Antirrhinum majus Rosea1 (Ros1) is an MYB-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. However, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. Therefore, viruses carrying the Ros1 marker might have a low fitness and consequently rapidly lose the marker. To compare the stability of the Ros1 marker, we generated Tobacco etch virus (TEV) based constructs containing either Ros1 or the enhanced green fluorescent protein (eGFP) between the NIb and CP cistrons . We measured the within-host competitive fitness of both viruses by direct competitions with a common competitor during infection of Nicotiana tabacum. The fitness of TEV-Ros1 was significantly lower than that of TEV-eGFP, and both recombinant viruses had a significantly lower fitness than the wild-type virus. Nevertheless, after seven weeks of infection in N. tabacum, similar levels of marker gene instability where found for both viruses. Despite lower fitness of the marked virus, Ros1 is therefore a viable alternative marker for tracking viral infection in plants. Antirrhinum majus (snapdragon) Rosea1 (Ros1) transcription factor, which induces biosynthesis of colored anthocyanins in infected tissues. A wide range of markers for tracking virus infection is therefore available. The strengths and weaknesses of these markers have, however, often not been documented in detail, with the exception of key attributes such as detection sensitivity and ease of use. To enable rational choices with respect to marker use in research and industry, we here consider potential weaknesses of the Ros1 marker. A comparison in terms of the effects on viral fitness and stability is made with the enhanced green fluorescent protein (eGFP) [Viruses carrying markers have been widely used to study plant-virus infection dynamics, at spatial scales ranging from individual cells to whole plants ,6,7,8,9.A. majus Ros1 is a 25.7-kDa  R2R3 MYB transcription factor [Tobacco etch virus : the effects on within-host competitive fitness and on marker stability. Within-host competitive fitness is a measure of the ability of a virus to directly compete with conspecific viruses infecting the same host. This fitness measure therefore indicates to what extent the expression of a marker protein affects the virus capacity to replicate and move. Moreover, when compared to the within-host fitness of the wild-type virus, it will give an indication of the strength of selection for those virus variants that lose the marker gene by means of recombination. However, the actual stability of the virus will also depend on the mutation supply  and virus-host interactions. Experimental measures of marker stability are therefore also relevant. In order to assess stability, we therefore analyzed the integrity of the marker sequence after seven weeks of infection in a single plant. These conditions were chosen because it has been shown that deletions in a marker gene are most likely to occur during long passages [n factor . It is an factor . In termpassages . FinallyOur aim was to compare some of the properties of the recently developed Ros1 marker with the more classical eGFP as reporters of plant virus infection. We therefore constructed a TEV recombinant clone with an eGFP cDNA inserted between NIb and CP cistrons: TEV-eGFP . This neNicotiana tabacum L. cv. Xanthi plants were initially agroinoculated with TEV-Ros1 and TEV-eGFP. Symptomatic tissues were collected at 7 dpi and aliquoted to more easily produce infectious extracts of both virus variants. When new N. tabacum plants were mechanically inoculated with these infectious extracts, individual primary infection foci were detected in the inoculated leaves. In the case of TEV‑eGFP, infection foci were fluorescent and they were detected starting from 3 dpi using a fluorescent stereomicroscope .croscope a. In thecroscope b. When t plotted c, the nu plotted . The DA  plotted . This re plotted . This reeN) for virus infection of a plant [eN can be approximated as the smallest census size for rapidly expanding and contracting population [The number of primary infection foci renders an estimate of virus effective population size . TEV-wt had a significantly higher within-host fitness than the TEV-eGFP , whereas TEV-Ros1 had a significantly lower fitness than TEV-eGFP . Therefore, even though TEV-Ros1 has a slightly shorter marker sequence than TEV‑eGFP, its fitness was significantly lower. The difference between TEV-eGFP and TEV-Ros1 is, however, small compared to differences between these two viruses and TEV-wt.Overall, there were highly significant differences between the viruses in fitness . Our data therefore suggest that the stability of TEV-Ros1 and TEV-eGFP are similar, despite the minor difference in within-host competitive fitness.After seven weeks of infection in 0th leaf . Visual 0th leaf a–d. Note0th leaf e. We the0th leaf . We also0th leaf . With thi.e., output is the raw ratio of the signal of the two amplicons on gel). Therefore, under the conditions used the assay appears to work well because the higher efficiency of PCR amplification for smaller products [During a PCR, smaller products may be amplified with a greater efficiency than larger products . Selectiproducts  is countχ2 test for the presence of the ancestral virus and a second procedure developed here, a Mann-Whitney U-test on the mean relative amplicon length for the genomic region containing the marker. The latter procedure may in principle be useful for detecting more subtle differences between evolved virus populations than simply the presence or absence of the ancestral virus . For example, the amplicon-length-based procedure might still detect differences if (i) the frequency of the ancestral virus tends to be higher in the evolved populations of one virus type, but (ii) the number of lineages in which deletions of the marker gene has occurred is the same for both virus types. In practice, however, for both procedures we did not find a difference in instability between the two viruses, and there is therefore not yet any evidence that the amplicon-length-based procedure has greater power for detecting differences in instability.Two tests of marker instability were performed: a U-test: P < 0.001), suggesting that Ros1 expression is adversely affecting plant growth. When the Ros1 maker was lost during virus expansion, it appeared as if plants grew faster in those regions of the plant in which virus expressing Ros1 were no longer present. However, this effect was not statistically significant for either virus; those plants in which we sampled only viruses that did not express the marker gene in the 30th leaf were not significantly larger than plants in which marker genes were detected .After seven weeks of infection plants infected with TEV-Ros1 were significantly shorter than those infected with TEV-eGFP  or eGFP (AAB08060) were inserted in between the TEV nuclear inclusion b (NIb) and coat protein (CP) cistrons by standard techniques based on the use of the type IIS restriction enzyme Eco31I (Thermo Scientific) and T4 DNA ligase (Thermo Scientific) [Plasmid pGTEVa  containsentific) . pGTEV-Rentific) . The comAgrobacterium tumefaciens C58C1 containing the pCLEAN-S48 helper plasmid [2 [Four-week-old tobacco plants cultivated in a growth chamber under a photoperiod of 16 h day and 8 h night at 24°C were agroinoculated in the third true leaf with cultures of  plasmid  and plasN. tabacum plants (as described above) were mechanically inoculated by gently rubbing 5 μL virus extract and a 2-µL drop of 10% Carborundum in inoculation buffer onto the third true leaf.Infectious extracts were obtained by homogenizing infected tissue with five volumes of inoculation buffer . in vitro from the pTEV-mCherry plasmid [8 to 3.2 × 104 virus genome copies per µL, in a final concentration of 50 ng/µL RNA from a non-virus control plant, were used as the template for the standard curve [Total RNA was extracted from approximately 100 mg of virus-infected tissue using the Invitrap Spin Plant RNA Mini Kit (Stratec Molecular). The Primescript RT-PCR Kit II (Takara) and a Step One Plus thermocycler (Applied Biosystems) were then used to perform a SYBR Green I-based one-step RT-qPCR, with TEV-specific CP primers: forward primer 5'-TTGGTCTTGATGGCAACGTG-3' and reverse primer 5'-TGTGCCGTTCAGTGTCTTCCT-3'. Absolute quantification using the standard curve method was performed, using the Step One v 2.2 software  to analy plasmid . This RNrd curve . Using t7 genome equivalents per μL. Three-fold serial dilutions were then made in inoculation buffer. Five 4-week-old N. tabacum plants were then mechanically inoculated on the third true leaf with each virus dilution or inoculation buffer for non-virus controls. Plants were then kept in a growth chamber at 24 °C with 16 h light. For quantifying TEV-eGFP primary infection foci, a Leica MZ16F stereomicroscope with 0.5× objective and GFP2 filters (Leica) was used [Extracts from tissues infected with TEV-eGFP and TEV-Ros1 were diluted with inoculation buffer to a concentration of 1.67 × 10was used . TEV‑Rosf) is the product of the probability of infection per virion (ρ) and virion dose (d). This model results in a linear relationship between dose and the number of foci. The dependent action (DA) model [, where κ is constant that determines the strength of dose-dependent effects. When κ > 1 there are synergistic effects, when κ < 1 there are antagonistic effects, and when κ = 1 the DA model collapses to the IAH model without dose‑dependent effects on the probability of infection per virion. Both models were fitted to the data using a maximum-likelihood approach. Given that the distribution of primary infection foci for a given dose follows approximately a Poisson distribution for low and intermediate doses [k is . The negative log likelihood was then minimized by grid searches using R 2.14 [To analyze the data, we fit two simple infection models to the data. The independent action hypothesis (IAH) model ,27 assumA) model  assumes te doses ,9, the lg R 2.14 , renderi7 genome equivalents per μL using inoculation buffer. TEV-wt, TEV-Ros1 and TEV-eGFP were then mixed with TEV-mCherry in 3:1 mixtures, and five 4-week-old N. tabacum plants were mechanically inoculated with each mixture. Under these conditions, the TEV‑eGFP:TEV-mCherry mixture rendered approximately a 3:1 ratio of primary infection foci at 3 dpi.Competition experiments were performed following an approach proposed elsewhere . RT‑qPCRR of the virus being tested  to the common competitor  is then , where CPn and mCherryn are the copy numbers of CP and mCherry cistrons, respectively, as measured using the RT-qPCR assay. We then use the approach of [W) such that . Here t is the time in days, tR is the virus ratio at the end of the experiment, and 0R is the virus ratio at the start of the experiment. Note that we used a 3:1 mixture of the two viruses in the inoculum, instead of a 1:1 mixture, for methodological reasons. R can only be accurately measured when mCherryn levels are considerably lower  than CPn levels. We anticipated that the within-host competitive fitness of TEV-Ros1 might be low, and starting with a 3:1 ratio ensures mCherryn levels remain lower than CPn levels.Infected tissue was harvested 7 dpi, and RNA was extracted, again using the Invitrap Spin Plant RNA Mini Kit. We then performed RT-qPCR with the CP primers , which wroach of  to measuN. tabacum plants were mechanically inoculated with inoculation buffer (mock-inoculated control), TEV-wt, TEV-eGFP and TEV-Ros1, and infection was allowed to proceed for 7 weeks. We then measured plant height and counted the number of fully formed leaves. We collected the 30th leaf, or if plants did not have 30 leaves yet, the highest fully formed leaf. RNA was extracted from each leaf using the Invitrap Spin Plant RNA Mini Kit. RT was performed using M-MuLV (Thermo Scientific) and the primer 5'-CGCACTACATAGGAGAATTAG-3'. A Taq DNA polymerase (Roche Applied Science) based PCR was then performed using the forward primer 5'-TACGATATTCCAACGACTG-3' and reverse primer 5'-GCAAACTGCTCATGTGTGG-3'. These primers flank the site of Ros1/eGFP insertion in the TEV genome. PCR products were then resolved by electrophoresis on 1% agarose gels, and ImageJ [i.e., viruses with an intact marker gene) was present or absent was constructed. A χ2 test was then performed to test for differences between the two viruses. Note that a contingency table also including ‘only ancestral virus’ category was not used because this latter category would have less than 5 observations for both viruses, precluding the use of a χ2 test.d ImageJ  softwareα) was first estimated from the length of the PCR product, rounded off to the nearest 100 bp. We use the proportion of marker instead of simply the length of the PCR product, since eGFP is slightly longer than Ros1 and a significant difference could otherwise simply reflect the length of the marker gene in the ancestral viruses. The ImageJ-derived estimates of the relative intensity of a particular PCR amplicon on the gel within a sample (β) were then used to generate a weighted mean (μ) for the z bands in each individual sample . A Mann-Whitney U-test was then performed to test for significant differences in μ values for the two viruses.In an attempt to increase the statistical power of the analysis of marker gene stability, we also performed a second test. The proportion of the respective marker gene present (10 plasmids). We then performed the PCR and quantification of amplicons on agarose gel as described above. Based on the results obtained, we performed a second experiment in which the molar mixtures 30:1, 3:1 and 1:3 were used. Two independent replicates of both series of mixtures were performed.This second test of stability assumes that the PCR products are at least a qualitative representation of the different virus variants in the sample. In order to test this assumption, we made mixtures of the purified and Nanodrop-quantified plasmids p15TV3 (to represent TEV-wt) and p15TV3-eGFP (to represent TEV-eGFP). These plasmids contain inserts with the approximately last third (from restriction site Eco81I) of the TEV genome. p15TV3-eGFP includes the eGFP marker in a similar situation as the TEV-eGFP recombinant clone. The following molar mixtures (p15TV3:p15TV3-eGFP) were made: 1,000:1, 100:1, 10:1, 1:1, 1:10, 1:100 and 1:1,000, whilst the total amount of DNA was the same for each reaction (10We found that TEV-Ros1 had a significantly lower within-host competitive fitness than TEV-eGFP —although the difference in fitness was small—and both viruses clearly had a lower fitness than TEV‑wt. However, our data indicate that there is not a significant difference in marker loss in TEV‑eGFP and TEV-Ros1. This result suggests that the dynamics of marker loss in a virus population will probably also depend on the mutation supply and virus-host interactions. Therefore, a relatively small difference in within-host competitive fitness may not lead to appreciable differences in marker stability. Previously, it was shown that the Ros1 marker is stable in the TEV genome after six plant-to-plant passages of 9 days . A relat


Sweet potato chlorotic stunt virus (SPCSV) is probably the most important virus infecting sweetpotato worldwide, causing severe synergistic disease complexes with several co-infecting viruses. To date only one isolate (Ug), corresponding to the EA strain has been completely sequenced. It was later shown to be unusual in that, in contrast to most isolates, it encoded an additional p22 protein at the 3′ end of RNA1. We report the complete sequence and genome organization of a Peruvian isolate of SPCSV (m2-47) as determined by siRNA deep sequencing. We confirm that the ORF encoding p22 is lacking from m2-47 and all tested Peruvian and South American isolates, whereas additional isolates containing p22 were identified from Uganda. Other potentially important genomic differences such as two small ORFs encoding putative small hydrophobic proteins instead of one, upstream the hsp70h gene and a more divergent sequence at its RNA1 3′-UTR in contrast to SPCSV isolates that contain p22 are discussed and a model for recent acquisition of p22 in Uganda is proposed. A role for p22 as a pathogenicity enhancer of SPCSV is also provided by complementary expression of p22 in transgenic sweetpotato plants. Sweet potato chlorotic stunt virus (SPCSV) is a bipartite member of the family Closteroviridae, genus Crinivirus. It is phloem-limited, whitefly transmitted and among the largest of the single-stranded positive-sense RNA viruses with a total genome size of ∼17.6 kbp  which causes ‘sweetpotato virus disease’ (SPVD), the economically most important disease of this crop, associated with yield losses of >60%  activity  than co-infection of SPFMV with SPCSV isolates lacking p22 in the indicator plant Ipomoea setosa , Sweet potato virus 2 , Sweet potato latent virus , Sweet potato mild mottle virus , Sweet potato mild speckling virus (SPMSV), Sweet potato chlorotic fleck , C-6 virus, Cucumber mosaic virus  and Sweet potato caulimo-like virus  to confirm their absence. The plants were also checked to confirm the absence of begomoviruses by PCR (Ipomoea leaves using Trizol (Invitrogen) following the manufacturer's instructions. RNA was resuspended in 250 μl sterile Milli-Q water (Sigma–Aldrich). The amount and quality of the RNA were checked using a spectrophotometer  and agarose gel electrophoresis. For isolate m2-47, RNA was lyophilized and sent to Fasteris Life Sciences SA  for processing and high throughput sequencing (deep sequencing) on the Illumina Genome Analyzer II and short RNA viral sequences were assembled in silico using the programs Velvet and MAQ (http://maq.sourceforge.net) as previously described  and Escherichia coli DH5α chemically competent cells. The sequence of two or more clones obtained from independent PCR reactions were confirmed by Sanger sequencing (Macrogen). Extension of these contigs, including sequences obtained by RT-PCR, was done using the program ContigExpress included in the Vector NTI package (Invitrogen). Transmembrane helices in p6 proteins were predicted with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM-2.0/) as previously described (® Taq polymerase (Invitrogen) according to the manufacturers recommendations. A specific fragment of the expected size of ∼310 bp was obtained, purified from gel, cloned into E. coli and sent for Sanger sequencing as described above. To determine the RNA1 and RNA2 3′-UTRs of remaining SPCSV isolates  and of two EA and EAp22 isolates each by sequencing (p6-SHP (see below) using the primers SPCSV2-44f (5′-TAA GCT CGT ATC ATT GGT TGT CGT CA-3′) and SPCSV2-1046r (5′-GAC CTT CAT CGT ACC CCC GAC-3′). To study a possible role of p22 during SPCSV infection we used non-transgenic and p22-expressing (under control of the phloem specific RolC promoter) transgenic sweetpotato lines of cultivar ‘Huachano’  and previous partial sequence characterization of SPCSV m2-47 indicating the absence of p22  upstream the hsp70h gene: Isolates m2-47 and Ug contain a p6 gene at the 5′ end of RNA2 showing 100% nt and aa identity. Although similar small ORFs found in the Closteroviridae encode SHP, no evidence of transmembrane domains in p6 have been detected 3′-UTR ofRNase3 RSS (Closteroviridae (hsp70h) and RNase III-like genes with marginal aa sequence similarity to cellular RNase III enzymes  as compared to other EA isolates  and considering reports on East Africa as a region hosting a high variability of sweetpotato viruses and virus strains, including SPCSV , a region where it is possible to find both EA and EAp22 isolates (this study) and where breeding for virus-resistance in sweetpotato has been an intense activity virus.Given that isolates containing t Africa  includinng SPCSV  it is liactivity . Strong


To achieve globally or regionally defined accelerated disease control, elimination and eradication (ADC/E/E) goals against vaccine-preventable diseases requires complementing national routine immunization programs with intensive, time-limited, and targeted Supplementary Immunization Activities (SIAs). Many global and country-level SIA costing efforts have historically relied on what are now outdated benchmark figures. Mobilizing adequate resources for successful implementation of SIAs requires updated estimates of non-vaccine costs per target population.This assessment updates the evidence base on the SIA operational costs through a review of literature between 1992 and 2012, and an analysis of actual expenditures from 142 SIAs conducted between 2004 and 2011 and documented in country immunization plans. These are complemented with an analysis of budgets from 31 SIAs conducted between 2006 and 2011 in order to assess the proportion of total SIA costs per person associated with various cost components. All results are presented in 2010 US dollars.Existing evidence indicate that average SIA operational costs were usually less than US$0.50 per person in 2010 dollars. However, the evidence is sparse, non-standardized, and largely out of date. Average operational costs per person generated from our analysis of country immunization plans are consistently higher than published estimates, approaching US$1.00 for injectable vaccines. The results illustrate that the benchmarks often used to project needs underestimate the true costs of SIAs and the analysis suggests that SIA operational costs have been increasing over time in real terms. Our assessment also illustrates that operational costs vary across several dimensions. Variations in the actual costs of SIAs likely to reflect the extents to which economies of scale associated with campaign-based delivery can be attained, the underlying strength of the immunization program, sensitivities to the relative ease of vaccine administration , and differences in disease-specific programmatic approaches. The assessment of SIA budgets by cost component illustrates that four cost drivers make up the largest proportion of costs across all vaccines: human resources, program management, social mobilization, and vehicles and transportation. These findings suggest that SIAs leverage existing health system infrastructure, reinforcing the fact that strong routine immunization programs are an important pre-requisite for achieving ADC/E/E goals.The results presented here will be useful for national and global-level actors involved in planning, budgeting, resource mobilization, and financing of SIAs in order to create more realistic assessments of resource requirements for both existing ADC/E/E efforts as well as for new vaccines that may deploy a catch-up campaign-based delivery component. However, limitations of our analysis suggest a need to conduct further research into operational costs of SIAs. Understanding the changing face of delivery costs and cost structures for SIAs will continue to be critical to avoid funding gaps and in order to improve vaccination coverage, reduce health inequities, and achieve the ADC/E/E goals many of which have been endorsed by the World Health Assembly and are included in the Decade of Vaccines Global Vaccine Action Plan. However, studies were less consistent about acknowledging and distinguishing between immunization-specific versus shared costs, and whether/how depreciation for capital equipment was factored into their analyses. In some instances (notably for OPV + measles + VitA SIAs) campaigns were performed in an integrated fashion. In these instances, authors assumed that aside from the costs of vaccines and supplies, other cost components could not be assigned to each antigen/intervention and assumed they would have been the same regardless of whether one intervention or multiple interventions were delivered through the SIA. Variability in these definitions and costing practices makes comparing across the published literature difficult. Furthermore, the information detailed in Table Given the low number of observations for menA, yellow fever, MR and ‘Other’ SIAs in the dataset, average per person operational costs for these vaccines were not analyzed separately. The observations for these vaccines are included in other aggregate operational cost indicators presented – for the following groups of vaccines:● Vaccines in GAVI’s portfolio – Based on 51 SIA expenditure observations for measles (n=38), MR (n=3), menA (n=6), yellow fever (n=1), JE (n=2), and typhoid (n=1) vaccines.● Injectable vaccines – Based on the 51 observations listed above for ‘Vaccines included in the GAVI portfolio’ plus a further 28 observations for tetanus (26), hepatitis B (n=1) and pertussis (n=1) vaccines.● Orally administered vaccines/interventions – Based on 63 observations for OPV (n=53), vitamin A (n=8) and albendazole/mebendazole (n=2) tablets.Table Focusing on the results for ‘All countries’ in Table  and outswithin the sample of cMYPs themselves, and specifically comparing in aggregate more recent operational SIA costs from campaigns conducted over the period 2009–2011 to older estimates from campaigns conducted over the period 2004–2008, we find that across vaccines, more recent campaigns are consistently higher, particularly for OPV SIAs  and therefore may receive lower salaries, require less training and less costly supervision, all of which can reduce the operational costs.Operational costs for measles vaccine SIAs are, on average, higher than for OPV and tetanus vaccine SIAs; and since measles vaccine observations dominate the sample used to define operational costs of vaccines in GAVI’s portfolio, these costs are also higher. By contrast, average per person operational costs of tetanus vaccine SIAs appear to be lower than other injectable vaccines. Since cMYP data do not offer breakdowns of cost drivers it is not possible to explain definitively from a programmatic standpoint what might be driving these differences. While further research would be needed to provide a definitive answer, we hypothesize that the lower operational costs associated with tetanus vaccine SIAs may in part relate to the district-specific targeting strategy used in MNT elimination SIAs known as the “high risk approach”– WCBA living in specific high risk districts (HRDs) are targeted with three properly spaced doses of TT . Since tAverage operational costs are consistently higher in the African region Figure  and it iFigure The breakdown of SIA operational costs by cost component illustrates that four cost drivers make up the largest proportion of costs across all vaccines: human resources , program management, social mobilization , and vehicles and transportation (e.g. fuel to transport vaccinators). The fact that cold chain equipment makes up such a small proportion of operational costs suggests that SIAs build on existing routine immunization systems and do not require significant additional capital investment Figure .Compared to other vaccine SIAs, as a proportion of total operational costs Tetanus vaccine SIAs tend to be associated with higher human resources and training costs, and lower vehicles and transportation costs, and virtually no cold chain cost. This finding supports the notion that the HRD approach used in MNT elimination SIAs rely on training local health staff rather than bringing in external vaccinators. While Yellow Fever budget breakdowns also differ from other vaccine SIAs, this variance may be more associated with outliers in the small number of observations in the budget sample.Our analysis of operational costs of SIAs represents a minority of the total SIAs conducted worldwide over the analytical period. For example, our analysis of country-specific plans covered around 11% (41/360) of the measles/rubella-containing vaccine follow-up and catch-up SIAs conducted anywhere in the world between 2004 and 2011 . Since mcore costs” in agency budgets but are rarely factored into estimates of operational cost estimates to illustrate the true nature of SIAsh.The information documented in both existing literature and possibly the data from cMYPs may not be reflective of the full costs of implementing an SIA. In addition to the operational costs incurred mainly by national payers and reported in the literature and cMYPs, implementation of SIAs that are part of global ADC/E/E efforts often rely on additional expertise from global agents such as WHO, UNICEF, and the United States Center for Disease Control and Prevention (US CDC). These agencies provide highly specialized assistance to governments—undertaking disease surveillance and laboratory testing to monitor epidemiologic dynamics and to help target SIA efforts; developing/implementing social mobilization programs to strengthen community demand for vaccination; and providing other technical support  — ultimately to improve the quality of the campaigns. The costs associated with these additional support activities are often referred to as “Finally, the results of the cost analyses presented here are only as strong as the data on which they rest. There are many benefits of cMYPs; e.g., from a global perspective these country plans provide data on immunization program costs  across a wide array of countries in a standardized form and using a comparable methodology. However, cMYP data are not systematically validated and may be of variable quality. It is difficult to know what is included in each country’s cMYP estimate of SIA operational costs since breakdowns by cost components are not provided. Thus, despite the availability of guidelines and templates for detailed SIA budgetary planning ,52, it iElimination and eradication of vaccine preventable disease represent the ultimate in addressing health inequities on a local and global scale respectively, while accelerated disease control represents the next best alternative. Achieving ADC/E/E goals is seen as a public health imperative but success since smallpox eradication has been elusive. Robust planning and budgeting, adequate financing, good governance, and effective implementation of SIAs building on strong routine immunization programs are critical to achieving these goals. Our analyses illustrate that existing evidence that often informs budgeting, planning and financing decisions, is out of date, and for many programs, operational costs may be higher than previously thought. Underestimation of costs and funding shortfalls can lead to SIA implementation delays, and can force implementers to target narrower age groups undermining efforts to reach program goals. Therefore, the results of the analysis presented here may provide national and global-level actors with newer SIA operational cost benchmarks to inform planning, budgeting, and financing decisions in absence of better information.Our analyses also illustrate where program implementers might focus efforts to seek out efficiencies to manage the upward trends in SIA operational costs. For example, with a significant proportion of costs related to human resources, given that there is often criticism of the culture of per diems related to development assistance , new assWhile the information presented here updates the evidence base, there is a need for continued monitoring and additional research into SIA operational costs and expenditures. These efforts might address the deficiencies and discrepancies in monitoring and reporting of operational costs that are highlighted in our review of the literature. Future research might build on the work presented here using more sophisticated multivariate analysis techniques to explore the extent to which each of the factors identified above  explain and influence operational costs. Future research efforts might also seek to tie operational costs observed to intermediate outcomes such as coverage, or better still final outcomes like confirmed levels of population immunity so as to understand how different levels of investment in SIAs affect outcomes.Health systems cost of delivering routine vaccination in low & middle-income countries: What is needed over the next decade?, Submitted), but where successful, these efforts should provide a broader platform for ADC/E/E programs to reach all communities – ultimately reducing the marginal costs of vaccinating each additional child as illustrated by our findings. By contrast however, there are also calls to exploit the visibility and resources of SIAs in order to strengthen routine immunization , and there is evidence that such approaches are feasible and can have a positive impact [follow-up SIAs – where system strengthening efforts are often embedded – to wide age range catch-up SIAs where no system strengthening is undertaken) that strengthening routine programs through SIAs will increase the per person costs of those campaigns .There are a number of areas that have yet to be fully explored where future research/monitoring efforts may focus – most notably to tease out the impact on SIA operational costs of routine system strengthening efforts embedded within SIAs. ADC/E/E program strategies are again pointing to the fact that the key to their success lies not just in high-quality and well-funded SIAs, but in effective routine immunization programs built of strong health systems . Creatine impact -57. WhilNew research might also focus on the effect of growing security needs on operational costs. Recent attacks on vaccinators, particularly those conducting OPV SIAs in remote areas of the remaining polio endemic countries, are hampering ADC/E/E efforts ,59. AddiFinally, the focus of new studies may need to go beyond existing ADC/E/E programs discussed here. As aforementioned, several underutilized and new vaccines that seem likely to be introduced in the coming years have an SIA delivery component as one-time catch-up strategies, or to periodically maintain herd immunity. Better understanding of the costs of SIAs and how these are likely to change over time will enable more robust assessment of future ADC/E/E policies  and helpaEncompassing epidemic prevention.bOperational costs are normally expressed on a “per person” basis. In some instances, these data are expressed on a “per dose” basis. Where there was insufficient information in the paper to express the information on a per person basis, we excluded the paper.cThese were the vaccines and programs for which we had sufficient data to draw out vaccine delivery -specific findings; i.e. for menA SIAs targeting the populations below 29 years of age, or yellow fever SIAs targeting those below 60 years of age, there were insufficient observations to assess how operational costs for these vaccines/age groups compared to OPV, measles and TT/Td vaccines.dSince Albendazole/Mebendazole for de-worming and Vitamin A (VitA) are often delivered in campaigns and because many countries have estimated the operational costs of delivering these interventions within their cMYPs, these were included in the per person average costs of orally administered interventions along with OPV.eThese population grouping cut-offs were determined based on the countries included in the analysis dataset, balancing a need to ensure sufficient observations in each grouping to explore meaningful differences in operational costs for small versus midsize/large country both by vaccine, route of administration, and overall while simultaneously grouping similar sized countries together .fThese cut-offs were selected based on published policy/strategy targets associated with the third dose of Diphtheria-Tetanus-Pertussis (DTP3): The GAVI eligibility policy permits countries to apply for most of the routine new/underutilized vaccines so long as they have DTP3 coverage equal to, or greater than 70% [than 70% . Separatthan 70% ,63. HowegAs aforementioned, and unpublished analysis of a larger dataset including actual expenditures and projected operational cost data is presented elsewhere . The larger dataset covered 257 SIAs and was extracted from 87 unique cMYPs produced by 55 countries – Comparing the results generated from the larger but more heterogeneous dataset of 257 observations with the data/results presented in this paper, the estimates for the former were mostly lower than from the latter.hAlthough investments to cover core costs are often reported and/or projected by the global coordinators of ADC/E/E programs as part of financial resource requirements (FRRs). [Measles and Measles-Rubella SIA Forecast 2013–2018. Unpublished). (FRRs). ,  cMYP data-- the country- reported data extracted from individual cMYPs and adjusted for inflation. Used for the analysis of operational costs; (2) SIA budget data-- the country-reported data extracted from various sources and adjusted for inflation. Used for the analysis of the cost components that drive SIA costs; (3) Reference-style data—the demographic, immunization system performance, or economic data  from third party multilateral sources. Used to categorize country level data and/or create population-weighted average benchmarks.Click here for filecontains additional summary results and data used in analysis of SIA operation costs and SIA budget breakdowns.Click here for file


Viral diseases constitute obstacles to pepper production in the world. In Nigeria, pepper plants are primarily affected by pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), Pepper leaf curl Virus (TLCV), Tobacco mosaic virus (TMV), Pepper mottle virus (PMV) and a host of other viruses. The experiment was carried out with a diagnostic survey on the experimental field of the National Horticultural Research Institute, Ibadan, Nigeria and on pepper farms in six local government areas within Ibadan Oyo State, Nigeria, forty samples were collected from each of the farms. Diseased samples were obtained from the field and taken to the laboratory for indexing. In ELISA test some of the samples from the pepper farms showed positive reaction to single infection with PVMV (36.79%), CMV (22.14%) while some others showed positive reaction to mixed infection of the two viruses (10%) but some also negative reaction to PVMV and CMV antisera (31.07). Plant virus diseases cause major losses to agricultural crops around the world. Chemical agents similar to fungicides and bactericides are not effective in controlling virus diseases. Strategies for virus management in plant are mostly aimed at eradicating the source of infection to prevent it from reaching the crop and interfering with the movement of vectors in order to prevent the spread of the disease. However, the most effective means of controlling virus diseases is through cultivating the virus-resistant varieties.Generally, viral infection causes visible symptoms such as various forms of mosaic and distortions in plants with consequent reductions in crop growth and yield. While reduction in plant size is the most general symptom induced by virus infection, there is probably some stunting of growth even with 'masked' or 'latent' infections, where the systemically infected plant shows no obvious sign of disease . In natuPotato virus Y group but was later renamed the Potyvirus group [Capsicum annuum L. crops in Afghanistan [Capsicum spp. in Sierra Leone and Zaire, [Telfairea occidentalis (Cucurbitaceae) in Nigeria [Pepper veinal mottle virus (PVMV) was first recognized as a distinct member of a group of viruses which was originally designated the us group . PVMV ochanistan  and Indihanistan . PVMV ald Zaire, . PVMV had Zaire, ,9. There Nigeria . Strains Nigeria -14. Symp Nigeria . There h Nigeria .Myzus persicae, and by Aphis gossypii, but it can be transmittedby other species of aphids [Cucumber mosaic virus (CMV) is worldwide in distribution. The virus causing cucumber mosaic has perhaps a wider range of host and attacks a greater variety of vegetables, ornamentals, weeds, and other plants than other viruses . CMV is f aphids .In this paper we report the distribution of PVMV and CMV infection in pepper plants within Ibadan, Oyo state, Nigeria.Pepper veinal mottle virus, genus Potyvirus, and Cucumber mosaic virus, genus Cucumovirus was conducted during the 2009 planting season in seven locations in Ibadan, Oyo State in the southwest agro-ecological zone of Nigeria.Survey for The locations were the experimental field of the National Horticultural Research Institute (NIHORT), Lagelu Local government area, Akinyele Local government area, Egbeda Local government area, Ona Ara Local government area, Oluyole Local government area and Ido Local government area.Diseased Leaf Samples were randomly collected from the experimental fields containing pepper in NIHORT while in the Local government areas two cultivated pepper farms were randomly surveyed per Local government. On each site 20 plants were randomly sampled from the population of plants on the field. Forty plants were sampled per location from cultivated pepper plants showing symptoms of mosaic, chlorosis, yellowing, stunting and mottle making a total of two hundred and eighty samples in all. The sampled leaves were then stored under Calcium chloride and were placed in the refrigerator prior to indexing.2 PO4, 1.1 g Na2 HPO4 0.2 g KCl, 0.2 g NaNO3 in 1 l H2O + 0.5 ml Tween 20 (0.05%)) from the Virology Laboratory of the International Institute of Tropical Agriculture (IITA) Ibadan.International Iinstitute of Tropical Agriculture (IITA) Virology laboratory modified protocols for Direct antigen coating (DAC - ELISA) was used for the detection of the presence of PVMV and CMV both from the forty infected pepper leaf samples collected per location. The PVMV and CMV antibodies used were AAB 328 antiserum diluted in the ratio 1:1000 and 1:3000 respectively with Phosphate Buffered Saline (PBS-T)  was added per well and incubated at room temperature for one hour. For all incubations plates were covered with ELISA cover plates to avoid edge effects and to maintain uniform temperature. Healthy pepper plants (Capsicum sp.) were used as negative control while PVMV and CMV infected Capsicum sp were used as positive control. After one hour the absorbance was measured at 405 nm using multiscan ELISA reader. The samples were considered positive when the ELISA reading exceeded that of the healthy control or was at least twice the reading for the healthy control.One hundred micro litre of antigen (e.g. sap) ground at 0.1 g of leaf sample in 1 ml of coating buffer was dispensed into each well of ELISA plate. The plate was then incubated at 37°C for 1 h and later washed three times with PBS-T after the incubation period. Cross adsorption was made by grinding 1 g of healthy pepper leaf in 20 mls of conjugate buffer conjugate buffer . 100 μl of PVMV and CMV polyclonal (AAB 328) antisera diluted 1:1000 and 1:3000 in the adsorption solution was added to each of the ELISA plate and then incubated at 37°C for 1 h. After incubation the ELISA plate was washed three times with PBS-T. 100 μl of protein, A- alkaline phosphatase conjugate diluted in the ratio 1:15000 in conjugate buffer  was added per well and the plate incubated at 37°C for 1 h. The plate was washed three times with PBS-T. 100 μl of 0.001 g/ml of p-nitrophenyl phosphate substrate in substrate buffer (97 ml diethanolamine + 800 ml HAll the farms visited recorded the presence of PVMV and CMV as a single infection as well as mixed infection in some cases. Out of the two hundred and eighty leaf samples suspected to be virus infected collected from the farmers field in Ibadan, Oyo State of Nigeria 36.79% tested positive to PVMV, 22.14% tested positive to CMV, 10% tested positive to both PVMV and CMV through the serological test used, while 31.07% tested negative to the two antisera (Figure All the pepper farms surveyed within Ibadan showed differences in PVMV and CMV as observed in the percentage viral incidence Figure . The perPepper is highly susceptible to virus diseases in Ibadan, Oyo state, Nigeria as all samples picked from all the surveyed locations showed different viral symptoms ranging from different degree of mosaic, mottle, yellowing, stunting, puckering and in some cases reduction in leaf size which was similar to the earlier described symptoms of PVMV and CMV by Atiri  and ArogSolanum nigrum, S. gracil, Physalis angulata, Vigna rosea, Vigna sinensis, Commelina nudiflora, Petunia hybrida, Physalis floridana, P. micrantha and Solanum incanum.The high percentage incidence of PVMV and CMV in the experimental field of NIHORT could be as a result of numerous alternative host species surrounding the pepper field such as tomato, solanum, cucurbits and a host of other vegetables. This corroborates the findings of Alegbejo  who repo


There are insufficient data available on utilization and health care costs of non-alcoholic fatty liver disease. The cost data for different health conditions and services is a major gap in Iranian health system. So this study is the primary or first step towards filling this gap.This study aims to estimate the diagnosis and treatment costs of Non-alcoholic Fatty Liver.This cross-sectional study was conducted on 528 subjects. The subjects had been diagnosed with non-alcoholic fatty liver. All the subjects had been referred to the Tehran Fatty Liver Clinic, a clinic of the Baqiyatallah Research Center for Gastroenterology and Liver Diseases, in 2009 and they had been observed for 2 years to determine the frequency of health care utilization . The costs of diagnosis and treatment for each person were estimated in Purchasing Power Parity dollars (PPP$).The average total cost was 5,043 PPP$ per person in the 2 years of observation. Majority of these 528 patients (87.9%) had a BMI ≥ 25 (kg/m2). Also, 33.9% were diagnosed with comorbid diseases such as Diabetes Mellitus (DM), Coronary Artery Disease (CAD), hypertension (HTN) and hypothyroidism (HYPO).The results confirmed that the total costs for non-alcoholic fatty liver among the Iranian adult urban population alone exceeded 1 billion PPP$ per year. These costs can be saved or reduced by effective disease management and early prevention. Non-Alcoholic Fatty Liver Disease (NAFLD) is increasingly becoming the common cause of chronic liver diseases. One type of progressive NAFLD is known as Non-Alcoholic Steato-Hepatitis (NASH), which can lead to cirrhosis, hepatocellular carcinoma (HCC), liver dysfunction and ultimately death , 2. NonaObesity and metabolic syndrome, the most predictive factors of fatty liver disease, are increasing in Iran; therefore, the prevalence of NAFLD/NASH and related complications are also expected to increase in the future . AccordiFor this cross-sectional study, 528 patients with NAFLD were selected from patients' referred to the Tehran Fatty Liver Clinic, a private clinic of the Baqiyatallah Research Center for Gastroenterology and Liver Diseases during 2009. The patient selection criteria were based on:No history of alcohol consumptionNo history of drug consumption No history of chronic liver diseaseHad regular visits for check upsHad laboratory tests done regularlyPassed and completed a two year treatment periodThe inclusion criteria were the presence of NAFLD as suggested by ultrasonography and rule out of alcohol or drug consumption and liver disease. The ultrasonography criterion was a hyper echoic texture or a bright liver because of diffuse fatty infiltration. Sensitivity and specificity for the ultrasound were 85 and 94 percent, respectively. The procedure for the diagnosis of NAFLD was as follows:Evaluation of the serum aminotransferase Evaluation of BMI > 25 (kg/m2); BMI over 25 is for overweight and obese people Evaluation of some anthropometric measurement criteria including body weight and height as well as the size of the wristEvaluation of the metabolic syndrome such as Diabetes Mellitus (DM), Coronary Artery Disease (CAD), hypertension (HTN) and hypothyroidism (HYPO)And finally performance of ultrasound scan to identify NAFLDIn addition, some other variables including age, gender, behavioral risk factors like physical activity were also taken into consideration.In this cross-sectional study, the diagnosis and treatment costs of NAFLD were estimated. The factors used for cost estimation included frequency of health resource utilization and unitary costs. Health resource utilization included such categories as physician visits (Patients were followed every 3-4 months by repeated laboratory studies based on primary laboratory disorders), laboratory tests, medication and cost of ultrasonography. The unit cost of different health resources including physician  visits, laboratory tests, medication fees and the costs of ultrasonography were calculated based on the price lists approved in 2009-2011 by the Iranian Cabinet for the Public and Private Health Centers . The priDescriptive statistics and frequency distribution such as mean, standard deviation and percentage were employed. T-test and one-way ANOVA was used to test the differences between means of NAFLD-related costs. All statistical analyses were carried out using SPSS, version 17.0 . P < 0.05 was considered as statistically significant.In total, 528 patients with NAFLD were enrolled in this study out of which 320 patients (60.6%) were male and 208 (39.4%) were female. The mean age was 45.9 ± 10.8 (± standard deviation) years. The majority of them, 464 patients (87.9%), had a BMI ≥ 25 (Kg/m2). Also 179 individuals (33.9%) were diagnosed with comorbid diseases including DM, CAD, HTN and HYPO .Total costs of NAFLD patients with and without comorbid diseases, and also with BMIs over 25 (over-weight and obese people) have been shown in Although the results indicated that the charges were higher in overweight and obese patients, yet these differences did not reach statistical significance . This table also indicates no relationship between comorbid diseases and health care utilization and NAFLD-related costs (P = 0.649).In this study, data was collected from NAFLD patients’ files from a Tehran fatty liver private clinic from which costs of physician visit, laboratory tests, medication and ultrasonography was extracted from the price lists approved by the government and/or food and drug organization. It is worth noting that in estimating the unit costs of each health service, the minimum applicable cost was taken into account. This might have caused these estimated costs to be slightly lower than the actual costs. Although the charges were higher in overweight and obese patients, these differences did not reach statistical significant. One might assume that changes in patients' lifestyles, after the diagnosis of NAFLD, might have affected the results. The reason is that overweight and obese people, in this study, were first recommended to change their lifestyles by physicians. Also, the costs of the prescribed drugs, such as Orlistat, which reduce fat absorption and promote weight loss are much lower in comparison with the costs of other drugs in other countries , 22. In


Controlling a pandemic with vaccine and antiviral drugs will require a coordinated international approach to determine how the least amount of virus can immunize the largest segment of a population. While measures such as closing schools and social distancing may slow the effects of pandemic influenza, only vaccines and antiviral drugs are clearly efficacious in preventing infection or treating illness. Unless the pandemic strain closely resembles one already recognized, vaccine will not be available early. However, studies can be conducted beforehand to address questions concerning vaccine dose, frequency of inoculation, and need for adjuvants. In contrast, antiviral drugs, particularly the neuraminidase inhibitors, will be effective for treatment and available if stockpiling takes place. Special questions need to be answered if a highly lethal virus, such as influenza A (H5N1), produces the pandemic. Both vaccines and antiviral drugs will be required for a coordinated strategy. Prevention of influenza, particularly during a pandemic, may be attempted by many measures, such as closing schools, using facemasks, and keeping infected persons away from those susceptible, now termed social distancing. However, none of these measures are of clear value in preventing infection, even if they could be accomplished. A principal reason little effort has been made to determine their usefulness in the interpandemic period is the usual availability of vaccine, which is of known value in prevention. Thus, few studies have been undertaken. Similarly, symptomatic therapy is possible and perhaps appropriate in treating milder illnesses. Antimicrobial drugs are necessary when bacterial complications occur. However, antiviral drugs are specific and can not only prevent infection but also treat illness , will be effective against any pandemic virus, and stockpiling is possible  return, in keeping with the recycling theory? Much of the population will now be susceptible. Type A (H9N2), a less pathogenic avian virus, has transmitted occasionally to humans, with little or no further transmission, but has not produced disease with high case fatality , a known quantity, because of its presence from 1957 to 1968, in terms of immune response, population likely to be infected, and expected disease characteristics. Also, that virus presents the fewest issues about vaccine production, for the same reasons. However, the basic question relates to producing the best immune response with the least amount of antigen and avoiding if possible the need for a second injection, which would use additional antigen and delay production of protective immunity. One approach, already studied, is to leave the harvested virus particles intact, the modern equivalent of the whole-virus vaccines evaluated in 1976  virus presents many more problems in vaccine development and evaluation. The first one, already solved, involves removal of the molecular motif of high pathogenicity, the multibasic cleavage site, from the hemagglutinin. The virus is then reassembled by using reverse genetics, but on a background of the high-growth type A virus, PR8  and A (H9N2), treatment success in the interpandemic period would be more likely relevant to the pandemic. Such may not be the case with the type A (H5N1) virus. The virus has evolved since the 1997 Hong Kong outbreak, and some evidence of a systemic infection involving the brain and gastrointestinal tract exists  virus produces the pandemic and does not change in virulence or its tendency to infect the young, vaccination priorities would have to be changed radically.Once the pandemic virus is available, a rapid evaluation will be needed to address questions of dosage, need for adjuvants, and booster vaccination. However, this evaluation will need to be done quickly, especially for regions of the world close to the pandemic origin, so as much work as possible should be done before the pandemic. First, though, a virus for vaccine production will need to be created from the pandemic strain, with appropriate manipulation to make it high yielding. In the process, the molecular and antigenic differences between this virus and those of the same subtype already available will need to be defined. With luck, the pandemic virus may be similar enough to one already studied so that any available concentrates can be used. However, similarity is unlikely because of the antigenic variation of influenza strains within a subtype. Rather than stockpiling, another strategy needs to be considered for vaccines containing a virus such as A (H5N1) for which vaccine development has already begun. That virus can be included in vaccines in use before the pandemic. Although influenza A (H5N1) virus has been evolving, even a poorly matched vaccine might provide some protection, especially against a variant with such high lethality  virus, has assumed that adamantanes would not be useful. This assumption means that if the disease is systemic and case fatality is high, among the NAIs only oseltamivir would be useful, since it is absorbed (Ro or basic reproductive number is not high (Given the threat of a virulent virus such as A (H5N1) and the suggestion that adaptation to transmissibility may occur gradually, the concept has emerged that antiviral drugs may be used to interrupt early, local transmission. The aim would be to prevent spread out of the region of origin, in other words, extinguishing the epidemic at its source  virus similar to those currently circulating. Some are specific to the particular intervention, but others are more generic. Long-term needs exist, such as developing innovative technologies for vaccine prevention and designing antiviral drugs to affect different targets. However, immediate attention for vaccines must be directed to a coordinated international approach to vaccine evaluation, paying attention to ways in which the least amount of virus can immunize the largest number of persons. Use of a possibly unmatched A (H5N1) vaccine for priming should be considered, especially in Southeast Asia, or other areas with the most pressing need. In those regions, antiviral strategies need to be evaluated; drug studies in animal models will be necessary, given the sporadic nature of the disease in humans. Overall, developing countries will have limited access to vaccines and antiviral drugs, and their needs must not be forgotten. With marginal healthcare infrastructures, they will suffer the most, whatever the severity of the pandemic.


Duvenhage virus was isolated from a patient who died of a rabieslike disease after being scratched by a bat early in 2006. This occurred ≈80 km from the site where the only other known human infection with the virus had occurred 36 years earlier. The genus Lyssavirus within the family Rhabdoviridae currently includes rabies virus (RABV) (genotype 1) and 6 rabies-related viruses: 3 from Africa, Lagos bat virus (LBV) (genotype 2), Mokola virus (MOKV) (genotype 3), and Duvenhage virus (DUVV) (genotype 4); European bat lyssaviruses 1 and 2 (EBLV1 and 2) (genotypes 5 and 6); and Australian bat lyssavirus (ABLV) (genotype 7)  has never been isolated from bats outside North and South America, but rabies-related viruses have been isolated from bats elsewhere. In Africa, LBV and DUVV are associated with bats, but MOKV is uniquely associated with shrews and rodents, not bats. Fifteen isolations of LBV have been reported, including 8 from fruit bats and a cat in KwaZulu-Natal Province of South Africa, but the virus has never been associated with human disease , tachycardia, neck and general limb rigidity, hyperreflexia, facial fasciculation, and involuntary grimacing. Within 24 hours generalized tonic-clonic seizures had developed with status epilepticus supervening. These necessitated intubation, sedation, and mechanical ventilation. He died on day 14 of his illness.Heminested reverse transcription–PCR was performed as previously described , and liv,,,–The ability to distinguish between various lyssaviruses and monitor their relative distribution and prevalence has important implications for implementation of control measures. It was recognized in 1932 that mongoose-associated rabies in South Africa differs from classic dog rabies (


Genotype specific deletions in the untranslated region between the non-structural and structural open reading frames were observed for genotypes 2B and genotype 1G. No evidence was seen for recombination events among the 30 viruses. The analysis presented here is consistent with previous reports on the genetic characterization of rubella virus genomes. Conserved and variable regions were identified and additional evidence for genotype specific nucleotide deletions in the intergenic region was found. Phylogenetic analysis confirmed genotype groupings originally based on structural protein coding region sequences, which provides support for the WHO nomenclature for genetic characterization of wild-type rubella viruses.Rubella virus is the causative agent of rubella, a mild rash illness, and a potent teratogenic agent when contracted by a pregnant woman. Global rubella control programs target the reduction and elimination of congenital rubella syndrome. Phylogenetic analysis of partial sequences of rubella viruses has contributed to virus surveillance efforts and played an important role in demonstrating that indigenous rubella viruses have been eliminated in the United States. Sixteen wild-type rubella viruses were chosen for whole genome sequencing. All 16 viruses were collected in the United States from 1961 to 2009 and are from 8 of the 13 known rubella genotypes. Phylogenetic analysis of 30 whole genome sequences produced a maximum likelihood tree giving high bootstrap values for all genotypes except provisional genotype 1a. Comparison of the 16 new complete sequences and 14 previously sequenced wild-type viruses found regions with clusters of variable amino acids. The 5 Rubivirus genus of the Togaviridae family. The virus causes a mild childhood disease, but is also a potent teratogenic agent when contracted by a pregnant woman. Therefore, the goal of rubella control and elimination programs is the reduction or elimination of the congenital rubella syndrome (CRS) that occurs in 90% of infants whose mothers were infected with rubella in their first trimester [′ proximal ORF encodes the non-structural proteins (NSP) P150 and P90. The 3′ proximal ORF encodes the structural proteins (SP), the capsid (C) and two glycoproteins, E2 and E1. There are 3 untranslated regions (UTRs) in the rubella virus genome: a 40-nucleotide (nt) sequence at the 5′ terminus, an approximately 120-nt intergenic region (IR) between the two ORFs, and a 59-nt region at the 3′ terminus.Rubella virus (RV) is a positive-polarity, single-stranded RNA virus and the sole member of the rimester . The gen′s, a decade when a world-wide epidemic of rubella occurred, resulting in 20,000 cases of CRS in the United States alone [′s, mainly in industrialized countries. No further global epidemics were recorded and interest in collecting and characterizing rubella viruses waned. As a result, very few virus isolates exist from the 1970′s, 1980′s and the early 1990′s. In the 1990′s efforts to improve rubella control were strengthened globally as evidenced by an increase in the number of countries including rubella-containing vaccine in their national immunization programs (from 43% of WHO member states in 1996 to 67% of member states in 2009) [There are currently whole genome sequences for 21 RVs in Genbank . Almost half of the sequences (11/21) are from wild-type and vaccine viruses from the 1960′s and 2000′s. In 2005, a systematic nomenclature was adopted by the WHO [The use of molecular epidemiology was recognized as a powerful tool for virus surveillance and isolations of RVs to be used for this purpose greatly increased in the late 1990 the WHO ,5. Brief the WHO  and mole the WHO . Importe the WHO ,5, obtai′ 21 nts) were compared with 14 wild-type genomic sequences available from Genbank  possesses 31% amino acid variability while the amino terminus of Q-domain (aa 497–717) was less variable with changes less than 20%. Cluster III began at the junction between the X-domain (X), also known as the Macro domain, and the protease domain (Pro) and ends near the N-terminus of Pro  overlapped with the major RNA binding domain of the C protein (aa 28–56) . ClusterIn addition to clusters of variable amino acids, regions with little amino acid variation were also identified among the 30 viruses. In general, the putative methyltransferase (MT), helicase (Hel) and RNA-dependent RNA polymerase (RdRp) were less variable and E1 was the least variable among the SP. Among these 30 sequences at the nucleotide level, the percentage of variable nt positions (at least one virus differed from the consensus sequence) across the whole genome was about 26%; approximately 23% of the nucleic acids were variable in the Hel, RdRp and E1. The MT, containing 12% variable nucleic acids, was the most conserved region  while t′ terminus of the genome, while the nucleotide sequence was more variable in the C and E1 coding regions, with only 73% identity. The presence of highly conserved nucleotide sequences suggests their importance in virus replication and may potentially be used as targets for molecular detection of RV. It is not known whether the highly conserved 5′ 250-nt region contains critical secondary structures. Two conserved regions in the IR were also noticed which could be critical for the regulation of subgenomic RNA synthesis. Nevertheless, analysis of the 30 sequences also revealed genotype-specific deletions in this region: 6 genotype 2B viruses in this report had a single deletion at 6422 and two 1G viruses had nucleotides 6480 and 6481 deleted, indicating the flexibility of the IR in length. This dinucleotide deletion has also been found in 1h and 1i viruses, providing strong evidence that genotypes G, h, and i diverged from a common ancestor. Unlike two previous reports [′ termini for three viruses; however, the evidence was not considered significant.In addition to variable regions, regions containing conserved peptides of more than 30 consecutive identical amino acids were identified among the 30 viruses. The p90 is more conserved than p150 and SP. This includes a region of 114 consecutive identical amino acids in the putative RNA-dependent RNA polymerase domain. The nucleotide sequences coding for these conserved peptides were 80% identical overall; however the nucleotide identity was greater than 90% near the 5′s and 2000′s. One of the virus isolates is a laboratory strain (NJ.USA/61/1a), commonly known as M33  which was the first rubella virus isolate [Sixteen wild-type rubella virus isolates from the Centers for Disease Control and Prevention (CDC) were chosen for whole genome sequencing at the J. Craig Venter Institute (JCVI) Table . All vir isolate . There a NJ.USA/6/1a, commStocks of the 16 viruses were inoculated into T75 flasks of confluent Vero cells. Five to seven days post-infection, the culture medium was removed and total cellular RNA was extracted using Tri-Reagent  according to the manufacturer’s protocol. The RNA was resuspended in nuclease-free water and stored at −80°C.′ and 3′ termini were determined using the 5′/3′ RACE kit as directed by the manufacturer  and oligo dT priming, respectively. The termini sequences were incorporated into the whole genome sequences as described above.Oligonucleotide primers were designed using an automated primer design tool ,25. CladThe sequences were aligned by ClustalW in the MEGA 4.0 software . PhylogeRV: Rubella virus; CRS: Congenital rubella syndrome; ORF: Open reading frame; NSP: Non-structural proteins; SP: Structural proteins; C: Capsid protein; UTR: Un-translated region; nt: Nucleotide; IR: Intergenic region; HVR: Hyper-variable region; aa: Amino acid; X: X-domain; Pro: Protease domain; MT: Methytransferase domain; Hel: Helicase domain; RdRp: RNA dependent RNA polymerase; JCVI: J.Craig Venter Institute; CDC: Centers for Disease Control and Prevention.The authors declare that they have no competing interests.Virus RNA preparations were done by EA. Sequencing was performed by JB, EK, and EA. Sequence analysis was done by MHC, EA, SS, and QZ. The manuscript was prepared by EA, MHC, WB and JPI. All authors read and approved the final manuscript.The findings and conclusions in this report are those of authors and do not necessarily represent the views of the U.S. Department of Health and Human Services.Primer sets designed for sequencing of rubella virus clades 1 and 2.Click here for file


Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers. Filoviridae); Lassa virus (LASV) and the South American hemorrhagic fever viruses Guanarito virus, Junín virus, Machupo virus, and Sabiá virus (Arenaviridae); Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and hantaviruses (Bunyaviridae); and Kyasanur Forest disease virus (KFDV), Omsk hemorrhagic fever virus, yellow fever virus (YFV), and dengue viruses (Flaviviridae)  warrant specific emphasis because of their high rates of illness and death, and the potential for rapid dissemination by human-to-human transmission. The term "viral hemorrhagic fever" characterizes a severe multisystem syndrome associated with fever, shock, and bleeding diathesis caused by infection with any of several RNA viruses, including Ebola virus and Marburg virus (MARV)  in the context of differential diagnosis of respiratory disease , Ebola Sudan virus (SEBOV), MARV, LASV, RVFV, CCHFV, Hantaan virus (HNTV), Seoul virus (SEOV), YFV, and KFDV. Oligonucleotide primers were designed in conserved genomic regions to detect the broadest number of members for a given pathogen species. We developed a software program that culls sequence information from GenBank, performs multiple alignments with ClustalW, and designs primers optimized for multiplex PCR. The program uses a greedy algorithm to identify conserved sequences and create the minimum set of primers for amplification of all sequences in the alignment. Primers are selected within standard design constraints whenever possible ; degenerate positions are introduced in cases where template divergence requires more flexibility. Although degeneracy is not tolerated in the five 3´ nucleotides, MassTag PCR allows up to 4 nonneighboring variable positions per primer. Primers are checked by the basic local alignment search tool for potential hybridization to sequenced vertebrate genomes .Because only released mass tags are analyzed, staggering the size of amplification products created in multiplex reactions is unnecessary; thus, primers are selected for efficient and consistent performance irrespective of amplicon size . Before committing to synthesis of tagged primers, the functionality of candidate multiplex primer panels is examined in a series of amplification reactions that use prototype templates representing individual microbial targets. Primers that fail to yield a single, specific product band in agarose gel analysis are replaced. Target sequence standards for evaluation are cloned into pCR2.1-TOPO  by using PCR amplification of cDNA templates obtained by reverse transcription (RT) of extracts from infected, cultured cells or by assembly of overlapping synthetic polynucleotides.The agents assayed in the VHF panel have RNA genomes; thus, assay sensitivity was determined by using synthetic RNA standards. Synthetic RNA standards were generated from linearized target sequence plasmids by using T7 polymerase . After quantitation by UV spectrometry, RNA was serially diluted in 2.5 μg/mL yeast tRNA , reverse transcribed with random hexamers by using Superscript II (Invitrogen), and analyzed by MassTag PCR as previously described . SensitiTissue culture extracts were used to examine assay specificity. Random primed cDNA obtained from cultures of ZEBOV, SEBOV, MARV, YFV isolates from the Gambia and Côte d’Ivoire, RVFV, CCHFV, HTNV, SEOV, and LASV strains Josiah, NL, and AV were subjected to mass tag analysis. In all instances, only the appropriate cognate mass tags were detected (data not shown). No spurious signal was identified in assays with water or RNA controls.Performance with clinical materials was tested by using blood, sera, or oral swabs from 24 human patients of VHF previously diagnosed through virus isolation, RT-PCR, or antigen detection enzyme-linked immunosorbent assay. Differential diagnosis by blinded MassTag PCR analysis was accurate in all cases . For theThese results confirm earlier work in respiratory diseases that show that MassTag PCR offers a rapid, sensitive, specific, and economic approach to differential diagnosis of infectious diseases. Small, low-cost, or mobile APCI-MS units extend the applicability of this technique beyond selected reference laboratories. Given the capacity of the method to code for up to 32 genetic targets, we are expanding the hemorrhagic fever panel to include additional viruses (dengue and South American hemorrhagic fever viruses) and are exploring the inclusion of bacterial and parasitic agents that may result in similar clinical signs and symptoms and, thus, have to be considered in differential diagnosis.


In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.During November-December 2009, we collected leftover serum from a blood bank, a pediatric children's hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults. The 2009 pandemic influenza A (H1N1) virus (pH1N1) was first identified in April 2009 and caused widespread illness in the United States and around the world During the 2009 pandemic, Florida employed a surveillance system that tracked the percentage of Emergency Department (ED) visits for influenza-like illness (ILI) throughout the state. According to surveillance data, Tampa Bay experienced a gradual increase in influenza activity in the spring and summer of 2009, followed by a large fall wave of influenza activity that peaked in late October and decreased steadily thereafter . EstimatSerosurveys, which estimate the prevalence of antibodies to a specific pathogen, can be a valuable tool in determining the proportion of the population infected with a novel virus. Unlike most influenza surveillance, which relies on presentation of clinical illness, serosurveys capture persons that experienced symptomatic or asymptomatic illness, and can provide information on total infections which may be underestimated with traditional surveillance methodologies. However, serosurveys are limited by the sensitivity and specificity of the assay employed to detect antibody titers To date, one published study has reported on the prevalence of pH1N1 antibodies among residents in one region of the United States In November and December 2009, after pH1N1 virus activity in Tampa Bay had peaked , we collWe sought to collect 160 specimens from each of six age groups: <5 years, 5–17 years, 18–24 years, 25–49 years, 50–64 years and ≥65 years. Infants less than 6 months were excluded due to the potential for maternal antibody transmission. The required sample size was calculated using relative standard error measurements. We estimated that the lowest seroprevalence for all groups would be among adults aged ≥65 years. At the time of the serosurvey, we estimated that 15% of this age group would be seropositive, requiring a sample size of approximately 160 to maintain a relative standard error less than 20%. We were able to collect at least 160 samples from all ages groups except for children aged <5 years for which we were only able to collect 60 samples. We estimated that the seroprevalence among this age group would be high (30%), and therefore despite the small sample size, would meet the relative standard error criteria of less than 20%.This study was proposed to the Florida Department of Health Institutional Review Board (IRB) and Centers for Disease Control and Prevention (CDC) IRB who considered the investigation as public health response, and therefore not subject to IRB review and approval.Leftover serum specimens for Tampa Bay residents aged ≥16 years were collected from a large blood bank testing facility during a 4-day period from November 30 -December 3, 2009 . For resAntibodies against pH1N1 virus were detected by the hemagglutination-inhibition (HI) assay as previously described using A/California/07/2009 virus Using sera from patients with pH1N1 laboratory-confirmed infections and non-exposed United States residents, a previous study determined that a threshold HI titer ≥40 yielded a sensitivity of 75% and specificity of 97% in determining previous infection with the pH1N1 virus among persons <60 years of age Because serology cannot differentiate between antibodies produced by virus infection and response to vaccination, we developed a simple statistical model to estimate the proportion of seropositive results due to pH1N1 vaccination coverage  . MonthlyOverall, 27% of the study sample had pH1N1 antibody titers ≥40 with an age-standardized seroprevalence of 25% . A cumulThe overall BRFSS/NHFS vaccination coverage estimate for Florida two weeks prior to the time of specimen collection was 9.0%, with the highest vaccination coverage among children aged <5 years (17%) and school-aged children aged 5–17 years (15%) . Based oThe proportion of Tampa Bay residents that were estimated to be seropositive due to infection with pH1N1 virus after the peak of the fall wave was 25% . The higBy December 2009, an estimated 30% of Tampa Bay's population had elevated levels of antibodies against the pH1N1 virus (25% from infection and 5.9% from vaccination). Our estimates indicate that half of young adults aged 18–24 years and more than half of school-aged children had antibodies to the pH1N1 virus at titers of ≥40 at that time. Thus, the proportion of the Tampa Bay population among these age groups that remained susceptible to pH1N1 virus infection by December 2009 was markedly decreased.Our seroprevalence estimates are similar to previously published studies from Pittsburgh, PA Though the availability of pH1N1 vaccine was still limited by the time of the serosurvey , school-aged children comprised one of the target groups for initial vaccination campaigns, and thus were more likely to receive the vaccine earlier Our survey has several potential limitations. First, we did not have baseline serum specimens prior to the 2009 pandemic for comparison and therefore were not able to test for a four-fold rise in antibody titers or adjust for pre-existing, cross-reactive antibody titers. Previous studies have suggested that cross-reactive antibodies were most common among persons aged ≥60 years In summary, we performed a serosurvey in Tampa Bay to determine the prevalence of antibodies to the pH1N1 virus after the fall wave of the pandemic, and during the early phases of the pH1N1 vaccination campaign in Florida. We adjusted seroprevalence results to account for HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination. Our results provide evidence for substantial immunity against the pH1N1 virus among the Tampa Bay population. Though disease activity decreased after December 2009, vaccination levels continued to increase in Florida; thus the proportion of the population with immunity to the pH1N1 virus by the end of the pandemic was probably higher than the estimates presented herein.Appendix S1Method of seroprevalence adjustment to account for hemagglutination-inhibition (HI) assay sensitivity and specificity.(DOCX)Click here for additional data file.Appendix S2Simple statistical model used to estimate the proportion of seropositive results due to vaccination.(DOCX)Click here for additional data file.Table S1Statistical model to estimate the proportion of Tampa Bay residents with vaccine-induced pH1N1 virus seropositivity in November- December 2009, including all components and equations.1 Estimated from Behavioral Risk Factors Surveillance System (BRFSS) and National pH1N1 Flu Survey (NHFS)2 Vaccine immunogenicity estimates based on published immunogenicity studies 3 Estimated proportion of population with vaccine-induced seropositivity (≥1∶40 GMT)  =  (vaccine coverage) x (proportion with ≥1∶40 antibody response)4 Seroprevalence adjusted for assay sensitivity and specificity. For children and adults aged <65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 97%. For adults aged ≥ 65 years, assay-adjusted seroprevalence was calculated using a sensitivity of 75% and a specificity of 94% 5 Estimated proportion with pH1N1 virus infection prior to vaccination  =   x (estimated proportion of population with vaccine-induced seropositivity)6 Proportion with vaccine-induced seropositivity not infected prior to vaccination  =  (estimated proportion of population with vaccine-induced seropositivity) minus (estimated proportion of population with pH1N1 virus infection prior to vaccination)7 Proportion infected with pH1N1 virus  =   minus (proportion with vaccine-induced seropositivity not infected prior to vaccination).(DOCX)Click here for additional data file.


To the Editor: In recent years, Aedes albopictus, a mosquito vector of dengue and chikungunya viruses, has rapidly expanded in Europe. Since 2007, the presence of viremic patients with imported cases of dengue and chikungunya virus infection has resulted in several incidences of autochthonous transmission of the viruses in Italy, France, and Croatia  instructions. For chikungunya diagnosis, microchip technology  was performed to detect specific IgM and IgG antibodies. Paired acute- and convalescent-phase samples with a Laboratory diagnosis was matched to clinical observations; travel history, along with previous vaccination for yellow fever; and tickborne or Japanese encephalitis. Local infection was differentiated from imported infection by interpretation of a questionnaire that is required from treating physicians before serologic testing. Testing for West Nile virus and Sindbis virus were done to rule out cross-reactions with endemic flaviviruses and alphaviruses, respectively.A. albopictus mosquitoes in Israel during 2008–2009 was observed by monitoring the presence of eggs, larvae, and adult mosquitoes in ovitraps and by monitoring reports from municipalities and pest management professionals  dengue and chikungunya patients to prevent the establishment of autochthonous transmission.In summary, we report conditions in Israel suitable for autochthonous transmission of dengue and chikungunya viruses. Although no autochthonous cases have been reported in Israel, they have been reported from other countries where


Headache occipital neuralgia is an example of pain-disease for which treatment both pharmacological protocols and invasive methods are used. Among the latter, the RF (Radiofrequency) pulse-dose has been of interest for the prospects of analgesic efficacy, safety and patient compliance, although at the moment only data concerning the pulsed RF and not the RF pulse-dose, that represents its evolution, are discussed in scientific literature. The purpose of this study is a “simple” economic evaluation of this method in headache occipital neuralgia. I.Essential headache recognizes a significant economic impact, the assessment of which of course are not only included the costs of treatment. There is no single therapeutic option and, if on the one hand the drug therapy is to be distinguished in therapy of acute and prophylactic therapy, the other for the latter no protocol showed a clear superiority over other -2-3. ThiII.The aim of our study was to verify the presence of any economic advantage for the Health System of the Campania region resulting from the RF pulse dose compared to headaches drug protocols. In our economic analysis we have used the break even analysis, which identifies the point (= break even point), in which the revenues obtained (avoided costs) equal the total costs incurred for a period of 24 months. We considered as avoided costs the prices of drugs administered to a group of 22 patients , afferent (from January to September 2011) to Pain clinic of the University “Federico II” of Naples, suffering from occipital neuralgia and subjected first to drug therapy and, in a second time, the pulse dose RF. The total costs, although generally comprising a fixed part and a variable part, in our case, detailing the costs of treatment with Pulse Dose RF, are only fixed. . Regarding drug therapy, we considered the monthly cost per patient, the weighted average based on three different protocols followed by our patients . Cost are reported by  15 patieIII.At the end of our economic evaluation, the fixed costs amount to 2,115 euros, while the avoided costs for 12 months amounted to Euro 1416, where the break even point is set at 18 months .In practice this means that the Pulse Dose RF would be economically advantageous for the Campania Region SS with respect to drugs only if the term had analgesic efficacy equal to or greater than 18 months.In our patients the Pulse Dose RF resulted in a reduction of VAS greater than 60% compared to baseline with a significant improvement in quality of life within three and six months from performing the procedure.IV.From a strictly economic point of view, our study presents a simple model of cost-effectiveness and indicates that in practice if you want to conduct efficiency studies of the Pulse Dose RF, you should perform a follow-up of at least 18 months. On the other hand, for the specialist in pain management, the minimum time limit of 18 months has a relative weight if it occurred to her patients a significant reduction in pain with improved quality of life and overall satisfaction for a significant shorter period. In fact, considering the socio-economic impact of a disease and its treatment, medication-economic studies should take into account several aspects: Van Zundert , in an a- Direct costs, specifically attributable to its diagnosis and/or therapy .- Indirect costs, which relate to the psychological and social scope of the patient , and the impact caused to the company (decreased job activity).In describing this method, the Author follows the pattern presented previously by Tugwell and coll. that conIt ’s clear that the former are easily quantifiable, while the latter can be investigated only through the administration of particular questionnaires that assume a statistically significant value only on large patient populations, in the condition of homogeneity. Improving the quality of life and patient satisfaction ultimately represents a reduction of indirect costs and, if you had the opportunity to monetize QoLi score improvement, this would affect positively on the economic advantage. Currently, the tests used to measure various aspects of quality of life are subjective and, to have statistical significance, requires a large and homogeneous sample.V.Currently, this work aims only propose some reflections on a promising technique used for occipital headache as other painful conditions, but for which the scientific literature needs better and more systematic studies. Finally, considering the limited data in the literature, we could evaluate the efficacy and efficiency of the pulse dose RF and continuous RF in occipital headache. The pulse dose RF is a technique of neuromodulation, so that its effect is reversible and repeatable. Giving a response limited in time, however, it will be necessary for the patient to undergo several sessions after months (the duration of effect is subjective). The continue RF, instead, is a neurolesive technique: it offers the advantage of a long-lasting analgesic effect but also the risk of developing deafferentation pain.


In Cambodia, influenza A(H5N1) virus surveillance at live poultry markets (LPMs) relies on virus isolation from poultry specimens; however, virus is rarely detected by this method. We tested 502 environmental LPM samples: 90 were positive by PCR, 10 by virus isolation. Virus circulation could be better monitored by environmental sampling of LPMs. Highly pathogenic avian influenza (HPAI) A(H5N1) virus was first detected in 2004 in Cambodia. Since then, outbreaks of subtype H5N1 infection among poultry have been regularly detected, and 21 human cases associated with 19 deaths have been recorded  dropped by birds were gathered and placed in sterile plastic bags.Virus in water, soil, and mud samples was concentrated, as described  were positive for subtype H5N1 virus by qRT-PCR, and 10 (2%) were positive by virus isolation . We did Compared with the market in Kampong Cham (M4), the markets in Phnom Penh and Takeo (M1–3) had a higher percentage of samples positive for H5N1virus . Overall, the level of environmental viral contamination in LPMs was highest at the beginning of the study  and corresponded with intense movement of poultry within the country and higher densities of poultry populations on farms .The full genomic sequence of 4 strains and the hemagglutinin sequence of 3 other isolates were generated (GenBank accession nos. JQ673600–JQ673606). Phylogenetic analyses showed that the virus strains detected during this study belong to lineage 6, a group of viruses that seems to be endemic to Cambodia , and notHPAI A(H5N1) virus circulation in Cambodia has traditionally been monitored by using the moderately sensitive egg inoculation method to test cloacal and tracheal swab samples from birds randomly selected from LPMs or farms. Our results show that a more effective approach—especially before and during the main annual festivals, when the movement of poultry within the country is increased—would be to use highly sensitive qRT-PCR to test environmental samples from LPMs. In our study, water samples proved to be the best choice for isolation of infectious subtype H5N1 virus . The lowIn Cambodia, birds not sold the same day they arrive at a live poultry market are transported to an overnight resting place, which is sometimes another market (