Lynx lynx) from Sweden. Thymidine kinase gene amplicon sequences (339 bp) from 21 lynx were all identical to those from cowpox virus isolated from a person in Norway and phylogenetically closer to monkeypox virus than to vaccinia virus and isolates from 2 persons with cowpox virus in Sweden. Prevalence was higher among animals from regions with dense, rather than rural, human populations. Lynx are probably exposed to OPV through predation on small mammal reservoir species. We conclude that OPV is widely distributed in Sweden and may represent a threat to humans. Further studies are needed to verify whether this lynx OPV is cowpox virus.Cowpox virus, which has been used to protect humans against smallpox but may cause severe disease in immunocompromised persons, has reemerged in humans, domestic cats, and other animal species in Europe. Orthopoxvirus (OPV) DNA was detected in tissues  in 24 (9%) of 263 free-ranging Eurasian lynx ( Poxviridae, genus Orthopoxvirus [OPV]) was originally considered to infect milking cows and to have zoonotic potential. Because of its relationship to variola virus and immunologic cross-reaction, cowpox virus was used to protect humans against smallpox (––Cowpox virus (family Panthera leo), cheetahs (Acinonyx jubatus), black panthers (Pantherapardus), ocelots , jaguars (Panthera onca), pumas (Felis concolor), leopard cat , Pallas cat (Otocolobus manul), and domestic cat (Felis catus)—during an outbreak in the Moscow Zoo in 1973–1974 (–––Cowpox virus was first isolated from felid species—lions (Lynx lynx), red fox (Vulpes vulpes), and a brown bear (Ursus arctos)  , and emaciated .Most (245) animals submitted were apparently healthy , 15 had sarcoptic mange, and 3 died of starvation caused by noninfectious or unclear etiology. Lynx killed by hunters were submitted for necropsy without their fur. Body condition was evaluated by body weight, fat deposits in the abdomen and on the heart, and appearance of the bone marrow of the femur. The animals were categorized into 3 groups: normal body condition , poor body condition , subadult , and adult . Numbers of males and females were equal in the juvenile and adult age classes, but within subadults there were more males (n = 68) than females (n = 27). According to the time of death, animals were grouped into seasons relevant to the biology of the lynx: delivery ; small kittens and lactation ; large kittens hunting with the dam ; and breeding season, separation of kittens from dam, and main hunting season .For 228 animals, age was determined by counting cementum annuli of a canine tooth ; for the others, it was estimated on the basis of body size and weight, tooth wear, development of genital organs, and, if appropriate, morphologic characteristics and ossification of the skull. Investigated lynx with exact age known were 0–13 years of age (mean 2.5 years ± 2.6 SD). On the basis of sexual maturity, and thus reflecting social behavior, 3 age classes were established: juvenile (= 262), 25 mg kidney (n = 263), and 10 mg spleen (n = 261) were cut in small pieces and homogenized in phosphate-buffered saline. DNA was extracted by using a QIAamp tissue kit ; mean DNA concentration was 39.7 μg/mL ± 23.8 SD for lung, 99.0 μg/mL ± 46.4 SD for kidney, and 101.0 μg/mL ± 49.0 SD for spleen. Five μL of the DNA eluate was used as template for the PCR.From each sampled lynx, 25 mg lung (n tk) were used as described (2 (25 mmol/L), 5 μL GeneAmp 10X Gold Buffer , 2.5 U AmpliTaq Gold polymerase (Applied Biosystems), and 19 μL water. The tubes were placed in a preheated block (95°C) and held for 5 min before cycling. Five cycles of denaturation (95°C for 30 s), annealing (53°C for 2 min), and primer extension (72°C for 30 s) were followed by 35 cycles with 30 s annealing time. After the last cycle, the temperature was held at 72°C for 10 min.PCR primers  from the thymidine kinase gene (www.mbio.ncsu.edu/BioEdit/bioedit.html). Multiple sequence alignment was performed by using ClustalX 1.181 (www.megasoftware.net). The reliability of the phylogenetic relationship in the tree topology was statistically evaluated from 1,000 bootstrap replicates. The lynx orthopoxvirus DNA sequences obtained in this study have been submitted to the GenBank nucelotide sequence database  in a 2% agarose gel  with TAE buffer  and ethidium bromide for DNA staining. Fifteen μL of PCR product mixed with 3 μL 6X loading buffer (0.25% [wt/vol] bromphenol blue and 40% [wt/vol] sucrose) were loaded in each well. The gels were run in TAE buffer at 150 V for 1.5 h and examined by using a Gel Doc 2000 Documentation System . Primers and dNTP were removed from amplicons by using ExoSapIT reagent , adding 1 µL/5µL PCR product, incubating 45 min at 37ºC, and by conducting enzyme inactivation for 20 min at 80ºC. Cycle sequencing was conducted in both directions by using Big Dye 3.1 reagents . Two microliters EDTA (125 mmol/L), 2 μL sodium acetate (3 mol/L), and 50 μL of ethanol were added to the 20 μL sequencing product. Electrophoresis of the cycle sequencing extension products was conducted in an ABI Prism 377 DNA Analyzer (Applied Biosystems). Raw sequence data were edited by Chromas Pro software version 1.41  and BioEdit Sequence Alignment Editor version 7.0.4 (database .www.ncss.com). Statistical significance of differences was analyzed by using the 2-tailed Fisher exact test; level of significance was set at p<0.05.Statistical calculations regarding differences in prevalence were performed by using NCSS 2007 Statistical Software  and differed by1 nt substitution from a cowpox virus isolate from a felid in Norway (No.F1) (tk gene sequences differed from those of CPXV-BR and of cowpox virus isolates from Sweden (Swe.H1 and Swe.H2) by multiple nucleotide substitutions  . In addiitutions . Phylogeirus GRI .On the basis of previous serologic investigations that found anti-OPV antibodies in lynx in Sweden   . Given tUnexpectedly, the OPV sequences obtained from the lynx in Sweden were similar to the 2 isolates from a person and a felid in Norway and less similar to 2 isolates from persons in Sweden. A potential explanation is that none of these 4 clinical cases occurred near the sites in which the lynx were sampled for this study. In Sweden, the 2 human cases appeared in Skåne, the southernmost part of the country, 150–200 km from the southernmost region where the lynx were sampled. In Norway, all feline cases and all but 1 human case occurred in the southwestern part of the country, far from the common mountainous Sweden–Norway border, the region where most lynx are found. Recently, only 1 case of cowpox virus in a human has been identified in this region .>1 internal organs of the lynx is evidence of a virus infection and indicates that the host most likely has entered a viremic phase. No specific pathologic lesions in skin or lungs were recorded, but only a limited number of skins were submitted with the carcasses. This finding calls for increased efforts to document possible clinical signs, pathologic changes, and the effects of cowpox virus infection in lynx, taking into account the clinical signs and organ lesions documented in domestic cats and in large felid species in zoos  had sarcoptic mange, whereas the total prevalence of mange among the 264 animals included in this study was 6.4%; this finding indicates a possible relationship between OPV infection and infestation with We found no seasonal differences in OPV prevalence, although one could assume that autumn, with peak populations of rodents and shrews, would be when contact rate between these animals and lynx is highest. It could also be expected that the presence of OPVs such as cowpox virus would fluctuate between years, reflecting the fluctuations of rodent populations, but we found no significant differences in prevalence between the years in this study. These findings could be the result of the low number of samples that were included from the nonhunting seasons and from some of the years represented in the study.,,,Cowpox virus infection is a zoonosis, capable of being transmitted from rodents to humans, often by domestic cats. Cowpox virus infection in humans is usually characterized by single lesions on the infection site , but it sometimes spreads and cause secondary lesions and complications and can be especially severe in immunocompromised persons (In conclusion, our results support the hypothesis that carnivores that prey on OPV reservoir species can be used as indicator species for the presence of such viruses in the ecosystem (


The preferential transmission of ancestral virus needs to be integrated into evolutionary models if we are to accurately predict the evolution of immune escape, drug resistance and virulence in HIV-1 at the population level. Moreover, early infection viruses should be the major target for vaccine design, because these are the viral strains primarily involved in transmission.Over calendar time, HIV-1 evolves considerably faster within individuals than it does at the epidemic level. This is a surprising observation since, from basic population genetic theory, we would expect the genetic substitution rate to be similar across different levels of biological organization. Three different mechanisms could potentially cause the observed mismatch in phylogenetic rates of divergence: temporal changes in selection pressure during the course of infection; frequent reversion of adaptive mutations after transmission; and the storage of the virus in the body followed by the preferential transmission of stored ancestral virus. We evaluate each of these mechanisms to determine whether they are likely to make a major contribution to the mismatch in phylogenetic rates. We conclude that the cycling of the virus through very long-lived memory CD4 HowevFirst, under stage-specific selection, it is argued that the rate mismatch occurs because selection is weaker in early infection, resulting in a lower rate of diversification per unit time when measured from the time of infection to transmission (the period that determines between-host rates of divergence), than when measured during chronic infection (the period during which within-host rates of divergence are measured). In the absence of stage-specific selection, we would expect the rate of evolution per unit time to be independent of when transmission occurs. If transmission tends to occur during early infection, the rate of divergence per transmission event will be slower than if transmission tends to occur late, but when measured over calendar time, the two rates will be the same.Second, under adapt and revert, it is argued that mutations that are adaptive in one individual are likely to be maladaptive in another owing to, for example, different human leukocyte antigen (HLA) backgrounds, and thus will revert after transmission. If a sufficient proportion of mutations that are fixed within an infected host revert once a new host is infected, then a mismatch in phylogenetic rates is likely to emerge because not all mutations accumulating at the within-host level will accumulate at the between-host level.Finally, under store and retrieve, it is argued that ancestral sequences  are stored in the body and are preferentially transmitted, resulting in faster rates of divergence when measured at the within-host level compared with the between-host level. Preferential transmission of ancestral strains could occur either because ancestral strains have an intrinsic transmission advantage, or because virus is more likely to be stored in the genital tract, thus leading to preferential transmission during sexual transmission.Our aim is to establish which, if any, of these mechanisms are likely to make major contributions to the observed mismatch in phylogenetic rates, or whether additional or alternative mechanisms are required. We select among plausible mechanisms by a process of elimination. By comparing previously published estimates of the rate of synonymous and non-synonymous mutations at the within- and between-host levels, we argue that neither stage-specific selection nor adapt and revert is likely to explain a substantial proportion of the mismatch, especially among synonymous mutations. Of the mechanisms we consider, store and retrieve is the only mechanism likely to make a major contribution to the mismatch in phylogenetic rates. Moreover, by comparing rates of divergence between virus circulating in different host populations, we argue that stored ancestral virus is probably preferentially transmitted because it has an inherent transmission advantage, rather than because virus is more likely to be stored in the genital tract than in other parts of the body.To test whether the store and retrieve mechanism, with an intrinsic transmission advantage to ancestral virus, is sufficient to quantitatively explain the observed mismatch in phylogenetic rates, we develop a simple mathematical model of within-host HIV evolution coupled to store and retrieve transmission dynamics. We find that if virus is stored in latent form in long-lived cells for a sufficient amount of time, in the order of years, then the store and retrieve mechanism can explain the mismatch in phylogenetic rates of divergence. We thus conclude that the store and retrieve model of viral evolution can qualitatively and quantitatively explain observed trends, and we explore some evolutionary and public health consequences of these findings.2.env at the within-host than at the between-host level in populations where the virus is transmitted sexually [env at the within- and between-host levels  or from different patients during the course of an epidemic . In general, it is difficult to compare estimates of the rate of divergence of HIV-1 at the within- and between-host levels because of differences in study design, such as the use of different HIV-1 subtypes, different host populations, different segments of the viral genome, different alignment techniques and different statistical methods. However, where authors have controlled for these differences a strong rate mismatch has been observed, with a 2× to 6× faster rate of divergence for sexually ,6. Moreo levels .Preliminary analyses of whole viral genomes appear to confirm that the mismatch in rates is present for all genes, though may be greater for 3.(a)et al. [Stage-specific selection has been invoked as a mechanism explaining why, when measured over calendar time, viruses from similar clades evolve at very different rates in different epidemics . In rapiet al.  hypotheset al.  put forwAs a consequence of stage-specific selection, there will be a mismatch in the measured within- and between-host rates of diversification; the between-host rate of diversification is determined by the number of genetic substitutions accumulated by the viral population between the time of infection and the time of onward transmission (i.e. including the period during which selection is weak and adaptation slow), whereas the within-host rate of diversification tends to be measured only during chronic infection (i.e. when selection is strong and adaptation fast). If there is no stage-specific selection, then we would expect the evolutionary clock to tick at a similar rate regardless of whether transmission tends to occur early or late in infection because the clock is measuring divergence over calendar time, not per transmission.Direct evidence for this proposition is equivocal. Studies following the evolution of the virus within-hosts do not show clear evidence of an early ‘eclipse’ phase of slow within-host viral evolution . More reHowever, because the mismatch in phylogenetic rates at the within- and between-host levels is observed for synonymous as well as non-synonymous mutations  and tabl, it is uIt is also worth noting that the comparisons of the rates of divergence at the within- and between-host levels have focused on populations where the virus is sexually transmitted, and therefore, the effect of slow divergence during early infection would be likely to be swamped by adaptive processes occurring later on in infection, and cannot explain the mismatch that we reported in (b)After transmission to a new host, HIV-1 partially reverts towards a consensus wild-type sequence, though in a heterogeneous manner that is difficult to predict ,19–21. RDirect evidence of reversion is complex to interpret. Reversion appears to be fast for only the most costly adaptive mutations, which are rare, while other adaptive mutations revert slowly or not at all ,20,22.env than for other genes.In terms of explaining the mismatch in rates summarized in (c)+ T cells [+ T cells with integrated provirus will enter a resting phase; these latently infected memory T cells effectively store virus creating a very stable viral archive [Ancestral HIV sequences can be ‘stored’ within a host for long periods of time, creating within-host heterogeneity in the amount of evolution that viral lineages have undergone within a single host at any given moment in time ,24. HIV- T cells . The dou T cells . A small T cells . Occasio archive . Months, archive –31. In a archive .Irrespective of the exact mechanism of storage, if ancestral virus is preferentially transmitted, then the evolutionary clock is predicted to tick rapidly within-hosts, but at transmission, the hands of the clock are metaphorically turned back. Viral storage and retrieval through preferential transmission would lead to slower rates of divergence when measured at the population level.Evidence is accumulating that ancestral virus is at least sometimes preferentially transmitted. By studying viruses in many transmitting couples, it has been shown that virus circulating in newly infected heterosexual recipients (within a year of infection) tends to be more closely related to donor ancestral sequences than contemporary sequences circulating within the donor at the time of infection , and thaet al. [et al. [Recent evidence from high-resolution phylogenies is more equivocal; data from Herbeck et al.  clearly  [et al. , two werA key prediction of the store and retrieve mechanism is that it should affect synonymous and non-synonymous sites equally. Therefore, the observation that the mismatch in phylogenetic rates affects synonymous and non-synonymous mutations to a similar extent provides strong support that store and retrieve is a major mechanism affecting the rate mismatch.For store and retrieve to result in a mismatch in phylogenetic rates, there must be a mechanism allowing for the preferential transmission and/or establishment of ancestral viral sequences in new hosts. This might be because ancestral viruses have an inherent transmission and/or establishment advantage, and/or because virus is preferentially stored in, and transmitted from, the genital tract as a consequence of compartmentalization.A recent study has shown that although compartmentalization of the virus in the genital tract is apparent, this compartmentalized virus does not appear to be preferentially transmitted , suggestIf the virus has an inherent transmission and/or establishment advantage, however, then we speculate that we might well expect the rate of divergence to be slower in populations where transmission is intravenous rather than heterosexual. For a strain of HIV-1 to have a transmission and/or establishment advantage, it must be better than other strains at negotiating the mucosal barrier , be better at gut homing, and/or have a faster growth rate during the first few days of infection. Ancestral virus is likely to have an advantage during this process because it will be very similar to the already successfully transmitted strain and will not have accumulated costly cytotoxic T-lymphocyte (CTL)-escape mutations. Larger inoculum sizes during high-dose rectal and intravenous transmission ,36, the α4β7 is a marker for gut homing of CD4+ T cells [In conclusion, the store and retrieve mechanism, with an inherent transmission and/or establishment advantage of ancestral virus is, of the mechanisms we considered, the only one leading to a mismatch in phylogenetic rates that alone is consistent with all of the available data. Such an inherent transmission and/or establishment advantage of ancestral virus is certainly plausible. During the course of infection, HIV-1 accumulates CTL-escape mutations that are likely to be deleterious to the virus when transferred into a new host ,38,39. A T cells . It has  T cells ,43,44. H T cells .It is of course possible that the mechanisms described earlier act together to generate the mismatches we see, and we do not rule that out, but given the evidence currently available, the most parsimonious explanation is store and retrieve. Having discussed the conceptual model's qualitative ability to reproduce the data, we now test the concept further by quantitatively comparing the predictions of a mathematical formation of the store and retrieve model with the data reported in 4.i, a virus is removed from the founding strain . We usey et al. , but als+ T cells , latently infected memory CD4+ T cells (iL) and macrophage . The subscript i indicates cells infected by ith generation virus. In addition, the model tracks the number ith generation virus, iV.Our model follows the numbers of three host-cell types: active CD4For a full list of parameters and variables see + T cells is 1 day  is 5 × 106 and susceptible macrophage (BM) is 5 × 104. CD4+ T cells and macrophage are assumed to have the same infection rate (βT = βM = 1 × 10−7) and the same viral growth rate when infected (κ = 100), consistent with the observation that about 10 per cent of virus produced in the body are derived from macrophage [+ T cell enters the latent phase, k, is 0.001 and that the activation rate of latently infected memory CD4+ T cells, α, is 0.001 [k = 0; that is, where virus is not stored in memory CD4+ T cells.Here, we assume that the generation time of the virus when infecting active CD4δT = 1; ,46) and  and + T M = 0.1; ). The prcrophage . We assuis 0.001 ,29. We aiT: step function ; and exponential decline .As a consequence of the virus circulating through different host-cell types, we see increasing heterogeneity in the amount of evolution circulating viral strains have undergone as infection progresses . For thit)), the mean number of generations transmitted virus have gone though since infection (MGT(t)) and the mean transmissibility of the viral population (MT(t)), where the maximum transmissibility is 1 and where t is the time since infection:Using these loss of transmissibility functions, we calculate the mean number of generations viruses circulating with the host have gone through since infection . However, once we include storage the difference between the two populations becomes appreciable, reaching factors of four or more . We also see a concomitant drop in transmissibility for the viral population as a whole, which is plausible given the large drop in empirical estimates of infectiousness following acute infection [For the store and retrieve model to explain a slower rate of divergence at the between-host level than at the within-host level, the transmitted viral population must be less evolved than the general viral population circulating within the host. We find that when there is no storage of virus in memory CD4pulation a,c. Howe or more b,d. We anfection .et al. [et al. results were derived from Rakai cohort data involving HIV-1 heterosexual sero-discordant couples [+ T cells. However, when storage of the virus is allowed, we see a threefold difference in the rate of accumulation of viral generations when transmissibility declines in a step-like fashion, and a sixfold difference when transmissibility declines exponentially .It is worth noting that within- and between-host rates of evolution have only been compared for Our study establishes the importance of acute infection in determining the evolutionary course of the HIV-1 pandemic: the viruses present in acute infection are stored and then preferentially transmitted. A major consequence of this observation is that what happens after acute infection, in the extreme case where only viruses stored during acute infection are transmitted, could be considered an evolutionary dead end at the population level (while remaining important for pathogenesis). This observation needs to be integrated into our thinking if we are to accurately model population level evolution of immune escape, drug resistance and virulence in HIV-1. Specifically, mutations selected for in early infection would be more likely to be transmitted than those selected for later. This could help explain the slow spread of many drug resistance mutations to date , even wh


To the Editor: On August 5, 1994, a northern spotted owl, Strix occidentalis caurina, was found dead in Kittitas County, Washington, USA  captured July 9 at Yellow Bay on the east shore of the lake  contained spirochetes detected when stained with Giemsa and examined by microscopy (600× brightfield with oil immersion). Whole blood from the squirrel contained live spirochetes visible by dark-field microscopy, and ≈50 µL of this blood was injected intraperitoneally into a laboratory mouse. The next day, a few spirochetes were observed in the peripheral blood of the mouse, and during the next 3 days, the density of spirochetes increased. We used intracardiac puncture to collect blood from the mouse for spirochete isolation in BSK-H medium  and for analysis by PCR and DNA sequencing of multiple bacterial loci as described elsewhere .The spirochetes observed in the squirrel’s blood failed to grow in BSK-H medium after passage in the laboratory mouse; however, we acquired DNA sequences from infected squirrel and mouse blood from PCR amplicons for 6 spirochete loci including www.ncbi.nlm.nih.gov), including those sequences reported for the spirochete found in the spotted owl  , flaB (467 bases), and IGS (665 bases) from the squirrel spirochete were identical to those of the owl spirochete; no base differences were found among the 2,422 bases compared. We also examined DNA extracted from the spotted owl’s liver during the first investigation (16S rDNA and the complete flaB, gyrB, glpQ, and vtp genes from the owl spirochete DNA and determined their sequences. The complete sequences of the first 4 loci from the owl and squirrel spirochetes were identical and differed from all other B. hermsii sequences. A phylogram of the concatenated sequences totaling 5,188 bases demonstrated that the owl and pine squirrel spirochetes were identical and were divergent members of B. hermsii genomic group I (Next, we compared the sequences from the squirrel spirochete with those available in the National Center for Biotechnology Information database ( group I .B. hermsii, separated by ≈525 km, in a pine squirrel and a spotted owl demonstrates a broader geographic distribution and host range for this spirochete than what could have been envisaged previously. The possible role of birds as hosts for the vector Ornithodoros hermsi ticks has been demonstrated elsewhere (B. hermsii that includes a broader host range for this group of relapsing fever spirochetes than previously appreciated.Finding the same strain of


To the Editor: An outbreak of Cryptococcus gattii cryptococcosis has been ongoing in the US Pacific Northwest (PNW) since 1999 , a sentinel public health surveillance system of infectious disease clinicians that is supported by the Centers for Disease Control and Prevention and sponsored by the Infectious Diseases Society of America  respondents (representing 43 states) who treated a cryptococcosis patient during the past year. We compared answers from respondents in the 4 US census regions . ResultsC. gattii infections in the West  respondents reported that they were aware of the C. gattii infections. When respondents were asked if their clinical laboratory could differentiate C. neoformans from C. gattii isolates, 131 (46%) responded “yes, either routinely or when requested”; 68 (24%) responded “no”; 87 (30%) did not know. When we excluded respondents who did not know, only 66% of respondents from the West indicated that their laboratory could differentiate species. This finding is concerning because outbreak-associated C. gattii is clearly endemic to the region. A better understanding of which laboratories perform this service and which send specimens to a reference laboratory will help identify where additional capacity is needed.Of the respondents, 94% reported that they most often use the cryptococcal antigen test for diagnosis, although 73% of respondents report that they commonly request a culture. Furthermore, 76 (27%) of respondents report using a combination of tests  that does not include culture. Tests that do not differentiate between cryptococcal species represent missed opportunities for diagnosis of C. gattii infection. This may reflect a low incidence of C. gattii infections in these regions, or it may be a result of decreased clinical suspicion for C. gattii infections outside the PNW.A lower percentage of respondents from the Northeast (10%), Midwest (5%), and South (3%), compared with those from the West (44%), reported having ever consulted on a case of C. gattii and the ongoing outbreak in the PNW, missed opportunities for diagnosis still exist. To understand the true incidence of C. gattii inside and outside the PNW, vigilance among physicians nationwide is necessary. Clinicians and laboratorians should be aware of the need to obtain specimens for culture and of the need to develop methods to differentiate cryptococcal species. An accurate diagnosis of cryptococcosis cases in the United States will lead to a better understanding of the epidemiology and incidence of C. gattii in this country and may result in improved treatment.Results from this study suggest that although most EIN members are aware of Cryptococcus gattii infection, Emerging Infections Network survey, March–February 2011.Distribution of survey respondents who had seen any patient(s) with cryptococcosis during the past year and who have ever treated a patient with


The demand for induced abortions in Uganda is high despite legal and moral proscriptions. Abortion seekers usually go to illegal, hidden clinics where procedures are performed in unhygienic environments by under-trained practitioners. These abortions, which are usually unsafe, lead to a high rate of severe complications and use of substantial, scarce healthcare resources. This study was performed to estimate the costs associated with induced abortions in Uganda.A decision tree was developed to represent the consequences of induced abortion and estimate the costs of an average case. Data were obtained from a primary chart abstraction study, an on-going prospective study, and the published literature. Societal costs, direct medical costs, direct non-medical costs, indirect (productivity) costs, costs to patients, and costs to the government were estimated. Monte Carlo simulation was used to account for uncertainty.The average societal cost per induced abortion (95% credibility range) was $177 ($140-$223). This is equivalent to $64 million in annual national costs. Of this, the average direct medical cost was $65 ($49-86) and the average direct non-medical cost was $19 ($16-$23). The average indirect cost was $92 ($57-$139). Patients incurred $62 ($46-$83) on average while government incurred $14 ($10-$20) on average.Induced abortions are associated with substantial costs in Uganda and patients incur the bulk of the healthcare costs. This reinforces the case made by other researchers--that efforts by the government to reduce unsafe abortions by increasing contraceptive coverage or providing safe, legal abortions are critical. Induced abortion is illegal in Uganda except to save the life of a pregnant mother and to preserve her physical and mental health . InducedYet the demand for induced abortion remains high, probably because of a high number of unintended pregnancies, at least 700,000 annually , which aWomen who decide to abort often resort to untrained and usually unskilled practitioners who practice in illegal and hidden clinics and often provide unsafe abortion procedures that result in a high rate of complications and sometimes death. Unsafe abortions are estimated to be the cause of 21% of all maternal deaths in Uganda  comparedIllegally-performed unsafe abortions in Uganda pose a large health risk for women because of inadequate skills of the providers, unsanitary environments, and hazardous techniques  which inPrevious studies of the cost of induced abortion in Uganda did not consider the consequences of failed induction and the impact of abortion provider on healthcare costs  or did nWe performed a descriptive cost-of-illness study to assess the economic burden of induced abortion in Uganda. A decision tree was developed to represent the consequences of induced abortion and to estimate the cost of an average case in Uganda from a societal perspective. Data to inform the model were obtained from a primary chart abstraction study, an on-going prospective study, and the published literature. The on-going prospective study is a cohort of women enrolled following discharge after post-abortion complications, discharge after child birth, and clinic visit for contraception. It was designed to compare the women discharged following post-abortion complications with the other women groups with regard to health and economic outcomes. The total national cost of induced abortion for 2010 was estimated by multiplying the average cost by an estimate of the annual incidence of induced abortion in Uganda.The decision tree showing the consequences of induced abortion is shown in Figure Women who receive abortion procedures from the different providers are further divided into those for whom induced abortion succeeds and those for whom it fails. Induced abortion rarely fails when performed by trained practitioners, and we found no studies that estimated its incidence in Uganda or similar countries, but studies in other settings have reported frequencies of 0.01% , 0.05% , and 0.0Women who have had successful induced abortions are divided in the model into those who develop complications and those who do not. The type of abortion provider has a direct influence on the probability of having abortion complications. A survey of health workers in Uganda estimated the proportion of induced abortion complications by provider ,28. It rWomen who develop complications following induced abortion were divided into those who need out-patient care and those who need hospital care. According to Prada et al . of the In the model, women who need and obtain hospital treatment following abortion complications both improve and are discharged alive, or they die in hospital. The in-hospital rate of abortion related mortality ranges from 1.3%  to 3.3% In the case of abortion failure after an attempt by a trained practitioner, the woman carries the pregnancy and faces the consequences of pregnancy. These include: a) miscarriage before 13 weeks gestation, b) miscarriage between 13 and 22 weeks which usually requires treatment, or c) birth of a child which includes preterm birth as well as term live or still birth. Miscarriages at 13-22 weeks account for 2.9% of all recognized pregnancies and live births account for 84.8% ,35. The We estimated the average cost of each outcome in the decision tree Figure . The oveThe cost of induced abortion was considered to include the following cost categories: 1) direct medical costs, 2) direct non-medical costs, and 3) indirect (productivity) costs. Direct medical costs included personnel, medical supplies, drugs, radiology tests, laboratory tests, and patient out-of-pocket costs. Direct non-medical costs included recurrent expenditures (such as utility bills) and capital expenditures , patient transportation, and patient upkeep while seeking healthcare. Indirect costs included lost productivity while seeking abortions and getting treatment for complications as well as productivity losses from abortion morbidity while convalescing and premature abortion-related maternal mortality. The total healthcare cost is the sum of the direct medical and direct non-medical costs.In a separate classification, the costs of induced abortion were also considered to include patient/family costs and government costs. Patient/family costs included the costs of procuring abortions, out-of-pocket costs, transportation, and upkeep while procuring abortions and seeking treatment for complications, and self-medication. Government costs included the costs of treating abortion complications and pregnancy-related costs when abortions fail, but excluded the healthcare costs associated with the procurement of abortions which are illegal in Uganda and are not provided by the national healthcare system.The societal cost estimate is the sum of all the different kinds of costs i.e. direct medical + direct non-medical + indirect/productivity costs or patient/family costs + government costs.The cost to women of abortion services by provider were obtained from a survey of health workers  costs of out-patient and hospital treatment of abortion complications were estimated from the World Health Organization Choosing Interventions that are Cost-Effective (WHO-CHOICE) database for Uganda . TranspoProductivity losses due to morbidity were estimated for both patients and caregivers using data from the prospective study by summing lost time spent in transit to hospitals (for patients and caregivers), seeking care, convalescing, and admitted to hospital (for patients and caregivers), and multiplying by wages. Wage data were obtained for formally-employed women in the same prospective study. The wage of the proportion of women who were unemployed (subsistence farmers) was valued at Uganda's gross domestic product per capita at the official exchange rate which was $474 in 2009 [Productivity losses due to mortality were estimated using the human capital approach  valuing The unit costs used in the analysis are summarized in Table All costs were converted into United States dollars ($US) using the Bank of Uganda official exchange rate on 1st June 2010  and wereThe average costs of induced abortion are shown in Table The annual incidence of induced abortion in Uganda is 362,000 cases. Therefore the national annual expenditure on induced abortion is projected to be $23.6 million in direct medical costs, $7.0 million in direct non-medical costs, $33.5 million in indirect/productivity costs, $5.1 million in costs to the government, $22.3 million in costs to patients, and $64.2 million in societal costs.Univariate sensitivity analysis Figure  showed tUsing a decision tree and data from multiple sources, we found that the average induced abortion in Uganda was associated with $177 in societal costs. This is over four times the per capita expenditure on health care in Uganda which is $44 . Given tThe annual abortion expenditure in Uganda is substantial and is a testament to the economic impact of abortion in countries where it is illegal (and likely to be unsafe), which has been previously described . The proThe largest contributor to the average societal cost of induced abortion was productivity costs 52%) but the healthcare component, of which direct medical costs were the largest, was also substantial (48%). The key driver of productivity costs is abortion-related mortality. The loss of young mothers from abortion exerts a substantial burden to society. The largest part of the healthcare costs can be attributed to the treatment of the complications of unsafe abortions. The majority of trained and untrained providers choose surgical techniques--such as evacuation and manual vacuum aspiration--to terminate pregnancies but not misoprostol combined with mifepristone which is the safest form of medical abortion [% but theThe average healthcare cost  of induced abortion was $85. This estimate included the cost of abortion procedures as well as the treatment of post-abortion complications. Levin et al . estimatVlassoff et al . estimatOne limitation of this study was the use of estimates of the proportion of abortions induced by different providers, the rate of complications, and costs of abortion procedures obtained from a survey of health workers  as opposAnother limitation of the study is that we did not include intangible societal costs of abortion such as the non-labor force value of women in taking care of children and the potential multi-generational effect of orphan hood. While these effects have been demonstrated previously, there is a need for Uganda-specific research to better inform policies aimed at reducing the impact of unsafe abortion. Additional data limitations to our study include the use of some resource use estimates from an on-going cohort study before completion of the protocol due to time and resource limitations, the failure to distinguish between the costs of legal and illegal induced abortions in the analysis as well as use of data on induced abortion failure from developed countries due to a lack of estimates from Uganda or similar countries. As mentioned above, the inherent uncertainty in these estimates was addressed by using Monte Carlo simulation.The results of the sensitivity analysis showed that the estimate of costs of induced abortion in Uganda were most sensitive to the uncertainty surrounding in-hospital mortality from abortion complications and the rate of hospitalization for complications. The sensitivity of the societal cost to a relatively narrow range (1-3%) of in-hospital mortality, as well as the fact that productivity losses contribute over half (52%) of the total societal cost, point to the substantial economic burden of abortion-related mortality. Data on the rate of abortion complications were derived from surveys of health workers as opposed to prospective studies. It will require innovative research methods to improve on the quality of these estimates in the prevailing legal environment or a change in the abortion law which seems like a remote possibility at the current time.The substantial cost associated with the average induced abortion in Uganda, reinforces the case made by other researchers--that efforts to reduce unsafe abortions by increasing contraceptive coverage or providing safe, legal abortions, are critical. Such efforts would contribute to achieving Millennium Development Goal number 5  and are in line with the United States Global Health Initiative which places an emphasis on improving the health of women. The study may also be used as a basis for estimation of the potential economic gains of policies that increase contraceptive coverage or provide safe, legal abortions.The authors declare that they have no competing interests.JBB conceived of the study and participated in the design of the study, collecting data, performing the analysis and drafting the manuscript. AS participated in designing the study and revising the manuscript. DLV participated in designing the study and revising the manuscript. JSG participated in designing the study and revising the manuscript. JN participated in collecting data and revising the manuscript. PM participated in collecting data and revising the manuscript. LPG participated in designing the study and revising the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/904/prepub


Culex quinquefasciatus. A 71.43% prevalence for neutralizing antibodies was found in domestic fowl in the homestead of a patient with encephalitis.Twenty-six years after it was last detected, Saint Louis encephalitis virus (SLEV) genotype III reemerged in 2005 in Córdoba, Argentina, where it caused an outbreak. Two genotype III SLEV strains were isolated from  Saint Louis encephalitis virus  emerged in Córdoba Province, Argentina, in 2002. A single case of human encephalitis occurred , Cx. interfor (13%), Cx. quinquefasciatus (84%), and Ochlerotatus albifasciatus (0.2%). Seven pools of female mosquitoes were organized and processed as previously described  were identical except for 8 silent substitutions  and were closely homologous to SLEV sequence AF013416, with a high bootstrapping value . Subsequently, the entire E glycoprotein gene was sequenced from the 2 cultured isolates (GenBank accession nos. DQ385451 and DQ385450), and a phylogenetic tree was derived . The cloBlood samples (0.2 mL) were taken from the jugular vein (chicks) or brachial vein  with a 27-gauge 3/8-inch needle attached to a 1-mL syringe and added to 0.9 mL sterile phosphate-buffered saline, for an ≈1:10 dilution of serum. Sera were tested for neutralizing antibodies by using the plaque-reduction neutralization test .The Córdoba outbreak in 2005 represents the first reported SLE outbreak in Central and South America. Before 2005, the only recorded outbreak of human encephalitis caused by flaviviruses in this region was in 1975 in Brazil, which was attributable to Rocío virus . The high neutralizing antibody titers we detected in chickens <3 months of age indicated recent infection and support the hypothesis that SLEV was responsible for the simultaneous outbreak.While the specific avian amplifying hosts involved in the Córdoba outbreak remain unknown, important amplifiers would include competent reservoir hosts that are abundant and frequently exposed to infectious mosquito bites (Finally, the reemergence of SLEV in Córdoba represents an opportunity to study the ecology of this virus. Further studies are needed on vector competence for local strains of Cx. quinquefasciatus and Cx. interfor and on the reservoir competence of the bird species mentioned above.


C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources . In addition, the molecular types VNII , VNIV , VGI  and VGII  were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498  Cryptococcus neoformans/C. gattii species complex, causes life-threatening infection in humans with either normal or compromised immunity worldwide. The molecular epidemiology of this fungus in developing countries is highly under-investigated. Thus, three standard molecular typing methods were performed on 498 cryptococcal strains isolated from humans, the environment and animals in Thailand. As in developed countries, C. neoformans was found to be correlated with HIV infected patients and C. gattii infected humans with apparent normal immune status. Combining current and previous data, a total of 20 sequence types (ST) were identified suggesting a greater spectrum of major molecular types and genotypes of the C. neoformans/C. gattii species complex in Thailand than previously reported. Moreover, the discovery of the minor genotype (ST7) of the Vancouver Island outbreak in Thailand indicates that Asia may be an important stepping stone in its global expansion. This study gives an insight in the molecular epidemiological picture of this important fungal pathogen and necessitates further detailed studies.The most common fungal pathogen of the central nervous system, members of the  Cryptococcus neoformans/C. gattii species complex are the causative agent of cryptococcosis, which is a systemic mycosis, in a wide range of animals and humans C. neoformans comprises two varieties and three serotypes: C. neoformans var. grubii (serotype A), C. neoformans var. neoformans (serotype D) and a hybrid (serotype AD), whereas C. gattii comprises two serotypes, B and C The members of the C. neoformans has been associated worldwide with soil enriched with pigeon excreta and decaying wood C. gattii was until recently thought to be geographically restricted to tropical and subtropical regions and thought to be related mainly to eucalyptus trees C. gattii such as oiti (Licania tomentosa), almond trees , cassia (Cassia grandis), pottery tree (Ficus microcarpa) and Syzygium cuminiC. gattii on Vancouver Island, Canada, a temperate area, indicated an environmental shift of this species C. gattii has been found in association with a number of native tree species  on Vancouver Island rather than with eucalypt trees Extensive surveys of the yeast have shown that the ecological niches of both species are different. C. neoformans/C. gattii species complex C. neoformans var. grubii, serotype A; VNIII/AFLP2 corresponds to the AD hybrid; serotype AD, and VNIV/AFLP3 corresponds to C. neoformans var. neoformans, serotype D. The molecular types VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5 and VGIV/AFLP7 correspond all to C. gattii, serotypes B and C. These eight major molecular types have been subsequently confirmed using restriction fragment length polymorphism (RFLP) analysis of the orotidine monophosphate pyrophosphorylase (URA5) or phospholipase B (PLB1) genes, Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) analysis A number of molecular typing techniques have been used to study the molecular epidemiology of the C. neoformans has been known to be the major cause of the infection in immunocompromised hosts, while the immunocompetent hosts were virtually always affected by C. gattiiC. gattiiCryptococcosis is among the most prevalent life-threatening mycoses, especially in hosts with an impaired immune system such as HIV positive patients. C. gattii, serotypes B and C C. gattii has never been reported from the environment in Thailand C. neoformans var. grubii serotype A being the major cause of infection C. neoformans var. neoformans (VNIV) and C. gattii (VG) strains had been isolated before in Thailand C. neoformans/C. gattii species complex.Before the AIDS era, cryptococcosis in Thailand was mainly caused by The 498 cryptococcal isolates were recovered from the culture collections of the Molecular Mycology and Mycobacteriology Laboratory, Faculty of Medicine, Siriraj Hospital, Mahidol University, the Mycology Unit Laboratory, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, the Mycology Laboratory of the National Institute of Health, Nonthaburi, Thailand C. neoformans via india-ink preparation, urease test and phenoloxidase production test C. neoformans/C. gattii species complex and included into the study. Two additional environmental strains isolated from pigeon droppings were kindly provided from the mycology laboratory, Chiang Mai University. The strain information is listed in The environmental strains were collected from pigeon droppings in Bangkok during the years 2002–2005 using a method described previously URA5-RFLP analysis as previously described URA5 gene was amplified with the following primers URA5 (5′ATGTCCTCCCAAGCCCTCGACTCCG3′) and SJ01 (5′TTAAGACC TCTGAACACCGTACTC3′). The obtained amplification products were digested with the restriction enzymes HhaI and Sau96I. The digested PCR products were visualized and compared to the reference strains on a 3% agarose gel after staining with ethidium bromide.Genomic DNA was isolated as described previously 5′ GAGGGTGGCGGTTCT 3′). The PCR-fingerprinting profiles were visualized and compared on 1.4% agarose gels containing ethidium bromide using the 1D gel analysis module BioGalaxy in the software package BioloMICS ver. 7.5.30 . Strains with identical M13 PCR-fingerprints were grouped in the same M13 type , WM 626 , WM 628 , WM629 , WM 179 , WM 178 , WM 175  WM 779 , R265 (VGIIa) and R272 (VGIIb) The following set of laboratory standard reference strains representing each of the eight major molecular type of the Demographic and clinical data for each isolate, including isolation site and date, patient's residence, age, gender and HIV status, were retrieved from the clinical records if they were available and are listed in Statistical analysis was performed using the SPSS software package ver. 18.0.0 . Unknown data were regarded as missing data and excluded from the calculations.C. neoformans and 13 C. gattii strains were studied. Most of the C. neoformans clinical strains were from HIV positive patients (88.5%). In comparison, only 37.5% of the C. gattii strains were from HIV positive patients. The clinical strains were collected from all areas of Thailand with most strains originating from Bangkok (47.3%) and the central part of Thailand (27.0%). From the clinical strains, 80% were recovered from CSF, 17.5% from blood and 2.5% from other sites. All environmental isolates were obtained from pigeon droppings. The most common site of cryptococcal isolation in veterinary cases was the nasal cavity of cats (72.4%). For further information, see Among the 498 strains collected, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. Of the clinical strains, 68.9% were from male and 31.1% from female patients, with an average age of 37.97 years. A total of 485 URA5-RFLP analysis To examine the genetic diversity of Thai cryptococcal strains, the major molecular types were determined by M13 PCR-fingerprinting and/or As the majority of VNI Thai isolates were reported previously to be clonal C. neoformans isolates when compared with a previous report, which had identified the following STs: ST4, ST5, ST6, ST81, ST82, ST83, ST85, ST93 and ST141 for VNI; and ST173 for VNII C. neoformans isolates are an integral part of the global population structure of this species, with nine ST's being unique to Thailand, but closely related to other global isolates  based on neighbor joining analysis of the concatenated seven ISHAM consensus MLST loci. Bold numbers on the branches indicate bootstrap support above 75%. Letters in brackets indicate the M13 type. WM148 = VNI standard, WM626 = VNII standard.(TIF)Click here for additional data file.Table S1Strains used in this study and associated demographic and molecular data.(DOC)Click here for additional data file.Table S2Correlation between old and new allele and sequence type numbering from the Simwami et al. 2011 (28) publication and the new C. gattii MLST database at mlst.mycologylab.com for the MLST data used in the current study.(DOC)Click here for additional data file.Table S3MLST data for the additional published C. neoformans strains used in this study.(DOC)Click here for additional data file.Table S4GenBank accession numbers for all sequences of the MLST alleles obtained from Thai C. neoformans and C. gattii isolates used in this study.(DOC)Click here for additional data file.


To the Editor: Since the discovery of Toscana virus (TOSV) in 1971 in Tuscany  with 100% lymphocytes, a protein level of 126 mg/dL (reference range 15–45 mg/dL), and a glucose level of 63 mg/dL (reference range 50–80 mg/dL). A nasopharyngeal swab specimen was negative for influenza A and B virus antigens. Other than a decreased thrombocyte count and an elevated serum glucose level, the results of complete blood count, comprehensive chemical panel, and coagulation studies were within normal limits. PCR results for CSF were negative for herpes simplex virus, enterovirus, and parechovirus. Test results for acute-phase and convalescent-phase serum specimens performed at the Washington State Department of Health Laboratory were negative for West Nile virus and St. Louis encephalitis virus immunoglobulin M.Computed tomographic scan and magnetic resonance imaging of the brain showed no mass lesions or abnormality of the cerebral vessels. A sample of cerebrospinal fluid (CSF) obtained at admission showed 14 leukocytes/mmSerum and CSF were sent to the Centers for Disease Control and Prevention in Fort Collins, Colorado. TOSV RNA was detected in a CSF sample collected on day 1 of illness by using reverse transcription–PCR  (Although the clinical course varies from asymptomatic infection to severe meningoencephalitis, TOSV should be included in the differential list of viral pathogens among patients who seek treatment with symptoms consistent with meningitis or encephalitis if the patients have recently traveled to Mediterranean areas, including Sicily. Because neither a vaccine nor specific antiviral drug treatment is available to prevent or treat TOSV infection, travelers to TOSV-endemic areas should be advised to take all precautions to prevent insect bites.


To the Editor: Bats have been associated with a wide diversity of viruses, including lyssaviruses, which can cause rabies. Currently, 12 distinct species of lyssaviruses have been classified worldwide; 3 of these were isolated from bats in northern and central Asia  strain CVS-11, European bat lyssavirus-1 (EBLV-1), and Duvenhage virus (DUVV). We also tested for neutralizing antibodies against Lagos bat virus (LBV) and Mokola virus in 535 samples by using a modified rapid fluorescent focus inhibition test  were positive for neutralizing antibodies against lyssaviruses. Ninety (11.4%) of 789 bat serum samples had neutralizing antibodies against RABV, 71 (9.0%) against DUVV, 142 (18.0%) against EBLV-1, and 4 (0.75%) against LBV. No bat serum was positive for neutralizing antibodies against Mokola virus. Neutralizing antibodies against the 5 lyssavirus genotypes tested were found in 16 Microchiropteran and 1 Megachiropteran species .Of the 193 serum samples positive for neutralizing antibodies against lyssaviruses, 65 (33.7%) also neutralized ≥1 of the remaining viruses tested. Twenty-five samples that were positive for RABV were negative for the other viruses; 103 samples were negative for RABV but positive for ≥1 of EBLV-1, DUVV, and LBV. Different titers of neutralizing antibodies against different lyssaviruses were found in some bats  Table.This study provides serologic evidence of lyssavirus-neutralizing antibodies in bats in northern Vietnam. Because no virus was isolated, we could not conclude to which virus or viruses these bats had been exposed. Positive results for antibodies to multiple lyssaviruses, including RABV, found in some bats might have been caused by cross-neutralization of other viruses. The absence of consistent reactivity patterns suggests exposure of these bats to the tested lyssaviruses or another unknown lyssavirus. These findings are similar to findings reported from other parts of Asia (Information on lyssavirus circulation in bat populations in Vietnam should be made available to public health authorities, clinicians, and the general public to increase awareness of the risk for rabies transmission from bats; improve recognition, documentation, and reporting of bat exposure to rabies surveillance systems; and increase consideration of the need for post exposure prophylaxis after receiving a bat bite. Our data suggest that several lyssaviruses are circulating among bats in northern Vietnam, and a substantial proportion have neutralizing antibodies to RABV. Further investigations are required, particularly of sick and dying bats, to determine the implications of these findings for human health.Neutralization antibody titers against lyssaviruses in bats, by bat species and location, and bat collection sites and serum samples tested for lyssavirus, northern Vietnam.


By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses  env genes and their progeny in three subjects by single genome sequencing and performed a dynamic assessment of Nab responses based on env evolution and phenotypic changes in the Env glycoprotein over time. Surprisingly, we found genetic evidence of Nab activity as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. Although delayed in comparison with autologous CD8 T-cell responses, Nabs appeared earlier in HIV-1 infection than previously recognized and impeded virus entry at low titers. This raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.Characterizing early adaptive immune responses to HIV-1 can inform studies of virus persistence, pathogenesis and natural history and can guide rational vaccine design. Previous studies examined the role of neutralizing antibodies (Nab) in acute and chronic HIV-1 infection but not against the precise envelope (Env) glycoproteins of transmitted/founder (T/F) viruses and not in direct comparison with autologous cellular immune responses in the same subjects. Here, we identified T/F HIV-1  Taq-polymerase mediated recombination and nucleotide misincorporation errors in finished sequences, it allows for the analysis of mutational linkage across complete viral genes and genomes Much of what is known about virus-host interactions underlying HIV-1 persistence and pathogenesis in humans has come from quantitative measurements and mathematical modeling of viral replication dynamics and virus evolution in response to selective pressures, including antiretroviral drug therapy and adaptive immune responses in vivo have not been determined, although it is clear that moderate and high titers of Nabs can lead to HIV-1 selection and escape in vivo; and (iv) viral replication fitness costs associated with Nab escape.Nabs constrain the replication of most viruses and are essential to the efficacy of most viral vaccines trans-complementation assays to detect autologous strain-specific Nab responses, but neither study used SGA to identify T/F viruses or to look for genetic linkage of mutational escape pathways, nor did they use deep sequencing methods to detect the earliest escape mutations. Other investigators examined Nab responses against early viruses but without deep sequencing or a detailed kinetic analysis of low-titer antibody effects env genes could provide for the most sensitive detection of Nab pressure on the replicating virus quasispecies — even before direct phenotypic detection of virus neutralization in vitro — and that such findings could be corroborated and extended by deep sequencing, site-directed mutagenesis, antibody adsorptions and in vitro testing of T/F and escape variant Env proteins for neutralization by autologous antibodies.Previous studies have addressed some of these same questions but with different experimental strategies that allowed for lesser degrees of molecular and dynamic resolution. Wei 50) at 3, 6 and 12 month time points were 1∶1446/1∶2432/1∶1282 (CH40), 1∶38/1∶100/1∶239 (CH77) and <1∶20/1∶48/1∶243 (CH58)  ; Table 13 (CH58) , were tyIn each subject, we examined the neutralization sensitivity of full-length infectious molecular clones (IMCs) of the T/F virus compared with IMCs of consensus 6 month sequences . The latenv sequences. env mutations in subjects CH40 . Sequences from the earliest sample 45 days post-seroconversion were highly homogeneous  and conformed to a Poisson distribution of random changes and “star-like” phylogeny For subject CH40, 83 SGA-derived sequence . The medIn addition to changes in V1, selected mutations emerged early in the N- and C-terminal regions of V3 . These menv sequence analyses were also performed on CH77 (days 32–592) and CH58 (days 45–350). For CH77, 47 SGA env sequences  were obtained at four time points. Previously, Goonetilleke Sequential gp160 env gp160 sequences . By targeting SGA on shorter env fragments, we were able to obtain a robust set of sequences (n = 95) for analysis of viral env quasispecies evolution . A different V1 substitution at the same position, E146G, which was present at low frequency at day 111 but later expanded to become the majority sequence at days 132 and 181, also conferred significant escape from day 111 plasma (IC50 = 1∶69). An infrequent G145E substitution at day 111 had only an intermediate effect on virus neutralization (IC50 = 1∶523) and did not expand to greater prevalence at later dates. Surprisingly, amino acid substitutions at the amino- and carboxy-terminal base of V3 , which did not appear until day 132 in the SGA sequences and did not predominate until day 181, also led to complete neutralization escape from day 111 plasma (IC50 = 1:<20–1∶24). Other V3 base mutations, including T295N, and R327K and E322K individually, conferred partial escape (IC50 = 1∶332–1∶532). These findings raised the possibility that the base of V3 could comprise a discontinuous epitope recognized by the initial Nab response with V1 contributing directly or indirectly to Nab recognition and escape.Autologous plasma Nabs against the T/F virus were first detected for subject CH40 111 days after seroconversion with titers of 1∶1400 ; Table 150 = 1∶2432) and the Env clones containing V1 mutations  that conferred resistance to day 111 plasma Nabs. In contrast, mutations located in the V3 base , with or without the V1 mutations, conferred escape from day 181 plasma. Subsequent mutations in the V1 sequence motif at day 412 (N139T/E146T/M147T), however, also led to escape from earlier plasma antibodies including those in day 111 and day 181 plasmas . These findings are consistent with a well-described cycle of Nab development and rapid virus escape Neutralization patterns of day 181 plasma revealed continued evolution of fine Nab specificities . Day 181In contrast to the robust autologous Nab titers directed against CH40's T/F Env, CH77's plasma Nab neutralized its autologous T/F virus with a relatively low titer of 1∶38 at day 102 and only modestly higher at day 159 (1∶100) . Despiteenv gp160 SGA sequences and inferred from the partial env SGA sequences. These results suggest that early CH58 Nabs, despite their low titers of 1∶48 or less, recognized a conformational epitope involving C2, C3 and V4 and selected for virtually complete virus escape by day 154.Subject CH58 also developed a relatively delayed and low titer autologous Nab response; the first detectable plasma neutralization occurred at day 154 post-seroconversion with titers of 1∶48 . Again, 50 = 0.2 and 0.07 ug/ml, respectively) and both neutralized the CH40 T/F Env pseudovirus potently (IC50 = 0.075 and 0.034 ug/ml) . Both AbCH83 and AbCH84 bound well to CH40 T/F Env gp140 (EC4 ug/ml) . AbCH83 4 ug/ml) . For exa4 ug/ml) . Mutatio4 ug/ml) . Interes50 = 0.7–1.5 ug/ml), served as a positive control to assess the conformational and antigenic integrity of the synthesized gp120 and gp140 glycoproteins. As shown in To distinguish Nab reactivity targeting linear versus conformational (discontinuous) epitopes, we performed competition and adsorption assays with linear Env peptides, full-length gp120 proteins, and full-length tethered gp140 proteins corresponding to the sequences of the autologous T/F viruses, and as controls, heterologous peptides and proteins and randomly shuffled peptide sequences. First, we used overlapping linear peptides spanning complete variable loop and constant region sequences where Nab escape mutations first arose . At concentrations of 25 ug/ml, none of the linear peptides reduced the neutralizing activity in patient plasma (data not shown). As a positive control, we showed that 25 ug/ml of HIV-1 V3 peptides could inhibit V3-targeted Nabs in the same TZM-bl assay For CH77 and CH58, weaker autologous Nab titers made the effects of adsorption more difficult to discern, but in both cases, the Env monomers and Env trimers were equally effective at adsorbing neutralizing activity . For CH7Escape mutations were analyzed in the context of a relatively complete model of gp120 that was assembled from crystal structures of core gp120 with V3 and core gp120 with N and C termini  [50]–. Autologous gp120 and gp140-tethered Env glycoproteins were generated as described The gp120 Env monomers and tethered gp140 trimers were coupled to a solid phase tosylactivated magnetic Dynabeads MyOne beads (Invitrogen) as previously described Competition assays were performed with linear peptides having sequences described above. Neutralization assays were performed with additional peptide and plasma incubation for 30 minutes at 37°C prior the addition of the 2,000 infectious units of each virus for 1 hour at 37°C. The neutralization assay was then completed as described above with a final concentration/well of each peptide of 25 ug/ml. For the polyprotein adsorption studies, the plasmas were incubated with 12 uls of the protein-bead complex (or BSA-bound beads) for 30 minutes. The beads were removed with a magnet and discarded. This process was repeated an additional 2 times, using a total of 36 uls (0.036 mg) of bead slurry. In previous reports, three rounds of bead adsorption resulted in nearly complete removal of Env-specific antibodies from serum/plasma samples The PASS assay was performed as previously described 5′Acr-TTTCCCTGGTCCCATGGGTATACTTTTTC-3′ orCH40-rev: 5′Acr-ATTATTGCCGGGTCTCATACATTTG-3′CH77-rev: 5′-CCACAGACCCCAACCCACAAGAAG-3′CH40/77-for: PASS sequencing primer sequences,CH40:5′-TACTAATACCACTAATAGTAACGGG-3′ (nt 6635–6659), 5′-TACTAATACCACTAATAGTAACGGGG-3′, 5′-TACTAATACCACTAATAGTAACGGGA-3′, 5′-TACTAATACCACTAATAGTAACGGGGAA-3′, 5′-TACTAATACCACTAATAGTAACGGGACA-3′, 5′-TACTAATACCACTAATAGTAACGGGAAA-3′, 5′-TACTAATACCACTAATAGTAACGGGGGA-3′CH77:5′-TTATAAACTTGATGTAGTACCAATAGATACA-3′ (nt 6752–6782)5′-TTATAAACTTGATGTAGTACCAATAGATACAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAG-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAC-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAGA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACACA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAC-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAC-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAGAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACACAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAACA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAAAA-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAAAAG-3′5′-TTATAAACTTGATGTAGTACCAATAGATACAGAAA-3′RNA was extracted from pelleted virions containing at least 200,000 viral RNA copies using EZ-1 viral RNA kit (Qiagen) from CH40 plasma from days 16, 45, 181, and 412 post-seroconversion. cDNA was synthesized using superscript III reverse transcriptase (Invitrogen) in 5 replicates with the antisense primer 1.R3.B3R. The cDNA was immediately subjected to nested PCR amplification using Platinum Taq DNA Polymerase High Fidelity (Invitrogen). For each time point, 96 replicate PCR reactions (40 µl each) were performed with 5 µl cDNA, using forward primer BKB3F2 and the same reverse primer used for cDNA synthesis (1.R3.B3R). All 96 first round reactions for each time point were pooled and used as template for three inner PCR reactions. Each inner PCR reaction was performed with 32 replicates (40 µl each) using 5 µl of pooled first round template, and specific primers that incorporated a 4 base identification tag as well as a 19 base 454 adaptor sequence. Agarose gel-run PCR amplicons were visualized with crystal violet/white light and then subjected to 454 sequencing. Each sample was run on a separate picotiter plate with GS-FLX titanium reagents. All amplicons were agar-gel purified, eluted in EB buffer (QIAquick gel extraction kit), and visualized with gentian violet. Directional reads were codon-aligned from two amplicons that span the V1 loop to the T/F sequence and reviewed for sequencing errors as described previously Bulk PCR primers:5′-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′; nt 9642-9611 HXB2).1.R3.B3R BKB3F2 (454 patient-specific primer pairs for two amplicons (A & B):GTGGGTCACAGTCTATTATGGG HXB2 nt 6326–6347A–F: GGCTCAAAGGATACCTTTGGAC HXB2 nt 6859-6838A–R: GGGATCAAAGCTTAAAACCATG HXB2 nt 6015–6036B–F: GCATTGTCACTGAAATTGACTG HXB2 nt 6522-6501B–R: Specific adapter sequences ligated to 5′ end of each primer for directional sequencing:CGTATCGCCTCCCTCGCGCCATCAGF: CTATGCGCCTTGCCAGCCCGCTCAGR: 5 cells were incubated with 50,000 IU of virus (multiplicity of infection 0.1) overnight at 37°C in 250 µl RPMI 1640 with 15% FBS and 30 U/ml IL-2. Cells were washed three times and plated in 24-well polystyrene tissue culture plates in a volume of 500 µl RPMI 1640 with 15% FBS and 30 U IL-2/ml. 50 µl of media was removed for day 1 p24 baseline analysis. Every 2 days, 50 µl lf media was removed and frozen for p24 analysis. For viral replication competition assays, cells were isolated and activated as described above. 1×106 cells were incubated with 50,000 IU of each virus (for a combined multiplicity of infection of 0.1) overnight at 37°C in 250 µl RPMI 1640 with 15% FBS and 30 U/ml IL-2. Cells were washed three times and plated in 24-well polystyrene tissue culture plates in a volume of 1 ml RPMI 1640 with 15% FBS and 30 U IL-2/ml. 50 µl of media was removed for day 1 p24 baseline analysis, and 80 µl for an estimate of the input stock for PASS (see above). Every 2 days, 50 µl lf media was removed and frozen for p24 analysis and 80 µl was frozen for PASS analysis.Viral replication was assessed in activated primary CD4+ cells from normal human donors as previously described To investigate quantitative relationship between virus replication, diversification, and antibody-mediated selection, we extended a previous model of virus dynamics in HIV-1 infection in vivo is larger than the fitness cost associated with escape, The change in frequency of the WT, or T/F virus in the population, de novo infections by the Emax model commonly used in pharmacodynamic modeling, i.e.The efficacy of the Nab response may change over time, for example, because of an increase in the level of Nabs. To describe the kinetics of the Nab response to the WT virus, we use a simple model where the level of Nabs begins increasing after a time delay, A model of HIV-1 gp120 for all regions except V1/V2 was constructed from crystal structures of HIV-1 gp120 core with complete N and C termini Dataset S1Fitness costs of Nab escape mutations and mathematical modeling of Nab efficacy.(DOCX)Click here for additional data file.Figure S1Kinetics of autologous Nab responses, plasma viral load and CD4+ T lymphocyte counts. Autologous plasma Nab titers against the respective T/F Envs for subjects CH40, CH77 and CH58, as measured by TZM assay and expressed as reciprocal IC50s, are shown in bold lines. Each value is the average of four independent experiments, performed in duplicate. The minimal plasma dilution used in the assay was 1∶20, and for this plot Nab titers of <1∶20 are denoted as zero. Each subject's plasma viral load is color-coded and represented by dashed lines; the CD4+T lymphocyte count is represented in black with the scale to the right.(EPS)Click here for additional data file.Figure S2Replicative fitness of Nab escape mutants. A. Replication kinetics of CH40 T/F, 6 mo, and 6 mo-Nab IMCs cultured in parallel. Virus stocks were generated on CD4+ T cells and equal M.O.I.s were inoculated onto parallel cultures of CD4+T cells from two seronegative donors. Viral p24 antigen was measured every 2 days. B. Replication kinetics of CH40 IMCs grown in direct competition. Equal quantities of viruses derived from CH40 6 mo IMC and CH40 6 mo-Nab IMC were co-cultured in the same CD4+ T lymphocytes. 50 ul of supernatant was removed every two days and assayed for viral growth by p24 antigen ELISA and tested for relative proportions by PASS. Replication kinetics of CH77 (C) and CH58 (D) T/F, 6 mo, and 6 mo-Nab IMCs. Results represent mean (+/− SD) of three independently performed experiments.(EPS)Click here for additional data file.Figure S3Model of HIV infection and Nab escape in acute infection. In this model, wild-type (WT) or T/F virus, (EPS)Click here for additional data file.Figure S4Estimation of Nab efficacy to block de novo infections. Using the data on the loss of the T/F sequence, we estimate the average efficacy of the Nab response at blocking de novo infections by the T/F virus. Our model assumed that the Nab response started at in vivo efficacy of the Nab response, in vivo efficacy of the Nab response (panel D) and the loss of the T/F virus (panel E) using Eqns. (4) and (7). The estimated parameters for the kinetics of the Nab response are (EPS)Click here for additional data file.Table S1Frequency (%) of CH40 V1 Nab epitope sequence variants measured by SGA and PASS.(DOCX)Click here for additional data file.Table S2Frequency (%) of CH77 V2 Nab epitope sequence variants measured by SGA and PASS.(DOCX)Click here for additional data file.