has been worked out and optimized while implementing the global smallpox eradication program under the aegis of the WHO as well as anti-epidemic measures and methods for mass vaccination


Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus and cross-species infection of HEV. Up to now, the mechanism of cross-species infection of HEV from swine to humans is still unclear. This study sought to identify receptor element for genotype 4 HEV on swine liver cells using the viral overlay protein binding assay (VOPBA) technique and Mass Spectrometry fingerprinting. A single virus binding band with natural molecular weight about 55 kDa was observed, and mass spectrometry revealed that this virus binding band contained 31 different proteins. Infection inhibition assay suggested that this 55 kDa protein could prevent HEV from infecting its susceptible A549 cell line, which was further confirmed by the HEV genome detecting in the inoculated cells. Further research should be performed to elucidate the accurate receptor of HEV on the swine liver cells. Hepatitis E virus (HEV), a member of the genus Hepevirus, is a non-enveloped virus with a positive-stranded RNA genome approximately 7.2 kb in length, which consists three open reading frames ORF1-3)[-3[1]. ORViral overlay protein binding assay (VOPBA) has been used to identify receptor proteins for various viruses, such as Bovine adenovirus , Dengue EF570133). This sample was proved to be negative for PEVs (including PTV and PEV1-10), haemagglutinating encephalomyelitis virus, Aujeszky's disease virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus, Japanese encephalitis virus, porcine transmissible gastroenteritis virus, porcine epidemic diarrhoea virus, porcine rotavirus, porcine sapovirus, cytomegalovirus, porcine Torque-Teno virus and porcine parvovirus by RT-PCR/PCR methods. The fecal sample was converted to 10% (w/v) suspensions in PBS (pH7.4) and clarified by centrifugation at 15, 000 g for 30 min. Supernatants were purified by passage through 0.22 μm microfilters  before virus inoculation or purification for viral overlay protein binding assay (VOPBA). For purification of viral particles, 150 ml of the purified virus suspension were mixed with polyethylene glycol 6000 (PEG 6000) and NaCl to final concentrations of 6% and 0.3 M, respectively. The mixture was stirred at 4°C for 6 h, then centrifuged at 20, 000 g for 90 min at 4°C. The precipitated sediment was resuspended in 15 ml PBS. A 10-60% (w/v) discontinuous sucrose density gradient was made in a Beckman 25 × 76 mm Ultraclear tube  using a modification of the method described previously [HEV positive swine fecal sample was form an experimentally infected pig with a genotype 4 HEV strain  in accordance with the manufacturer's protocol. The concentration of protein was quantified by the Bradford method .Membrane proteins (60 to 100 μg) were subjected to electrophoresis through an 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to PVDF membrane . The membrane containing transferred proteins was blocked with 5% skim milk in TBS at room temperature for 1 h. The membranes were incubated with suitable amount of purified HEV particles in 1% skim milk in TBS for 12 h at room temperature and washed three times with TBS buffer. Subsequently, the membranes were incubated with a specific rabbit anit-HEV-ORF2 antibody  at a dilution of 1:100 in 5% skim milk in TBS buffer. The viral binding band was visualized by incubation with a secondary horseradish peroxidase-conjugated sheep anti-rabbit IgG. Finally, the signal was developed using the ECL Advanced™ Western Blotting Detection kit  in accordance with the manufacturer's protocol.Mass spectrometry was undertaken commercially by the Research Centre for Proteome Analysis, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Band was subjected to tryptic digestion for 16 h followed by Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry and was performed with a Micromass Maldi Time of Flight (MALDI-TOF) Mass Spectrometer. Spectra were acquired in the mass range 400-2000 Da. Spectra were searched against Suina Protein Data Bank in NCBI.Proteins in band were electroeluted using D-Tube Dialyzers & D-Tube Electroelution Kit  in accordance with the manufacturer's protocol. The purified HEV supernatant was pre-incubated with eluted proteins, or purified swine fecal suspension which was collected from SPF pigs, and was then incubated with the human lung carcinoma A549 cell line according to the previous report . The cytTo confirm the existence of the HEV particles and antigens, the sucrose density gradient centrifugation purified sample were subjected to EM and western blotting observation. Results were presented in Figure To preliminarily identify the molecules on swine liver cells involved in binding HEVs, the virus overlay protein binding assay (VOPBA) methodology was utilized. Membrane proteins from swine liver cells were isolated and separated by SDS-PAGE on two parallel 8% gels. One gel was stained with Coomassie brilliant blue R-250 Figure , while tTo investigate the protein content of the virus binding band, the position equivalent to the major virus binding band was extracted from the duplicate gel and sent for commercial mass spectrometry fingerprint analysis. The basepeak and peptide mass fingerprinting of MALDI-TOF analysis of HEV binding band from VOPBA experiment were shown in Figure th day results were indicated in Figure To investigate whether the electroeluted protein from the virus binding band could inhibit the HEV infection of the susceptible cell line A549, an infection inhibiting assay was performed. Before infection, HEV suspension was pre-incubated with the electroeluted protein, and then the mixture was used to infect the A549 cell line. The cell shape was investigated every 24 hours and the 7There is gathering evidence that HEV is enzootic and pigs are considered one of the major reservoirs of human infection with this virus. The zoonotic transmission of HEV from pigs to humans has been suggested previously, particularly for cases in non-endemic areas. This hypothesis was mainly based on phylogenetic analysis, which showed that swine and human HEV strains from same geographic regions share a high genomic similarity ,7,20-22.Using a combination of VOPBA and MS analysis, we have identified a virus binding protein on the swine liver cell with nature molecular weight about 55kDa. The infection inhibition analysis indicated that this virus binding protein could prevent the HEV from infecting the A549 cell line Figure ,25, MoesIn the present study, we only used genotype 4 HEV for identifying the virus binding proteins in swine liver cells, because among the four genotypes of HEV, only genotype 3 and 4 belong to zoonotic pathogens. This study will provide some reference for the research of identifying the binding proteins of the other HEV genotypes. Although the aim of our study was identifying the binding proteins on the surface of swine cell so as to finding the candidate receptor of HEV, some of proteins in the aim band were not membrane proteins, which may be due to slight solubility of membrane proteins. Therefore, more effective method for membrane protein extraction should used during the future research for identifying the receptors of virus using VOPBA methods.Taken together, we have identified a virus binding protein on the swine liver cell with nature molecular weight about 55kDa by using a combination of VOPBA and MS analysis, and this virus binding protein could prevent the HEV from infecting the A549 cell line. The peptide fingerprint analysis and protein search showed that this virus binding band containing 31 different proteins. Although the accurate receptor of HEV has not been identified, the present study will lay a foundation for further research of the receptor of HEV.The authors declare that they have no competing interests.WZ, LC, and XH conceived the study. WZ, QS, SY, and HY performed all the experiments. WZ and LC wrote the paper. All authors read and approved the final manuscript.Protein component of the HEV binding band from VOPBA. The protein band was subjected to MALDI-TOF and the resulted mass spectra were searched against Suina Protein Data Bank in NCBI. The table listed the proteins in the binding band which was rather complicated and contained 31 different proteins.Click here for file


Clinician dependent costs are the costs of care that are under the discretion of the healthcare provider. These costs include the costs of drugs, tests and investigations, and discretionary outpatient visits and impatient stays. The purpose of this review was to summarize recent evidence, relevant to both developed and developing countries on whether evidence based clinical guidelines can change hospitals variable costs which are clinician dependent, and the degree of financial savings achieved at hospital level. Potential studies for inclusion were identified using structured searches of Econlit, J-Stor, and Pubmed databases. Two reviewers independently evaluated retrieved studies for inclusion. The methodological quality of the selected articles was assessed using the Oxford Centre for Evidence- Based Medicine (CEBM) levels of evidence. The results suggest that 10 of the 11 interventions were successful reducing financial costs. Most of the interventions, either in modeling studies or real interventions generate significant financial saving, although the former reported higher savings because the studies assumed 100 percent compliance. Globally, health care expenditures have increased tremendously in the last decade raising concerns over their sustainability-4and valto describe the costs of care that are under the discretion of the healthcare provider. These costs include the costs of drugs, tests and investigations, and discretionary outpatient visits and inpatient stays. Changes in CDCs may arise from interventions such as clinical guidelines that aim to alter health workers use of available resources. We are therefore interested in examining the evidence that supports or refutes the proposition that CDCs can be altered by interventions aimed at changing health worker practices.The cost consequence of such interventions has received less attention in the literature. The degree to which costs can be expected to change in response to clinical guideline adoption will depend on both the responsiveness of health worker behaviour, and on the share of patient costs that are attributable to decisions made by health workers. We have coined the term "clinician-dependent costs"This review aimed to address the question of whether and to what degree could interventions change costs that are clinician dependent. The particular focus was on studies reporting interventions designed to act through clinicians' behaviour change. We did not include studies describing the modeled difference in costs of care after assuming one drug or combination of drugs were substituted by an alternative drug or combination of drugs of equal or varying efficacy. Similarly we did not include studies that reported the cost-difference (or cost effectiveness difference) between alternative drug regimens whose efficacy was being compared in randomized controlled trials. Rather the focus was on interventions that aimed at influencing the clinician to follow a recommended process or procedure expected to influence CDCs. In this case the effect on costs was influenced by both the actual cost of the resources used and the degree to which the clinician opts to follow the advised procedures. Specifically this paper examines the degree of financial savings attributed to the interventions identified together with the resultant magnitude expressed as the proportionate reduction in total or consumable costs.Potential studies for inclusion were identified by direct searches of the online databases such as; PubMed, J-Stor and Econlit and the grey literature to ensure a comprehensive literature review. The grey literature was sought at several libraries including those of the Kenya Medical Research Institute, Kenya Institute for Public Policy Research and Analysis, Institute of Policy Analysis and Research and the African Population and Health Research Center, Searches were also conducted through the websites of: The World bank, Department for International Development, United States Agency for International Development, Policy Project- Kenya, Health Policy Initiative and German Agency for Technical Co-operation (GTZ). Literature retrieved for the period 1995 to 2010 using the following combination of search terms:■ Clinical guidelines and cost savings■ Clinical prescribing behaviour and costs■ Costs, decision making and prescribing variation■ Prescription behaviour and costs■ Prescribing behaviour and financial/costs savingsThe titles and abstracts of the retrieved articles were read by two independent reviewers and those that met the pre-inclusion criteria were selected  clinicalFor example, the IMCI intervention study in Tanzania involved an eleven day training period of which 30% of the time was spent on clinical practice with one follow up visit by an IMCI supervisor that occurred one month after training. This intervention changed prescribing behaviour which resulted in lower costs per child for the total resources used in treatment but increased drug costs. In anotThe studies based on modeling behavior change covered topics including: adherence to a practice guideline around malaria microscopy to overcome malaria over diagnosis and apprThe success of any intervention aimed at altering clinician prescribing depends on compliance which translates to the effectiveness in changing health worker behavior. In a systematic review on effectiveness and efficiency of guideline dissemination and implementation strategies, clinical guidelines were found to result in observed improvements in care and absolute improvements in performance. HoweverThis review further evaluates the success of interventions that are aimed at reducing clinician dependent costs, and thus generate some financial savings to the health system. On this note, interventions as opposed to modeling studies will be considered because they did not assume a high degree of compliance and they were more pragmatic. However, 10 out of the 11 interventions included in this review brought out the desired effect . However, several issues stood out with respect to changing clinician dependent costs.There is a possibility that studies are only conducted and reported in areas where they already feel that costs are unreasonably high,20,24,25Modeling studies assume a 100 percent success rate whereas the intervention studies have a success rate of less than 100 percent. This gives intervention studies greater appeal for use in policy making because they use real life scenarios. Furthermore, cost effectiveness studies of various interventions would be desired provided quality of care is not disregarded. In this review, one real intervention study evaluating the multidisciplinary clinical pathway for oxygen management led to an increase in health care costs, but improved the quality of care significantly. BesidesMinimization of costs through use of cheap or poor quality resources may compromise health care quality. An optimal balance is required based on cost effectiveness of the particular intervention. It is necessary to compare the costs of an intervention with the effectiveness in order to justify resource use.This review is aimed at establishing the degree of financial saving on clinician dependent costs brought about by clinical guidelines. However, there was relatively little specific literature on the cost consequences of approaches to improving clinical practice despite the broad literature exploring means to change clinician behavior and improve practice. The methodological quality of the selected articles was assessed using the Oxford Centre for Evidence- Based Medicine (CEBM) levels of evidence and in general the quality of studies  Table S3 Summary of studies included in the systematic review (Modeling studies).Click here for file