Cost calculations were based on average daily treatment costs for intensive care unit (ICU) and normal medical wards as well as on the specific costs of the ECCO2R system.Retrospective ancillary cost analysis of data extracted from a recently published multicentre case–control-study  was shorter than in the matched control group treated with IMV (23.0 vs. 42.0 days). The overall average hospital treatment costs did not differ between the two groups . A subgroup analysis of patients with chronic obstructive pulmonary disease (COPD) revealed significantly lower median ICU length of stay (11.0 vs. 35.0 days), hospital length of stay (17.5 vs. 51.5 days) and treatment costs for the ECCO2R group .In the group treated with ECCO2R to avoid IMV in patients with acute-on-chronic ventilatory insufficiency failing NIV may be offset by a cost reducing effect of a shorter length of ICU and hospital stay.Additional costs for the use of arterio-venous ECCO At the 2R) . Recentlescribed –10. The escribed . Once IMescribed –18.2R in patients with NIV refractory acute on chronic hypercapnic ventilatory failure to avoid intubation [2R (av-ECCO2R), which has been licensed in 2006 [Our research group has recently published a matched case–control study on the feasibility and safety of ECCOtubation . The und in 2006  and sinc in 2006 .Table 1D2R. Two patients in the av-ECCO2R group (9.5 %) had major bleeding complications. Ventilator-associated complications, such as VILI, VAP and VIDD, could not be recorded for methodological reasons. The median duration of av-ECCO2R support and mechanical ventilation in the av-ECCO2R group was 9 days (range 1–116) and the median duration of mechanical ventilation in the control group was 21 days . Length of stay (LOS) in the ICU and in hospital was shorter in the av-ECCO2R group than that of the matched control group treated with invasive mechanical ventilation . Six month mortality was 33 % in both groups.Intubation was avoided in 19 of 21 patients (90 %) treated with av-ECCODespite its increasing use, nothing is known about the economic impact of the use of this complex and costly technology to avoid intubation and IMV. Thus the purpose of this ancillary economic analysis was to evaluate if the reduction in hospital length of stay also translated to reduced treatment costs.2R) to avoid invasive mechanical ventilation in patients with acute on chronic respiratory insufficiency failing NIV [Department of Intensive Care Medicine, University Medical Center Hamburg-Eppendorf; Department of Internal Medicine, Infectious Diseases and Respiratory Medicine, Charité-Universitaetsmedizin Berlin; Department of Medicine III, University of Halle ; Department of Cardiology and Intensive Care, Klinikum Bogenhausen, Munich). The data collected in the case–control-study were routine clinical data from medical records. Because of the retrospective study design patient consent to participate in the study was not applicable. The institutional ethics committees of all four participating centres approved anonymised data collection and analyses.This ancillary retrospective economic evaluation was undertaken using data from a recently published multicentre case–control-study on the use of an arterio-venous extracorporeal carbon dioxide removal device  underlying diagnosis, 2) age, 3) simplified acute physiology score (SAPS-2), and 4) arterial pH before ECCO2R or intubation [In 21 non-intubated patients with acute on chronic hypercapnic respiratory failure not responding to NIV and fulfilling criteria for intubation the iLA® device for ECCOtubation . All con2R cases and their corresponding controls, which subsequently were not lung transplanted. Because no control patient underwent lung transplantation costs were deemed not to be more comparable in this subgroup analysis. The second subgroup was further limited to all 12 cases with COPD not undergoing lung transplantation and their matched controls. The rationale for this was to evaluate a homogeneous patient group with the most common underlying diagnosis for acute on chronic ventilatory failure.In addition to the economic evaluation of all 21 cases and their matched controls two subgroups were further analysed: The first subgroup was limited to those 17 ECCOThe costs were analysed from the hospital’s perspective and only direct treatment costs were evaluated . All cosAverage daily costs for treatment in the normal medical ward were derived from the 2013 cost calculations of the study centres administrative data. These costs also included both variable and fixed costs. Applied average daily costs for ICU treatment and treatment on the medical ward are shown in Table 2R treatment were calculated for each case based on the actual utilisation of consumables (cannulas and membranes) and length of treatment   Fig. . Costs f2R treatment were added to the ICU costs. Costs for treatment on the normal wards were calculated by multiplying average daily costs for a normal medical ward with the number of days actually spent on the normal ward after ICU discharge until hospital discharge or death. Finally, treatment costs for ICU and the normal ward were added to estimate the total hospital treatment costs.ICU treatment costs for each case and control were calculated by multiplying the average daily ICU costs by the number of days spent in the ICU. In the case group specific costs for av-ECCOTo test the robustness of these cost estimates and to assess the impact of variations of daily ICU and normal ward costs, sensitivity analyses were performed. Average daily ICU costs calculated by Moerer et al.  were varDiagnosis-related groups (DRG) and specific reimbursement plans for mechanical ventilation and extracorporeal lung support were not analysed.t-test or the Mann–Whitney U test depending on the data distribution. Two-sided p < 0.05 values were considered significant. The software used was SPSS® .Results are presented as absolute numbers and percentages, as means with standard deviations for continuous variables if normally distributed, and as medians with ranges if not normally distributed. Comparisons between the two groups were performed using the 2R group than in the matched control group treated with IMV . The overall mean hospital treatment costs did not differ significantly between the two groups . A sensitivity analysis did not reveal any significant differences when applying higher margins  or lower margins . Average ICU costs of the av-ECCO2R and the control group were 38.459 €  and 33.290 € , respectively. Average costs for av-ECCO2R, included in the ICU costs of the case group, were 7.717 € (18.8 %).Median hospital length of stay (LOS) was shorter in the av-ECCOp = 1.0). The median hospital LOS and ICU-LOS were 22 vs. 42 days (p = 0.41) and 13 vs. 30 days (p = 0.23). Overall mean hospital treatment costs did not differ significantly between av-ECCO2R cases and controls  and a sensitivity analysis did not change these non-significant differences.In the subgroup of 17 cases and controls without lung transplantation 6-months mortality rates were 41.2 and 35.3 % respectively (p = 1.0). Hospital LOS  and ICU-LOS  were significantly shorter in the av-ECCO2R group. Mean hospital treatment costs were also significantly lower in the ECCO2R group compared to the control group  of absolute cost difference 5.769–48.113 €). A sensitivity analysis also showed significant differences. Detailed results of all groups and subgroups are presented in Table A subgroup analysis of each 12 non-lung-transplanted COPD cases and their matched COPD controls revealed no significant differences in 6-months mortality  despite additional average costs of 7717 € per av-ECCO2R treatment. This did not change after excluding four ECCO2R patients who underwent lung transplantation and their controls. Thus, in this study the extra costs for av-ECCO2R treatment were offset by lower hospital costs due to shorter average hospital length of stay in the av-ECCO2R group. A short length of stay in the ICU may per se result in a reduction of treatment costs, as it has previously been shown that length of ICU stay explains approximately 85 to 90 % of interpatient variation in hospital costs [This study is the first economic evaluation to compare the costs of an av-ECCOal costs .2R group (19.610 vs. 46.552 €) suggesting that ECCO2R to avoid intubation in patients with AECOPD and NIV failure may lead to a reduction of overall treatment costs. The potential economical relevance of this hypothesis is augmented by the fact that COPD has a high prevalence projected to be the fifth leading burden of disease worldwide by the year 2020 [Analysis of all COPD patients, the biggest and most homogenous diagnostic subgroup, revealed significantly lower overall hospital treatment costs in the av-ECCOear 2020 , 25.2R. Itemised cost evaluation of all other treatment costs was not feasible due to the retrospective study design. Instead, further cost analysis was performed by means of macro-costing. Average daily costs for ICU treatment used for cost analysis in this study were based on a previous study by Moerer et al. who calculated the average daily ICU costs in Germany by cost analyses of 51 German ICUs [Micro-costing was used for cost calculation of material consumption for av-ECCOman ICUs . Their cman ICUs , 26–29. 2R patients despite the fact that the majority this group were not intubated and mechanically ventilated was, that from the experience of the participating study centres staff resource utilisation for management of invasive MV and av-ECCO2R with or without non-invasive ventilation is comparable. In addition, costs for material consumption from MV were proposed to be relatively low and not significantly contributing to the overall treatment costs. Costs for treatment of side effects of av-ECCO2R observed in the original case–control study, such as bleeding and vascular surgery in one case, were not itemised separately. These costs were included in the average daily ICU costs as were treatment costs in the control group for side effects caused by invasive MV, such as treatment of ventilator-induced pneumonia. There is evidence that costs for patients on MV are significantly higher than costs for non-ventilated patients in ICU [2R into average daily ICU costs and may even suggest that a potential cost saving effect of avoiding MV with its complications and associated costs by applying av-ECCO2R may have been underestimated. On the other hand, additional costs for complications caused by av-ECCO2R may also be associated with significant costs. The incidence of major, potentially costly complications was low in the av-ECCO2R group (9.5 %), but may have been underestimated because of low case numbers. Excluding 4 case-controls where the av-ECCO2R cases subsequently underwent lung transplantation with potentially higher costs did not change the results.The rationale for applying the above mentioned data on average ICU costs also to ECCOs in ICU , 30, 31 s in ICU . These dSince there are no valid national data on average daily costs for treatment of patients on normal wards in Germany who have previously been in ICU with respiratory failure, estimating these costs was based on the study centres local average cost calculations. These locally derived calculations may be less generalizable. However, as only less than 25 % of the total hospital costs were attributable to treatment on the normal ward, this potential error may be negligible.This relationship is in line with findings of other studies that have shown that length of ICU stay was the main determinant of overall hospital cost variations , 33. Aga2R were based on the pumpless arterio-venous ECCO2R device applied in the original study, these results cannot directly be applied to veno-venous, pump-driven devices for ECCO2R since their technology is more complex and extra cost apply for more expensive circuits and consoles. In addition, the clinical characteristics of patients and their controls of this study are quite specific as they had actually failed NIV and all patients in the control group had been intubated. Therefore they differ from the population of a recently published case–control-study in which patients at risk of NIV failure where treated with ECCO2R to avoid intubation [2R device revealed a significantly lower intubation rate in comparison to their 21 controls treated with NIV alone (12 % vs. 33 %). The LOS in ICU and hospital did not differ significantly between the two groups .Since cost calculations for ECCOtubation . In this2R paradoxically may lead to an economic disadvantage for a specific health care provider if “lost” reimbursement rates for (prolonged) MV are not compensated for by reimbursement rates for ECCO2R treatment. Therefore further prospective and larger clinical and economic studies on strategies to avoid MV by means of ECCO2R are warranted to provide more evidence for rational decision making both in clinical practice and on a health care system level.Further, this cost analysis did not take into account the health care provider’s perspective on reimbursement plans, because aspects of diagnosis-related groups (DRG) or specific reimbursements for costly treatments and procedures such as invasive mechanical ventilation and extracorporeal lung support were not addressed. The rational for this was that reimbursement plans are individually negotiated between health care providers and healthcare payers on a local, regional and/or national level. Reimbursements do not necessarily represent factual costs and are highly variable between health care systems and over time , 34. In 2R to avoid IMV in NIV failure in a mixed group of patients with acute-on-chronic ventilatory insufficiency may be offset by a potential cost reduction through a shorter length of hospital and ICU stay. Moreover, in patients with acute exacerbation of COPD this novel treatment strategy may not only hold clinical advantages, but may even reduce overall resource utilization and treatment costs. However, the preliminary and hypothesis-generating results of this pilot study must be validated in larger, prospective, multicentre, high quality randomised clinical trials not only evaluating clinical benefits but also paralleled by evaluation of cost-effectiveness applying a micro-costing approach.The additional costs of the use of ECCOAdditional costs associated with the use of arterio-venous extracorporeal carbon dioxide removal to avoid invasive mechanical ventilation in patients with acute-on-chronic ventilatory insufficiency failing non-invasive ventilation may be offset by a potential cost reduction through a shorter length of ICU and hospital stay.


Through the course of their evolution, viruses with large genomes have acquired numerous host genes, most of which perform function in virus reproduction in a manner that is related to their original activities in the cells, but some are exapted for new roles. Here we report the unexpected finding that protein F12, which is conserved among the chordopoxviruses and is implicated in the morphogenesis of enveloped intracellular virions, is a derived DNA polymerase, possibly of bacteriophage origin, in which the polymerase domain and probably the exonuclease domain have been inactivated. Thus, F12 appears to present a rare example of a drastic, exaptive functional change in virus evolution.Reviewers: This article was reviewed by Frank Eisenhaber and Juergen Brosius. For complete reviews, go the Reviewers’ Reports section. Genomes of large viruses, in addition to a small core of viral hallmark genes, encompass numerous genes that apparently have been acquired from the hosts at different stages of evolution -3. Some Bacillus subtilis phage Phi29 DNAP (pdb 2py5), with a probability values greater than 98.5, which is considered strong evidence of homology, and a close correspondence between secondary structure elements  initiated with the amino acid sequence of Vaccinia virus (VACV) protein F12 (GenBank Accession No Q80HX6) detected, in addition to the highly significant similarity to the homologs from all chordopoxviruses, a marginal, not statistically significant similarity to several identified or putative DNA-dependent DNA polymerases (DNAPs) from plant and fungal mitochondrial plasmids and bacteriophages. To further investigate the possible homology of F12 and DNAPs, we used the sequence of the F12 homolog encoded by the most distant from VACV, early branching chordopoxvirus, the Nile Crocodile Virus (NCV) (YP_784228), as the query for a new PSI-BLAST search. This third iteration of this search identified statistically significant similarity  between F12 and a variety of organellar plasmid and phage DNAPs. Further sequence analysis was performed using the HHPred method which compares Hidden Markov Model profiles derived from the multiple alignment of readily detectable homologs of the query protein to databases of profiles of structurally characterized protein families. The HHPred search initiated with the sequence of either VACV F12 or the NCV homolog of F12 consistently yielded alignments with the Taken together, these observations indicate that chordopoxvirus F12 proteins are homologs of family B DNAPs, with the strongest sequence similarity observed with the protein-primed DNAPs of phages and organellar plasmids. The family B DNAPs consist of an N-terminal 3′-5′-exonuclease (Exo) domain and the C-terminal polymerase moiety that encompasses the Palm, Fingers and Thumb domains ,15. The Poxviruses encode their own, functional family B DNAPs that is essential for virus replication . The PFAvia horizontal gene transfer. This acquisition was then followed by exaptation for a role in IEV morphogenesis and transport along microtubules [The multiple alignment of various DNAPs and chordopoxvirus F12 proteins  region of kinesin light chains (KLC) although no quantitative evidence has been presented in support of this connection . HoweverIt has been reported that F12 is required for the recruitment of kinesin-1 which enables the movement of IEV along microtubules in VACV-infected cells and that deletion of the purported TPRs in F12 abrogated kinesin binding . The finWe report here the unexpected finding that a chordopoxvirus protein implicated in IEV morphogenesis and intracellular motility is a derived, inactivated DNAP. The results of sequence comparison and phylogenetic analysis suggest an evolutionary scenario in which the F12 gene evolved from an acquired bacteriophage DNAP gene, with both exonuclease and polymerase activities apparently abrogated as indicated by the disruption of the respective catalytic sites. Alternative routes of evolution, such as duplication and subsequent inactivation of the ancestral poxvirus DNAP, cannot be formally ruled out but appear much less likely. Inactivated DNAPs has been described previously in archaea and eukaryotes ,17,18, aThe non-redundant database of protein sequences at the NCBI was searched using the PSI-BLAST program . ProteinThe proposed MS describes the discovery of chordopoxvirus F12 phylogenetic history as a inactivated DNA polymerase. The MS is well written, logically rigorous and worth being published.We appreciate this laconic yet encouraging review.Authors’ response: The finding that chordopoxvirus protein F12 is an acquisition of a DNA polymerase of likely bacterial origin and a subsequent exaptation as a protein with different viral function(s) is very interesting.Unlike other examples of DNA polymerases (DNAPs) that lost their central function but still are involved in DNA replication or repair, the F12 protein has been recruited (fortuitously) into an entirely different viral task, namely intracellular virus morphogenesis and transport. As an aside, the term inactivated DNAPs is somewhat imprecise and misleading as they still exhibit a function. Perhaps, partially inactivated would be a better term.The term “inactivated” pertains to enzymatic activities not to the loss of any functionality. We thought this should be clear but in order to make the text unequivocal in this regard, we modified the penultimate sentence of the introductory paragraph as follows:Authors’ response: Language to the same effect was also inserted into the Conclusions.“We report here that poxvirus protein F12 that has been implicated in intracellular enveloped virus (IEV) morphogenesis, and in particular IEV movement along microtubules -13, is aIt is not clear whether any of the bona fide family B DNAPs of chordopoxviruses are shown in any of the alignments, although it is stated that there is most likely no relationship to invoke a viral gene duplication event.The alignment in Figure 1actually includes 5 poxvirus DNAP sequences that are explicitly marked as such. The problem probably is with the truncation of the figure in the original pdf file. We are taking care of this in the revision.Authors’ response: The term horizontal transfer is missing from the text. Does it mean that acquisition of an originally bacteriophage DNAP gene by a virus infecting eukaryotic cells cannot be considered HGT?We agree that horizontal gene transfer is an appropriate term to describe this case and include it in the revised text.In the pdf version of the manuscript, Figure Authors’ response: We regret the inconvenience caused by poor formatting of the figure. We include a version with wider margins which should eliminate the problem.Authors’ response: DNAP: DNA polymerase; Exo: Exonuclease; IEV: Intracellular enveloped virus; KLC: Kinesin light chain; NCV: Nile crocodile virus; TPR: TetratricoPeptide repeat; VACV: Vaccinia virus.The authors declare that they have no competing interests.ARM made the original observation and incepted the study; NY, GF and EVK performed data analysis; EVK wrote the manuscript that was read and approved by all authors. All authors read and approved the final manuscript.Arcady R Mushegian: The views expressed in this article are those of the author in his personal capacity and do not necessarily represent the view of the National Science Foundation or the Government of the United States.The HHPred output for the F12 homolog of CNV.Click here for filePhyre 2 results for VACV F12.Click here for fileMultiple alignment used for the construction of the phylogenetic tree in Figure 2.Click here for fileTPRpred results for the relevant poxvirus proteins and kinesin light chains.Click here for file


Pseudorabies virus (PRV) is a neurotropic herpesvirus that causes Aujeszky’s disease in pigs. PRV strains are widely used as transsynaptic tracers for mapping neural circuits. We present here the complete and fully annotated genome sequence of strain Kaplan of PRV, determined by Pacific Biosciences RSII long-read sequencing technology. Alphaherpesvirinae subfamily, causes significant abortion and morbidity in pigs, the natural host of the virus (––Pseudorabies virus (PRV), also known as Aujeszky’s disease virus or suid herpesvirus 1, a member of the he virus –6, are te virus –, and sere virus –, 4 and ce virus –; PRVs hae virus ––11. Furt virus ––.The currently available genome sequences of PRV contain several discrepancies, mainly in intergenic repetitive regions (GenBank accession no. JF797218.1), and the totally annotated version of genome sequence is a composite of six different PRV strains (GenBank accession no. NC_006151.1). We have sequenced the PRV Kaplan genome with Pacific Biosciences single-molecule long-read sequencing technology  in order to upgrade the draft sequences, reconstruct the GC-rich and repetitive regions of the genome, and extract epigenetic information. The availability of the completely annotated genome and the single-base resolution methylation map of strain Kaplan will aid in understanding the control of viral gene expression at different levels. Investigations of the PRV genome and gene functions are expected to result in the development of effective vaccines and direct practical applications in gene, cancer, and antiviral therapies.Sequencing of purified virion DNA was carried out on the Pacific Biosciences RSII sequencer. SMRTbell template libraries were prepared from the DNA, as previously described , 14, usihttps://github.com/PacificBiosciences/blasr) and the Pacific Biosciences SMRT Analysis pipeline  using the standard mapping protocol.The sequencing reads were processed and mapped to the respective reference sequences with the BLASR mapper , two latency-associated transcripts, and a long noncoding RNA, and its genome predicts 16 miRNAs.KJ717942.The complete genome of strain Kaplan of pseudorabies virus was assigned DDBJ/EMBL/GenBank accession no.


Glossina hytrosavirus (family Hytrosaviridae) is a double-stranded DNA virus with rod-shaped, enveloped virions. Its 190 kbp genome encodes 160 putative open reading frames. The virus replicates in the nucleus, and acquires a fragile envelope in the cell cytoplasm. Glossina hytrosavirus was first isolated from hypertrophied salivary glands of the tsetse fly, Glossina pallidipes Austen  collected in Kenya in 1986. A certain proportion of laboratory G. pallidipes flies infected by Glossina hytrosavirus develop hypertrophied salivary glands and midgut epithelial cells, gonadal anomalies and distorted sex-ratios associated with reduced insemination rates, fecundity and lifespan. These symptoms are rare in wild tsetse populations. In East Africa, G. pallidipes is one of the most important vectors of African trypanosomosis, a debilitating zoonotic disease that afflicts 37 sub-Saharan African countries. There is a large arsenal of control tactics available to manage tsetse flies and the disease they transmit. The sterile insect technique (SIT) is a robust control tactic that has shown to be effective in eradicating tsetse populations when integrated with other control tactics in an area-wide integrated approach. The SIT requires production of sterile male flies in large production facilities. To supply sufficient numbers of sterile males for the SIT component against G. pallidipes, strategies have to be developed that enable the management of the Glossina hytrosavirus in the colonies. This review provides a historic chronology of the emergence and biogeography of Glossina hytrosavirus, and includes researches on the infectomics  and pathobiology of the virus. Standard operation procedures for viral management in tsetse mass-rearing facilities are proposed and a future outlook is sketched.The  Glossina and most of which can transmit trypanosomoses, only 8–10 tsetse species are of medical and agricultural importance. The most important tsetse vectors are the riverine species  in Western and Central Africa and the savannah species  in Eastern and Southern Africa [Tsetse flies (Glossinidae: Diptera ) are impn Africa . Althougn Africa , to-daten Africa .HAT is one of the most serious of the so-called “neglected tropical diseases” (NTDs ). NTDs aThe people at the highest risk of tsetse bites, and of contracting HAT are the rural populations that primarily depend on small-scale agriculture, fishing, animal husbandry and hunting. Resurgence and epidemics of HAT are often associated with economic decline, civil disturbance/wars, population movements and refugees . The preT. vivax group only stays in the proboscis, the T. congolense group has a lifecycle involving the proboscis and the midguts, while only the T. brucei group has a cycle involving the salivary glands [HAT is difficult to treat, and there are no effective vaccines are available against either AAT or HAT. None of the available trypanocidal drugs for HAT is ideal; their treatment schedules are prolonged, excruciatingly painful (often described by patients as “fire in the veins”), and require continuous hospitalization . The tsey glands .T. b. gambiensis cause a chronic HAT that can take many years to kill a patient, while in East Africa, T. b. rhodiensis cause an acute HAT that can kill a patient within weeks [Without treatment, HAT can be fatal. However, fatalities of trypanosomes differ from one group to another. For instance, in West Africa in weeks ,17. The in weeks , is lethin weeks ,20. It iin weeks  and drugin weeks . For allin weeks .G. pallidipes are infected by Glossina hytrosavirus, a double-stranded (ds) DNA virus recently classified into the Hytrosaviridae family of insect viruses [G. pallidipes colonies [Glossina hytrosavirus, and consider the virology, epidemiology, pathology, and prospects for control of viral infections in laboratory colonies of G. pallidipes.In this review, we describe the “evolution” of tsetse control methods: from traditional methods used in the early 20th Century, to modern control methods. The sterile insect technology (SIT) is considered an effective control method to eradicate tsetse populations within the frame of area-wide integrated pest management (AW-IPM) approaches . Success viruses ,26,27. Vcolonies . In thisk-strategists) [r-strategist) [Two main characteristics of tsetse render them suitable for eradication. Firstly, compared to other insects of medical and agricultural importance, tsetse flies have a very low reproduction rate (tegists) ,30. Therategist) .Tsetse control methods have evolved from discriminate bush clearing and wild game culling at the beginning of the 20th Century, to broadcast insecticide applications after the Second World War , traps, 60Co or 137Ce source [Ceratitis capitata (Diptera: Tephritidae) populations in Chile, Argentina, Mexico, Central America, South Africa, Israel etc. [Cydia pomonella (L.) (Lepidoptera: Tortricidae) in the Okanagan Valley of Canada [Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa [Teia anartoides Walker (Lepidoptera: Lymantriidae) in New Zealand [Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in Texas, New Mexico, Arizona, California (US) and in Sonora and Chihuahua of northern Mexico [In 1937, Knipling developed the theory of controlling insect pest population by manipulating their reproductive capacity. He likewise modeled that a target population could be eradicated when the release of sterile males was applied on an area-wide basis against an entire insect pest populations in a delineated area ,37. Thise source ,39. The e source . After tael etc. ,42. Latef Canada , the falh Africa , the Aus Zealand , and then Mexico ,46.Glossina austeni from the Unguja Island (Zanzibar) [G. palpalis gambiensis and G. tachinoides in the Sideradougou area in Burkina Faso, and against G. palpalis palpalis in the Lafia area of Nigeria [G. austeni the island was declared tsetse-free in 1997, it still is to-date.The SIT played a pivotal role in the sustainable eradication of the tsetse fly anzibar) ,48. This Nigeria ,50. The G. austeni and trypanosomosis from Unguja Island inspired African Governments to call for increased efforts to manage the tsetse fly and trypanosomosis on mainland Africa. Consequently, an AW-IMP program with an SIT component was initiated in 1997 to eradicate G. pallidipes from a 25,000 square kilometers of under-utilized fertile land in the Southern Rift Valley of Ethiopia [G. pallidipes was established in 1997 at the Insect Pest Control Laboratories (IPCL) of the Joint FAO/IAEA Seibersdorf Laboratories, Austria. This colony was initiated from pupae originating from Arba Minch, Ethiopia. However, the colony collapsed in 2002. After the collapse of the colony, more pupae were shipped from Arba Minch to IPCL, Seibersdorf in an attempt to re-establish the colony. However, these attempts have been largely unsuccessful: currently, only a few of these flies (n = 25) are surviving, and their fecundity is very low. Further investigations revealed that the colony collapse was due to infection by a virus that caused salivary gland hypertrophy (SGH) syndrome [The success of SIT in eradicating Ethiopia . For thisyndrome ,53,54. AG. pallidipes individuals collected in the Umfolosi Game Reserve, Zululand, South Africa, had grossly enlarged salivary glands [G. fuscipes and G. morsitans [Drosophila, mosquitoes, and nematodes [In the 1930s, Whitnall reported that some y glands . In the orsitans ,57,58,59There were several notable features that supported the suggestion of the tsetse virus as an arbovirus. For instance, the rod-shaped viral particles detected in tsetse were thought to resemble one non-typical arbovirus group, the vesicular stomatitis Indiana virus, which was found to be rod-shaped with distinctive helical symmetry ,96. OtheRhabdoviridae), the apparent failure of father-to-progeny transmission of the sigma virus in Drosophila was attributed to transfer of very low viral titers to the developing embryo via the sperm [Glossina hytrosavirus is transferred from mother to her progeny, father-to-progeny transmission cannot be totally ruled out. This revelation implies that, under undefined conditions, the virus is reactivated from a “latent” state to a symptomatic state [In the 1980s and 90s, several researchers suggested that the tsetse virus is maintained in nature via transmission from the mother to her progeny and not from the male parent ,75,83,85he sperm . In the he sperm . Althougic state . It was ic state . This suic state .Merodon equestris  [Musca domestica L.  collected at a dairy in Florida, USA [Merodon virus. Although the Glossina and Musca viruses share pathological impacts on their respective hosts such as seasonal fluctuations in the incidence of SGH symptoms [Stomoxys calcitrans  [S. calcitrans [SGH symptoms have been reported in two other dipteran insects: in adult populations of the narcissus bulb fly, rphidae) ,64, and ida, USA . Notablyida, USA . To datesymptoms ,91 and ssymptoms ,82,85,86symptoms ,105. Notuscidae) , but it lcitrans .Diachasmimorpha longicuadata (Ashmead)  [D. longicuadata that originated from Hawaii and released in Thailand and subsequently introduced to southern China as a bio-control agent for the oriental fruit fly, Bactrocera dorsalis (Hendel)  [B. dorsalis is distributed throughout Southeast Asia and the Pacific, and is considered to be one of the most prominent agricultural pests in this part of the globe [D. longicuadata is known to introduce D. longicuadata entomopoxvirus (DlEPV) into Caribbean fruit fly Anastrepha suspensa (Diptera: Tephritidae) during oviposition [D. longicuadata may potentially be an endosymbiont to the wasp. Unfortunately, similar to the Merodon virus, the virus detected in the AGFs of D. longicuadata has not been further investigated.Recently, a virus was fortuitously detected in hypertrophied accessory gland filaments (AGFs) of the parasitic wasp, conidae) . The virconidae) . The wasritidae) . Inferrihe globe . D. longposition . DlEPV, position . ConsequAs indicated in Glossina mainly results from cellular hypertrophy (enlargement), SGH in Musca reflects a combined effect of both cellular and nuclear hypertrophy. Pathological effects of the Glossina hytrosavirus in colonized G. pallidipes were first documented the early 1980s. In 1979, a G. pallidipes colony was initiated at the Kenya Trypanosomosis Research Institute (KETRI) using flies caught from the Kibwezi forest, Kenya. This colony collapsed within two years of its establishment due to poor productivity [G. pallidipes colonies have been reported over the last two decades [Notably, whereas the SGH syndrome in uctivity . Investiuctivity . The res decades ,52. The Glossina hytrosavirus cause such serious problems in colonized G. pallidipes? It should be noted that such serious impacts are yet to be reported in other tsetse species. Further, other insect cultures  do not seem to experience such virus-induced problems. Although this question needs more research, inferences can be made from other viral-insect systems to understand the case of Glossina hytrosavirus pathology. It is important to keep in mind that insect viruses hardly “walk alone”: viruses co-infect (simultaneously) their hosts with other micro-organisms such as the vertically-transmitted bacterial endosymbionts [Drosophila by sigma viruses. Boucias et al., have recently suggested that the pathological effects of Glossina hytrosavirus on colonized G. pallidipes flies are modulated by interplay between the virus and tsetse endosymbionts [Wolbachia in the laboratory stock of G. pallidipes that was used in the study. Although Glossina hytrosavirus has been reported in other Wolbachia-harboring tsetse species [Wolbachia. It has been reported that the levels of PCR-detectable Glossina hytrosavirus in G. fuscipes fuscipes populations are influenced by the tsetse genotype, and inversely correlate with Wolbachia prevalence [Wolbachia is also absent from some of the major economically and medically important species of mosquitoes such as Anopheles spp. [Wolbachia-infected Ae. aegypti tested positive for Dengue virus after oral infection, 30%–100% of Wolbachia-free mosquitoes were virus-infected [Wolbachia in the G. pallidipes colonies explains the severe negative impact of Glossina hytrosavirus on large-scale colonies. Considering the shared intracellular locations of Glossina hytrosavirus and Wolbachia, this endosymbiont may influence the outcome of viral infection in tsetse hosts.One of the outstanding questions to be answered is: why does the ymbionts ,113,114.ymbionts . Specifi species , so far,evalence . Notablyles spp. . Studiesinfected . It can Glossina hytrosavirus, the virus was purified from hypertrophied salivary glands dissected from G. pallidipes flies originating from Tororo, Uganda in 1975. This colony was initially maintained at Leiden University, The Netherlands, and subsequently transferred to IPCL, Seibersdorf, Austria in 1982. Twenty-six years later, the genome of the virus was fully sequenced (NC_010356.1) [Glossina virus ORFs was the presence of homologues to five of the so-called per os infectivity factor genes (pifs)  encoded by baculoviruses, nudiviruses and whispoviruses [Glossina virus genome is characterized by an inverted repeat (ir) sequence, and fourteen direct repeat sequences (drs) composed of 51–246 bp.To understand the pathobiology of 10356.1) . The 19010356.1)   . Thirty-oviruses . Other noviruses . ApproxiMusca virus genome has also been sequenced [Glossina virus, over 70% (76/108) of the putative Musca virus ORFs had no significant homologies to proteins available in various databases. Notably, the two viruses share at least eight homologs of baculovirus core genes, including homologues to the pif genes, and to cellular genes [drs were distributed throughout the viral genome [dr18), and the number of copies of the drs ranged from 1.9 to 7.4, making the total sizes of the drs from 30 bp–380 bp-long. The G + C content of Musca virus is 43.5%, a ratio similar to that found in several nudiviruses: Heliothis zea nudivirus-1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV), but significantly higher than that of Gryllus bimaculatus nudivirus  and the Glossina virus (28%) [The equenced . The virequenced , with a ar genes . Eighteel genome . The sizus (28%) ,122,123.pifs among all the baculoviruses that have so far been sequenced [Glossina and Musca hytrosavirus ORFs do encode functional cellular orthologues, and what their functional roles are.The above-mentioned hytrosavirus gene homologies to other insect viral genes have several implications. Firstly, the conserved nature of the equenced , and theequenced , may refequenced . Thirdlyequenced . It remaGlossina virus could not be assigned to any of the families of insect DNA viruses described at that time [Hytrosaviridae [Hypertrophia sialoadenitis”, the Greek word for “salivary gland inflammation”. The Glossina virus is now a member of the newly-established Hytrosaviridae family, genus Glossinavirus, as the species Glossina hytrosavirus [Glossina and Musca viruses, the Musca virus is the second member of Hytrosaviridae family, but accommodated in the genus Muscavirus, and species Musca hytrosavirus. Hereafter, the Glossina and Musca hytrosaviruses are abbreviated as GpSGHV and MdSGHV, respectively.Initially, the hat time . Accumulaviridae , a name osavirus ,128. Simdna pol), which is present in all large dsDNA viruses, does not cluster these hytrosaviruses with other insect dsDNA viruses [dna pol of GpSGHV and MdSGHV clusters more closely to that of herpesviruses and other viruses with linear dsDNA genomes. On the other hand, the alignments free method using whole proteome phylogenetic analyses of dsDNA viruses shows close association of the hytrosaviruses and nimaviruses  [Phylogenetic analysis of GpSGHV and MdSGHV based on the DNA polymerase gene  ,136,137.s; WSSV) ,139, sups; WSSV) ,139. Thes; WSSV) .The presence of protein-coding regions or ORFs in a viral genome does not necessarily imply the presence of functional proteins. For instance, frame shift mutations caused by “indels” (insertions/deletions) in viral genomes can alter the structure and functions of the encoded proteins resulting in decrease or complete loss in protein expression . FurtherProteomics can be used to validate presence of virally-encoded proteins. Although the repertoire of biological processes controlled by viral gene products is complex , proteomG. pallidipes revealed that up to twenty of the secreted host proteins potentially interact with at least twenty-five viral proteins [G. pallidipes colonies, the response to GpSGHV can be highly symptomatic. This suggests that in the wild tsetse populations, GpSGHV is mainly in covert (latent or persistent) infection state, and the virus is only reactivated to symptomatic state under certain specific ecological and/or environmental conditions. It is not clear which viral and/or host proteins may be expressed during viral latency. However, Lee et al. reported that latency can be caused by the infection of Spodoptera frugiperda (Sf) cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that lack the anti-apoptosis gene p35 [Melanoplus sanguinipes entomopoxvirus anti-apoptotic gene, iap [Equally important to the repertoire of viral proteins composing the viral proteome are the interactions encrypted by both the viral and host genomes. Analyses of the secretome of GpSGHV-infected proteins . GpSGHV proteins . The GpSgene p35 . Notablyene, iap . It shouene, iap . In addiLatency of insect virus infections is probably widespread. Latency can be defined as a viral infection that does not produce visible disease symptoms , but the virus may be transmitted, either vertically or horizontally . This phet al. argued that in the adults of the armyworm S frugiperda (Lepidoptera: Noctuidae) latency may provide dispersal and reproductive advantages to S frugiperda nucleopolyhedrovirus (SfNPV) [et al. described a latent infection in a laboratory colony of the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae) that was vertically transferred from one generation to another without disease symptoms [The first question about latency is: is latency advantageous to the virus? Possibly, latency is an evolutionary viral strategy to utilize cues from the host and/or environment as opportunities for dispersal and transmission. In 1992, Fuxa  (SfNPV) . As the symptoms . Latencysymptoms . Latencysymptoms ,148. Latsymptoms ,150,151.symptoms .gypsy elements in D. melanogaster, which are transmitted as infectious particles from mother to progeny via oocytes [Leptopilina boulardi filamentous virus (LbFV) that infects the solitary parasitic wasp L. boulardi . When both an LbFV-infected and a non-infected female wasp lay eggs in Drosophila larvae during oviposition, LbFV is horizontally transmitted to the offspring of the non-infected wasp parent [The second question on viral latency is: how is viral latency established? One strategy in which viruses are vertically transmitted is by integrating into the host genome. For instance, the dsDNA polydnaviruses (PDVs) are vertically transmitted as proviruses stably integrated into the genomes of the parasitic wasps, ichneumonid  and braconid  . Another oocytes . Other ip parent . Consequp parent . Other ip parent ,157. WheG. pallidipes colonies, and are infectious per os to healthy flies [The GpSGHV replicates in the nucleus of host cells, where it induces formation of virogenic stroma (chromatin-like network of electron-dense filaments) . After nhy flies ,142. A rhy flies ,134. UnlG. pallidipes samples that were randomly collected from eleven geographical locations in six countries of eastern and central Africa [G. pallidipes samples were GpSGHV-infected: viral prevalence ranged from 2% to 100% [Early epidemiological surveys established that the prevalence of SGH symptoms in wild tsetse populations depends on the geographical location, seasonality, the distribution, age and species tsetse flies ,68,72,73l Africa . The PCR to 100% . Moreovei.e., from North America, Europe, Asia, the Caribbean, and the south-western Pacific [Unlike tsetse flies, house flies are among the most widely distributed insects, found in all inhabited areas of the world . The glo Pacific ,159. The Pacific . Further Pacific . To date Pacific ,91.G. pallidipes should be noted. Firstly, all males with SGH symptoms are fully sterile. Secondly, females with SGH symptoms do produce progeny flies; most  of these progeny flies exhibit SGH symptoms. Thirdly, asymptomatic females produce a small proportion of SGH-positive progeny flies; if such progeny flies are female, the F2 generation is sterile. The large numbers of virus particles released via saliva into the blood meals by the GpSGHV-infected flies during the in vitro membrane feeding  imply that asymptomatic flies may have more influence on the dynamics of GpSGHV transmission in the colonies compared to symptomatic flies [G. pallidipes flies were orally-infected with GpSGHV, the viral titers increased with the number of contaminated blood meals taken by the flies [G. pallidipes flies that secreted viral particles via saliva during the feeding events increased from 20% in the first blood meal to 100% in the seventh meal [Taken together, the following implications are apparent. Firstly, as hypothesized by Jaenson in 1986 , survivaic flies . The higic flies . These she flies . In markhe flies . It can he flies . Notablynth meal . So, whaper os [et al., [Aedes aegypti because of the barrier effect of the PM [G. pallidipes for at least 48 h-post-blood meal. Thereafter, the PM starts to decay and its disintegration may be completed a few days after, depending on availability of blood meal as has been suggested in sandflies [Plasmodium secretes chitinase to penetrate the PM in Ae. aegypti (inhibition of chitinase blocks transmission of Plasmodium) [G. pallidipes. The potency of anti-chitinase antibodies to block transmission of pathogens has been demonstrated with the creation of Plasmodium-refractory mosquitoes [G. pallidipes colonies.The peritrophic membrane (PM) of the intestinal tissue constitutes a physical barrier for viral diffusion into the salivary glands via the hemolymph. There is still conflicting information about the function of the PM in insects. Possible functions include protection of the stomach epithelium , ultrafiper os . Further[et al.,  suggestef the PM . Subsequf the PM . Likewisandflies . It has andflies . There iandflies ,90, probandflies . This fusmodium) . The impsquitoes . A similG. pallidipes flies secrete infectious GpSGHV particles via saliva [1 progeny produced by virus-injected mothers increased exponentially from 0–3% in the first larviposition cycle to 100% in the fourth cycle [1 progeny produced in the early larviposition cycles? Possibly, if oocyte GpSGHV infection did not occur early in oogenesis, the developing egg membrane (chorion) might protect eggs from infection. Since the mothers ovipositioning later were blood-fed a few days post-infection, their ovaries probably became infected before egg maturation. It should be noted that in tsetse flies, larviposition is influenced by blood feeding, and is correlated to the digestion stages of the blood meal [1 progeny. Orally-infected a saliva . In conta saliva . Notablyth cycle . So, whaood meal . Unlike G. pallidipes ovaries by GpSGHV could take place only during the later larviposition cycles. Since maturation of eggs begins shortly after blood meal ingestion [Taken together, the invasion of ngestion , it is uG. pallidipes, infection was observed in 13.3% and 30.0% of treated male and female parent insects, respectively. The prevalence of SGH in the F1 progeny adults was much higher than in the parents . Whereas all infected females were fertile, all infected males had SGH syndrome and were sterile. Maternal larviposition, F1 pupae weight, and F1 pupae incubation periods were normal regardless of treatments. Two other studies reported that, although GpSGHV-infected males had reduced reproductive potential [Glossina hytrosavirus could be applied as a tsetse bio-control: the sterile male parents would compete with normal wild males in mating, and the fertile but infected females would transmit the virus trans-ovarially to subsequent generations, since such females produce only infected progeny [Many insect-pathogenic viruses such as baculoviruses are effective bio-control agents against insect pests ,176. Theotential , such maotential . Althoug progeny , where mper os infection of the virus in G. pallidipes result in SGH syndrome in the same  generation, rather, the syndrome is only detectable in the third (~65%) and fourth (~100%) larviposition cycles of the F1 generation [G. pallidipes colonies reduces the mating propensity and competitiveness of males thus affecting the stability and performance of tsetse colonies [in vitro mass production of GpSGHV for field applications is currently impossible due to limitations such as the absence of a cell culture system permissive to the virus. Currently, the only alternative method to multiply GpSGHV is by intra hemocoelic injection of the virus into colony flies, or by feeding the flies with virus-contaminated blood meals. Even if it were feasible to multiply GpSGHV by the injection method, it is a laborious process, and would require maintenance of huge number of flies. Fourthly, there is no available evidence for horizontal transmission of GpSGHV through contact between flies, mating, or fecal contamination, thus limiting the modes of how the virus would be dispersed in the field. Finally, GpSGHV does not produce occlusion bodies, as for instance baculoviruses do to achieve prolonged stability in the environment [Application of GpSGHV as a bio-pesticide for tsetse control however, is technically challenging for several reasons. Firstly, recent findings show that neither micro-injection nor neration . So, thecolonies . The colironment . Besidesironment , with moironment , possibli.e., neutralizing antibodies, phage display technologies, and RNAi have shown potential to significantly reduce GpSGHV infections. Currently, these immune-intervention strategies are being optimized.In view of the current understanding of GpSGHV epidemiology under laboratory conditions, two potential strategies to manage the viral infections in the colonies were considered. The first strategy was to reduce or inhibit horizontal GpSGHV transmission by either (a) modifying the feeding system currently used in the colony rearing , or (b) G. pallidipes colonies. This was finally achieved mainly by modification of the in vitro membrane feeding regime routinely practiced in tsetse mass-rearing facilities. Since the membrane feeding favored horizontal viral transmission, it was conceptualized that modifications of the colony maintenance protocol(s) would significantly reduce GpSGHV transmission. The routine in vitro feeding regime involved feeding up to 10 sets of fly cages in succession [G. pallidipes colonies are discussed in sub-The objective of various research reviewed in this paper was the development of a cost-effective strategy to manage the GpSGHV infections in ccession , thus siccession . Within ccession , was comccession . Additioccession , or suppG. austeni population in the Unguja Island [G. pallidipes for the SIT component of AW-IMP programs on mainland Africa were faced with difficulties because this tsetse species is susceptible to infections by GpSGHV. The virus has caused collapse of G. pallidipes colonies that were initiated in Ethiopia and Seibersdorf, Austria, and thus prevented full implementation of the release component. Now that strategies are available to successfully manage GpSGHV infections that can result in complete elimination of SGH from G. pallidipes colonies, production of sufficient numbers of sterile males is within reach. This success provides opportunity to revive the SIT component of the program to eradicate G. pallidipes from the fertile Rift Valley lands of Ethiopia [G. fuscipes fuscipes, G. morsitans and G. swynnertoni, irrespective of their ages, sex, and season of the year [G. pallidipes can be used to make recommendations on standard operational procedures (SOPs) for the management of GpSGHV in large-scale tsetse fly production facilities. The proposed SOP is detailed in The membrane feeding system used in the tsetse mass-production facility located in Tanga, Tanzania greatly contributed to the production and release of ~8.5 million sterile males between 1994 to 1997, resulting in a crash of the a Island . HoweverEthiopia . This wothe year . TherefoThe aim of this review was to highlight the intricacies associated with the occurrence of a newly described group of viruses, the hytrosaviruses, and the interaction with their dipteran hosts. The common denominators are the structure and genome content of these viruses and their potential to induce the SGH syndrome. Despite the knowledge of the genome and proteome, very little is known about the regulation of viral gene expression and the interaction of viral proteins with the dipteran host and its microbiome and parasites. Nevertheless, the wealth of information available in a relatively short period through the concerted efforts of many research groups  allowed


The enterovirus (EV) types echovirus (E-) 5, E-9, and E-18, and coxsackievirus (CV-) A9 are infrequently reported in human diseases and their epidemiologic features are poorly defined. Virus transmission patterns between countries have been estimated with phylogenetic data derived from the 1D/VP1 and 3CD gene sequences of a sample of 74 strains obtained in France (2000–2012) and Tunisia (2011–2013) and from the publicly available sequences. The EV types  exhibited a lower worldwide genetic diversity  in comparison to CV-A9 (n = 10). The phylogenetic trees estimated with both 1D/VP1 and 3CD sequence data showed variations in the number of co-circulating lineages over the last 20 years among the four EV types. Despite the low number of genogroups in E-18, the virus exhibited the highest number of recombinant 3CD lineages (n = 10) versus 4 (E-5) to 8 (E-9). The phylogenies provided evidence of multiple transportation events between France and Tunisia involving E-5, E-9, E-18, and CV-A9 strains. Virus spread events between France and 17 other countries in five continents had high probabilities of occurrence as those between Tunisia and two European countries other than France. All transportation events were supported by BF values > 10. Inferring the source of virus transmission from phylogenetic data may provide insights into the patterns of sporadic and epidemic diseases caused by EVs. Picornaviridae family (a group of non-enveloped viruses with a positive RNA genome) and they comprise genetically and antigenically distinct (sero/geno)types . A second example involving close relatives of EV-A71 is the recent increase in the reported number of HFMD outbreaks caused by coxsackievirus (CV)-A6 in Europe and Asia [9]. Thirdly, in 2014, two health alerts focused attention on an outbreak of severe respiratory infections in children in Colorado, USA, caused by a disregarded EV type designated EV-D68 [10]. Sporadic cases were reported in European countries [13]. The epidemiologic factors involved in these geographic and temporal variations are multifactorial and still incompletely understood.Human enteroviruses (EVs) belong to a large genus within the 18]. In an earlier study, we showed that virus transportation events were involved in the sporadic dissemination of EV-A71 between European countries in the 2000s [19].The EV epidemic patterns at global and local geographic scales are mainly determined by herd immunity against each EV type. Other non-selective factors such as random transportation events of virus strains between countries by infected individuals may also be involved but have not been analyzed. Statistical phylogenetic methods developed recently have been used to address this question and applied to investigate virus migration of several virus groups other than EVs [21]. They were reported in acute neurological manifestations, of which acute meningitis was the most frequent [23], while meningo-encephalitis, encephalitis, and flaccid paralysis were occasional [25]. Severe infections were reported in both immunocompetent and immunocompromised individuals [27]. CV-A9 had an endemic pattern of circulation in the USA between 1970 and 2005, while the other three types displayed epidemic features [28]. E-18 and CV-A9 were involved in recent outbreaks of acute meningitis in Asia [30]. In France and Tunisia, sporadic detection of the four EV types has been reported [35]. Previous molecular epidemiology studies involved only E-9 and CV-A9 but did not address the issue of inferring their geographic spread from phylogenetic data [37].To investigate this question further, the present collaborative study focused on two countries, France and Tunisia, and four EV types, echovirus 5 (E-5), E-9, and E-18, and CV-A9. The four EV types were usually associated with mild febrile infections and exanthemas [The aim of the study was to investigate virus transportation patterns between France and Tunisia as estimated by phylogeographic analyses of two viral gene sequences (1D/VP1 and 3CD) and also to explore virus spread between other countries by combining the sequences determined with publicly available sequences.S1 Table). The sample comprised 74 strains . A sample of 7 E-6 strains was also included to analyze the recombination patterns (see below). The study was performed with the leftover material of specimens collected for diagnostic purposes during hospitalization of patients. The specimens were used after de-identification of clinical data and analyzed anonymously. The study and use of stored samples for testing were approved by the local committee for the protection of human subjects  with a waiver of informed consent  and by the local Tunisian Ethic and Research Committee .The virus sample consisted of EV strains identified in clinical specimens  obtained during routine virologic diagnosis in patients admitted to the hospitals of Clermont-Ferrand  and Monastir  (38]. The 1D/VP1 sequences of 24 virus isolates in the sample have been reported previously (see S1 Table). The nt sequences determined in this study  were submitted to the European Molecular Biology Laboratory database (accession numbers LN876128–LN876255).The 1D/VP1 and 3CD viral sequences were determined with previously described methods . The country of virus sampling was checked for each sequence to infer the geographic locations of nodes within the 1D/VP1 genealogies. A phylogenetic pattern indicative of a probable virus transportation event between two nodes was defined as follows: (1) the nodes exhibited pp>0.9, (2) the probabilities of inferred locations (p) were >0.5 at both nodes, and (3) the difference between the times of the most recent common ancestor (TMRCA) or the 95% highest posterior density (HPD) intervals estimated at the nodes were in a range of one year. This empirical interval was chosen because the dates of viral sequences were only defined by the sampling year whereas the month and day of virus isolation for publicly available sequences are commonly missing. The virus transportation events assessed over this short time interval were considered epidemiologically more relevant than those estimated over larger temporal intervals. The spatial diffusion history of each EV type was reconstructed by the phylogenetic analyses of complete (near-complete) and partial sequence datasets. A Bayesian stochastic search variable selection (BSSVS) approach was used to find a parsimonious set of rates explaining the migrations between discrete geographic locations in the phylogenies [. The phylogenetic data were analyzed with the Bayes factor (BF) test implemented in the SPREAD v1.0.4 program to determine the most probable virus migration events [45].A discrete phylogeographic model was used to analyze virus spread between countries [46] to detect recombination events. The analysis provided statistical evidence that only one 3CD sequence (namely HF948106_E18_CF1442_FRA02) was recombinant. The 1D/VP1 and 3CD loci are 2260 nt apart . Positions are given relative to the genome of the E-18/Metcalf strain (accession number AF317694). The occurrence of recombination between the two loci was investigated by analyzing partitioning of lineages in the 1D/VP1 and 3CD phylogenetic trees. On the basis of a previous study [47], a phylogenetic pattern indicated a recombination event between two distinct VP1 lineages when they clustered into a single 3CD lineage. When the 1D/VP1 lineages were from two EV types, the recombination was intertypic; otherwise, the recombination was intratypic. The recent recombination events were identified with the following additional conditions: (1) the 3CD sequences had >95% nt similarity. This threshold was chosen because it is generally accepted that the evolutionary rate of EV genomes is 1% per year and the molecular footprints of recombination events are rarely conserved more than five years. (2) the common ancestral node exhibited pp>0.9, and (3) the 95% HPD intervals of TMRCA values estimated for nodes that grouped equivalent clades in the 3CD and VP1 trees should overlap to infer probable intratypic recombination events. When the above criteria were not met, partitioning of lineages was considered to reflect an ancient, most probably intertypic, recombination.The 1D/VP1 and 3CD sequence datasets were analyzed with the RDP computer program . The spatial diffusion patterns inferred were dependent on the sequence datasets devised for a given EV type . The conservative method used for assessing the most likely virus transportation events that occurred between countries over short periods was robust to this sample bias. All the virus transportation events assessed with the empirical method exhibited BFs > 10, a value which was used as a threshold to select the most probable virus migrations. Phylogenetic data inferred from non-structural genes provided further support of virus migrations: the analysis of 3CD gene suggested a migration event between France and Tunisia involving strains of the EV type E-6. The overall analyses of the four EV types showed that France was connected to a range of 1 to 10 countries including Tunisia: the larger the size of the sequence sample analyzed, the higher the number of countries. A survey of 156 French patients with EV meningitis reported between 2008 and 2013 showed that only 2.6% of cases were caused by E-5 and E-9 [34]. In the same survey, CV-A9 and E-18 infections were reported more frequently . This epidemiological pattern of infrequent infections is in agreement with the phylogenetic data, which suggest occasional transfers of virus strains between France and other countries.The phylogeographic patterns were influenced by missing data, a common bias present in sequence samples [52], also designated lineages d to h in [53]) has been reported in multiple worldwide outbreaks of acute meningitis over the last 15 years, in contrast to the re-emerging genogroup III (lineage a), which exhibited ‘endemic features’ in Russia [54]. Similar observations were derived from molecular epidemiology studies of two other infrequent EV types, E-3 and CV-B1. In E-3, all genogroups were isolated worldwide but genogroup C was more frequently isolated in acute flaccid paralysis in India [55]. In CV-B1, genogroup IV has a wide geographic distribution compared to genogroup III, which has been more frequently reported in India [57]. Similarly, our phylogenetic data for E-9 showed that a genogroup c virus was spread between France and Australia while a few years later genogroup d strains were involved in virus migration between France and Tunisia. The varying feature of CV-A9 showed that distinct but closely related strains of the same genogroup i1 were involved in virus migrations between different countries, at about the same period. The epidemic and endemic patterns seen in different EV types might be related to these differences in virus transportation but other features could also be involved (see below).The present study provides phylogenetic evidence of earlier hypotheses suggesting that different lineages of a given EV type may have different transmission features and geographic distributions. The EV type E-30 genogroup VI  was reported in a range of three to five continents. In this respect, CV-A9 was similar to the other three types although it was considered as an endemic EV in the USA [28]. In addition, CV-A9 and E-9 shared a common feature as they were phylogenetically structured into a greater number of genogroups  in comparison to the other two types (n = 3 and 4). An earlier study reported 12 CV-A9 genogroups by analysis of the partial VP1-2A genomic region [37]. More specifically, CV-A9 and E-9 differed from the other two types by the number of epidemiologically active genogroups. Their phylogenies showed that the infections reported over the last 20 years resulted from the worldwide co-circulation of lineages among five and four genogroups respectively. In contrast, the E-5 and E-18 infections mainly arose from the co-circulation of lineages of one predominant genogroup. Whether or not these variations in co-circulation are involved in endemic or epidemic features will need to be established for a greater number of EV types.The overall data reported in this study have additional interest beyond estimations of a number of virus migration events between different countries: they are evidence that EV phylogenies had no strong spatial structure even over short periods of time. The global persistence of EV lineages and the local seasonal recurrence of infections are underlined by worldwide transportation of virus strains, as seen for instance in human influenza A [Geographic distance was not a barrier to virus transmission of EV strains as transportations of E-5, E-9, E-18, and CV-A9 were observed between distant countries. Virus migration is necessary but not sufficient for a virus lineage to initiate a successful transmission chain in the receiver population. The epidemiological consequences of virus transportation are also determined by herd immunity against the imported virus strain. Future research is needed on other infrequently reported (EV-D68) and epidemic (E-30 and EV-A71) EV types to determine which factor among virus transportation and local virus transmission is involved in the occurrence of outbreaks.S1 FigThe phylogenetic clusters in the 1D/VP1 trees (left panels) were traced back to the 3CD tree (right panels) to investigate the involvement of recombination in the evolutionary pathway of each EV type. The 1D/VP1 trees are monophyletic  while the lineages within the 3CD phylogeny are polyphyletic . The 3CD tree was inferred with 117 sequences from five EV types, those analyzed in the study plus E-6.(PDF)Click here for additional data file.S2 FigThe phylogeny was estimated with 117 3CD gene sequences by a Bayesian Markov chain Monte Carlo analysis implementing a discrete phylogeographic model. The branches are colored according to the geographic location that had the highest probability; the color code is indicated in the Figure. The tree nodes are indicated with circles colored according to the most probable geographic location; circle size is proportional to probability. A node shown with an open dark circle (and letter T followed by a number) indicates the source of a transportation event of a virus strain between two different countries see also . The sca(TIF)Click here for additional data file.S3 FigThe diffusion patterns were inferred through phylogenetic analyses of sequence samples derived from the 3’ part of the 1D/VP1 gene. The alignments were constructed by selecting the largest number of sequences that shared as many nucleotide positions as possible within the 3’ part of the 1D/VP1 gene. The general features of the sequence datasets used are indicated on each panel. The nucleotide positions common to all partial sequences are indicated . The sampling countries are indicated with full white circles. The lines connecting countries were colored according to the intensity scales indicating increasing BF values. The virus transportation events assessed with the complete (or near-complete) sequence samples are indicated.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


To the Editor: Enterovirus infections are associated with many clinical manifestations, and specific virus groups or serotypes are associated with specific manifestations. Coxsackievirus A16, a common cause of hand, foot and mouth disease, rarely causes encephalitis. Although most enterovirus infections are cleared by cellular immune responses, invasive enterovirus disease is prevented or controlled by neutralizing antibodies  have been reported after therapy with rituximab, a monoclonal antibody (MAb) that causes secondary hypogammaglobulinemia  and low serum immunoglobulin levels . Cerebrospinal fluid (CSF) samples were collected on days 1, 4, and 6. CSF leukocyte counts increased from 14 cells/mm3 (day 1) to 60 cells/mm3 (day 4) . Cytologic and immunophenotypic analyses showed no cerebromeningeal lymphoma infiltration. Total protein levels in CSF increased from 561 mg/L on day 1 to 771 mg/L on days 4 and 6.The patient was hospitalized because of a history of high-grade fever that did not respond to antimicrobial drugs, confusion, general weakness, and urinary incontinence. She had a neutrophil count of 3.1 × 10Borrelia spp., Listeria spp., parvovirus B19, measles virus, and galactomannan. PCR results for CSF were repeatedly negative for herpes simplex virus, varicella zoster virus, cytomegalovirus, Toxoplasma gondii, JC polyomavirus, human herpesvirus 6, Epstein-Barr virus, and mumps virus. PCR results and culture were negative for Mycobacterium spp. Serum samples were negative for antibodies against neuronal nuclear  antigens. However, enterovirus RNA was detected by reverse transcription PCR in all CSF samples. Sequencing of the virion protein 1 gene obtained directly from RNA extracted from CSF identified the virus as coxsackievirus A16  was started on day 4 and given for 5 consecutive days, which resulted in marked and continued neurologic improvement. Monthly doses of IVIG (500 mg/kg) resulted in normal serum IgG levels. Four months after initial examination, the virus has been cleared but the patient still has intermittent confusion and language defects. Treatment with IVIG will be continued for an additional 6 months.Use of MAbs against CD20 B-cell antigen has become standard treatment for B-cell lymphomas and an increasing number of autoimmune disorders (We anticipate that more cases of enteroviral encephalitis might develop, given the increasingly frequent use of MAbs against CD20 and widespread occurrence of enteroviruses. However, in view of the few reported cases (Any patient receiving MAbs against CD20 who has neurologic symptoms should be screened for infection with enterovirus RNA. In contrast to JC polyomavirus–associated progressive multifocal leukoencephalopathy, enteroviral encephalitis can be successfully treated by early administration of IVIG, which might contain neutralizing antibodies, albeit in variable amounts (


To the Editor: On February 1, 2016, the World Health Organization designated the Zika virus disease outbreak in Latin America as a Public Health Emergency of International Concern , diagnosed 8 cases of Zika virus disease in travelers returning to Israel. The Center is part of the Central Virology Laboratory of the Israel Ministry of Health and is the reference laboratory for the diagnosis of Zika, dengue, and chikungunya virus infections in Israel. During the same period, 4 cases of dengue and 1 of chikungunya were also diagnosed. Of the 8 cases of Zika virus disease, 7 were in patients returning from South and Central America and the Caribbean  and 1 waThe patient was a 61-year-old man from Israel who spent 10 days in Vietnam during December 2015: 3 days in Hội-An, 3 in Hue, and 4 in Ho Chi Minh City. After spending 2 more days in Hong Kong, he returned to Israel. On the third day after his return, he experienced fever, malaise, and headache; he had no rash or conjunctivitis. Laboratory studies showed only lymphopenia and mildly elevated liver enzymes. Symptoms continued for 8 days and then resolved completely. His illness was initially suspected to be dengue; however, test results for dengue  and chikungunya  viruses were negative.In Israel, Zika virus diagnostic tests were introduced in December 2015 and are available only through the National Center for Zoonotic Viruses. Serologic testing for Zika virus is performed by using an ELISA IgM and IgG kit (Euroimmun AG), which detects antibodies against the Zika nonstructural protein NS1 and is therefore considered very specific for Zika virus infection (http://beast.bio.ed.ac.uk/Main_Page) of the 231-nt fragment obtained from this patient and from 3 other patients from Israel who acquired their infection in South and Central America  and Zika virus 1162c (5′-CCACTAACGTTCTTTTGCAGACAT-3′) and an ABI 3500 Genetic Analyzer . A Bayesian maximum clade credibility time-scaled phylogenetic tree (BEAST,  America  was perf America . HoweverThis case illustrates the role of returning travelers as potential disease sentinels and the inadequacy of information about Zika virus circulation in Asia. During December 2015–January 2016, when this patient was evaluated and followed up, no cases of Zika virus disease had yet been reported from Vietnam. Since then, a case in a traveler from Australia has been reported (Until more thorough epidemiologic data from Asia become available, testing all travelers returning from Southeast Asia with exanthema, fever, or other signs or symptoms suggestive of Zika virus disease is justified. In addition, because during this period Zika virus had become the most frequent arbovirus isolated from travelers returning to Israel, Zika virus now seems to be a substantial cause of febrile illness in travelers returning from Zika virus–endemic regions.Epidemiology and diagnosis of Zika virus disease and case descriptions for travelers returning to Israel during December 2015–February 2016.


Ducks and seabirds are natural hosts for influenza A viruses (IAV). On oceanic islands, the ecology of IAV could be affected by the relative diversity, abundance and density of seabirds and ducks. Seabirds are the most abundant and widespread avifauna in the Western Indian Ocean and, in this region, oceanic islands represent major breeding sites for a large diversity of potential IAV host species. Based on serological assays, we assessed the host range of IAV and the virus subtype diversity in terns of the islands of the Western Indian Ocean. We further investigated the spatial variation in virus transmission patterns between islands and identified the origin of circulating viruses using a molecular approach. Our findings indicate that terns represent a major host for IAV on oceanic islands, not only for seabird-related virus subtypes such as H16, but also for those commonly isolated in wild and domestic ducks . We also identified strong species-associated variation in virus exposure that may be associated to differences in the ecology and behaviour of terns. We discuss the role of tern migrations in the spread of viruses to and between oceanic islands, in particular for the H2 and H9 IAV subtypes. Avian influenza viruses circulate in wild birds, worldwide, in particular in ducks and seabirds from which a large diversity of viruses have been described. The continued emergence of influenza viruses in poultry and humans has stimulated both research activities and surveillance programs; however, there are still many gaps in our knowledge on virus ecology and epidemiology, in particular in the Southern Hemisphere. In this study we investigated influenza virus circulation in seabirds in the islands of the Western Indian Ocean. We demonstrate that terns act as a major host for influenza viruses on oceanic islands and that, in addition to being infected with virus subtypes usually associated to wild birds, they also could regularly be in contact with viruses that represent a significant threat to veterinary and human health. This study demonstrates that the spatial isolation of these oceanic islands does not limit connectivity with the global avian influenza virus epidemiology and that it may create opportunities for local viral maintenance in wild bird communities. Spatial isolation represents a major barrier to the introduction and transmission of infectious agents on oceanic islands. Animal migration is a key mechanism for the dispersal of infectious agents over long distances  and coulWild birds are the reservoir for a large diversity of infectious agents that threaten human and veterinary health . Ducks aOn oceanic islands, species diversity, host abundance and density are generally higher for seabirds than for ducks, and is likely to affect the relative importance of these hosts in the ecology and evolution of IAV. In addition to spatial isolation, the small duck community size could limit opportunities for virus transmission and also negatively affect virus subtype diversity on these islands . In addition, sample collection on Reunion and the Éparses Islands  were conducted under the approval of the “Direction de l'Environnement, de l'Aménagement et du Logement de la Réunion” and of the “Terres Australes and Antarctiques Françaises”. Bird capture, handling and collection of biological material in the Seychelles , as well as sample export to Reunion Island, were performed under the approval of the Seychelles Bureau of Standards and Department of Environment.e.g. on Reunion Island) or for species highly sensitive to human disturbance (e.g. frigatebirds).We focused on seven islands of the Western Indian Ocean : Aride . To takeTemporal variation in seabird breeding was also considered as it strongly affects the time of sample collection. In the tropics, seabirds species and populations do not breed at the same time of the year. For example, in the Western Indian Ocean, populations of sooty terns breed at different times on different islands. On Bird and Aride islands, breeding occurs during the austral winter (in June-July) while on Juan de Nova, it occurs during the austral summer (in December-January). Because of this temporal pattern, not all populations of the same species were sampled at the same time, and not all species of each island could be sampled during a single sampling session.Anous stolidus), Lesser noddy (Anous tenuirostris); Suliformes: Great frigatebird (Fregata minor), Masked booby (Sula dactylatra), Red-footed booby (Sula sula); Phaethontiformes: Red-tailed tropicbird (Phaethon rubricauda), White-tailed tropicbird (Phaethon lepturus); Procellariformes: Wedge-tailed shearwater (Puffinus pacificus). Details that relate to the number of collected samples for each species and island is presented in In total, 2997 samples (1647 sera and 1350 swabs) were collected from nine seabird species representing four different orders; Charadriiformes: Sooty tern, Brown noddy  was collected from the medial metatarsal or basilic veins, as appropriate for each species. In the field, blood samples were placed in 2 ml Eppendorf tubes in a cooler with ice packs and centrifuged within 12 hours after collection. Sera were transfered in cryotubes and stored at -20°C. Samples were shipped to the laboratory within 48 hours and held at -80°C until tested.Serum samples were tested with the IDvet ID Screen Influenza A Antibody Competition  enzyme-linked immunosorbent assay (ELISA), following an optimized protocol for the detection of antibodies to IAV nucleoprotein (NP) in wild birds . Sample Samples that tested positive for the presence of IAV NP antibodies with the ELISA were tested for the presence of HA-specific antibodies using virus neutralization  and hemagglutination inhibition  assays. Serum samples were heat-inactivated for 30 min at 60°C prior to the VN and HI assay. IAV strains used as antigens were: A/Mallard/NJ/AI10-4263/2010(H1N1), A/Mallard/MN/AI08-2755/2008(H2N3), A/Mallard/MN/AI10-2593/2010(H3N8), A/Mallard/MN/AI10-3208/2010(H4N6), A/Mallard/MN/AI11-3933/2011(H5N1), A/Mallard/MN/SG-00570/2008(H6N1), A/Mallard/MN/AI08-3770/2009(H7N9), A/Mallard/MN/SG-01048/2008(H8N4), A/Ruddy_turnstone/DE/AI11-809/2011(H9N2), A/Mallard/MN/SG-00999/2008(H10N7), A/Mallard/MN/SG-00930/2008(H11N9), A/Mallard/MN/SG-3285/2007(H12N5), A/Ringed-billed gull/MN/AI10-1823/2010(H13N6), A/Blue-winged teal/TX/AI13-1028/2013(H14N5), A/Wedge-tailed_shearwater/Western_Australia/2327/1983(H15N6), A/Ringed-billed gull/DE/AI10-1144/2010(H16N3). The HI assay was done as described in .50) per 25 μL) diluted in MEM supplemented with antibiotics and TPCK-trypsin  were added in each well and the plate was incubated at room temperature for two hours. After incubation, 25 μl from each well were transferred to a 96-well cell culture plate with a confluent monolayer of Maden Darby canine kidney (MDCK) cells  and incubated for 3 days at 37°C under 5% CO2. Prior to transfer, MDCK plates were washed twice with DPBS (150 μL per well) and media in each well was replaced with 150 μL of trypsin-supplemented MEM (as described above). Examination for cytopathic effects was performed with light microscopy; samples were considered positive when no cytopathic effect was observed. A single well without antigen was included for each sample to test for toxicity. In addition, a positive and negative well (MEM only) was run for each antigen and all antigens were back titrated to confirm the proper dilution. Positive reference antisera for the 16 HA subtypes were provided by National Veterinary Services Laboratory, APHIS, USDA.For the VN assay, 25 μL of sample diluted 1:10 in minimal essential medium (MEM) supplemented with antibiotics  were placed in a 96-well plastic V-bottomed microtitre plate. Twenty-five microlitres of virus suspension (100 median tissue culture infectious dose (TCIDAll samples testing positive at the 1:20 dilution were tested again at dilutions ranging from 1:20 to 1:640. Fifty microlitres of each previously diluted sample (1:10) was placed in the first line of a 96-well plastic V-bottomed microtitre plate and serially diluted in 25 μl of trypsin supplemented MEM. Antigen (25 μl) was then added in each well and the plate was incubated as described before; all previously described controls and back-titrations were also performed. The VN titre was considered to be the highest dilution without cytopathic effects and if testing was negative at the 1:20 dilution the samples were considered negative.versus chick), on the probability of successful detection IAV NP antibodies in bird serum. GLMs were also used to test the effect of the bird species and island on the probability of successful detection of IAV NP antibodies specifically in Charadriiformes. A second-order Akaike information criterion (AIC) was used to select the most parsimonious model. The effect of variables included in the most parsimonious model was tested using a Chi square test (χ2). Analyses were conducted in R 2.15.2 , suggesting recent gene flow between Eurasia and the Southwestern Indian Ocean.Partial nucleotide sequences of HA of seven H2 IAV detected on Reunion Island were obtained (sequence lengths of either 910 bp or 1013 bp). The limited genetic diversity observed (nucleotide sequence similarity ranging from 99.8% to 100%) indicate that a single H2 IAV subtype was likely circulating at the time of bird sampling. Phylogenetic analyses suggested that these viruses were closely related to those that have been circulating in wild birds in Eurasia since 2009 , and IAV subtypes that represent a significant threat to veterinary and human health .e.g. shorebirds). Secondly, as North American IAV isolates were used as reference viruses in the VN and HI assays (except for H15), we cannot exclude that they may be antigenically different to IAV circulating in the Western Indian Ocean, and this may have resulted in underestimating subtype prevalence. Thirdly, failure to isolate viruses from positive samples precluded the sequencing of the full genomes of the H2 IAVs from Reunion Island, therefore limiting possible inferences on their phylogenetic and geographic origins.Several limitations in our study need nevertheless to be taken into consideration. Firstly, although our sampling strategy was designed to provide representative numbers of samples as a function of colony size and from a maximum number of species, a subset of seabird species and populations were included in our analysis and one cannot exclude that other species may also be actively involved in IAV epidemiology on oceanic islands (Sterna hirundo) that occurred in South Africa [The first isolation of an IAV in wild birds was reported in 1961 following an epizootic among common terns  and other wild duck species that are recognized as natural host reservoirs for IAV , may play a central role in the epidemiology of IAV on oceanic islands.The global prevalences of infected  and serAmerica; ). This re.g. six nests per square meter on Bird Island [Particularities of the ecology of terns might account for the variation in virus exposure and transmission between closely related species. Species-related differences in life history such as colonial nesting, social behaviour, migration and foraging characteristics are indeed likely to affect opportunities for virus transmission. For instance, Sooty terns breed in colonies of hundreds of thousands of pairs, with high bird densities , potentid Island ). A potePopulation size and density may not be the only ecological drivers for IAV transmission in tern colonies and other life history characteristics, such as behaviour during the post-breeding migration, could play a more significant role. Sooty terns remain at-sea without land stop-over during the non-breeding period. In contrast, brown and lesser noddies tend to visit and roost on a large number of islands in the Indian Ocean after the breeding season . These pet al. reported very high prevalence of infections (up to 72%) in nesting and fledging black-headed gulls [Spatial and temporal variation in virus transmission may also exist between tern populations of the same species. This is suggested by the significant differences in seroprevalences estimated at different time of the year, in the populations of lesser noddies sampled on Bird and Reunion islands. In a recent study, Verhagen ed gulls . The auted gulls . In our Our findings stress that terns are exposed to a high diversity of IAV subtypes, and that this diversity differs between species. Similarly to gulls , noddiesIn both sooty terns and lesser noddies, H16 was the most common subtype, supporting that, together with gulls, terns could be a major host reservoir for this virus subtype. None of the samples that tested ELISA positive for antibodies tested positive with the HI assay for the H13 HA subtype. As previously mentioned, we cannot exclude that this negative result merely reflects a technical limitation with our HI assay because of significant antigenic differences between the North American origin H13 IAV used in the serological assays and H13 IAV circulating in the Indian Ocean.The variations reported in the detected subtypes as well as in their number also reflects significant species-related differences in virus transmission. As compared to sooty terns, noddies may be more frequently exposed to a wider diversity of IAV as result of gregarious behaviour and mixing with conspecific and interspecific individuals during the post-breeding season. The detection of IAV subtypes usually associated with wild ducks and poultry  further suggests that noddies might share habitats with ducks at some stage during their life cycle. Mixing between species and populations originating from different geographic areas favour virus gene flow between hosts and facilitates cross-species transmission, in particular during the course of migration ,45 and, Anas erythrorhyncha) may for instance be involved in local IAV circulation [Anas querquedula) is also present on the eastern coast of Africa and is an occasional non-breeding visitor to several Western Indian Ocean islands [In the Western Indian Ocean, however, the absence of indigenous duck species on oceanic islands and the limited influx of migrant ducks , potenticulation . The Gar islands . This lo islands –52; one  islands ,53.The detection of the H2 IAV on Reunion Island further supports that gene flow between Eurasia and the Western Indian Ocean can occur. Genetic analyses indeed showed that the virus detected on Reunion Island was closely related to H2 IAV isolated in black-headed gulls in the Republic of Georgia, and suggested that this virus might have been introduced to the Western Indian Ocean by long-distance migrants in the East-Asia—East-Africa or Black Sea—Mediterranean migratory flyway. Overlap between the spatial ranges of black-headed gulls and noddies could have favoured direct virus transmission of this virus subtype, usually isolated in ducks. The limited information on IAV circulation in wild birds in Eastern Africa, as well as the limited knowledge on seabird migrations in this area, however, precludes a more precise conclusion on the geographic and host origins of the Reunion Island viruses.e.g. 40% of the birds that tested positive for the presence of IAV NP antibodies on Bird Island) indicates that this species is regularly in contact with this IAV subtype. The H9 IAV subtype is relatively uncommon in wild ducks [e.g. [The high detection rate of H9 HA-specific antibodies in sooty terns . Theks [e.g.  and was ks [e.g. . This viks [e.g. , and hasks [e.g. ,62 and iks [e.g. . Interesks [e.g. . AlthougOur study stresses the potential role of seabird migrations and behaviour during the non-breeding period in the spread of IAV on oceanic islands. We highlight that the spatial isolation of oceanic islands is unlikely to disconnect virus transmission from the global IAV epidemiology and that it may create opportunities for their local maintenance in wild bird communities. Future investigations will have to focus specifically on tern population structure and migratory pattern to better assess the risk associated to virus transmission within and between islands.S1 TableNumbers are expressed in breeding pairs.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableTitres are indicated for each tested sample and highlighted in red when considered positive. NT: not tested.(PDF)Click here for additional data file.S1 FigComputations were realized with the GTR+I+ Γ evolutionary model . Blue branches highlight viruses isolated in humans and green branches viruses recovered from birds, swines and from the environment. Red branches highlight the genetic lineage of Reunion Island H2 influenza A viruses for which the detailed evolutionary history was investigated with coalescent analyses . Bootstr(PDF)Click here for additional data file.


To the Editor: Zika virus (ZIKV), a mosquitoborne flavivirus, was first isolated from a rhesus monkey in Uganda in 1947  has a 10,794-nt genome and is most closely related to Spondweni virus . Participants contacted study staff to report fever and were also contacted weekly by staff to determine if they had fever during the previous 7 days. Fever episodes triggered an acute-illness visit by a study nurse, who performed a clinical assessment of the patient and collected an acute-phase blood sample. During the first year of surveillance, 270 acute febrile illnesses were detected; 267 of the patients had samples available for serologic testing for evidence of influenza, dengue, chikungunya, Japanese encephalitis, and Zika virus infections.In May 2012, a 15-year-old boy in Cebu City reported a subjective fever; an acute-illness investigation followed. Other symptoms included headache, conjunctivitis, sore throat, myalgias, stomach pain, anorexia, nausea, and vomiting, but no rash. The boy did not seek medical care or require hospitalization; his only treatment was acetaminophen. He had no recent travel history, and no other members of his household were ill. The boy recovered fully by the 3-week study follow-up visit. An acute-phase blood sample, collected 2 days after symptom onset, was negative for dengue and chikungunya viruses by reverse transcription PCR. An in-house dengue/Japanese encephalitis IgM/IgG capture ELISA and chikungunya ELISA were used to test paired acute- and convalescent-phase blood samples; all results were negative. ZIKV ELISA was not available at the testing laboratory. However, by using real-time reverse transcription PCR targeting the gene that encodes the precursor of membrane protein, we detected ZIKV RNA in the patient’s serum sample  was used to obtain sequence reads by next-generation genomic sequencing, which identified a 789-bp contig as a partial sequence of the ZIKV gene that encodes the nonstructural 5 protein (GenBank accession no. KM851038).Virus was then isolated by intrathoracically inoculating Maximum-likelihood phylogenetic analysis of the gene encoding the nonstructural 5 protein sequence showed that the isolate belonged to the ZIKV Asian lineage . Pair-wiAedes spp. mosquitoes are present and are known to transmit ZIKV; most notable among these vectors are A. aegypti mosquitoes, which are widespread throughout the Philippines , Thailand (2013), or French Polynesia (2013). It is possible that the ZIKV infection in our study was an isolated case (the virus was confirmed in only 1/270 episodes of acute febrile illness). However, because the symptoms of disease are similar to those for other known endemic arboviruses, it is also possible that the strain was introduced into the Philippines before 2012 and remained undetected.The spectrum of ZIKV-associated clinical disease remains uncertain. Although reports indicate most cases of infection are mild, infections may be associated with more severe disease outcomes, such as Guillain-Barre syndrome (