During 2008–2009 in Israel, equine encephalosis virus (EEV) caused febrile outbreaks in horses. Phylogenetic analysis of segment 10 of the virus strains showed that they form a new cluster; analysis of segment 2 showed ≈92% sequence identity to EEV-3, the reference isolate. Thus, the source of this emerging EEV remains uncertain. Equine encephalosis virus   proteins from 10 linear dsRNA genome segments  in Israel were collected into EDTA tubes and analyzed at the Koret School of Veterinary Medicine . Vero cell  culture results of blood from H3, H5, and H8 were positive for EEV .1GTT AAG TTT CTG CGC CAT GT23-3′, 5′-741GTA ACA CGT TTC CGC CAC G760-3′. Thermal cycling conditions for the PCR were as previously described (http://cmgm.stanford.edu/phylip/dnaml.html) to construct the trees. Finally, the Consense program version 3.5c (http://cmgm.stanford.edu/phylip/consense.html) was used to create a final consensus tree for our dataset. Broadhaven virus, a tick-borne orbivirus, was used as the outgroup in the phylogram for the gene encoding NS3.Total RNA was extracted from the fifth and sixth passages of all 3 samples by using the QIAamp Viral RNA Mini Kit  to obtain sufficient viral load for the subsequent analyses and replications. RNA was reverse transcribed into cDNA by using the Verso cDNA Kit . PCR amplification of the gene encoding NS3 (Seg-10) was performed on the 3 isolates by using GoTaq Green Master Mix  with the following primers: 5′-escribed . PhylogeThe phylogenetic analyses of EEV Seg-10 grouped the Israeli isolates with other EEV isolates but as a distinct group with no close relation to African horse sickness virus, BTV, or epizootic hemorrhagic disease virus. Within the EEV group, 3 discrete clusters  were recognized; the Israeli isolates formed one of these clusters C; . The Isr,In addition, full-length cDNA copies of individual EEV (from H3 and H8) genome segments were synthesized and amplified by reverse transcription PCR by using the anchor spacer–ligation method as described ,. PreviouEquine encephalosis virus has long been enzootic to southern Africa, but it has not been isolated in other parts of the world. We report the characterization of an EEV strain isolated outside of Africa. Phylogenetic analysis of Seg-2 showed 92% sequence identity to EEV-3 .Culicoides spp. midge vectors, C. imicola (senso stricto) and C. bolitinos. The former is the most abundant Culicoides spp. midge in Israel  of different orbiviruses showed 2 clusters of South African EEV strains (A and B), in agreement with previously published studies (,The question of how and when the virus was initially introduced to Israel remains unanswered. Because the clinical manifestations of equine encephalosis are usually mild, it is often overlooked and underdiagnosed. EEV could have been introduced to Israel before the virus was first isolated in 2009. Alternatively, the virus might have been introduced into neighboring countries and transmitted into Israel by infected vectors carried by winds, as described for other orbiviruses (


Intensive insulin therapy (IIT) has been shown to reduce morbidity and mortality of critically ill patients ,2. A surThree ICUs developed and implemented an evidence-based guideline for IIT. For 1 year before and 1 year after implementation, all disposables and devices explicitly used for IIT were identified in each hospital. Local costs were calculated, based on costs for disposables and devices. Variable cost included costs associated with disposables. Fixed cost included costs associated with syringe pumps and point-of-care devices for blood glucose level (BGL) measurements.P < 0.001). The number of BGL measurements per patient per day doubled from 4 (3 to 7) to 9 (5 to 12) per day (P < 0.001). Yearly variable costs increased from €58.574 to €118.624 (P < 0.001), yearly fixed costs increased from €450 to €14.282 (P < 0.001). Importantly, costs differed substantially from one centre to another: variable costs per patient increased from €34 (€13 to 75) to €116 (€61 to 212) (P < 0.001), from €13 (€5 to 44) to €48 (€32 to 88) (P < 0.001) and from €15 (€7 to 34) to €31 (€15 to 70) (P < 0.001) for the three ICUs, respectively. Fixed costs per bed per year increased from €0 to €250 (P < 0.001), from €13 to €384 (P < 0.001) and from €25 to €544 (P < 0.001) for the three ICUs, respectively.A total of 2,490 patients were subjected to IIT. Patient demographics did not differ among the three ICUs and did not change over time. Median BGL declined from 119 (99 to 150) to 105 (85 to 130) mg/dl (Glucose control-associated costs rise with the implementation of IIT. Major differences in costs are noticed when comparing ICUs with similar patient cohorts and similar blood glucose control metrics after implementation of IIT.


Orthopoxviruses, including variola virus, vaccinia virus, and monkeypox virus, have previously been documented in humans in West Africa, however, no cases of human orthopoxvirus infection have been reported in the region since 1986. We conducted a serosurvey to determine whether human exposure to orthopoxviruses continues to occur in eastern Sierra Leone.To examine evidence of exposure to orthopoxviruses in the Kenema District of Sierra Leone, we collected and tested sera from 1596 persons by IgG ELISA and a subset of 313 by IgM capture ELISA. Eleven persons born after the cessation of smallpox vaccination had high orthopoxvirus-specific IgG values, and an additional 6 persons had positive IgM responses. No geographic clustering was noted.These data suggest that orthopoxviruses continue to circulate in Sierra Leone. Studies aimed at obtaining orthopoxvirus isolates and/or genetic sequences from rodents and symptomatic humans in the area are indicated. Poxviridae [Orthopoxvirus genus are variola virus, the causative agent of smallpox; vaccinia virus, the live virus component of the smallpox vaccine; monkeypox virus, the causative agent of monkeypox; and as well as other zoonotic viruses, including many not known to cause human disease. Smallpox vaccination results in broadly reactive anti-orthopoxvirus serologic responses that persist for decades [Orthopoxviruses are large DNA viruses in the family xviridae . Include decades .Sierra Leone is a country of 6 million inhabitants on the west coast of Africa Figure . In respDespite the absence of reported human orthopoxvirus infections in West Africa in recent decades, the introduction of monkeypox virus into the United States from West Africa demonstrated that orthopoxviruses are still present in the region, at least in small mammals. We conducted a large serosurvey in Kenema District in eastern Sierra Leone, to determine whether human exposure to orthopoxviruses continues to occur in that region. This is the most populous district in the country, with an estimated 482,000 inhabitants . ExtensiThe study was approved by the institutional review boards of CDC and Tulane University Health Sciences Center and the Ethics Committee of the Ministry of Health of Sierra Leone. Sera were collected as part of a study on Lassa fever in Kenema District in the Eastern Province of Sierra Leone.Statistics Sierra Leone, the main body responsible for government statistics in Sierra Leone, administratively divides the country into "enumeration areas." For the survey, 40 enumeration areas identified through a 2004 national census were selected with probability proportional to population size . Eightee5 PFU/well), a goat anti-human IgG horseradish peroxidase conjugate (1:2000 dilution), and developed with tetramethybenzidine one-component substrate; optical density was read at 450 nm. The IgM ELISA is an indirect capture assay using goat anti-human IgM (1:800 dilution) coated plates, purified vaccinia virus (6.2*105 PFU/well), anti-variola virus hyperimmune mouse polyclonal ascitic fluid (1:250 dilution), goat anti-mouse IgG horseradish peroxidase conjugate (1:6000 dilution), and developed with tetramethybenzidine one-component substrate; optical density was read at 450 nm. Sera were run in parallel using a quality-controlled lot of reagents on the same day at dilutions of 1:100 for the IgG assay and 1:50 for the IgM assay. Known anti-orthopoxvirus positive and negative human sera were used for assay standardization and as controls. Assay cut off values (COV) were defined as the mean plus three standard deviations of 5 known negative specimens. Results are reported as the optical density minus the COV (OD-COV). Based on previous studies, an equivocal range for IgM results was established as OD-COV of 0.0 to 0.04, with values above 0.04 considered positive [Orthopoxvirus, they cannot differentiate between antibodies elicited by different orthopoxvirus species or from antibodies induced by previous vaccination.All samples were tested by ELISA for anti-orthopoxvirus IgG antibodies and a subset for IgM antibodies, as previously described ,15. Briepositive ,15. AlthSerum samples were obtained from 1596 persons residing in 18 different villages in eastern Sierra Leone Figure . BecauseWhile it is apparent that IgG seroreactivity is higher on aggregate in the older age groups, the distribution plots did not display a clear demarcation for designation of negative and positive seroreactivity. However, 11 persons (1.3%) below vaccination age  showed exceptionally strong positive IgG results (OD-COV > 0.5), suggesting exposure to orthopoxvirus in the absence of smallpox vaccination. Nine (82%) of the seropositive persons were female; however, the proportion of seropositive females and males was not significantly different , which was not surprising, given the small sample size. There was no apparent geographic clustering of these 11 persons   was positive. Given the indication of possible recent orthopoxvirus exposure at this site, we decided to test all 46 remaining samples from Sawula (n = 46) as well as all persons below vaccination age with an IgG OD-COV > 0.2 (n = 57), and 50 randomly selected persons from the remaining pool of all samples. Five (3%) of these 153 samples were IgM positive . Four of the five persons were below vaccination age while the age of the fifth, from the randomly selected group, was unknown. Although 4 of these 5 IgM positive persons had IgG OD-COV values > 0.0, all were < 0.5.Our data suggest that, although likely infrequent, human exposure to orthopoxviruses continues to occur in eastern Sierra Leone even after the eradication of smallpox. The rarity of exposure is surprising considering the historic occurrence of monkeypox in the region ,6, and eDespite the low seroprevalence, the finding of high anti-orthopoxvirus IgG titers in 11 persons below vaccination age and positive IgM titers in 6 persons, 4 of whom were below vaccination age, suggest that human exposure to orthopoxviruses in Sierra Leone does occur. Although we do not have a detailed migration history for each study participant, 15 (88%) of the 17 heads of households of these sero-positive persons reported living in the same village for their entire lives, with the exception of some displacement during the civil war. This observation suggests that infection may indeed have occurred in Sierra Leone.There are multiple possible explanations for the finding of serological evidence of orthopoxvirus infection, but no reported disease, in the study population: First, human illnesses may be occurring but not being detected or reported. The Sierra Leone civil war has severely impeded surveillance and reporting over the past few decades. It is also possible that cases of monkeypox infection continue to occur but, like in Central Africa, are mistaken for chickenpox . No laboRecent serologic evidence from Ghana showed orthopoxvirus infection to be frequent in rodents and humans with rodent exposure despite the absence of any reported human disease in the area . SimilarPresumably the circulating orthopoxvirus in Sierra Leone is not a human pathogen, however due to broad serologic cross-reactivity between orthopoxviruses, we cannot definitively identify the virus (or viruses) associated with these exposures. While other serologic assays  may be more specific for certain viruses, without a presumptive virus species interpretation of other assays may be equally challenging. Taterapoxvirus, which is closely related to variola virus, is not known to cause human infection but has been found in gerbils in West Africa .We recognize various limitations in our study; interpretation of serosurvey results, particularly with regard to setting cut-off values for a positive serologic response, is always challenging and open to debate. Background smallpox vaccination inevitably further clouds the picture when attempting to assess possible exposure to zoonotic orthopoxviruses. Because of these challenges, we took a conservative approach to the interpretation of our data, choosing to focus our analysis on those with exceptionally high IgG OD-COV values rather than simply reporting the overall orthopoxvirus seroprevalence. However, the 11 IgG positive individuals we identified had OD-COV values that were higher than the majority of OD-COV values in the 29 years and older population , strongly supporting the notion that this seroreactivity is the result of previous exposure to wild-type zoonotic orthopoxviruses.In conclusion, despite the low number of positives, the findings from our study, the historic occurrence of monkeypox in the region ,6, and eThe authors declare that they have no competing interests.AM, MR, ID, KK, and DB developed the study and obtained protocol approvals, RF, LK, WR, VL, AG, and LM assisted in design, sampling, and implementation of the serologic survey, JA performed all study assays, AM and AJ performed analysis and interpretation of serosurvey data, RL and LM engaged in collection and analysis of geographic data, AM, MR, ID, KK, and DB drafted the manuscript. All authors read and approved the final manuscript.


To the Editor: The newly identified human bocavirus (hBoV), a member of the Parvovirus family, is suspected to infect the cells of the respiratory tract and thus may be an etiologic agent of respiratory disease in humans  that changed into a highly malignant blastic B-cell lymphoma . The patient was previously treated with vincristine and prednisone, followed by chemotherapy according to the R-CHOEP-14 protocol (3 cycles) (November 2003 through January 2004). From January through February 2004, chemotherapy was combined with antimicrobial drug therapy according to the R-DHAP protocol  for persisting symptoms from the B-cell lymphoma. This regimen was followed by a therapy switch to alemtuzumab with ifosfamid, carboplatin, and etoposide (March 2004), which led to a therapy-induced leukopenia, thrombocytopenia, and high fever >40°C by the end of March and the beginning of April 2004. In May 2004, a second round of alemtuzumab with ifosfamid, carboplatin, and etoposide chemotherapy was initiated. In June 2004, a therapy-induced hemorrhagic cystitis occurred. During July 2004, the patient had ongoing high fever and aplasia of bone marrow with unclear etiology. On July 22, hospital treatment was initiated; it consisted of antimicrobial drug treatment with ceftriaxone  and gentamicin (320 mg once daily), and antimycotic therapy was started with caspofungin (50 mg once daily).Since cytomegalovirus (CMV) infection was suspected, ganciclovir (250 mg twice daily) was administered IV for 2 weeks. Although the patient reported an ongoing cough and pneumonialike symptoms, a severe atypical pneumonia that was refractive to antibacterial and antimycotic treatment was diagnosed for the first time during this hospital treatment. Computed tomography scan showed bilateral atypical reticulonodular infiltrations predominant in the lower zones of the lungs .Mycobacterium tuberculosis, Chlamydia pneumoniae, Pneumocystis jirovecii, Aspergillus sp., Candida sp., Cryptococcus neoformans, CMV, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, HIV, herpes simplex virus, and varicella-zoster virus by PCR and culture cultivation. Results were negative, except for a temporarily weak reactivity for Aspergillus antigen in serum and for CMV DNA in peripheral blood lymphocytes, which was positive before and became negative after ganciclovir therapy. An archived portion of the BAL was assayed retrospectively by PCR/reverse transcriptase–PCR for human bocavirus, respiratory syncytial virus, human coronaviruses including severe acute respiratory syndrome–associated coronavirus, influenza virus, and human metapneumovirus (hMPV). The only positive result was obtained for human bocavirus, which was confirmed by sequence analysis of the PCR product.The BAL obtained during exacerbation of the pulmonary symptoms was tested for Within a few days, the patient's symptoms decreased, and she was discharged from hospital on day 41, despite ongoing bone marrow aplasia with antimicrobial and antimycotic prophylaxis, including trimethoprim/sulfamethoxazole (160 mg/800 mg once daily) and (voriconazole 200 mg twice daily). Clinical observations led to the primary assumption that the fever, cough, and pulmonary symptoms were likely caused by the postchemotherapeutic extended bone marrow aplasia and CMV infection accompanied by an unclear bacterial but fungus-typical infection. Retrospectively, however, human bocavirus DNA in the archived BAL strongly suggests that pulmonary symptoms were caused by this agent rather than by a yet unknown bacterial or fungal infection. Thus, in the clinical episode described here, the likely causative agent responsible for the severe pneumonia was the recently described bocavirus.––Respiratory viruses such as respiratory syncytial virus, hMPV, and hBoV seem to be the most prevalent etiologic agents of acute lower respiratory tract infection in children. Recently, evidence of human bocavirus infection was reported for 3.1% to 5.7% of children <3 years of age (


Of the eight human herpes viruses, varicella-zoster virus, which causes chickenpox and zoster, has a unique epidemiology. Primary infection is much less common in children in the tropics compared with temperate areas. This results in increased adult susceptibility causing outbreaks, for example in health-care workers migrating from tropical to temperate countries. The recent demonstration that there are different genotypes of varicella-zoster virus and their geographic segregation into tropical and temperate areas suggests a distinct, yet previously unconsidered climatic factor may be responsible for both the clinical and molecular epidemiological features of this virus infection.Unlike other human herpes viruses, varicella-zoster virus does not require intimate contact for infection to occur indicating that transmission may be interrupted by a geographically restricted climatic factor. The factor with the largest difference between tropical and temperate zones is ultra-violet radiation. This could reduce the infectiousness of chickenpox cases by inactivating virus in vesicles, before or after rupture. This would explain decreased transmissibility in the tropics and why the peak chickenpox incidence in temperate zones occurs during winter and spring, when ultra-violet radiation is at its lowest. The evolution of geographically restricted genotypes is also explained by ultra-violet radiation driving natural selection of different virus genotypes with varying degrees of resistance to inactivation, tropical genotypes being the most resistant. Consequently, temperate viruses should be more sensitive to its effects. This is supported by the observation that temperate genotypes are found in the tropics only in specific circumstances, namely where ultra-violet radiation has either been excluded or significantly reduced in intensity.The hypothesis is testable by exposing different virus genotypes to ultra-violet radiation and quantifying virus survival by plaque forming units or quantitative mRNA RT-PCR.The ancestral varicella-zoster virus, most probably a tropical genotype, co-migrated with man as he left Africa approximately 200,000 years ago. For this virus to have lost the selective advantage of resistance to ultra-violet radiation, the hypothesis would predict that the temperate, ultra-violet sensitive virus should have acquired another selective advantage as an evolutionary trade-off. One obvious advantage could be an increased reactivation rate as zoster to set up more rounds of chickenpox transmission. If this were so, the mechanism responsible for resistance to ultra-violet radiation might also be involved in reactivation and latency. This could then provide the first insight into a genetic correlate of the survival strategy of this virus. Chickenpox epidemiology is unique among human herpes viruses. In the tropics primary infection is often delayed into later childhood whereas in temperate zones most infection occurs before leaving school. Indeed, in some tropical countries 30-50% of adults are susceptible, compared with only 5-10% from temperate areas [Conventionally, transmission has been considered to occur by shedding of virus from the upper respiratory tract 1-2 days before the rash . The papWhy such a common, global infection should be less common in children from the tropics when infections are generally more common remains unknown. Although previously suggested factors such as heat, humidity, viral interference, population density or infection with cross-protecting viruses, have been suggested as possible causes of the epidemiological differences, a unified, coherent explanation has eluded discovery ,12. The 2 ≈ 0.5) was demonstrated across all age groups of children >5 years  and the United Nations (http://www.fao.org/WAICENT/FAOINFO/SUSTDEV/EIdirect/CLIMATE/EIsp0002.htm), showed no consistent correlation  using the terms "varicella", "chickenpox" and "seroepidemiology" produced a total of 25 papers. From these publications other relevant references were also located giving a total of 42 articles, reviewed in . Whilst s Figure . The sam Figures , 4 and 5Chickenpox is seasonal in temperate zones, with the highest incidence seen in winter and spring . One expFurther support for the hypothesis derives from sequence analysis which has classified VZV into distinct genotypes. In the largest published study, 348 genotypes of VZV were given geographic locations based on where the virus was originally detected . In the In Australia widespread preventative measures are taken limit exposure to UVR in schools by having large, shaded playground areas.In urban Brazil, man-made biomass burning and in rural areas, the forest canopy and high humidity act together to reduce UVR .In the Congo, the first ever demonstration of transmission of temperate virus, occurred in only one family all living in the same house, the implication being that temperate virus is rapidly inactivated by UVR after leaving the confines of the family home .Finally, the detection of temperate virus genotypes from cases of chickenpox in Mexico City may be explained because it is one of the most heavily polluted cities in the world which reduces UVR, allowing temperate genotypes to survive .in vitro has never been tested [in vivo was demonstrated over 60 years ago when artificial UVR was used successfully to reduce virus transmission in US schools to limit spread of chickenpox [The hypothesis is biologically plausible because UVR is virucidal against many viruses, yet the effect of UVR on survival of VZV n tested . Howeverickenpox . Epidemiin vitro by exposing virus to UVR and quantifying the surviving virus by either plaque forming units or quantitative mRNA RT-PCR. Finally, it may also be possible to make hybrid viruses by exchanging those regions of the VZV genome which are significantly different between genotypes and determine for the first time the molecular markers that underlie transmission or reactivation of VZV.If different genotypes of VZV possess different tolerances to UVR this could be demonstrated The principal difficulty with the hypothesis is explaining how an ancestral tropical virus genotype, inherently more resistant to UVR, migrated with man out of Africa 200,000 years ago only to lose the selective advantage of resistance to UVR, form a temperate virus genotype lineage and as result become less transmissible. The solution to this paradox could be that loss of the selective advantage of resistance to UVR and reduced transmissibility was offset by an increased propensity to reactivate as zoster. This could indicate that the areas of the VZV genome which confer resistance to UVR are the same as are involved in latency and reactivation.I suspect this to be the case because as the transmission environment is so harsh in the tropics, random mutation and natural selection should have brought about a tropical virus genotype which reactivates much more frequently to counter-act the lower transmissibility of chickenpox. The fact that the data on zoster epidemiology from tropical countries (in the pre-AIDS era) are virtually absent suggests that the tropical genotype reactivates only in severely immune suppressed individuals. Potentially it may have implications for VZV vaccine since if it was made from a tropical genotype which reactivated much less frequently, it might be possible, in years to come, to significantly reduce the disease burden from zoster.UVR: Ultra-violet radiation; VZV: Varicella-zoster virusThe authors declare that they have no competing interests.Solely responsible for developing, researching and writing the hypothesis.


Potato virus Y (PVY) variants differing at most by two substitutions involved in pathogenicity properties. Model selection procedures were used to compare experimental results to six hypotheses regarding competitiveness and intensity of genetic drift experienced by viruses during host plant colonization. Results indicated that the frequencies of variants were well described using Lotka-Volterra models where the competition coefficients ijβ exerted by variant j on variant i are equal to their fitness ratio, jr/ir. Statistical inference allowed the estimation of the effect of each mutation on fitness, revealing slight (s = −0.45%) and high (s = −13.2%) fitness costs and a negative epistasis between them. Results also indicated that only 1 to 4 infectious units initiated the population of one apical leaf. The between-host variances of the variant frequencies were described using Dirichlet-multinomial distributions whose scale parameters, closely related to the fixation index FST, were shown to vary with time. The genetic differentiation of virus populations among plants increased from 0 to 10 days post-inoculation and then decreased until 35 days. Overall, this study showed that mathematical models can accurately describe both selection and genetic drift processes shaping the evolutionary dynamics of viruses within their hosts.Uncovering how natural selection and genetic drift shape the evolutionary dynamics of virus populations within their hosts can pave the way to a better understanding of virus emergence. Mathematical models already play a leading role in these studies and are intended to predict future emergences. Here, using high-throughput sequencing, we analyzed the within-host population dynamics of four  Potato virus Y variants differing at most by two substitutions involved in pathogenicity properties. The competitiveness costs induced by these mutations as well as the mathematical expressions of the competition coefficients of virus variants were derived from Lotka–Volterra equations. High genetic differentiation of virus populations between hosts was evidenced as well as its hump-shaped behaviour with time. The modelling framework proposed here was intended to help design control strategies aiming to prevent virus emergences.Natural selection and genetic drift drive the evolution of virus populations within their hosts and therefore influence strongly virus emergences. To help predict future virus emergences, we developed a model that estimates simultaneously genetic drift and selection intensities using high-throughput sequence data representing the within-host population dynamics of  Plant virus emergences represent near half of emerging plant infectious diseases Natural selection is a deterministic process by which the frequencies of the fittest variants in a given environment increase Potato virus Y (PVY) variants simultaneously submitted to genetic drift and natural selection. The four variants differ by one or two mutations that change their pathogenicity properties towards pepper genotypes carrying resistance alleles at a single locus While within-host genetic drift and selection act simultaneously, and thus jointly determine emergence, their intensities have rarely been estimated and modelled jointly from experimental data. Drift intensity was often measured using populations of pathogen variants with equal multiplicative fitness Capsicum annuum L.) genotype used in this study was Yolo Wonder, a bell pepper cultivar susceptible to all PVY isolates. The SON41p infectious cDNA clone 2pvr2 resistance gene (The pepper (nce gene  [20].Nicotiana clevelandii plants (four week old) N. clevelandii plants were calibrated using DAS-ELISA i.e. at two-leaf stage). The conformity of each variant was checked by direct sequencing of the RT-PCR product corresponding to the entire VPg cistron of the PVY populations present in the four plants used for the inoculum Inoculations were carried out under insect-proof greenhouse conditions. First, separate inoculations with the cDNA clones were realized by DNA-coated tungsten particle bombardments of juvenile Avian myeloblastosis virus reverse transcriptase (Promega), the high-fidelity Herculase II fusion DNA polymerase (Stratagene) and primers PYRO-FOR  and PYRO-REV . Emulsion-PCR and high-throughput 454 sequencing were realized by GATC-Biotech . The genome region sequenced encompasses notably codons 101 to 123 of the VPg cistron which has been demonstrated by reverse genetics to be the only region involved in breakdown of the pvr2 resistance genes in pepper Virus populations were separately sampled from eight plants at five successive dates: 6 days post-inoculation (dpi) corresponding to the 3–4 leaf stage, 10 dpi (5–6 leaf stage), 15 dpi (7–8 leaf stage), 24 dpi (11–12 leaf stage) and 35 dpi (22–23 leaf stage). Additionally, a sample of the mixed inoculum used for mechanical inoculations on pepper plants was also collected. At each date, all leaves of each plant were harvested, homogenized in a buffer (0.03 M phosphate buffer (pH 7.0) supplemented with 2% (w ∶ v) diethyldithiocarbamate; 4 mL of buffer per gram of leaves) and total RNAs were purified with the Tri Reagent kit  from a 150 µL aliquot of each sample. RNAs were used to amplify the central part of the VPg cistron by RT-PCR with In addition, in order to estimate the effective population size during leaf colonization, the eight plants sampled at 15 dpi were kept till 50 dpi and then a single newly grown leaf was sampled randomly on each plant . These lWe obtained between 209 and 930 correctly assigned sequences per virus population of each pepper plant, corresponding to a total of 24,166 sequences. Alignment was done using default parameters of the software SeqMan . Because indels are the most frequent 454 pyrosequencing errors i(t)V of virus variants i = 1,…,4 at time t, within a host plant behaved as a deterministic interacting system of four ordinary differential equations (ODE):To investigate the time course of competing virus populations within host plants, we merged deterministic and stochastic aspects into a single model. The deterministic aspect relied on the assumption that the mean numbers (or densities) K is the virus carrying capacity of a plant,ir is the intrinsic rate of increase of variant i,i,jβ is the coefficient that accounts for the competition strength exerted by genotype j on genotype i,i,jμ is the mutation rate from genotype j to genotype i.μ denotes the point mutation rate per replication cycle and per nucleotide, we get i and j are distant from one mutation and i and j are distant from two mutations.Equation 1 extends Lotka-Volterra competition equations to four competitors i(t)V due to the heterogeneity of virus populations between plants and to the nature of samples (counts of virus sequences). To be more specific, let p sampled at time t (dpi) corresponding to virus variants NN, DN, NH and DH. Our second main assumption states that, conditionally to the total number i.e.:(i)t over all plants t.N (discarding time and plant indices t and p) is N results from a two-step random procedure. First, a non-observed random vector totN and random frequencies Λ, N is drawn according to a multinomial p at time t gives rise to a non-observed random vector θ(t) and on overall theoretic means λi(t) of the variant frequencies. The numbers p at time t is a multinomial θ(t) decrease the genetic differentiation among host plants. Asymptotically, if i.e. Λ is deterministic and equals λ. Besides, θ(t) can be actually related to the so-called fixation index FSTFor clarity, we recall that an integer valued vector t were given via the ODE system as Finally, the coupling between deterministic and stochastic components consisted in asserting that the overall frequencies of variants at time via the forms of the competition coefficients used to derive the overall theoretical frequencies i(t)λ and (ii) the intensity and temporal variation of genetic drift experienced by viruses during host plant colonization, which could be investigated via the time dependence of the scale parameter θ(t).Our goal was to determine (i) the forms of selection processes occurring between competing viruses within a host plant, which could be handled Accordingly, six models allowing four to 14 parameters  were con1) (C2) (C3)  (D2) , i.e. the number of founder infectious units initiating the systemic infection of a single leaf, we used a method based on FST statistics described in FST value at 15 dpi (resp. 50 dpi). A 95% confidence interval was calculated for eN with a bootstrap method by resampling data 1,000 times over plants.In order to estimate the effective population size . The detailed proof is provided in −5 and 4.34×10−7, respectively.The long term behaviour of the model that is best supported by the data  and at 50 dpi  among the same plants. At 15 dpi, three variants were systematically detected in all plants, although with varying frequencies .The scale of our study was the set of leaves of infected plants. It represents the epidemiologically-relevant part of the virus population since it is readily accessible to vectors that ensure plant-to-plant horizontal transmission. It is possible that the narrow bottlenecks and intense genetic drift observed at larger scales (whole plant and plant organs) are direct consequences of those incurred by virus populations at the smallest scale . Supporting this hypothesis, the time dependence of nt level  parallelFST values observed at later infection times are related to the sink-source transition undergone by leaves during plant growth. Source-to-sink translocation of carbohydrates through the phloem corresponds to the direction of the systemic movement of viruses within plants FST values. In the context of our experiments, the timing of the sink-source transition in pepper and its comparison with FST variation remain to be determined. The two above hypotheses are not mutually exclusive, since the increasing number of source leaves during infection could also increase the MOI FST values.It is also possible that decreasing 121H and N119D amounted to −0.45% and −13.2%, respectively. These figures agree with the distribution of the mutational effect of single nucleotide substitutions. Indeed non-lethal mutations reduce virus fitness by 10–13% on average 2pvr2 (mutation N121H followed by mutation N119D) −7). In natural context, the RB variant would most likely appear initially by mutation at a very low frequency in a virus population largely dominated by the fittest variants NN and NH. Note also that, although multidrug-resistant variants can be generated by recombination  and final  virus populations are represented in blue and red, respectively.Protocol used to estimate the effective population size during the colonization of a pepper leaf by (PDF)Click here for additional data file.Table S1Number of well-assigned and cleaned sequences to each sample (eight plants by date and the mixed inoculum) and number and frequency of each virus variant  in each population.(PDF)Click here for additional data file.Table S2eN during plant colonization.Estimation of (PDF)Click here for additional data file.Text S1Note on model identifiability and on likelihood-based inference.(PDF)Click here for additional data file.Text S2Mathematical analysis of the Lotka-Volterra model with mutation.(PDF)Click here for additional data file.


We report the accidental needlestick inoculation of a laboratory worker withvaccinia virus. Although the patient had previously been vaccinated againstsmallpox, severe lesions appeared on the fingers. Western blot and polymerasechain reaction–restriction fragment length polymorphism were used toanalyze the virus recovered from the lesions. The vacciniavirus–specific immunoglobulin G levels were measured by enzyme-linkedimmunosorbent assay. Our study supports the need for vaccination for laboratoryworkers that routinely handle orthopoxvirus. The smallpox vaccine, formulated with vaccinia virus, is a highly effective immunizingagent. In 1980, the World Health Organization certified that the world was free ofnaturally occurring smallpox, and smallpox immunization programs were subsequentlydiscontinued  virus as abioterrorism weapon. The vaccine is considered safe but can produce mild to moderatedisease in vaccinees and can be disseminated to their close contacts , as recommended bythe manufacturer. The variable region of vaccinia virus genome was investigated byamplifying the DNA segment from the B9R to B14R genes and digestion of the ampliconswith EcoR V and AluI , as recommended. The digestion products were analyzed by using1.2% agarose gels. The restriction patterns obtained for both regions in the testsample were identical to the profiles observed with the genome of vacciniavirus–WR (Pustular fluid from the lesions was collected and tested for the presence of bacteriaand virus. The Gram stain and cultures were negative for bacteria. When a dilutedsample of the pustular fluid was added to BSC-40 (monkey kidney) cell culture, apoxviruslike cytopathic effect was evident after 48 h of infection (data not shown).Vaccinia virus proteins were detected in infected cells by 12% sodiumdodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed byWestern Blot analysis with rabbit antiserum raised against total vaccinia virusproteins as described before . The previrus–WR .Serum collected from the patient day 20 after the initial inoculation was tested forvaccinia virus–specific immunoglobulin (Ig) G response by enzyme-linkedimmunosorbent assay (ELISA) as described . FurtherAccidental infection with live pathogens by healthcare and laboratory workers hasbeen frequently reported . The amount of virus in the needle before theaccident was approximately 1,000 times higher than the amount in the vaccinepreparations used for smallpox vaccination (


Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV.The family Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called “tymobox” or “marafibox”, the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus.The CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.The CuTLV is a novel virus related to members of the family  Tymovirus, Marafivirus, Maculavirus) in the family TymoviridaeTymoviridae differ according to the genus or individual viral species. The genomic RNA of tymoviruses is 6.0–6.7 kb in size, contains three ORFs, has a 16-nucleotide (nt) sequence known as the “tymobox” which functions as a subgenomic RNA promoter, and has a tRNA-like 3′-terminal structure Tymoviridae is thought to involve post-translational proteolytic cleavage of the polypeptide encoded by the large ORF by a papain-like virus-encoded protease, as well as coat protein expression via a subgenomic RNA There are three genera , referred to as CuTLV, and its phylogenetic relationships with known members of this family.To date, 26, 7, and 1 known species have been reported for the genera Culex spp.) captured in Xinjiang, China in 2005. The virus was found to induce cytopathic effect (CPE) in first-passage C6/36  cells on Day 3 after inoculation. The CPE was characterized by cell shrinking, shedding, fusion or cytolysis with eventual detachment from the growth surface . DNase-SISPA (sequence-independent single primer amplification) Tymoviridae following BLAST searches.Total RNA was extracted from CuTLV inoculated C6/36 cell lysates and analyzed independently in two laboratories . The extracted RNA was randomly PCR amplified and hybridized to a pan-viral DNA microarray as described To obtain the whole genome sequence of CuTLV, 5′ RACE and a modified 3′ RACE protocol were performed. The entire genomic sequence was confirmed by overlapping PCR amplification and sequencing with primers  designedLike other members of family Tymoviridae, the genomic sequence of CuTLV contains a high percentage of cytosines and a low percentage of guanines  Tymoviridae, ranging from 27.0% identity with Ononis yellow mosaic virus  to 37.9% identity with Bombyx mori macula-like virus .The second ORF  is 747 nt in length and encodes a putative 248-aa protein with a predicted molecular mass of 26.4 kDa. ORF2 is located at the 3′-end of the CuTLV genome. The putative CP of CuTLV has low-level sequence identity with the CPs of other members of family ORF3 overlaps the CP gene towards the 3′-terminus. ORF 3 starts at nt 5999 and ends with an opal stop codon (UGA) at nt 6446, which is 447 nt in length and encodes a putatively proline and serine rich 149-aa polypeptide (16 kDa). The putative p16 of CuTLV shares limited homology with the proline-rich p31 (21.8%) and p16 (35.2%) proteins Tymoviridae . The overall nucleotide identities of CuTLV with other members of family Tymoviridae ranged from 46.2% to 52.4%; the highest identity (52.4%) was with GFKV (NC_003347) and the lowest identity (46.2%) was with ChiYMV (NC_014127).Tymoviridae family based on the sequences of the RP and CP genes revealed that the topology of both trees was shown to coincide well. CuTLV is related to members of the Tymoviridae family, especially those of the Maculavirus including BmMLV (Bombyx mori macula-like latent virus) Maculavirus) Phylogenetic analyses of CuTLV and other members of the Tymoviridae. Among the chief characteristics is a large ORF that encodes proteins associated with replication of positive sense RNA viruses. Phylogenetic analysis of the predicted RP and CP genes demonstrated that CuTLV falls within the genus Maculavirus. This classification is supported by the absence of a tymobox or marafibox, the 16 nt conserved sequences that act as subgenomic promoters in the genera Tymovirus and Marafivirus. However, both nucleic acid and amino acid homology are very low compared CuTLV with BmMLV and GFkV, the reported macula-like virus which identified in Bombyx mori cultured cells and the type species of Maculavirus. Other distinctive features of CuTLV is the presence of three predicted stem loops structures in the 5′ UTR, and non-polyadenylated genomic RNA with a -AAC 3′-terminus.We identified a novel virus that shares many characteristics of the family Tymoviridae have been recorded from most parts of the world. Geographical distribution of individual species varies from restricted to widespread Tymoviruses and maculaviruses mainly infect dicotyledonous plants  marafiviruses preferentially infect Poaceae (Zea mays) Okra mosaic virus (Tymovirus), early infection of okra leads to yield losses of 12%–90% depending on the severity of the disease Marafivirus) is of great agronomic importance, as it produces significant yield losses in maize throughout Central and South America Marafivirus are transmitted by hemipteran insects  in a persistent manner Tymovirus are persistently or semipersistently transmitted by coleopteran insects (beetles) belonging to the families Chrysomelidae and Curculionidae, which are nonsucking insects. The vectors of Maculavirus are unknown Members of the family In conclusion, we identified a novel Tymoviridae-like virus from mosquitoes, CuTLV, and analyzed its genomic characterization. Long-term monitoring and further investigations should be performed to clarify the origin and replication strategy of this virus, and the potential risks that this virus poses for agriculture or public health.Aedes albopictus C6/36 cell lines were grown in minimal essential medium  with Hanks' salt solution supplemented with 10% FBS and 100 U/ml each of penicillin and streptomycin. The C6/36 cells were propagated and maintained at 28°C. BHK-21 and Vero cells were grown in MEM with a balanced salt solution supplemented with 10% FBS and 100 U/ml each of penicillin and streptomycin. The mammalian cells were maintained at 37°C in 5% CO2.Mayaro virus, Sagiyama virus, Ross River virus, Chikungunya virus, Eastern and Western equine encephalitis viruses, Getah virus, Sindbis virus, and Semliki Forest virus), flavivirus , bunyaviruses, and seadornaviruses (Banna virus and Liaoning virus).To investigate arboviruses in China, mosquitoes were collected from July to August 2005 in Jiashi , Xinjiang, China, using mosquito-trapping lamps  placed in resting sites of animal corrals in the evening (No specific permits were required for the described field studies. No specific permissions were required for collection of mosquitoes from this location. Field studies did not involve endangered or protected species). The mosquitoes were sorted and pooled. Pools of mosquitoes were homogenized, then inoculated and blind passaged three times (7 days per cycle) on monolayers of C6/36, Vero, and BHK-21 cells in 24-well plates, as previously described Csp6 I (Fermentas) were added, and the mixture was incubated for 1 h at 37°C. Then, the DNA fragments were extracted, precipitated, dissolved in water, and ligated to Adaptor_A (5′-AGGCAACTGTGCTATCCGAGGGAG-3′) and Adaptor_B (5′-TACTCCCTCGG-3′) in a 20-μl reaction that contained 20 pmol of each adaptor and 1 U of T4 DNA ligase (New England BioLabs) in a standard ligation buffer. The reaction mixture was incubated at 4°C for 1 h, followed by 16°C for 4 h. Then, 2 μl of the ligation reaction was used as a template for PCR, using Adaptor A as the primer. The PCR products were analyzed on a 1.5% agarose gel, and then extracted using the QIAquick Gel Extraction kit (Qiagen). The gel-purified fragments were cloned into the pGEM-T Easy Vector (Promega), and introduced into chemically competent Escherichia coli TOP-10 cells (Invitrogen). After transformation, single colonies were cultured and sequenced using the ABI 3730xl sequencing system and Big Dye Terminator chemistry (Applied Biosystems). Three clones were sequenced for each extracted and cloned band. The obtained sequences were subjected to homology searching using the BLAST software and the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/).DNase I  was added to 200 µl of culture fluids containing isolate CuTLV, and incubated for 2 h at 37°C, which was then used for RNA extraction using the QIAamp Viral RNA Mini kit (Qiagen) according to the manufacturer's protocol. Extracted RNA was converted to double-stranded cDNA with random hexamers. After completion of cDNA synthesis, the reaction mixtures (100 μl) were heat-inactivated at 72°C for 10 min, 10 U of the restriction endonuclease www.DNASTAR.com). Based on the sequences obtained from SISPA, the RACE method was used to identify the 5′-UTR and 3′-UTR of the CuTLV genome. The 5′-RACE method was employed using a commercially available kit (Invitrogen) with the Universal Amplification Primer (UAP) and CuTLV segment-specific primer R1  with all available databases. An ORF search was performed with the ORF Finder of the NCBI. Conserved domains in the amino acid sequences were identified using the CD-Search of the NCBI Tymoviridae were downloaded from GenBank and used to build the phylogenetic trees. Sequences were aligned using the CLUSTAL_X, version 2.0 software The nucleic acid sequences and deduced amino acid products were analyzed and assembled using the DNASTAR program (Lasergene). BLAST searches were carried out using the NCBI server (


Hendra virus is a recently emerged zoonotic agent in Australia. Since first described in 1994, the virus has spilled from its wildlife reservoir  on multiple occasions causing equine and human fatalities. We undertook a three-year longitudinal study to detect virus in the urine of free-living flying foxes (a putative route of excretion) to investigate Hendra virus infection dynamics. Pooled urine samples collected off plastic sheets placed beneath roosting flying foxes were screened for Hendra virus genome by quantitative RT-PCR, using a set of primers and probe derived from the matrix protein gene. A total of 1672 pooled urine samples from 67 sampling events was collected and tested between 1 July 2008 and 30 June 2011, with 25% of sampling events and 2.5% of urine samples yielding detections. The proportion of positive samples was statistically associated with year and location. The findings indicate that Hendra virus excretion occurs periodically rather than continuously, and in geographically disparate flying fox populations in the state of Queensland. The lack of any detection in the Northern Territory suggests prevalence may vary across the range of flying foxes in Australia. Finally, our findings suggest that flying foxes can excrete virus at any time of year, and that the apparent seasonal clustering of Hendra virus incidents in horses and associated humans (70% have occurred June to October) reflects factors other than the presence of virus. Identification of these factors will strengthen risk minimization strategies for horses and ultimately humans. Paramyxoviridae has spilled from its wildlife reservoir on 14 identified occasions (Pteropus (family Pteropodidae), colloquially known as flying foxes, are the natural host of the virus, and are asymptomatically infected Pteropus species occurring on mainland Australia , and across their geographic range Hendra virus is a sporadic, but highly lethal, recently emerged zoonotic agent in Australia. Case fatality rate in humans is 60%, and in horses, 75%. Since 1994, when it was first described, this novel member of the family ccasions , resultiWe undertook a three-year longitudinal study that used a quantitative RT-PCR assay to detect Hendra virus genome in pooled urine samples collected under flying fox roosts. The objectives of the study were two-fold: to investigate infection occurrence and frequency in flying fox populations, and to investigate the genetic diversity of Hendra viruses in flying fox populations. In this paper, we describe the former.Fieldwork was conducted under Department of Employment, Economic Development and Innovation Animal Ethics Committee permit SA 2008/10/270, Queensland Department of Environment and Resource Management Scientific Purposes permit WISP05810609, and the Northern Territory Parks and Wildlife Commission permit 37243.Flying fox colonies rather than individual flying foxes were targeted. Multiple roost sites were identified in three geographically disparate sampling locations (>1500 km apart) in south-east Queensland (SEQ), far north Queensland (FNQ) and in the Northern Territory (NT) . SampledAfter all samples were collected, 560 ul of urine was taken by graduated micropipette from each sample and added to an equal volume of lysis buffer  in a second 2 ml tube, to inactivate virus particles and preserve RNA. Then 500 ul of virus transport media (VTM) was added to the balance of the sample in the original tube. Samples were kept on wet ice or refrigerated until they could be packed and shipped on dry ice  to the Queensland Health Forensic and Scientific Services (QHFSS) laboratory (prior to March 2009) or the Biosecurity Sciences Laboratory (BSL) in Brisbane. At QHFSS/BSL, the samples were held in the PC3  laboratory, the lysis buffer samples at 4°C pending RNA extraction and PCR analysis, and the VTM samples in a biosafety container at −80°C pending PCR results.et al, unpublished). The extracted eluate was screened for Hendra virus RNA by TaqMan™ RT-PCR using a set of primers and probe derived from the matrix protein gene of Hendra virus At QHFSS/BSL, viral RNA was extracted using a QIAGEN viral RNA minikit™ and following a modified ‘large volume’ protocol Data were stored and managed in a Microsoft Office Access™ database. Analyses were performed on extracted data exported to a Microsoft Office Excel™ spreadsheet. P values of the chi square statistic are used to examine statistical association at the 95% confidence level. Where the nature of the data precludes the use of the chi square test, 95% binomial confidence intervals  are presented.Over the three-year study period, 25% (15/59) of sampling events yielded detections. There was no statistically significant association with year or location, the proportion of detections being 15.8% (3/19) in year one, 42.9% (6/14) in year two, and 23% (6/26) in year three (p = 0.20), and 26% (10/39) in SEQ, 41.7% (5/12) in FNQ, and 0% (0/8) in NT (p = 0.11). Of the 1460 pooled urine samples collected within the 59 sampling events, 2.5% (36/1460) yielded detections. The proportion of positive samples was statistically associated with both year and location, being 1.6% (5/311) in year one, 5% (21/416) in year two, and 1.4% (10/733) in year three (p = 0.0003), and 2.7% (25/926) in SEQ, 3.44% (11/320) in FNQ, and 0% (0/214) in NT (p = 0.03). In the collapsed within-year analysis , the nonP. alecto and P. polioceplalus; P. alecto and P. scapulatus; P. conspicillatus and P. scapulatus; P. alecto, P. polioceplalus and P. scapulatus) and single species roosts  (Infection was detected in mixed species roosts (illatus) .et al (2005) While serologic surveys have provided evidence of previous Hendra virus infection in Australian flying fox populations, successful attempts to demonstrate current infection have been limited P. alecto, P. conspicillatus or P. poliocephalus that (from our extensive observations) would roost within a 3 m.×2 m. perimeter (the dimensions of each sheet); the figure of 20 individuals per sheet reflected the typical number of the more densely roosting P. scapulatus that would roost within the same perimeter. However the calculations do not consider the often multiple vertical ‘layers’ of flying foxes above sheets that could result in two or three times as many individuals over sheets, significantly lowering the minimum prevalence detectable by 30 pooled samples.We believe that our 1.5–6% minimum detectable prevalence range, based on a typical 30 pooled urine samples from 10 sheets  is conservative; that is, we may sometimes be detecting infection present at lower prevalence. To elaborate, the figure of 5 individuals per sheet reflected the typical number of et al (2011) The number of sampling events per annum over the three-year study ranged from 16 to 32, the latter primarily reflecting an increased effort on FNQ and NT samplings in the third year. In all three years, the majority of the sampling events were in SEQ , due primarily to logistical constraints to sampling in FNQ and NT. However, in the third year, we were able to increase the number of FNQ and NT samplings to reach our target sampling frequency (every 7–8 weeks) in these locations. We also sought to increase our sampling efforts at all locations in January and February in the third year (these months coincide with the peak of the wet season in Queensland), and were successful in SEQ, but not in FNQ and NT . The number of SEQ samplings stayed relatively constant  across the three years, reaching or exceeding our target sampling frequency (every 4 weeks) for this location. The increased number of SEQ sampling events in the third year reflected a period of ‘follow-up’ sampling from January to early-April 2011, to gather additional information on infection dynamics in a colony in which infection was detected. A similar period of increased sampling frequency also occurred August to October 2009 in the same colony following detection of infection. Comparison of the two periods reveals contrasting infection dynamics, with the 2009 period indicating an explosive outbreak (peaking with 33.3% of urine samples positive), and the 2011 period suggesting a smoldering infection . While tThe proportion of positive sampling events and the proportion of positive samples provide two distinct measures of ‘prevalence’, the former between sampling events, and the latter within sampling events. We found that positive events were not significantly associated with location or year, whereas positive samples were. The wide 95% confidence intervals suggest the lack of association with sampling event (given the trend suggested by the point estimates) is primarily a reflection of the  limited number of sampling events, rather than a true absence of association. Because multiple samples were collected in each sampling event, sample size does not constrain statistical confidence, and we see a strong association between positive samples and both variables. Firstly considering location, we found that the proportion of positive samples was similar in SEQ and FNQ over the study period, and statistically different from NT, where no samples were positive. The proportion of positive sampling events showed the same pattern, but was not statistically significantly different. This finding suggests a real difference in the prevalence of Hendra virus infection in flying fox populations in Queensland and the Northern Territory. Whether this difference truly reflects location, or is confounded by species, population structure or some other variable cannot be ascertained from our study. Nonetheless, the finding of different infection prevalence in flying fox populations in different locations (for whatever reason) suggests a rationale for the (to date) geographic predominance of Hendra spillover events in Queensland, when flying foxes and horses exist in similar overlapping densities elsewhere in Australia.Considering detections over time, we found that the proportion of positive samples was significantly higher in year two than in years one and three, indicating between-year variation in Hendra virus infection prevalence in flying fox populations. This finding fits well with the observed variable annual frequency of equine cases of Hendra virus, and suggests that ecological, physiological or other factors moderate infection or excretion in flying foxes.et al (2005) et al (2011) We were interested to explore the ‘seasonal’ pattern of virus excretion from flying foxes to compare and contrast with the cumulative within-year occurrence of equine cases. When we collapsed the three years of data into a single 12-month period, we found positive sampling events in all months except April and December, and while statistical confidence was lacking (again evidently due to the modest number of sampling events), the over-lapping 95% confidence intervals suggest detections can occur in any month. To examine whether more detections might occur in some months, we sought an association between the proportion of positive samples and month. While the derived 95% confidence interval for September suggests at least a partial association, the collapsed September data actually represents a single positive sampling event in 2009 in which 33.3% of samples were positive (discussed above), thus this analysis should not be over-interpreted. Our finding of positive sampling events throughout the year is consistent with those of Walcharaplusadee P. alecto and P. conspicillatus demonstrates that Hendra virus is currently cycling in populations of these species. Where positive samples were obtained from mixed species roosts, it was not possible to conclusively attribute collected urine to a particular species. Previous serologic surveys have found anti-Hendra virus antibodies in all four species The detection of positive samples from single species roosts of Our findings indicate that Hendra virus is not present in all flying fox colonies all of the time, and that the level of excretion in any particular colony fluctuates over time. In this study, 8.5% of samples from positive sampling events were positive, with a range of 3 to 33%, again indicating the dynamic nature of infection in any particular colony. These figures may be an over-estimate of the true population prevalence, given the possibility that multiple positive (pooled) samples on individual sheets may reflect a single individual. Paradoxically, while the data indicate that infection in any given flying fox colony is evidently periodic and transient, from a risk management perspective, it is appropriate to assume that any colony could be infected at any time, and for horse-owners, veterinarians and para-veterinarians to adopt recommended exposure risk minimization strategies Screening populations rather than individuals, and seeking to maximize the likelihood of detection at each step has been an effective approach to investigating Hendra virus infection dynamics in flying fox populations, and our study has yielded a number of significant findings. Firstly, Hendra virus excretion occurs periodically, rather than continuously, and in geographically disparate populations in Queensland. Secondly, the lack of any detection in the Northern Territory suggests prevalence may be higher in Queensland than elsewhere. A proposed expanded comparative prevalence study between Queensland, the adjacent state of New South Wales, and the Northern Territory aims to clarify this. Additional years of data are needed in the current study to improve the robustness of statistical comparisons within-year and between-year, and a further three years are planned. Finally, the finding that detections in flying foxes did not temporally cluster with recorded spillover events to horses was illuminating, and suggests a ‘necessary and sufficient cause’ paradigm for Hendra virus spillovers. That is, for spillover to horses to occur, not only must virus be present, but also must one or more ‘pre-disposing’ factors that increase the likelihood of infection. Investigating such plausible host, agent or environmental factors was beyond the scope of this study, and underlines the need for on-going longitudinal studies.Since submission of the manuscript, the occurrence of an unprecedented cluster of incidents in Queensland and News South Wales (more than doubling the previously known number) dramatically illustrates our finding of between-year variation in virus excretion, and reinforces our key conclusions and recommendations, particularly the ‘necessary and sufficient cause’ paradigm, and the proposed expanded research on infection and transmission dynamics in Queensland and New South Wales.Table S1Details of the total 67 sampling events yielding 1672 pooled urine samples from July 2008 to June 2011.(DOC)Click here for additional data file.


The NanoSight LM10 with Nanoparticle tracking analysis (NTA) software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the method’s accuracy was demonstrated by using known concentrations of the subject particles. NTA analysis was also used to quantify chromatographic fractions of adenovirus and influenza virus after purification on a CIM monolithic column. NTA results were compared and evaluated against hemagglutination (HA) and end point dilution assay, determining total and infection virus particle number, respectively. The results demonstrated that nanoparticle tracking analysis is a method for fast estimation of virus concentration in different samples. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation. Vaccines play an important role in the prevention and treatment of disease. Although vaccines have been used for decades, there has only recently been a concerted effort to optimize their production and improve vaccine safety and efficacy-3. ApartThe downstream processing (DSP) of vaccines represents a considerable production cost. During these purification steps, it is critical to monitor impurity removal and virus recovery in order to stop production runs as early as possible if a problem is detected. Since DSP strongly depends on the consistency of the up-stream process (USP), virus titre monitoring during USP is just as critical and important as during DSP.The infective viral titre is determined by using the tissue culture infectious dose 50 (TCID50), plaque assay and end point dilution assay. In contrast to infectivity assays, which are laborious and time-consuming, total virus particles quantification methods are in general relatively quick, but have some other drawbacks. Spectrophotometric-based measurement of virus concentration (absorbance ratio 260/280 nm) can be applied only for highly purified virus preparations; quantitative real time polymerase chain reaction (qPCR) measures gene copy rather than particles and thus excludes empty capsids from the analysis; transmiNone of the above mentioned (infectivity and non-infectivity) methods enables the assessment of virus aggregation and complex formation. In a non-natural environment, such as low pH for influenza virus, virusesThe level of virus aggregation should be monitored throughout the downstream processing because this phenomenon can influence the behavior of some viruses like influenza A virus where aggregation inhibits virus hemagglutination activity and infectivity. This coFactors that contribute to particle aggregation and methods to prevent its occurrence during virus purification and formulation were highlighted by Wright et al.. Using rFor determination of virus particle size and distribution (aggregation), field flow fractionation connected with multiangle light scattering (FFF-MALS) and size exclusion chromatography coupled with MALS (SEC-MALS) were evaluated by Wei et al.. These mFor some applications, the ratio between total virus and infective virus particles is of great importance (e.g. gene therapy vectors), while for the others  the total virus particle count is sufficient. To speed up downstream process development while simultaneously reducing costs, a total virus particle quantification method should be employed that is quick, reliable, and robust to enable the quantification of virus throughout the purification process.Nanoparticle tracking analysis (NTA) is a system for sizing particles from about 30 to 1000 nm and ability to determine the concentration of particles in the solution within the concentration range from 10E+7 to 10E+9 particles/ ml depending on sample type. The technique combines laser light scattering microscopy with a charge-coupled device (CCD) camera operating at 30 frames per second, which enables the visualization and recording of nanoparticles in solution. The NTA software is then able to identify and track individual nanoparticles moving under Brownian motion and the results allow particle number concentration to be recovered. NTA was already evaluated for the measurement of nanoparticles, drug deOur goal was to evaluate the potential of the Nanoparticle tracking analysis for virus particle quantification and assessment of the level of virus particles aggregation. A direct comparison with hemagglutination assay and indirect comparison with End-point Dilution Assay was made in order to reveal the advantages and drawbacks of a technique.Latex particles  were purchased from Christine Grőpl . Approximate particle concentration (1.05E+12 LP/ml of latex particles) was provided by manufacturer.Adenoviruses are medium-sized (90–100 nm), non-enveloped, icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adenovirus 5 (Ad5) was propagated in HEK293 cells and released from the cells by three cycles of freezing in a dry ice/ethanol bath and thawing in a 37°C water bath. After centrifugation at 6.500 x g for 10 min, the supernatant was treated with benzonase  for 1h at 37°C and filtered using a 0.22 μm PES filter . The benzonase treated and filtered virus harvest was purified on a strong anion exchanger, CIM QA monolithic column, as previously reported by Lah et al..Influenza virus (H1N1) was propagated in Vero cells, using serum free medium. The virus was harvested, benzonase treated, and concentrated by tangential flow filtration . The concentrated virus harvest was then filtered using a 0.45 μm Chromafil® cellulose acetate (CA) membrane  and diluted 1:1 (v/v) with 50 mM HEPES pH 7.5 (equilibration buffer used for chromatography). The virus was purified on a strong cation exchanger, CIM SO3 monolithic column, using the method developed and described by Peterka et al..NTA measurements were performed using a NanoSight LM10 instrument , consisting of a conventional optical microscope, Marlin charged coupled device (CCD) camera, and a LM10 unit (sample unit) with a laser light source. LM10 is the first generation instrument from the NanoSight Company, which in the mean time has developed devices with additional features and upgraded software.Following the manufacturer’s instructions, we serially diluted all samples with sterile water or DPBS to reach a particle concentration suitable for analysis with NTA (1.0E+8 to 2.5E+9 particles/ml). We prepared at least two different sample dilutions for each sample and analyzed each one twice. The samples were injected into the LM unit (approximately 300 μl) with a 1 ml sterile syringe. The capturing settings (shutter and gain) and analyzing settings were manually set according to the protocol suggested in the Technical note ”How to make Concentration Measurements using NanoSight Equipment”  and then optimized for a specific virus or the latex particles. The NanoSight LM10 recorded 60 second sample videos which were than analyzed with the Nanoparticle Tracking Analysis (NTA) 2.0 Analytical software release version build 0125.To estimate the total number of influenza virus particles, a standard hemagglutination assay (HA) using standardized concentration of avian red blood cells (0.5% RBC) as previously described by John Hierholzer et al.,24 was uA serial twofold dilution of the virus was prepared in U-bottomed 96 well microtiter plates containing 50 μl DPBS. Into each well, a 50 μl of 0.5% solution of chicken red blood cells was added and the plate was incubated for 60 minutes at room temperature. HA titres (HA/ml) were read as the reciprocal dilution of the last well showing hemagglutination.The infective virus titre of Ad 5 was determined by End-point Dilution (EPD) Assay. The assay is based on observing the cytopathic effect (CPE) of serially diluted adenovirus samples in HEK293 cells cultured in 96-well plates. CPE was examined 7–10 days after infection and the virus titre was calculated based on the dilution factors.2 and 0.4 M NaCl, pH 8 equilibration buffer. The influenza virus was purified on a strong cation exchanger (CIM SO3 monolithic column) using a 50 mM HEPES equilibration buffer, pH 7.5. Both viruses were eluted from the column using high NaCl concentration in equilibration buffer.A CIM® monolithic column  with a dimension of 12 mm x 3 mm i.d. and bed volume of 0.34 ml were used for purification of adenovirus and influenza virus. The column was attached to a Knauer  gradient HPLC system, consisting of two K-500 pumps, a UV–VIS detector K-2500 set to 280 nm, and a data acquisition and control station with a PEEK capillary tube (i.d. 0.75 mm). A conductivity meter  was added to the system. The adenovirus was purified on a strong anion exchanger (CIM QA monolithic column) using a 50 mM Tris containing 2 mM MgClThe NTA method was first characterized by quantifying latex particles with a diameter of 120 nm at a concentration of 1.05E+12 LP/ml. These latex particles were used as a model nanoparticle since their size is similar to that of the two tested viruses (adenovirus and influenza). We assumed that the NTA method settings determined for the latex particles would be applicable for the viruses as well.The settings selected for the video capturing of the latex particles were set according to the manufacturer’s instructions. The videos were analyzed under different conditions by changing brightness, blur, gain, and detection threshold. By comparing the concentration of latex particles in differently diluted samples, analyzed with different settings, we were able to determine which settings have a smaller or greater effect on the result (data not shown). Similar to the previously published findings by Filipe et al., it was The videos were analyzed using the optimal settings determined for latex particle quantification with NTA: brightness −15, gain 2, blur 5x5 and detection threshold 30  and it was determined that it is within the acceptable range of 15-20% for the accuracy of bioanalytical methods.Since the particle size of an adenovirus is 100 nm in diameter which is similar to the 120 nm latex particles, the parameters used for the analysis were similar to those of the latex particles. The video capture used a shutter speed of between 1340 and 1500 and the gain was set to the maximal value. The videos were processed with the brightness set to −15, gain 2, blur 3x3 and detection threshold 30.To establish the inter-day variability/repeatability of the NTA method, the same adenovirus sample was assayed for 4 consecutive days. Between the measurements, the adenovirus sample was stored at −82°C. Each day the adenovirus sample was thawed, two dilutions were prepared (with DPBS), and each dilution was analyzed twice. The inter-day variability of the method was determined to be 14%  solution, containing particles with a diameter of 120 nm at a concentration of 1.05E+12 LP/ml, was diluted 10-fold with DPBS. The resulting theoretical concentration was 1.05E+11 LP/ml which was then used as a spike.Since 5 μl of the 10-fold diluted latex solution was added to the adenovirus sample as a spike, the total particle titre added corresponds to 5.25E+8 LP/5 μl. In TableSince the adenovirus particle size is relatively close to the latex particles, we used the same shutter speed and gain mode for the NT analysis of spiked and non-spiked samples.Two adenovirus samples, differing in virus purity and concentration, were chosen for the analysis. The first adenovirus sample was treated with benzonase and filtered through a 0.22 μm filter and the second was purified using an anion exchange chromatography resin, CIM QA monolithic column. Both samples were analyzed with and without the addition of the spike and the concentrations obtained were compared and spike recovery was calculated Table. Spike rTo minimize the inter-day and operator to operator variability of NTA, a set of chromatographic fractions  obtained during influenza virus purification on a CIM monolithic column were analyzed by the NTA method and hemagglutination assay.The hemagglutination assay is based on the binding (agglutination) of red blood cells (RBCs) which occurs after a certain concentration of viruses have attached to the RBCs surface. The ability of viruses to agglutinate RBCs is rarely linked to their infectivity thereforIn the majority of the samples tested, the virus presence (compared on the level of the virus recovery) obtained by the HA and NTA method were comparable. In 6 out of 7 samples analyzed, the NTA method determined a slightly higher virus content and consequently virus recovery compared to the HA assay. This was to some extent expected since the NTA sample analysis showed a broader size distribution of peaks than expected based upon the size of the influenza virus Figure. This inThe main difference between the results obtained by both methods is the virus presence determined in the flow-through fraction (in both chromatographic purifications of influenza virus). While no virus (or less than 1 HA unit/ml) was detected with the HA assay, the NTA measurement showed the presence of the virus reflecting a virus recovery of 13% and 16%, respectively. This discrepancy between the measurements of the virus presence in the flow-through fraction resulted in a relatively big difference in total virus recovery determined by both methods.There are two hypothesis why no virus could be determined in the flow-through fraction by the HA method, while this was not the case with the NTA method. The first one is connected to virus aggregation in the flow-through fraction resulting in an underestimation of virus particles determined with the HA assay. This phenomenon was previously described by Kalbfuss et al.. But thiIn any case, the NTA method proved to be suitable for a quick estimation of total virus particles in chromatographic samples of influenza virus and could be used as a sole or a complementary method for virus recovery estimations.In general, the data obtained by the NTA should be interpreted with care. It is very important that enough particles are tracked and that the right settings are chosen. After analysis only the “relevant” data should be used for the calculation of the final particle concentration, following the criteria discussed earlier .During downstream processing development, it is very important to monitor virus recovery. To determine the virus recovery, different virus samples (e.g. chromatographic fractions) containing different virus titres have to be analyzed and compared with the starting material. Titres of different virus samples can only be compared if they are analyzed under the same conditions. In the case of NTA, this means the analysis is performed using the same parameters. To determine this, the optimal settings to enable virus quantification over a large span of virus titters should be identified.Since the virus material during DSP development contains different concentration of viruses, linearity of the method is a key feature. The optimal set of NTA parameters should therefore enable virus quantification over a large span of virus titters. The method’s linearity can be determined by assaying different dilutions of the virus sample and calculating the ratio between the theoretical linearity factor and a measured one (TF/MF ratio).Inter-day variability of the method should also be taken into account by comparing several samples on different days. In addition, the accuracy of the NTA method, shown by spike and recovery method indicates that the NTA method underestimates the titre of adenovirus for approximately 15%.Comparison of NTA with HA assay showed that NTA can successfully substitute the HA method when infectivity of the virus is not an issue. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation.The authors work for the company BIA Separations  and/or for the Center of Excellence for Biosensors, lnstrumentation and Process Control . None of the authors have any relation to the technology tested. We have never received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. Such an organization does not finance this manuscript (article-processing charge). The authors do not hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. The authors do not hold or are currently applying for any patents relating to the content of the manuscript and none of the authors have received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. There are none non-financial competing interests  to declare in relation to this manuscript.MP and PK designed and coordinated the study, MC and PK carried out the NTA measurements, PK participated in the chromatography part and drafted the manuscript. MP and AŠ have been involved in revising the manuscript critically and made substantial contributions to the interpretation and discussion of the results. All authors read and approved the final manuscript.


The false-positive rate (FPR) is a percentage-score provided by Geno2Pheno-algorithm indicating the likelihood that a V3-sequence is falsely predicted as CXCR4-using. We evaluated the correlation between FPR obtained by V3 population-sequencing and the burden of CXCR4-using variants detected by V3 ultra-deep sequencing (UDPS) and Enhanced-Sensitivity Trofile assay (ESTA).54 HIV-1 B-subtype infected-patients , with viremia >10,000copies/ml, were analyzed. HIV-tropism was assessed by V3 population-sequencing, UDPS , and ESTA.By UDPS, CCR5-using variants were detected in 53/54 patients, irrespective of FPR values, and their intra-patient prevalence progressively increased by increasing the FPR obtained by V3 population-sequencing . Conversely, the intra-patient prevalence of CXCR4-using variants in the 54 patients analyzed progressively decreased by increasing the FPR . Indeed, no CXCR4-using variants were detected in 13/13 patients with FPR>60. They were present in 7/18 (38.8%) patients with FPR 20–60 , in 5/7 (71.4%) with FPR 10–20, in 4/6 (66.7%) with FPR 5–10, and in 10/10(100%) with FPR<5 .FPR by V3 population-sequencing can predict the burden of CXCR4-using variants. This information can be used to optimize the management of tropism determination in clinical practice. Due to its low cost and short turnaround time, V3 population-sequencing may represent the most feasible test for HIV-1 tropism determination. More sensitive methodologies (as UDPS) might be useful when V3 population-sequencing provides a FPR >20 (particularly in the range 20–60), allowing a more careful identification of patients harboring CXCR4-using variants. Human immunodeficiency virus type 1 (HIV-1) entry into host cells requires coordinated interactions of the envelope glycoprotein gp120 with the CD4 receptor and with one of the chemokine receptors, CCR5 or CXCR4. Pure CCR5-tropic and pure CXCR4-tropic virus use only the CCR5 and CXCR4 co-receptors to enter target-cells, respectively, while dual-tropic virus can use both co-receptors http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf) The impact of HIV-1 co-receptor usage has been correlated with the rate of disease progression in HIV-1 infected individuals http://www.aidsinfo.nih.gov/ContentFiles/Adultand AdolescentGL.pdf) HI V-1 co-receptor usage can be assessed with either phenotypic or genotypic approaches. The commercial Trofile assay , and its newer version the enhanced sensitivity Trofile assay (ESTA)  have been so far the most widely applied phenotypic test. Due to logistical and financial limitations of Trofile assays, different genotypic assays have been developed (http://coreceptor.bioinf.mpiinf.mpg.de/) is so far the most commonly used interpretation algorithm in clinical practice in Europe Two approaches have been used for V3 sequencing: V3 population sequencing and V3 ultra-deep pyrosequencing (UDPS). V3 population sequencing is currently used in routine clinical practice especially in Europe, while UDPS is mainly used for research purposes In this light, this study is aimed at: i) investigating the correlation between FPR by V3 population sequencing and the burden of X4-species, detected by UDPS; ii) analyzing the correlation between quasispecies diversity and frequency of CXCR4-using variants.pol sequences, and confirmed by V3 analysis Stored plasma samples derived by clinical routine assessment of HIV-1 resistance from fifty-four HIV-1 infected patients were retrospectively retrieved and included in the analysis. Ethic approval was deemed unnecessary because, under Italian law, biomedical research is subjected to previous approval by ethics committes only in the hypothesis of clinical trials on medicinal products for clinical use . The research also was conducted on RNA samples and data previously anonymized, according to the requirements set by Italian Data Protection Code (leg. decree 196/2003). All of the selected specimen had a viral load >10,000 copies/ml at the time of sampling, and they were all infected by HIV-1 subtype B, as determined by phylogenetic analysis of The protocol for V3 population sequencing based on single round of amplification has been generated and optimized as previously described UDPS was carried out with the 454 Life Sciences platform  as described in To estimate the UDPS error rate, a plasmid clone containing the region of interest was sequenced in parallel with the Sanger method http://coreceptor.bioinf.mpi-inf.mpg.de/. HIV-1 co-receptor usage of V3-sequences, obtained by both population and ultra-deep sequencing, was inferred by using the clonal version of geno2pheno set at FPR of 5.75. This cut-off, used in all the analyses carried out in this study, was chosen since it has been shown to be a good predictor of virological response to a maraviroc-containing regimen in both multi-experienced and drug-naïve patients HIV-1 co-receptor usage was inferred from the V3 nucleotide sequence by using the geno2pheno algorithm available at the following website The amino acid UDPS sequences resulting from the correction pipeline were analyzed to assess diversity and quasispecies complexity. To assess diversity, the mean genetic distance of amino acid sequences was calculated by PROTDIST using Jones-Taylor-Thornton matrix and with an in-house written code. Quasispecies complexity was calculated using normalized Shannon entropy (Sn = -Σ(pi ln pi)/ln N), where pi was the frequency of each distinct nucleotide sequence and N was the total number of sequences analyzed.Data were analyzed using the statistical software package SPSS . In particular, the correlation between the prevalence of X4 and R5 variants and the values of FPR at V3 population sequencing was assessed by Spearman’s rank correlation coefficient. P-values less than 0.05 were considered statistically significant.10 HIV-1 RNA copies/ml, and median (IQR) CD4 cell count was 254 (107–349) cells/ul  infected patients: 15 HAART-naïve and 39 HAART-experienced . All patcells/ul .V3 population sequencing (set at a FPR of 5.75) identified 10/54 (18.5%) samples as CXCR4-using , and had 76.6% concordance with ESTA, in line with other previous studies We observed that the FPR obtained by V3 population sequencing was directly correlated with the median FPR of V3 sequences detected by UDPS (p<0.001) , thus suOverall, UDPS detected CXCR4-using variants  in 26/54 (48.1%) patients, while 28/54 (51.9%) showed 100% CCR5-using viruses in their quasispecies population. The intra-patient prevalence of X4 variants in these 26 patients showed a wide range from 0.6% to 100% of the viral population (median [IQR]:27.4% [4.8%–81.4%]), corresponding to an X4-load  ranging from 186 copies/ml to 129,107 copies/ml (median [IQR]: 14838 [3715–43253] copies/ml). Only 1 patient showed 100% CXCR4-using variants with a FPR range of 1.5–5.3 by UDPS, and a FPR value of 2.7 at population sequencing.In drug-naïve patients, CXCR4-using variants were detected by UDPS in 8/15 (53.3%) of them, with intra-patient prevalence ranging from 2.3% to 99.1% (median [IQR]: 27.4% [4.2%–43.7%]), and with a median (IQR) X4-load of 21,972  copies/ml. Among them, 4 have an intra-patient prevalence of X4-variants <20% that is generally the limit of detection by population sequencing.Similarly, CXCR4-using variants were detected in 18/39 (46.1%) drug-experienced patients, with a median [IQR] X4-load of 8,754  copies/ml. Their intra patient prevalence was 47.7% [7.6%–92%], higher than that observed in drug-naive patients (27.4% [4.2%–43.7%]). The difference in the prevalence of CXCR4-using variants in drug-naive versus drug-experienced patients was not statistically significant .A next step of this study was to evaluate the correlation between the FPR detected by V3 population sequencing and the burden of CXCR4-using species detected by UDPS. In this analysis, at least 1 CCR5-using variant was detected in 53 out 54 patients, irrespective of FPR values obtained by population V3 sequencing. Their intra-patient prevalence progressively increased by increasing the FPR  , while iIn detail, in 13/13 (100%) patients with FPR >60 by V3 population sequencing, only CCR5-using variants were detected by UDPS (FPR range: 7.1–95.4), without any X4 variants  , Fig.3. Many patients (11/18 [61.1%]) with FPR ranging from 20 to 60 by V3 population sequencing were infected only by CCR5-using variants , Fig.3. The proportion of patients with CXCR4-using variants increased for values of FPR <20 by V3 population sequencing . In partThe quasispecies diversity and variability  at a protein level ranged from 0.012 subs/site to 0.222 subs/site and from 0.02 to 1.18, respectively, and significantly correlated with intra-patient prevalence of CXCR4-using variants  (data not shown). In particular, the median [IQR] diversity of amino acid sequences was higher in patients with X4 variants >2% (0.09 [0.03–0.16] subs/site) than in patients with only CCR5-using variants (0.03 [0.01–0.05]) (P<0.001 by Mann-Whitney test) , panel AThis study highlights a direct correlation between the FPR detected by V3 population sequencing and the burden of CXCR4-using species detected by UDPS in HIV-1 B-subtype infected patients.In particular, no CXCR4-using variants were detected in patients with FPR >60 by V3 population sequencing. These results were supported in an independent dataset of 15 HIV-1 infected patients tested for HIV-1 tropism by V3 ultra-deep sequencing (454 GS-Junior). In this dataset, none of the 3 patients with FPR>60 had X4 variants (0.1% cut-off) . These results can also explain a recent study aimed at determining the prevalence and the correlates of co-receptor switch in antiretroviral-naïve patients For 1 patient with FPR of 60.9 by V3 population sequencing, exceptionally, the ESTA result reported an X4-tropism while UDPS reported only the presence of R5-using species. Discordances between genotypic and phenotypic tropism testing have been previously described, and can be explained by the existence of additional positions in the env gp160, beyond those within the V3 loop, which may influence viral tropism Interestingly, in our study, the intra-patient prevalence of CXCR4-using variants by UDPS progressively decreased by increasing the FPR obtained by V3 population sequencing. In particular, CXCR4-using variants were observed in 38.9% (7/18) of patients with FPR ranging from 20 to 60 , in 75% (9/12) patients with FPR ranging from 5 to 20 , and in 100% (10/10) patients with FPR<5 . The presence of CXCR4-using variants in almost all patients with FPR <20 by population V3 sequencing is in line with the current guidelines Furthermore, for the specific set of patients with FPR ranging from 20 to 60, V3 population sequencing (based on single amplification) may also not be sufficient for proper determination of HIV-1 tropism, and thus, more sensitive methodologies, such as V3 UDPS or the phenotypic ESTA, might be used to identify more precisely patients candidate to maraviroc treatment. This is important since analyses from the MERIT and MOTIVATE trials have recently shown that the presence of as little as 2% of non-R5 viruses is independently associated with an increased risk of virological failure to maraviroc-containing regimens In particular, V3 UDPS has been shown to be highly predictive of clinical outcome to CCR5 antagonist in retrospective analyses of large clinical studies For all these reasons, genotypic testing based on V3 population sequencing still remains the preferred test for tropism determination in several clinical settings. In this light, this study contributes to further support the use of genotypic testing as valid testing for tropism determination in line with the recommendation of recent guidelines on clinical management of HIV-1 tropism testing Finally, it is intriguing that, in line with previous results In conclusion, this study shows that the FPR determined by V3 population sequencing can predict the burden of CXCR4-using variants in the infecting viral quasispecies, and suggests to use the FPR score with more attention before CCR5 antagonist prescription. Due to its low cost and short turnaround time, V3 population sequencing may represent the most feasible test for HIV-1 tropism determination. More sensitive methodologies might be useful when V3 population sequencing provides a FPR >20 and particularly in the range from 20 to 60, allowing a better identification of patients harboring CXCR4-using variants.Text S1V3 population-sequencing.(DOC)Click here for additional data file.


To the Editor: In 2011, Schmallenberg virus, a novel orthobunyavirus of the Simbu serogroup, emerged in Germany and the Netherlands and spread rapidly over large parts of central and western Europe , which is maintained by the Friedrich-Loeffler-Institut; the reports are made online. This database was analyzed for reported holdings with Schmallenberg virus infections that had been detected in adult animals from June 1, 2002 through October 31, 2012, and confirmed by PCR . Forty-fAlthough some cases were reported from the region in western and northern Germany where the epidemic had its center in 2011 , several new infections occurred in regions in southern Germany where no cases or only few cases of Schmallenberg virus infection had been detected before . This phenomenon may have occurred because of a high level of protective immunity at the population level in the region affected before transmission resumed in 2012, although a substantial proportion of the animals at the margin of the affected area remained susceptible. Schmallenberg virus that has overwintered in these areas may thus be transmitted to naive animals and has apparently spread to regions in southern Germany that were not affected or were less affected by the previous Schmallenberg virus epidemic. Schmallenberg virus could also be introduced into neighboring countries through infected arthropods. Although the respective reports may not have been formally published, indications were that Schmallenberg virus had spread at least to Austria, Ireland, Finland, Norway, Poland, Sweden, and Switzerland by summer/autumn 2012.Schmallenberg virus infection is often mild or clinically inapparent in adult animals and leads only to a short viremic period of ≈4–5 days (