Venezuelan equine encephalomyelitis (VEE) epizoodemics were reported at 6–10-year intervals in northern South America beginning in the 1920s. In 1937, epizootic VEE virus was isolated from infected horse brain and shown as distinct from the North American equine encephalomyelitis viruses. Subsequently, epizootic and sylvatic strains were isolated in distinct ecosystems; isolates were characterized serologically as epizootic subtype I, variants A/B and C; or sylvatic (enzootic) subtype I, variants D, E, and F, and subtypes II, III, and IV. In 1969, variant I-A/B virus was transported from a major outbreak in northern South America to the borders of El Salvador, Guatemala, and Honduras. This musical poem describes the history and ecology of VEE viruses and the epidemiology of an unprecedented 1969 movement of VEE viruses from South America to equids and humans in Central America from Costa Rica to Guatemala and Belize and in Mexico and the United States that continued until 1972. Refrain: Mosquito’s in hisC crab hole, bidin’ his time,Venezuela virus working up the line,D heard the news,Boys in BeltsvilleE got the crab hole blues.Horses in TexasDown in Maracay back in ’36,F found a virus doin’ tricks,Kubes and RiosHorses die, this one’s gotta be,GNew cause of ’cephalitis, V-E-E.Voice-over: Horses, mules, and donkeys are all susceptible.The years roll by, the virus makes a score,HIn Vene, Colombo, and Ecuador,Comes rain to the desert instead of dew,IAnd, VEE burns the coast of Peru.In Trinidad, Panama, they say “Hey, Hey,”We got this creature like every day,His swampy home, you can always tell,Culex (Mel).J”By finding some rats and the Voice-over: Mosquito, that is.Back at MARU, raggin’ the brains,We found that the virus had different strainsHorses convulse with the southern kind,K”Northern virus leaves them feelin’ fine.Refrain.’Cause virus hits man by aerosol,Could be used to cripple us all,In Maryland, they grew it in vats,LTested it out in monkeys and rats.The other Team outside the fence,Lookin’ for the answer that made some sense,Got a strain from an old donkey,MMade a vaccine, TC-83.The offensive team tried to make some noise,NA cloud for the Commies, but not our boys,Did not work, that is why,O said ‘declassify’.NixonVoice-over: The Pentagon papers, volume 25, page 324, 1970… the New York Times, Los Angeles Times, the Washington Post, and the Christian Science Monitor….Sleepy defense gets a Latin call,New outbreak and it ain’t small,Took the vaccine off the wall,PIt really worked to save them all.RefrainDown in Guatemala, year of ’69,Horses started dying on the borderline,Virus come down from Ecuador,QNobody knows the reservoir.Voice-over: But MackenzieR has an idea.SVirus hopped the mountains, got to the sea,Vaccine brought in and given out free,Virus kept movin’, had to go,TAll the way from here to Mexico.Cause everybody said, “Gotta stop that bug,Gotta use the vaccine because there ain’t no drug,”Beltsville brethren said “Gotta wait,”UAfraid that the vaccine was MF-8.Voice-over: That means reversion to virulence.Results today are plain to see,VStory’s hot on the wire of Associated P,A million horses all over the West,WAre living proof the vaccine was best.RefrainVoice-over: Addresses for reprints may be mailed to Karl M. Johnson.A) The presentation is a chronologically, scientifically, and factually correct poetic and musical history of Venezuelan equine encephalomyelitis (VEE) viruses and epidemics through 1971. The music is a traditional US jug band song, a type of music—bluegrass—popular in rural areas of the eastern United States. Using a background of a guitar and everyday musical, rhythm, and percussion devices, such as whistling, blowing air across the mouth of a 1-gallon glass jug, scraping of a scrub brush on a metal washboard, humming harmonically into a kazoo, and the “hambone” , the voices and lyrics convey a musical story to the audience.Deinocerites pseudes, the crab hole mosquito, found along the Pacific coast of Central America was a competent vector of the epidemic and epizootic VEE virus that was causing disease and death in equids and humans at the time  Douglas F. Antczak, vocals, guitar, kazoo; William H. Dietz, vocals, jug, kazoo; Karl M. Johnson, vocals, kazoo; David H. Martin, kazoo, recording engineer; and Thomas E. Walton, washboard and scrub brush, hambone, kazoo, vocals.C) Gender-specific error. Only female mosquitoes  take a blood meal, which is needed to provide protein necessary for ovulation; only female mosquitoes are infected with and transmit VEE viruses (and other mosquito-borne arboviruses). Male mosquitoes feed on plant source liquids and water.D) Beltsville, Maryland, USA, the headquarters at that time of the regulatory officials in the US Department of Agriculture (USDA) responsible for the decision to recommend application of preventive vaccines to the Secretary of Agriculture in the face of an epizootic. Hesitation to vaccinate was predicated on lack of evidence that reversion of virulence of the attenuated vaccine virus could not occur, international trade considerations, international practices and agreements, and authorities and responsibilities delegated only to the Secretary of Agriculture.E) From 1969, the epizoodemic moved southeast, eventually reaching northwestern Costa Rica in August 1970, and northward, eventually reaching Texas in late June 1971  The Venezuelan agricultural and veterinary research laboratories are located in Maracay, Aragua State, Venezuela. Kubes and Rios first isolated, identified, and named VEE virus, then sent the isolate to the United States for confirmation that the South American virus was antigenically distinct from the North American eastern and western equine encephalomyelitis viruses  The antigenically related and clinically similar eastern and western equine encephalomyelitis viruses had been isolated and identified early in the 1930s in the United States  Periodic outbreaks of VEE had occurred in Colombia, Ecuador, Peru, and Venezuela since at least the 1920s, with hundreds of thousands of equine illnesses and tens of thousands of deaths; equids are the primary virus amplifier hosts for human infections (I) Throughout the history of VEE in northern South America, periodic epizoodemics in tropical dry and tropical thorn forests were often associated with unseasonably heavy rainfall and flooding during the normal dry seasons; during interepizootic periods, epizoodemic virus could not be isolated. The great Atacama Desert stretches along the Pacific coast from northern Chile and along coastal Peru nearly to the border with Ecuador; rare, but occasional, rainfall interrupts the barrenness of this parched, hostile environment permitting infrequent but noteworthy incursions of mosquitoes and epizoodemic VEE virus , the vectors of sylvatic subtypes and variants of VEE virus. The epidemiologic cycle involves sylvatic virus transmission by species of terrestrial rodents and possibly birds and arboreal rodents (J) In contrast, in swampy or jungle areas  where a definable dry season does not occur normally in countries of Central America and eastern South America and in Panama, Mexico, the Florida Everglades, and several Caribbean islands, field studies by scientists at MARU (K) Sylvatic virus subtypes were of low or no virulence to experimentally infected equids. The virulence of epizootic subtypes was high, with fatality rates to >90% of infected equids L) Historically, VEE virus has been a pathogen studied for aerosol release as a potential biologic weapon. In the United States at the Army Research and Development Command, , Fort Detrick, MD, the former Soviet Union and perhaps, elsewhere, VEE virus was studied as a possible offensive weapon.  In the defensive research programs at USAMRIID, an equine-virulent isolate from a donkey was serially passed in fetal guinea pig heart cell cultures to produce an attenuated vaccine, strain TC-83, for use in at-risk laboratory and military personnel (N) “Commies” [communists] reflected Cold War–era concerns about military personnel of the former Union of Soviet Socialist Republics and their allies. Aerosol releases of biological, chemical, and radioactive weapons are notoriously difficult to control, leading to use of vaccine, if available, and other, more cumbersome measures to protect military personnel.O) Recognizing the difficulties in controlling and using biological weapons, unauthorized release of classified documents, and the moral outrage of US citizens and world public opinion against biological weapons, President Richard M. Nixon cancelled the offensive biological weapons development programs.P) Beginning in 1967, a major epizoodemic of VEE occurred across northern South America (Q) In an unprecedented biomedical event in the history of VEE, the epizoodemic VEE virus subtype was transported from northern South America to Central America  An internationally recognized clinical and field research expert on zoonotic diseases, Ronald B. Mackenzie was a visionary and astute medical scientist with The Rockefeller Foundation in Cali, Colombia.S) VEE virus was transported by mosquitoes and possibly through transportation of asymptomatic but infected horses from the disease or danger zones to unaffected zones where susceptible equids were subsequently infected. Disease was documented along the Pacific and Caribbean coasts in the tropical dry and thorn forest environments that have been the traditional cattle-raising areas of Central and South America and in Mexico, where tropical wet forest and swampy environments that support sylvatic VEE viruses are irregularly located and noncontiguous or do not occur.T) Because of the lack of understanding of the epidemiologic cycle and virus-vector incubation requirements, the epizoodemic wave of infected mosquitoes and equids incubating the virus had not been anticipated to precede vaccination teams routinely into new areas of susceptible equids by 2–3 weeks. The disease moved to the southeast along the Pacific coast to northwestern Costa Rica, where the advance finally was stopped, protecting Panama, probably because of a combination of vaccination, presence of larger sylvatic foci in which larger numbers of resident equids were already immune, and a belt of lowland and montane rain forests where there are fewer cattle and horses and that stretches along the Pacific coast of southwestern Costa Rica to northwestern Panama; antibody to sylvatic virus strains provides cross-protection against epizoodemic virus strains. The virus crossed the Isthmus of Tehuantepec in Mexico and moved up both the Caribbean and Pacific coasts, finally reaching Texas in late June 1971 (detection of VEE in Texas before July 4 was predicted months earlier by K.M. Johnson). Despite thorough vaccination and aerial application of insecticides, some disease activity persisted in Mexico until 1972. The last activity from the epizootic in the Western Hemisphere occurred in 1973.U) MF-8 is an isolate of the epizoodemic VEE virus subtype I-A/B from Honduras isolated by Miguel Figueroa, a Honduran scientist working at MARU. Despite proven efficacy and safety of the attenuated VEE virus vaccine in South and Central America and Mexico, USDA authorities delayed application of the vaccine until the first cases were diagnosed in Texas. Official USDA emergency response policies and regulations at that time did not include the option to use vaccines to interdict threats of foreign animal diseases in the absence of documented disease within the United States . Because of bilateral and international agreements and policies regarding emergency disease responses, the adverse impact of applying foreign animal disease vaccines on the exportation of US livestock and agricultural products internationally and the politically charged decision to change existing policy, an authority delegated only to the US Secretary of Agriculture, vaccine was acquired and stockpiled along the Mexico–US border but could not be applied until VEE was diagnosed.V) Associated P = the Associated Press news agency.,W) Attenuated VEE virus vaccine was safe, effective, and stable, and reversion to virulence did not occur  Detrick, Maryland; the Special Procedures program from which the Special Immunizations Program evolved was one of the earliest operations to open. The Special Immunizations Program is responsible for the investigational vaccines, including strain TC-83, which were developed and are used under the Investigational New Drug authority and guidelines of the Food and Drug Administration. Seed stock of strain TC-83 virus was made available to the biologics industries of Western Hemisphere countries. Strain TC-83 and other next-generation iterations of the original vaccine, including an inactivated strain TC-83 product (C-84), are used or available in many countries of the Western Hemisphere.


To the Editor: Rabies is caused by 12 recognized virus species within the Lyssavirus genus (family Rhabdoviridae) (Vulpes vulpes), followed by the raccoon dog (Nyctereutes procyonoides) in central and Baltic Europe . After the fox rabies case reported in FYROM in 2011, ≈0.3 km from Greece , and Bulgaria, where rabies is reported in wild and domestic animals  were rabies positive by fluorescent antibody test, rabies tissue culture infection test, and PCR. Positive samples were subjected to sequencing analysis of the N gene. The sequence analysis of the first 567 nt of the N gene of the 3 isolates  showed 99.8% nt identity with the N gene of a rabies virus (GenBank accession no. JQ973884) isolated from a red fox in FYROM in 2011. Nucleotide identity was 100% between the 6 isolates from Greece  and isolate JQ973884, as well as a Serbia strain (GenBank accession no. JF973785). This perfect nucleotide identity shown with the Serbia isolate collected >15 years ago suggests the persistence of some viral strains over time in the Balkans, in accordance with previous studies (Phylogenetic analysis of the partial sequences of the N gene of isolates from Greece compared with representative sequences from the Balkans  showed tThe perfect nucleotide identity shown between the 6 isolates from Greece and 2 strains from Serbia and FYROM demonstrates a close genetic relationship between them. This finding supports the hypothesis of movement of rabies-infected hosts in the western Balkan countries.Greece was rabies free for 25 years. In the 2012 outbreak, the rabies cycle appears to be sylvatic, whereas until 1987, dogs were the main reservoir of rabies. Measures including public awareness campaigns and intensified vaccination of stray animals and shepherd dogs, combined with control of stray dogs and cats, already have been implemented. In accordance with the current European Commission recommendations, Greece continued rabies surveillance and eradication program throughout the country in 2013, including the implementation of an oral rabies vaccination program for foxes in autumn 2013 (


The xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs) all rely on the XPR1 receptor for entry, but these viruses vary in tropism, distribution among wild and laboratory mice, pathogenicity, strategies used for transmission, and sensitivity to host restriction factors. Most, but not all, isolates have typical xenotropic or polytropic host range, and these two MLV tropism types have now been detected in humans as viral sequences or as infectious virus, termed XMRV, or xenotropic murine leukemia virus-related virus. The mouse xenotropic MLVs (X-MLVs) were originally defined by their inability to infect cells of their natural mouse hosts. It is now clear, however, that X-MLVs actually have the broadest host range of the MLVs. Nearly all nonrodent mammals are susceptible to X-MLVs, and all species of wild mice and several common strains of laboratory mice are X-MLV susceptible. The polytropic MLVs, named for their apparent broad host range, show a more limited host range than the X-MLVs in that they fail to infect cells of many mouse species as well as many nonrodent mammals. The co-evolution of these viruses with their receptor and other host factors that affect their replication has produced a heterogeneous group of viruses capable of inducing various diseases, as well as endogenized viral genomes, some of which have been domesticated by their hosts to serve in antiviral defense. Gammaretroviruses of three distinct host range tropisms have been isolated from the laboratory mouse Table . The firenv and in the host receptor gene. These genes evolved in concert, altering virus-receptor interactions and the biological properties of these viruses, and producing an unusually heterogenous set of retrovirus and receptor variants.The host range of these 3 MLV subtypes maps to the receptor binding domains (RBDs) of their envelope (Env) glycoproteins, and their RBDs govern the ability of these viruses to interact with their cognate receptors -11. WhilMus genome, and their interactions with and co-option as host restriction factors. This review will describe the evolutionary history of these viruses with special emphasis on tropism changes, the involvement of these viruses in disease induction in mice, and host factors that affect their replication and their recent transspecies transmission to humans.Analysis of X/P-MLVs in laboratory and wild mice has detailed their roles in pathogenesis, their acquisition as endogenous elements in the Mus genome is comprised of retroelements, and one-third of these are endogenous retroviruses (ERVs) [Approximately 37% of the s (ERVs) ,17. ERVsgag), enzymes  and envelope (env) that are flanked by long terminal repeat sequences (LTRs) that regulate transcription. The gammaretroviruses lack the accessory proteins of immunodeficiency viruses like HIV-1, have only one zinc-finger in nucleocapsid and rely on a translational strategy that reads through the gag termination codon. Many gammaretroviruses also uniquely produce a second, larger and glycosylated form of the Gag precursor polyprotein that uses an alternate, upstream initiation codon [The MLVs and their endogenous ERV counterparts have the simplest of retrovirus genomes . The MLVon codon -21.All three host-range MLV variants are present as ERVs in laboratory mice, many of which are full-length, with apparently nondefective protein-coding regions. Infectious viruses of all three host range classes can be isolated from mice, but not all ERVs produce virus, and those that do differ significantly in the timing and circumstances of their expression. Chromosomal locations for many of these ERVs in common inbred mouse strains were determined by conventional genetic methods -24 and tEmvs can produce infectious virus. Up to 6 Emvs are found in the inbred strains  and modified polytropic (Mpmvs or mPTs), and there is a smaller subgroup named intermediate polytropic MLVs (iPT), identified in NFS/N mice [M/Pmvs to refer to this subgroup of MLV ERVs collectively or when subtype is not known. Although the coding regions of many M/Pmvs have open reading frames [There are up to 40 copies of P-MLV ERVs in the laboratory mouse genome ,33. The S/N mice ,35. I wig frames , none arg frames  or to LTg frames .M/Pmvs to produce infectious virions, cell-to-cell transmission of this subgroup can be detected, and infectious P-MLVs can be produced in the presence of E-MLV infection. Thus, replicating E-MLVs can recombine with M/Pmv ERVs in mice to produce recombinant viruses with M/Pmv env sequences [M/Pmvs can also be packaged as homodimers into virions of exogenous ecotropic virus, and these "mobilized" M/Pmvs can infect cells, replicate in those new cells, and spread to other cells as pseudotyped virus [Despite the apparent inability of equences ,37-40; tequences ,42. In ted virus . Anothered virus ,45.Xmvs are present in 1-20 copies per mouse strain [Xmvs in the sequenced C57BL genome are highly polymorphic, and phylogenetic analysis suggests that these Xmvs fall into 3 clades which may have resulted from 3 separate infections [Xmvs produce high levels of virus and other Xmvs can be induced to produce virus, but most Xmvs are not readily capable of producing infectious virus  locus sensitive to chemical induction or stimulation by LPS [Bxv1 provirus has been identified [Bxv1 is low except in the F/St strain, where the high level of virus expression is linked to the major histocompatibility locus [Xmvs found in laboratory mice have not been characterized. The high virus NZB mouse carries two loci neither of which maps to Chr 1 [Nzv1 produces low levels of X-MLV, but Nzv2 is constitutively active [Xmv was identified in MA/My mice, a strain that also carries Bxv1 [Xmvs but are rarely or not capable of producing infectious X-MLV [There are four active proviruses capable of producing virus in laboratory mice Table . One of n by LPS , and is n by LPS . The Bxventified ,53, and entified . Expressty locus . The 3 ato Chr 1 -57. Nzv1y active . The fouus X-MLV ,33,53  on the Indian subcontinent, and the 4 Mus subgenera diverged from one another shortly after Mus diverged from other Murinae [Mus species, there are 3 commensal species, or house mice, that evolved 0.5-1.0 MYA, and a fourth house mouse population in Japan, M. molossinus, which represents a natural hybrid of M. castaneus and M. musculus [The presence of still active MLV ERVs in mice and the positional polymorphism of these loci among inbred mouse strains indicate that all 3 ERV types entered the  Murinae ,64  have ecotropic host range [env RBD [Not all laboratory mouse P-MLVs have polytropic host range. Some of these recombinant viruses , is fully permissive for all X/P-MLVs. The other 4 variants restrict infection by two or more members of this virus family. All variants except the XPR1 of NIH 3T3 cells support entry by X-MLVs, although with differences in efficiency. Only 2 of the 5 receptor variants are permissive for P-MLVs. The laboratory mouse allele, nXpr1, allows entry only by P-MLVs.variants ,96,101. Xpr1, ECL3 and ECL4  of 4 Figure  [78,79,1eceptors , althougeceptors   to the inbred mouse genome [M. domesticus would likely carry n Xpr1also makes biological sense, as these mice carry endogenous Pmvs but not Xmvs consistent with n Xpr1receptor function [M. domesticus mice trapped at various sites throughout its western European range and in the Americas all carry sxv Xpr1, and whng genes  further glycogag leader region, LTR and env. XMRV carries unusual deletions in glycogag, a region that in E-MLV influences virus release and sensitivity to interferon [Xpr1 ECL4 [The transmission of XMRV to humans was likely accompanied by adaptive changes, and the observed sequence and phenotypic differences of XMRV relative to the X/P-MLVs have focused particular attention on the terferon  and alsoterferon . XMRV diterferon -129. Finterferon . Also, wpr1 ECL4 . These mThe detection of XMRV and P-MLVs in various human patient groups and in blood donors raises questions about the pathogenic and mutagenic potential of these viruses in humans and concerns about the safety of the blood supply. While the involvement of these viruses in human disease is still under investigation, the MLVs were recognized as disease-inducing agents in mice almost 60 years ago . AlthougEmvs, like AKR, HRS, and C58, have a high incidence of spontaneous lymphomas, and mice inoculated with specific MLVs can develop diseases such as lymphocytic leukemia, erythroleukemia, immunodeficiencies, and neurological diseases. The naturally occurring and induced neoplastic diseases are generally induced, following a long latency period, by insertional mutagenesis. In this process, novel virus integrations activate genes involved in growth regulation or inactivate tumor suppressor genes [Mouse strains carrying active or genes ,131. Theor genes . While nde novo generation of infectious and pathogenic P-MLVs. The disease process generally begins with the establishment of chronic infection with E-MLVs. These viruses can recombine with M/Pmvs and Xmvs to generate recombinant infectious virus with P-MLV host range and increased virulence [Rmcf resistance gene that inhibits replication of P-MLV [In mice, MLV-induced neoplastic disease is often associated with the irulence ,134. Theirulence . Althougirulence . Also, iirulence , and disof P-MLV  or in miof P-MLV .env and LTR. The LTR sequences are contributed by the active Xmv, Bxv1 [Bxv1 proviral sequence [env segment in MCF MLVs can vary due to the sequence of the participating M/Pmv [env tend to cluster in 2 segments of the 3' half of SUenv or in the 5' end of TMenv [The recombinations that generate infectious pathogenic P-MLVs involve at least two segments of the viral genome, mv, Bxv1 ,139, andsequence . The recng M/Pmv  as well of TMenv ,85,86.env genes in the disease process is incompletely defined, but these substitutions can contribute to the target cell specificity and disease type induced by MCF MLVs. The most well-studied example of disease mediated by viral Env is the rapid erythroleukemia induced by Friend SFFV, a replication-defective MCF-type recombinant. SFFV encodes a unique 52/55 kDa Env-related protein that functions as an oncogene and induces disease by activating signal transduction pathways associated with the erythropoetin receptor and the receptor tyrosine kinase Stk [in vivo progression of tumors by hampering the immune response. In some cases, Env substitutions may facilitate virus evasion of the immune system [env genes expressed in tumors may contribute to a T-cell mediated subversion of immune surveillance that allows for tumor cell proliferation [The role of the recombinant nase Stk -142. Fore system , or the feration ,145.Preleukemic thymuses can contain large amounts of unintegrated MCF MLV DNA resulting from failure to establish superinfection interference ,146. SucThe acquisition of MLV ERVs, the time course and tissue specificity of their expression, and the transmission of these viruses to new hosts are governed by host factors that restrict or enhance virus replication and spread. These host factors include the innate and acquired immune systems, as well as numerous constitutively expressed antiviral factors that inhibit virus replication, many of which were initially identified in studies on the mouse gammaretroviruses. These factors can block or interfere with different stages in the viral life cycle, such as virus entry, uncoating and reverse transcription, integration, assembly and release. For this review, I will focus on the host factors that either specifically target the X/P-MLVs and XMRV, or factors that have been shown to have significant restrictive effects on these viruses Table . Among tMus restrict two or more viruses in the X/P-MLV family. These restrictions result from deletion mutations or replacements that have been shown to display a pattern of positive selection suggesting an evolutionary history of genetic conflicts [Xpr1 variants are found in non-Mus mammals [Receptor polymorphisms can clearly provide an especially effective antiviral defense. As already noted, 4 of the 5 XPR1 receptor variants in onflicts . Further mammals  , that inactivates X-MLVs . This faFv4, which blocks E-MLVs [Rmcf and Rmcf2 which restrict X/P-MLVs and, in the case of Rmcf, inhibit MCF MLV-induced disease [env genes: Fv4 and Rmcf have major deletions [Rmcf2 has a stop codon that prematurely terminates integrase [Fv4, Rcmf, and Rcmf2 reduce or downregulate activity of their cognate receptors, and Fv4 additionally has a defect in the fusion peptide of the TMenv, so incorporation of this Env into virions in virus infected cells results in their reduced infectivity [The mouse genome contains several resistance genes associated with production of MLV Env glycoproteins that are thought to restrict virus through receptor interference. These genes include s E-MLVs , and the disease ,162-164.eletions ,165, andntegrase . Fv4, Rcectivity .M. castaneus. Three breeding lines of this species show similar restriction of P-MLV infection. Two of these lines, CAST/Rp and CAST/EiJ, carry Rmcf2. Backcross mice of the third line, CAST/Ncr, show evidence of two unlinked dominant resistance genes , and neither of these genes maps to Chr 5 (Rmcf) or Chr 18 (Rmcf2) (unpublished data). These loci, together with Fv4 and Rmcf2, may therefore be representative of a larger set of interference genes found in virus infected mouse species. That such co-opted Env genes are effective as host antiviral factors is confirmed by the identification of comparable interfering env genes in chickens, sheep and cats [There is evidence of additional receptor blocking genes in and cats -169.Fv1 is the oldest known retrovirus resistance gene [gag gene of MuERV-L, a Class III ERV that is active in mice, but has no infectious virus counterparts [Fv1 sequence is found only in mice, and was acquired shortly after the origin of the Mus genus [Fv1 has three well-characterized restriction alleles, and there are additional Fv1-like restrictions found in inbred strains and wild mouse species [nFv1, bFv1, and nr Fv1produce characteristic patterns of resistance to N-, B-, and NR-tropic MLVs. Cells with the Fv1 null allele are nonrestrictive [Fv1. Fv1 targets the virus capsid; the major determinant that distinguishes N- and B-tropic viruses is at CA position 110, but other target residues in this CA region have also been identified [nce gene  and reprterparts ,172. Theus genus . The lab species -176. Thetrictive ,175, andentified .Fv1 restriction [n or Fv1bFv1. Infection of X-MLV susceptible inbred strains suggests that XMRV is somewhat more sensitive to nFv1, and also indicates that various mouse X-MLVs can be restricted by Fv1 [n Fv1restricted residue at capsid target site 110, but its sensitivity to both n Fv1and b Fv1and the presence of additional substitutions in its CA gene suggest that the Fv1 target is more complex than previously appreciated.Because X-MLVs fail to infect cells of many laboratory mice, early studies used pseudotypes and recombinant viruses to suggest that X-MLVs are subject to triction ,182. Grotriction  have mord by Fv1 . XMRV caFv1, is responsible for post-entry restriction of retroviral infection [Rfv3 [Pmvs but not Xmvs in the sequenced mouse genome show mutational patterns consistent with mAPOBEC3 silencing at the time of integration [APOBEC3, like nfection ,185. Thenfection ,187. Thenfection -190. mAPon [Rfv3 ,192, a gon [Rfv3 . Pmvs buegration . The senegration ,194.Xmv LTR inserted into an intron [Xmv, acquired in virus-infected mice [Fv1 and Rmcf.The 2 laboratory mouse alleles of mAPOBEC3 vary in their restriction of Friend E-MLV replication and virus-induced disease, and differ in protein sequence, splicing pattern and expression levels ,192,195.n intron . This inted mice , may thuTetherin, also termed BST2, CD317 or HM1.24, is an interferon-inducible host factor that blocks the release of enveloped viruses by tethering budding particles to the cell surface . TetheriMus species with permissive receptors, and subsequent co-evolutionary modifications produced, among other adaptive phenotypes, "xenotropic" MLVs. The receptor mutations responsible for resistance to these X-MLVs were only recently acquired, and these restrictive receptors are only found among the inbred strains descended from early 20th century fancy mouse colonies. It is now clear that the term "xenotropic" is somewhat of a misnomer for mouse viruses that actually infect cells of all Mus species and many common strains of laboratory mice, and that infect more non-Mus mammals than the so-called broad host range polytropic MLVs.Multiple examples of xenotropism exist among the retroviruses. In addition to X-MLV, viruses that fail to infect cells of their apparent "home" species include the cat RD114-related viruses ,204, GALXpr1 and Fv1. The battleground at the cell surface has produced at least 6 host range X/P-MLV variants that interact with different but overlapping sets of determinants on the XPR1 receptor. At the same time, other factors likely contribute to diversifying selection on the X/P-MLV viral Env glycoprotein, such as the Rmcf-type interfering ERVs and the inactivating LVIF serum factor. The resulting Envs not only vary in tropism, but these viruses have also evolved alternative mechanisms of transmission. Although germline PMV ERVs are effectively silenced, and the Env glycoproteins of infectious recombinant P-MLVs have narrow receptor requirements and reduced receptor binding efficiency, these multiple mechanisms ensure their transmission and also contribute to pathogenicity.The interacting interfaces of host and pathogen are co-evolutionary battlegrounds, and the effects of the ratchet-like mutational process affecting these entities are particularly obvious for restriction factors like Fv1, [Rmcf-like interference genes, and regulatory elements like the mAPOBEC associated Xmv LTR.MLV ERVs, as part of the host genome, are also shaped by evolutionary processes. ERV insertions introduce novel regulatory and protein coding sequences into the host genome. While most are silenced, some are co-opted for cellular functions, and the most easily recognized of these domesticated ERVs are those that are linked to antiviral functions. Such ERVs include the oldest recognized restriction factor, Fv1,  as well Mus species, like M. pahari, that are permissive for X-MLV and XMRV infection [The worldwide distribution of mice that carry MLVs and the broad host range of the X-MLVs suggest that we are only beginning to describe what may be common and widespread interspecies transmissions. The phenotypic diversity among the MLVs doubtless influences the likelihood of their transspecies transmission, and the invading viruses are then subject to additional co-evolutionary pressures. We do not know the evolutionary path taken by XMRV to humans, but multiple sequence and functional variations distinguish this virus from its MLV progenitors. The consequences of this transspecies MLV invasion are unclear, although XMRV pathophysiology is now being evaluated in other species, including primates  as well nfection . The facThe author declares that she has no competing interests.


Alfalfa mosaic virus (AlMV) RNAs 1 and 2 with deletions in their 3′ non‑translated regions (NTRs) have been previously shown to be encapsidated into virions by coat protein (CP) expressed from RNA3, indicating that the 3′ NTRs of RNAs 1 and 2 are not required for virion assembly. Here, we constructed various mutants by deleting sequences within the 3′ NTR of AlMV subgenomic (sg) RNA4 (same as of RNA3) and examined the effect of these deletions on replication and translation of chimeric Tobacco mosaic virus (TMV) expressing AlMV sgRNA4 from the TMV CP sg promoter (Av/A4) in tobacco protoplasts and Nicotiana benthamiana plants. While the Av/A4 mutants were as competent as the wild-type Av/A4 in RNA replication in protoplasts, their encapsidation, long-distance movement and virus accumulation varied significantly in N. benthamiana. These data suggest that the 3′ NTR of AlMV sgRNA4 contains potential elements necessary for virus encapsidation. Tobacco mosaic virus (TMV) has been studied most extensively and shown to be located within the movement protein (MP)-coding sequence [Brome mosaic virus [Citrus tristeza virus [Turnip crinkle virus [Peanut clump virus [Encapsidation of the viral genome is an essential step in the life cycle of many plant viruses because efficient virus infection and spreading require production of virions. Packaging of the viral genome occurs following specific interactions between viral RNA and coat protein (CP). However, RNA elements involved in encapsidation and thus representing the origin of assembly (OAS) have been investigated for only a few viruses. The OAS of sequence ,2. The Oic virus ,4, Citruza virus , Turnip le virus  and Peanmp virus . Alfalfa mosaic virus (AlMV), a member of the Bromoviridae family, has been elusive. Assembled AlMV forms four major bacilliform-shaped particles of 56 nm, 43 nm, 35 nm and 30 nm, depending on the size of the encapsidated RNA [Until now, identification of the OAS in the tripartite ated RNA . Each RNated RNA ,10. RNA3ated RNA , and CP ated RNA . CP is aated RNA , regulatated RNA , virus aated RNA , RNA staated RNA ,17, and ated RNA . Previouated RNA  have shoNicotiana benthamiana, Nicotiana tabacum and Spinacia oleracea, and the infection required formation of virions [Alternatively, viral chimeras containing heterologous CP can be used for identifying the RNA elements necessary for encapsidation. For this purpose, in the TMV-AlMV chimeric virus, Av/A4  Figure , the TMV virions . These rIn this study, we have generated mutants  of Av/A4 containing various deletions within the 3′ NTR of AlMV sgRNA4 to examine their effects on chimeric virus replication, encapsidation, movement and accumulation and to identify the RNA elements required for virus encapsidation.To investigate the effect of deletion of the AlMV sgRNA4 3′ NTR on replication of chimeric TMV (Av/A4), we generated Av/A4 mutants by deleting various length of sequences within CP binding site 2 (CPB2) and its downstream sequences, except from CPB1 which is needed for efficient RNA translation . The mutin vitro generated transcripts of each construct, and total RNAs were assessed by the Northern blot analysis at 24 h post inoculation. The results indicated that all the constructs produced genomic RNAs and CP sg mRNAs at comparable levels  had no impact on CP accumulation, whereas inclusion of an extended (p3) and a further extended (p8) sequence downstream of CPB2 had a strong positive effect on CP accumulation , whereas plants inoculated with p3 or p8, as well as with intact Av/A4, demonstrated visible curling and yellowing symptoms in upper non-inoculated leaves within 7 to 10 dpi. Moreover, these plants developed significant stunting. Although the deletions within the AlMV sgRNA4 3′ NTR had no effect on chimeric virus replication in inoculated tobacco protoplasts, it was unknown whether the symptom expression in plants inoculated with these mutants would be similar. To address this question, we inoculated To examine whether symptomatic or asymptomatic plants contained AlMV CP, total proteins were extracted from upper systemic leaves of the infected plants at 7 and 20 dpi and analyzed by Western blotting c. The reN. tabacum cv. Xanthi NN plants, and counting the number of lesions on each inoculated leaf at 2 to 3 dpi. We have found that the mutants p3 and p8 passaged infectivity at a high efficiency , whereas p0 and p1 did it at a very low efficiency (less than 20%). This marked reduction in the efficiency of passaging with p0 and p1 suggests low efficiency in encapsidation for these chimeric constructs compared to p3, p8 and Av/A4.Reduced encapsidation of p0 and p1 was further confirmed by passaging infectivity from the p0- and p1-infected plant sap to the local lesion host, Taken together, our findings indicate that deletions of the extended part of the CPB2 region . The wild-type AlMV virus preparation typically contains bacilliform-shaped particles of four major sizes—56, 43, 35 or 30 nm long and 18 nm wide , whereasTaken together, our purification results and TEM observations indicated that the longer the sequence deleted from the 3′ NTR of AlMV sgRNA4, the lower the yield of the mutant virus particles and the shorter their length.N. benthamiana plants inoculated with the deletion mutants of Av/A4 chimeric TMV virus, have demonstrated that the 3′ NTR of AlMV sgRNA4 is not essential for replication of Av/A4 but is required for efficient RNA encapsidation, long-distance movement and virus accumulation. Our long-term goal is to identify the AlMV OAS which appears to be within the 3′ NTR of sgRNA4 based on the current findings. However, additional research with more compelling evidence is needed to demonstrate this. In this study, we used the Av/A4 chimeric virus as a model, because it allows for studying functions of CP and 3′ NTR sequences of AlMV sgRNA4 independently. This has bypassed the need for creating viable CP mutants using AlMV RNAs 1, 2 and 3. Since the OAS of many viruses typically consists of stem-loop structures [The results of the presented study, using tobacco protoplasts and ructures ,23 and tBecause the sequences required for Av/A4 encapsidation were found to localize within the AlMV 3′ NTR, we hypothesized that Av/A4 would form at least three types of major-size virus particles. These particles were expected to encapsidate genomic RNA , MP sgRNA  or CP sgRNA . The TEM analysis of purified Av/A4 demonstrated the presence of bacilliform-shaped particles with rounded ends typical for AlMV, rather than the rigid rods typical for TMV. Moreover, the Av/A4 virus preparation was more heterogeneous than expected, showing at least six types of particles of different sizes . When siBrome mosaic virus CP replaced native CP revealed that in the hybrid virus only three sgRNAs, but not genomic RNA, were encapsidated. CP or sequences and/or structural features within the chimeric viral genome were thought to play a role in promoting virion assembly [Deletion of certain elements of the 3′ NTR has been also found to have an effect on the efficiency of encapsidation and size of Av/A4 mutant s and their ability to infect plants. Although the shorter particles (16–48 nm) were observed even in the purified preparations of the mutants p0 and p1, these preparations may have infected plants very inefficiently, indicating poor encapsidation of these mutants. Although we did not examine the encapsidated RNA, it is also possible that p0 or p1 could form empty particles, and this may be the reason why we observed CP expression on a Western blot. Studies with a TMV-BMV hybrid [Escherichia coli DH5α cells  were used for transformation. Av and Av/A4 constructs . Protoplasts were prepared from a N. tabacum cv. Xanthi suspension cell line and inoculated using procedures described previously [Plasmids were linearized with KpnI before performing eviously .N. benthamiana plants were inoculated with in vitro transcripts of various constructs as described previously [eviously . Virus weviously . Brieflyeviously . Northern blot hybridization of total nucleic acid extracted from protoplasts or virion RNA was performed as described previously , using aTotal protein from protoplasts or plants was analyzed by SDS-PAGE followed by Western blotting using a monoclonal antibody against AlMV CP . The blots were then incubated with a secondary antibody labeled with horseradish peroxidase and incubated with the enhanced chemiluminescence reagent . Chemiluminescence was detected and immunoblot images were obtained using the Gene Gnome Imaging System supported by the Gene Tools software package . For the purified virus analysis, the gels were stained with Coomassie Brilliant blue and then scanned for documentation.N. benthamiana plants were mechanically inoculated with in vitro transcripts of each construct. At 10 dpi, tissue from inoculated leaves was homogenized in 50 mM phosphate buffer (pH 6.5) and incubated at room temperature. Aliquots were taken at time 0 and 20 min after grinding. The inoculum was mechanically applied onto two half-leaves of N. tabacum cv. Xanti NN . At 2 to 3 dpi the number of lesions was counted. The experiment was performed in triplicate and the averages were calculated.The TEM analysis of the purified virus preparation was performed using carbon-coated EM grids . Briefly, one drop of the virus preparation was placed on the grid for 1 min and then blotted with filter paper, leaving a small amount of liquid on the grid. A drop of uranyl acetate or 5% ammonium molybdate was placed on the grid, and after 30–45 s the grid was blotted with filter paper and allowed to dry. Samples were studied using the Zeiss CEM 902 TEM. Morphology characterization and size measurements of virus particles were performed using the ‘Analysis’ software from Soft Imaging Systems, Muenster, Germany. in trans for encapsidation of AlMV RNAs 1 and 2. The latter is supported by the results of earlier studies which indicated that the 3′ termini of RNAs 1 and 2 are not required for the wild-type AlMV assembly [cis-acting 3′ tail-like structure (TLS) of BMV RNA3 was obligatory for encapsidation of BMV RNA3 but not RNAs 1 and 2. Therefore, when this 3′ TLS from BMV RNA3 was provided in trans to RNAs 1 and 2 that lacked their 3′ TLSs, these RNAs were encapsidated [The results of our experiments with the Av/A4 deletion mutants suggest that the sequences within the 3′ NTR of AlMV sgRNA4 (and RNA3) are required for efficient encapsidation of the TMV/AlMV hybrid, Av/A4. Therefore, one may hypothesize that the sequences within 3′ NTR of AlMV sgRNA4 (and RNA3) play an important role in encapsidation of wild-type AlMV and also function assembly . The autassembly . Other ipsidated . In addi


Journal of Hospital Medicine 2013;8:519–522. © 2013 Society of Hospital MedicineHealthcare-associated infections are common, costly, and potentially deadly. However, effective prevention strategies are underutilized, particularly for catheter-associated urinary tract infection (CAUTI), one of the most common healthcare-associated infections. Further, since 2008, the Centers for Medicare and Medicaid Services no longer reimburses hospitals for the additional costs of caring for patients who develop CAUTI during hospitalization. Given the resulting payment pressures on hospitals stemming from this decision, it is important to factor in cost implications when attempting to encourage decision makers to support infection prevention measures. To this end, we present a simple tool (with easy-to-use online implementation) that hospitals can use to estimate hospital costs due to CAUTI, both before and after an intervention, to reduce inappropriate urinary catheterization. Using previously published cost and risk estimates, we show that an intervention yielding clinically feasible reductions in catheter use can lead to an estimated 50% reduction in CAUTI-related costs. Our tool is meant to complement the Society of Hospital Medicine's Choosing Wisely campaign, which highlights avoiding placement or continued use of nonindicated urinary catheters as a key area for improving decision making and quality of care while decreasing costs.  Healthcare-associated infections affect 5% to 10% of all hospitalized patients each year in the United States, account for nearly *45 billion in direct hospital costs, and cause nearly 100,000 deaths annually.4http://www.apic.org/Resources/Cost-calculators), they do not provide estimates of potential cost savings. Our approach adds to the literature by providing estimates of a hospital's current costs based on a few simple inputs , and also yields projected costs after a hypothetical intervention to prevent infections. Results are derived by combining appropriate cost and risk estimates from the literature. Importantly, an online implementation of our approach is available that can be easily used by clinicians, hospital administrators, and national policymakers. Our implementation nicely complements efforts like the Society of Hospital Medicine's Choosing Wisely campaign, which highlights avoiding inappropriate urinary catheter use first on its list of Five Things Physicians and Patients Should Question, and aims to increase awareness about issues that could improve patient outcomes and reduce healthcare costs.To help motivate hospitals to reduce inappropriate urinary catheter use, we present a tool for estimating costs of CAUTI for individual hospitals. Although other tools for estimating the excess costs of healthcare-associated infections are available , bloodstream infection (BSI), and catheter removal. Conservatively, assuming that bacteriuria must precede SUTI and BSI, there are 5 possible trajectories for any hospitalized patient : (1) no We start with 2 minor simplifying assumptions. First, because the presence of asymptomatic bacteriuria is typically unknown, we only consider costs to the hospital due to SUTI and BSI We use per-patient hospital costs of SUTI and BSI of $911 and $3824, respectively, which were determined using a microcosting approach 6 and adjusted for inflation using the general Consumer Price Index.Specifically, for SUTI:(Proportion SUTI but no BSI) = {(SUTI risk among those catheterized with bacteriuria) − (BSI risk among those catheterized with bacteriuria)} × (bacteriuria risk among those catheterized) × (proportion catheterized).And for BSI:(Proportion BSI) = (BSI risk among those catheterized with bacteriuria) × (bacteriuria risk among those catheterized) × (proportion catheterized).The risks of SUTI and BSI among those catheterized with bacteriuria, along with the risk of bacteriuria among those catheterized, have been estimated previously via a meta-analytic approach.The approach described above permits estimation of current costs for managing patients with CAUTI for a particular hospital. To estimate projected costs after participation in an intervention to reduce infection risk, we characterize interventions of interest and introduce additional factorization. Specifically, following previous work,Because our estimators of current costs, projected costs, and savings are all formulated as functions of other estimators, we use the standard delta method approach http://cauti.umms.med.umich.edu/PHP/CAUTI_input.php. Although the work presented in this article incorporates risk and cost estimates from the literature whenever possible, the online implementation allows full user specification of input values.Customized results  can be computed using online implementation of our proposed method at After an intervention resulting in 29% and 37% reductions in placement and duration, respectively, reflecting reductions seen in practice,We have presented a tool for estimating customized hospital costs of CAUTI, both before and after a hypothetical intervention to reduce risk of infection. Our approach relies on mostly conservative assumptions, incorporates published risk estimates (properly accounting for their associated variability), and has easy-to-use online implementation. We believe this can play an important role in motivating hospitals to reduce inappropriate urinary catheter use.The methodology employed here does have a few limitations. First and foremost, our results depend on the reliability of the input values, which are either provided by users or are based on estimates from the literature (see Despite these limitations, we believe that our tool can help infection control professionals demonstrate the values of CAUTI prevention efforts to key administrators, particularly at a time where it has become increasingly necessary to develop a business case to initiate new interventions or justify the continued support for ongoing programs.


To the Editor: Recently, Murphy and Osburn (To the long list of bovine viruses cited in this paper, it seems necessary to add another, the pseudocowpox virus, a widespread parapoxvirus that may infect humans. During the 1960s, this virus was identified in vaccine lymph from a heifer at the Institut Pasteur, Paris (In humans, this virus is responsible for limited skin lesions, more frequently in immunocompromised patients. Mainly farmers and butchers are affected. Pseudocowpox virus is easily differentiated from orthopoxviruses such as vaccinia virus by the virus's peculiar form on transmission electron microscopy scan, but polymerase chain reaction is probably the best detection method (


Persons with longstanding substance abuse might become increasingly dependent on help by the public, eventually requiring permanent care. In 2006 the municipality of Stavanger established a so-called addiction ward for these clients, comprising 17 beds at the largest municipal nursing home. We assumed that the residents of this ward were high consumers of health care and social services during the last months preceding their admission. The aim of the study was to register the type and extent of services that were claimed by this client group during the last six months prior to admission, and to calculate the costs that were caused. Further, we estimated the incremental costs for nursing home placement.In 15 residents from the addiction ward the use of all welfare services during the six months prior to admission were registered. Costs were calculated by unit costs from a municipal, national and societal perspective.Mean total costs during this period were €32 474. Approximately half of these costs were borne by state-funded institutions, and half were borne by the municipality. The clients used a great variety of services aimed at subsistence, health care and support in independent living, while services aimed at drug withdrawal were not claimed. There was no correlation between costs and the level of functioning. The incremental costs for nursing home admission were borne by the municipalities.Persons with longstanding substance abuse represent a group with a high use of welfare resources and hence cause high costs. However, our findings do not indicate any correlation between the amount of services rendered and the level of functioning. Further research should focus on the identification of the clients’ need for support in order to facilitate targeted interventions that might prevent further deterioration and, finally, the need for permanent care. As compared to other European countries Norway has a low consume of alcohol and drugs . Though Substance abuse is a challenge for every health care system because of the deteriorating effect of drug and alcohol not only on the health status ,5, but oIn the municipality of Stavanger with approximately 125 000 inhabitants this client group is gathered in a so-called addiction ward, where the use of legal substances is unprohibited, and where the treatment aim is not substance withdrawal but a strengthening of the residents’ remaining resources and a good quality of life. It was established in 2006 at the largest municipal nursing home next to six regular nursing home wards and comprises today 17 beds. During the last five years the addiction ward has become an established part of the health care sector in the municipality of Stavanger, as it provides a well-functioning care option for clients who formerly had been difficult to place [However, public health care resources are scarce, and there is an ongoing discussion of whom to prioritize. Placement in a permanent care institution is one of the most expensive measures within a health care system and should be an effective step, either by meeting needs that cannot be satisfied in other ways or by replacing measures that are equally expensive. Therefore, the need for care is thoroughly evaluated in every patient, and clients have normally received various offers before, including home care and municipal housing supported by social workers. However, health care and social welfare are complex systems, and the financial sources might be municipal, governmental or private funding. There is no overstretching registration of services available, services rendered or costs involved, and there is little knowledge about the resources demanded by persons with longstanding substance abuse.We assumed that the residents of the addiction ward were high consumers of health care and social services prior to their admission to the nursing home. The aim of the study was to register the type and extent of services that were claimed by this client group during the last six months prior to admission, and to calculate the costs that were caused. Further, we estimated the incremental costs for nursing home placement.In Norway pension payments and specialist health care, including rehabilitation for substance abuse, are government-funded, while the responsibility for primary health care services and social services lies within the municipalities. Home care and support by social workers are issued by the municipalities and free of charge for the clients. The municipalities are required by law to offer accommodation adjusted to the clients’ housing abilities. This comprises housing programs for substance abusers, adjusted housing for physically or cognitive impaired persons, and, as the highest level of care, nursing homes. All of these measures require out-of pocket contributions. However, when a client has no financial means, social welfare will take over the costs until the client, eventually, might be able to pay back. As a measure for the client’s functioning in activities of daily living (ADL) the “IPLOS bistandsvariabler” questionnaire  is used, containing 17 items that cover personal and instrumental ADL, hearing, and vision. Every item is scored from one to five, with higher scores indicating worse states, and the mean of all scores gives the total score [In May 2010 there were 17 residents at the addiction ward of Stokka Teaching Nursing Home. One criterion for admission to the addiction ward is a severe substance abuse, meaning that the alcohol or drug addiction is the main reason for the president’s health problems. At the time of study inclusion all residents used alcohol. Some used other drugs as well, but only to a small extent and not regularly. All of them had a history of longstanding alcohol or drug abuse for several decades. Most of them had started to have a problematic use of alcohol in their late teens or early twenties. Sixteen residents were invited to participate in the study, while one resident could not give informed consent, due to dementia. One resident chose not to participate.The following services are administered and financed by the municipality of Stavanger: Home care, cleaning help, day care centre, social workers, municipal housing, nursing homes and social welfare payments. Information about the use of municipal services was collected from the municipal registration system CosDoc . CosDoc State-funded services included in-hospital stays and visits to the outpatient clinics at the Stavanger University Hospital, rehabilitation clinics for substance abuse and pension payments. Data about in-hospital stays and visits to the outpatient clinic were drawn from the files of the Stavanger University Hospital from the registry Nirvaco Medical Systems (NIMES). As this is the only hospital within a radius of two hours driving, we did not collect data from other hospitals. Costs for in-hospital stay and treatment in the outpatient department were estimated according to the Diagnosis-related group (DRG) refunding system. As the DRG system does not apply for stays at the mental ward, we estimated these costs based on average cost per in-hospital day which constituted €1182 per day (SAMDATA 2010). Data about pension payments were given by the Norwegian labour and welfare administration. We registered net payments after the deduction of taxes.Municipal housing, overnight stays in hospices, and visits to the general practitioner or community-based ED are paid by the client. Data was collected from the municipal registration system CosDoc. In case the costs were covered by social welfare payments, they are registered as services paid by the municipality.A list of unit costs is given in Table We described costs from the following viewpoints: Costs arising for the municipality, costs arising for the state, costs borne by the client, costs to society and total costs. Social welfare payments are included into costs to the municipality. Pension payments are included into costs to the state. To calculate costs borne by the client we deducted payments for specialized housing and hospice that were covered by social welfare. Costs to society do not include social welfare payments or pension payments as they are considered income transfers. Total costs include costs to the municipality, to the state and to the client.We calculated the incremental cost for the admission to the addiction ward based on the assumption that the residents used the same amount of visits to the community-based ED, specialist health care and police custody as prior to admission. However, our clinical experience shows that in most residents the use of these services is markedly reduced after admission to the ward. We therefore included a sensitivity analysis, based on the assumption that only 50% of these services were used as compared to prior to admission and that no services outside the addiction ward were used.The software program SPSS 15.0  was used for statistical analysis. The correlations between IPLOS score and total costs respectively costs to the municipality were evaluated by Spearman’s rho correlation coefficient.The study was approved by the Regional Committee for Medical Research Ethics, University of Bergen, Norway. All participants gave written consent.Fifteen residents agreed to participate in the study. Mean age in May 2010 was 62.3 years (49 to 76 years), fourteen males and one female. Date of admission to the addiction ward was between July 2006 and May 2010. Four residents were admitted from their own homes, four came from a specialized housing program, three were transferred from other institutions and four had lived in hospices during the last weeks before admission. None of them was in an employment relationship, but only one had no income at all, while the others either received retirement or disability pension.Services rendered, number of recipients and mean costs are shown in Table One patient was admitted to a rehabilitation clinic for substance withdrawal. However, this measurement was taken due to the patient’s low housing abilities, and he was transferred to the addiction ward as soon as possible.The mean functional level prior to admission as evaluated by the IPLOS- questionnaire was 3.01 (2.05 to 4.29). There was no significant correlation between these scores and total costs  or between the IPLOS scores and costs to the municipality .Costs per bed at the addiction ward were € 38 196 for six months, 93% resident aimed costs and 7% maintenance costs. Residents have to contribute with out-of-pocket payments adjusted to their income. Assuming that all residents received minimum pension, 85% of this income (€ 6753) were transfer payments to the municipality while 15% (€ 1192) were incremental costs for pension payments. Assuming that residents at the addiction ward still used the same amount of visits to the community-based ED, specialist health care and police custody as prior to their admission, this would add up to additional costs of €8923 per six months. Total costs for residents of the addiction ward would thus be €47 120 per half year and the incremental costs €14 646. Assuming that the residents used only 50% of the services as compared to prior to admission, these costs would decrease to €42 658 and incremental costs of €10 184. Assuming no services outside the addiction ward were used total costs were €38 196 and incremental costs €5722 per six months. Table In 15 persons with longstanding substance abuse the use of health care and social resources was registered during the six months prior to their admission to a specialized ward at a nursing home. Mean total costs during this period were € 32 474. Approximately half of these costs were borne by state-funded institutions, and half were borne by the municipality. We could show that these clients used a great variety of services aimed at subsistence, health care and support in independent living, while services aimed at drug withdrawal were not claimed. Our findings do not indicate any correlation between the amount of services rendered and the clients’ level of functioning. The incremental costs for nursing home admission were borne by the municipalities.The strength of this study is a detailed and robust evaluation of sources for welfare in this client group. However, as the patient cohort consisted only of fifteen patients of one single municipality the results might not be generalizable. In addition, staffing costs are relatively high in Norway, and the pricing for the various services might therefore vary substantially in other countries. We applied 2010 prices, even if some services and payments were already rendered some years earlier, and we might thus have overestimated costs slightly. Further, we gave an estimate on incremental costs due to nursing home admission that range from €5722 to €14 646. Unfortunately, we have no detailed recording of the actual use of resources after admission that would allow for precise calculations. However, our clinical experience shows that in most residents the use of in- and outpatient hospital services is markedly reduced after admission to the nursing home as they are regularly seen by a permanently assigned doctor at the ward.Costs caused by the residents prior to admission to the addiction ward showed a big variance with a maximum of €80 937 and a minimum of €6080. However, these costs might not reflect the actual health state and need for care of the clients, as there was no correlation between costs and functional level as measured by the IPLOS score. A study about the validity of the IPLOS score in evaluating the need for care in persons with dementia could show that this questionnaire was weak in assessing fluctuating presentations like neuropsychiatric symptoms . This miThere are few Nordic studies about the resource use and costs of persons with substance abuse, and most evaluations are reports with a nation-wide perspective -16. To tWe estimated the incremental costs for nursing home placement in order to set costs for health care and social services in perspective. However, one has to keep in mind that the placing in the addiction ward is not an economic decision. Main reason for admission is the loss of the ability to meet basic personal needs like nutrition and personal hygiene, and the aim of the treatment at the addiction ward is to stabilize physical and mental health while maintaining personal autonomy, and in consequence to improve the quality of life. In many ways these clients represent a fringe group as they are beyond the scope of rehabilitation and re-integration into a normal social life combined with a relatively short life expectancy. However, as substance abuse is an increasing problem, one might expect that this group will be growing over the next decades and require greater attention.We could show that persons with long standing substance abuse represent a group with a significant use of a great variety of health care and social services and hence cause high costs. However, our findings do not indicate any correlation between the amount of services rendered and the level of functioning. Further research should focus on the identification of the clients’ need for support in order to facilitate targeted interventions that might prevent further deterioration and, finally, the need for permanent care.ADL: Daily life activities; IPLOS: Individual-based care and support statistics; ED: Emergency department; DRG: Diagnosis related groups.The authors declare that they have no competing interests.CV participated in planning and designing the study, collected and analysed the data, and drafted the manuscript. IT participated in planning and designing the study and helped drafting the manuscript. RS participated in planning and designing the study and collecting the data. SN participated in planning and designing the study and helped drafting the manuscript. All authors read and approved the final manuscript.RS is the head nurse of the addiction ward at Stokka Teaching Nursing Home, while CV is the physician for the residents of the ward.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/185/prepub


In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota.  Avulavirus in the family Paramyxoviridae. The genome of NDV consists of six transcriptional units (3′-N–P-M-F–HN-L-5′) [Newcastle disease (ND) is a highly contagious avian disease with worldwide distribution . The cauHN-L-5′) . The F aHN-L-5′) ,5.All NDV strains belong to a single serotype ,7. HowevGRQGR↓L” by three amino acid substitution (underlined) [Recently, we developed a reverse genetics system for a highly virulent NDV strain Banjarmasin/010/10 (Ban/010) that was isolated from diseased chickens during an outbreak in Indonesia in 2010 . The Banerlined) . The rBain vivo and in vitro, and for immunogenicity and protective efficacy against challenge with virulent GB Texas or Ban/010 viruses. This study also evaluated the relative contributions of F versus HN in the superior protection provided by vaccine virus that is homologous to the challenge virus. These results will be useful for development of genotype-matched NDV vaccines.These results confirm that a genotype-matched vaccine generated by reverse genetics can provide better protection than commercial vaccines –17. HoweThe chicken embryo fibroblast cell line (DF1) and human epidermoid carcinoma cell line  were grown in Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. The modified vaccinia virus strain Ankara (MVA) expressing T7 RNA polymerase was kindly provided by Dr. Bernard Moss  and propagated in primary chicken embryo fibroblast cells in DMEM with 2% FBS. In experiments that required supplementations of exogenous protease for the cleavage of the F protein, normal SPF chicken egg allantoic fluid was added to a concentration of 10%.NDV strains rLaSota  and rBanThe F and HN ORFs of the avirulent strain rBan/AF were placed individually or together into a full-length antigenomic cDNA of NDV strain LaSota in place of the corresponding LaSota F and HN ORF(s) . These mInfectious viruses were generated using a reverse genetics system established in our laboratory . BrieflyTo evaluate cleavage of the F proteins of the chimeric viruses, DF1 cells were infected at an MOI of 0.1, and cell lysates were collected at 24 h post-infection (hpi), denatured under reducing conditions, subjected to electrophoresis on an 8% polyacrylamide gel, and subjected to Western blot analysis separately with anti-NDV F rabbit polyclonal antiserum  and an a50/ml) by the end-point method of Reed and Muench [The ability of the chimeric viruses to produce plaques was tested in DF1 cells under 0.8% methylcellulose overlay. The plaques were immunostained using polyclonal antibody raised against the N protein of NDV . The muld Muench .The pathogenicity of parental and chimeric viruses was determined by the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs . Briefly6 EID50) by the intranasal route. One group of chickens remained as unvaccinated controls. Three birds from each group were sacrificed at 4 days post-infection (dpi) and tissues samples (lung and trachea) were collected for vaccine virus titration. Virus titers were determined by limiting dilution and immunostaining in DF1 cells as described before. The presence of virus in the tissue samples also were determined by inoculation into 9-day-old SPF embryonated chicken eggs, which provides a more sensitive means of detection but does not provide a titer. In general, we obtained HA titers ranging from 24 to 27 from positive samples. At 3 dpi, the allantoic fluids were tested for virus growth by HA assay. For analysis of antibody responses, serum samples (3 ml from each chicken) were collected on weeks 2, 3, 4, and 5. Serum antibody titers were determined by hemagglutination inhibition (HI) assay using rLaSota or rBan/AF as antigen [Two-week-old SPF chickens in groups of eleven (11 birds each) were immunized with each virus  of virulent NDV strains GB-Texas (four birds) or Ban/010 (four birds) through the oculo-nasal route [Five weeks post immunization, the remaining chickens (eight per immunizing virus) were transferred to a USDA-certified BSL3containment facility for NDV challenge. The birds in each group were challenged with 100 chicken 50% lethal dose  .8 HAU/ml) and the sequences of the F and HN genes were confirmed, showing that the introduced ORFs were maintained without any adventitious mutations.We used reverse genetics to make three chimeric derivatives of the LaSota vaccine strain in which the complete ORF of the F and/or HN genes was replaced with that of the rBan/AF strain , which iWe further evaluated incorporation of heterologous glycoproteins into chimeric virions, since the presence of the compatible cytoplasmic tails and transmembrane regions is important for NDV replication . The virRRQKR↓F) to that of LaSota (GRQGR↓L), resulting in the avirulent rBan/AF derivative. Whereas the Ban/010 parent induced extensive syncytia and plaque formation in cell culture in the absence of added protease, the rBan/AF derivative caused only single-cell infections without syncytia or plaques in the presence or absence of extracellular protease [Strain LaSota lacks a polybasic sequence or furin motif at the F protein cleavage site, and depends on extracellular protease for cleavage . In our protease . Therefo7 TCID50/ml at 40 hpi. The other two chimeric viruses (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) replicated less efficiently than the other tested viruses and reached their maximum titers (106 TCID50/ml) at 56 hpi. The pattern of in vitro replication of chimeric viruses also showed the correlation with their ability to form syncytia in DF1 cells . To enhance the sensitivity of virus detection, the tissue homogenates also were inoculated into eggs  were collected for virus titration. Virus titration of the homogenates by limiting dilution assay showed that detection of parental and chimeric viruses was restricted to the trachea, and their titers were low, ranging from 1.6 to 2.4 lognto eggs . Replica2) against the two different parental viruses, namely the rLaSota  or the virulent wild-type Indonesian strain  via the oculo-nasal route. In addition, three chickens each from the unimmunized group were challenged with either of the two challenge NDVs. For the unimmunized chickens, challenge with the GB Texas virus resulted in clinical symptoms , and 100% of mortality at 5 dpc, whereas challenge with the Ban/010 virus resulted in 100% of mortality at 3 dpc (data not shown). The virus titers in the trachea of dead chickens were >7.0 log10 TCID50/g. In contrast, all of the previously-immunized chickens (5 chickens for each group) were completely protected from clinical disease and mortality against the GB-Texas and Ban/010 strains.To evaluate the protective efficacy of chimeric NDVs, at 5 weeks post-immunization, the remaining immunized chickens in each immunization group were divided into two challenge groups. Each challenge group (8 birds each) was challenged with 100 CLD6 TCID50/g) and lungs (1.8 × 105 TCID50/g)  were collected at 4 and 7 dpc and inoculated into eggs for sensitive detection of infectious virus. In addition, 3 birds from each challenge group were sacrificed at 4 dpi and tissue samples  were collected and tissue homogenates prepared. These were evaluated for the presence of virus by inoculation into eggs, and virus titers were determined by limiting dilution assay, as above. In the GB-Texas challenged groups, virus shedding was not detected in most swab samples , and no CID50/g) . SimilarThe two chimeric LaSota viruses containing rBan/AF F and F and HN together showed comparable levels of protective efficacy to that of rBan/AF. Our findings showed that rBan/AF vaccine and the two chimeric viruses containing the genotype-matched F gene (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) effectively prevented shedding of Indonesian challenge virus, whereas viruses lacking the Indonesian F gene, namely rLaSota and chimeric rLaSota-Ban/AF HN, were less effective in preventing shedding of Indonesian challenge virus. These results show that the genotype-matched vaccines provide better protection than genotype mismatched vaccines, and this superior efficacy is due to the F protein.All NDV isolates belong to a single serotype. Consistent with this, currently used vaccines, such as strains B1 and LaSota, are known to protect against morbidity and mortality caused by NDV isolates in different parts of the world . Howevervitro characterization of the parental and chimeric viruses showed that the F protein of the LaSota strain was cleaved more efficiently, in the presence of added allantoic fluid as a source of protease, than the F protein of rBan/AF, and the presence of this protein was associated with the formation of syncytia and plaques as well as better growth in vitro. This was true for both the parental and chimeric viruses. Thus, the rBan/AF HN virus had efficient cleavage of the F protein, induced syncytium and plaque formation, and had relatively efficient replication in DF1 cells, similar to rLaSota. Nonetheless, the virus was somewhat more attenuated (MDT 129 h) than rLaSota (MDT 112 h). In contrast, rLaSota-Ban/AF F and rLaSota-Ban/AF F HN showed inefficient cleavage of the F protein, single cell infection without syncytium or plaque formation, and reduced replication in infected DF1 cells. The finding that cleavage of the rBan/AF F protein was less efficient than that of the rLaSota F protein, even though their cleavage activation sequences were identical, indicated that structural features of the rBan/AF F protein additional to the cleavage site affect proteolytic processing of the F protein. Chimeric LaSota containing rBan/AF F and HN together resulted in a greater attenuation of the virus in ovo (MDT > 168 h) than either glycoprotein alone, showing that the rBan/AF HN protein also contributed to attenuation in ovo. One possible explanation could be that the glycoproteins from rBan/AF may not be perfectly compatible with the LaSota background. However, this did not appear to be true for the rBan/AF HN protein, at least, since the rLaSota-Ban/AF HN virus replicated more efficiently in vitro than either parent, and thus did not appear to be impaired.In our study, all the chimeric viruses were readily recovered, and the incorporation of the surface glycoproteins into virions and the yield of virions were similar between parental and chimeric viruses. This indicated that the amino acid sequence differences between the F and HN proteins of rLaSota versus rBan/AF did not detectably affect virus assembly and recovery. However, in in vitro (in fact, the rLaSota-Ban/AF F and rLaSota-Ban/AF F HN viruses replicated less efficiently in vitro than the others; in vivo (Immunization of 2-week-old chickens confirmed that all the chimeric viruses were highly attenuated. Replication of the three chimeric viruses was mostly limited to the trachea , and thu in vivo . These rPreviously, cross protection studies have suggested that chickens vaccinated with a live LaSota vaccine displayed disease symptoms after being challenged with antigenic variants of genotype VII . Inactiv


Assessing Screening Plus brief Intervention’s Resulting Efficacy (ASPIRE). An activity-based micro-costing approach was used to determine implementation costs and set-up costs. Implementation costs include variable costs (direct costs that directly vary by the number of services provided) and quasi-fixed costs (service-support costs that indirectly vary by the number of services provided). Set-up costs are fixed costs incurred to the program, such as the purchase of durable equipment and initial training. A taxonomy of activities and resource inputs  were identified. Quantity and most price values were obtained through primary data collection during the trial. Regional price estimates for wages and space were used where needed. Preliminary estimates indicate that variable costs to provide SBI for unhealthy drug use are similar to SBI programs focusing on alcohol. Quasi-fixed costs are substantially higher than variable costs for this program. Quasi-fixed costs are not usually estimated; the focus is usually on variable costs of service delivery. This study fills a gap in the economic literature on SBI regarding quasi-fixed costs. These findings have important implications for the adoption and sustainability of SBI programs, where focusing only on variable costs underestimates the fiscal impact of implementing SBI.In 2008, a World Health Organization multi-national clinical trial found screening and brief intervention (SBI) to be effective in reducing illicit drug use and many have called for its widespread dissemination. To gain policy support for widespread dissemination, however, a strong business case must be made to policymakers. In particular, policymaker concerns over implementation costs can act as a significant obstacle to widespread dissemination. This presentation estimates the various cost components of providing SBI to reduce illicit drug use in a primary care setting as part of the clinical trial


During May–July 2010 in Namibia, outbreaks of Rift Valley fever were reported to the National Veterinary Service. Analysis of animal specimens confirmed virus circulation on 7 farms. Molecular characterization showed that all outbreaks were caused by a strain of Rift Valley fever virus closely related to virus strains responsible for outbreaks in South Africa during 2009–2010. Bunyaviridae, genus Phlebovirus) is an enveloped RNA virus transmitted mainly by mosquitoes. This virus causes severe disease in humans and animals. The virus was identified in 1930 along the shores of Lake Naivasha in the Great Rift Valley in Kenya  have been considered vectors and reservoirs of the virus  outbreaks during the epidemic season; >14,000 cases and 8,000 deaths of animals occurred  were homogenized by using a mortar and sterile quartz pestle and diluted 1:10 in phosphate-buffered saline containing antimicrobial drugs . Tissue debris was removed by low-speed centrifugation.RNA was purified from blood samples and supernatants of homogenized tissues by using the High Pure Viral Nucleic Acid Extraction Kit  according to the manufacturer’s instructions. RVFV RNA was identified in samples by using the specific one-step reverse transcription PCR (RT-PCR) described by Battles and Dalrymple .Aliquots of samples were shipped to the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise in Teramo, Italy, where virus isolation was conducted on samples positive for virus by RT-PCR by infecting Vero E6 cell (ATCC CRL-1586 VERO C1008) monolayers  Table 1.www.ncbi.nlm.nih.gov) was used to identify homologous regions in sequence databases. Sequences were aligned by using ClustalW  and BioEdit Sequence Analysis Editor version 7.0.5.3   Table 1  7.0.5.3  Table 3  7.0.5.3  Figure 2Overall diversity of partial M segment sequences was low, and bootstrap values for tree nodes were weak in some instances. Phylogenetic analysis showed that isolate Namibia 2010 belongs to the same group of RVFV strains isolated in South Africa in 2009 (SA404/09) and 2010 . The cluster corresponds to lineage H of RVFV identified by Grobbelaar et al. (The high degree of sequence identity of related RVFV strains that co-circulated in South Africa and Namibia in 2004–2010 suggests that these strains probably originated from a virus population that circulated between these 2 countries. Molecular data suggest that RFV outbreaks in Namibia in 2010 were caused by possible disseminated infections from South Africa. This hypothesis is further supported by the temporal and geographic location of the outbreaks. Clinical signs were first observed at the beginning of May in southeastern Hardap near the border with South Africa . The AuoThe large RVF epidemic in South Africa in 2010 was attributed to heavy rainfall during January–February 2010  Figure 3Supplementary data for analysis of Rift Valley fever in Namibia, 2010.