The experiments followed recent recommendations for Ebola Treatment Centres. Three excreta matrices containing either raw municipal wastewater, or raw municipal wastewater plus 10% or 20% (w/v) added faecal sludge, were treated in 14 litre buckets at a ratio of 1:10 (chlorine solutions or lime suspensions: excreta matrix). The effects of mixing versus non-mixing and increasing contact time (10 and 30 mins) were also investigated. Bacterial  and intestinal enterococci (IE)) and viral (somatic coliphages (SOMPH), F+specific phages (F+PH) and Bacteroides fragilis phages (GB-124PH)) indicators were used to determine the efficacy of each approach. Lime-based approaches provided greater treatment efficacy than chlorine-based approaches, with lime (30% w/v) demonstrating the greatest efficacy . There was no statistical difference in efficacy between any of the chlorine-based approaches, and the highest log reduction values were: FC = 2.90, IE = 2.36, SOMPH = 3.01, F+PH = 2.36 and GB124PH = 0.74. No statistical difference was observed with respect to contact time for any of the approaches, and no statistical differences were observed with respect to mixing for the chlorine-based approaches. However, statistically significant increases in the efficacy of some lime-based approaches were observed following mixing. These findings provide evidence and practical advice to inform safe handling and containment of excreta and ensure more effective health protection in future emergency settings.Ebola and cholera treatment centres (ETC and CTC) generate considerable quantities of excreta that can further the transmission of disease amongst patients and health workers. Therefore, approaches for the safe handling, containment and removal of excreta within such settings are needed to minimise the likelihood of onward disease transmission. This study compared the performance and suitability of three chlorine-based approaches ) and three lime-based approaches  The recent West Africa Ebola virus disease (EVD) outbreak 2013–2016) led to the deaths of 11,310 people  [3–2016 leThe Ebola virus (EBV), is recognised to be relatively fragile and less resistant to environmental factors than enteric viruses  and therIn order to prevent on-going transmission at Ebola Treatment Centres (ETC), institutions involved with the Water, Sanitation and Hygiene (WASH) response, such as Doctors without Borders (MSF), the U.S. Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), took precautionary actions and recommended the addition of a 0.5%  chlorine solution to a bucket partially filled with human excreta –12. The Chlorine compounds commonly recommended for use in ETC have been powdered calcium hypochlorite (HTH); granular sodium dichloroisocyanurate (NaDCC or SDIC) and liquid sodium hypochlorite (NaOCl) (domestic bleach). For each of these compounds, treatment efficacy can vary, according to the concentration of the chlorine solution, contact time, temperature, pH level, and the presence of organic matter . PreviouChlorination is also commonly used to treat drinking water in emergency settings . HoweverAs a consequence, there remains a clear lack of information on how best to handle human excreta in emergency settings according to current protocols recommended by humanitarian agencies. In the absence of detailed information on the survival of the EBV within human excreta matrices, the WHO and UNICEF brought together a group of international experts in the field to support a critical review of existing WASH practices and to provide updated advice in light of questions raised by practitioners in the field. The resulting document–“Ebola Virus Disease (EVD): Key questions and answers concerning water, sanitation and hygiene”–suggested that a physico-chemical treatment using hydrated lime suspensions might be a potential alternative to the use of chlorine based approaches .2) is a white powder obtained when calcium oxide (CaO), or lime is mixed with water, which produces a highly alkaline suspension (c. pH 14). The use of hydrated lime to treat wastewater and faecal sludge dates back to Roman times, when it was mainly used to control odours arising from pit latrines [Hydrated lime, also known as slaked lime, or calcium hydroxide (Ca (OH)latrines . It has latrines , 23. In latrines .Hydrated lime has been suggested as an alternative treatment of municipal wastewater , and 27 Current WASH sector recommendations for excreta clean-up, containment and removal in Ebola Treatment Centres (ETC) are summarised in The aim of the research described here was to determine the suitability of bucket treatments as part of intervention protocols  and 20 fAll laboratory assessments were performed at the Environment and Public Health Research Group (EPHReG) facilities at the University of Brighton (UoB), UK. Excreta matrices (EM) were produced from dewatered faecal sludge and fresh untreated municipal wastewater collected weekly from a local municipal wastewater treatment plant (Hailsham North WWTP–with the permission of Southern Water Ltd (U.K)).2), also known as “high test hypochlorite” ; sodium dichloroisocyanurate ; and sodium hypochlorite (NaOCl) (Bleach 1.5% Sainsbury®). Performance assessments were undertaken within 14 litre high-density polyethylene (HDPE) plastic buckets supplied by the international NGO OXFAM®. Bacterial indicators  and viral indicators ) were used to evaluate performance. High titre bacteriophage stocks used to “spike” the excreta matrices were provided from the EPHReG repository, and made using phages purified and concentrated by a propagation method described elsewhere [All chlorine solutions  and hydrated lime suspensions  were prepared in the EPHReG laboratory. Chlorine solutions were prepared from calcium hypochlorite (Ca (ClO)lsewhere .Human excreta consist mainly of faecal material and urine, although blood and vomitus could also be present, especially in disease outbreaks. The physical and chemical characteristics, as well as daily output of human excreta depend on age, ethnicity, disease, diet, income levels and the geographical location of the excreting population , 36. PreTo produce excreta matrices for this study, raw wastewater and faecal sludge were used to simulate the liquid (urine) and solid (wet faeces) fractions, respectively. Three excreta matrices (EM), containing varying amounts of solid and dissolved organic matter, were produced. ‘EM 20%’ was composed of 80% raw wastewater plus 20% faecal sludge to represent excreta from healthy persons; ‘EM 10%’ was composed of 90% raw wastewater plus 10% faecal sludge to represent excreta from patients with mild diarrhoea; and ‘EM 0%’ was composed of 100% raw wastewater to represent excreta from patients with severe diarrhoea.® 8209 Iodometric titration method  was used in a series of tests to certify chlorine concentrations within HTH granules (65% available chlorine), NaDCC granules (65% available chlorine) and Sainsbury’s ® domestic thin bleach (containing less than 5% available chlorine). Results showed different percentages to those displayed on the product labels, in that HTH and NaDCC granules recorded available chlorine levels of 58.5% and 53.7% respectively (not 65%). The bleach solution recorded an available chlorine content of 1.58%. All reagents were recently purchased, stored according to the manufacturer’s instructions and well within their use by date.First, the HACH20 was added to glass Schott bottles. Using an OHAUS® Adventurer Pro digital scale (d = 0.01 g), 8.54 grammes of HTH or 9.30 grammes of NaDCC were weighed and 316.5 mL of bleach were measured. The chlorine solutions were added to the bottles and stirred using a plastic-coated stirring rod. Each solution was labelled according to the chlorine type and allowed to stand for at least 30 minutes  prior to use. A total chlorine concentration test (HACH® 8209) was always performed on each chlorine solution prior to treatment assessments to certify that solutions contained 0.5% (± 0.025%) available chlorine.Prior to each assessment, one litre of each 0.5% standard chlorine solution was prepared as follows: One litre of fresh deionised H®, CEMEX U.K.) with de-ionised H2O in glass Schott bottles, which were used within 24 hrs.The three hydrated lime (HL) suspensions were prepared as follows: ‘HL 10%’, ‘HL 20%’ and ‘HL 30%’ (w/v) by mixing a weighed quantity of lime . Proportional volumes of the previously described excreta matrices were used during the bucket experiments . Furthermore, 450 mLs of chlorine solution , or hydrated lime  suspensions were added to OXFAMa matrix . The eff® Ranger 3000 digital balance and volumes of wastewater were measured using measuring cylinders. Excreta matrices were then mixed and homogenized using an Arbeco® jar test machine (with a paddle speed of 200 rpm) for approximately 3 mins and the contents distributed into the various buckets (six treatments plus one control). Because naturally occurring concentrations of F-specific and GB124 phages are relatively low (compared with the other indicators) in UK municipal wastewaters, 5 mL of pre-prepared bacteriophage stock solutions of MS2 and B124 (± 109 PFU mL-1), respectively, were “spiked” into the buckets in order to raise the concentrations of these viral indicator to a level high enough to observe log reductions within the various excreta matrices.Faecal sludge was weighed using an OHAUS®), containing 45 mL of quarter strength Ringer’s (QSR) solution. For the ‘mixed’ method, the bucket contents were stirred gently for a period of ten seconds, using a plastic-coated stirring rod. Following subsequent contact times (Ct) of 10 and 30 mins, excreta matrices were subsequently and rapidly stirred for 1–2 seconds, before further 5 mL samples were withdrawn for analysis from all buckets (including the control bucket). Samples were poured into 50 ml self-standing centrifuge tubes (Corning®) containing either 45 mL of a sodium thiosulphate solution (300 mg/L)  for the chlorine-based treatments, or 45 ml of quarter strength Ringer’s (QSR) solution for lime-based treatments and control buckets. Dilution series  were produced from these master (10−1) 50 ml dilutions and analysed immediately.All bucket studies were conducted at room temperature (approx. 21 °C). Before the addition of any chlorine solution or lime suspension, and with the intention of quantifying the initial levels of all viral and bacterial indicators, a 5 ml sample was taken randomly from the buckets and poured into 50 mL self-standing centrifuge tubes (Corning®) in Ø 55mm Petri dishes. Results were expressed as colony-forming units (CFU) per mL. Somatic coliphages (SOMPH) were enumerated in accordance with ISO standard 10705–237 [E. coli (WG5) was used as the host bacterium [+PH) were enumerated according to ISO standard 10705–138 [Salmonella typhimurium (WG49) [Bacteroides fragilis (GB124PH) were enumerated according to ISO standard 10705–439 [−3, 10−4 and 10−5 dilutions were used, while for treated samples, 100, 10−1, 10−2 and 10−3 dilutions were used. The limit of detection for all microorganisms was either 1 PFU mL-1 or 1 CFU mL-1 for EM 0%; and 10 PFU mL-1 or 10 CFU mL-1 for EM 10% and EM 20%.In order to evaluate performance, viral and bacterial indicators were enumerated before and during treatment. Faecal coliforms (FC) and intestinal enterococci (IE) enumeration followed standard methods: namely ISO 9308/1:200035  and ISO 0705–237  and E. cacterium . F-speci0705–138  and Salmm (WG49)  was used0705–439  using st0705–439  as the h-1. COD was measured using method 5220 D, and results were expressed as mg O2.L-1. The concentration of ammonium ions was measured by the indophenol blue method; analyses were performed using a HACH® DR3900 bench-top spectrophotometer  and a HACH® LCK 303 kit  according to the manufacturer’s instructions. pH levels were measured using a Mettler Toledo FE20-ATC Kit FiveEasy™ benchtop pH meter.Chemical oxygen demand (COD), suspended solids (TSS), ammonium ions and pH were determined in order to assess the basic characteristics of the excreta matrices. TSS and COD analyses were conducted according to APHA Standard Methods for the Examination of Water and Wastewater . TSS was-1) prior to use in the full-scale bucket experiments, the HACH® Total Chlorine Iodometric Method Using Sodium Thiosulphate 8209  was used according to the manufacturer’s instructions. Miniaturised treatment experiments were performed to assess chlorine demand exerted by each excreta matrix. Excreta matrices and compound volumes were proportional and a hundred-times smaller than those used in the full-scale bucket experiments previously described. Therefore, 4.5 ml of chlorine solutions or lime suspensions were added to 50 mL centrifuge tubes containing the excreta matrices . Tubes were inverted three-times to facilitate mixing. After 10 and 30 min contact times, 1 mL of sample was withdrawn and HACH® method 8021 (USEPA DPD Method) was used to determine the concentration of free residual chlorine. (Note: this is a low range method (0.02 to 2.00 mg L-1 CL2) and samples were serially diluted, with 9 mL volumes of deionised H2O, when results were ‘over-range’. Results were expressed as mg L-1 CL2.In order to certify chlorine solutions were at a concentration of 0.5%  23.0’, and ‘Microsoft Excel 2013’. Non-parametric statistical tests were used to analyse the main data from this research  and median values chosen to express more accurately the average levels. Parametric statistical tests were used analyse physico-chemical data. The criterion of 95% confidence, or a 0.05 probability (p), was applied to test the significance of the various statistical tests used during this study.post hoc paired-comparison test was performed to identify differences in efficacy between all six approaches. The Mann–Whitney two-sample rank-sum test, was used to compare differences between two independent groups of variables (namely: Ct10 vs. Ct 30 mins and mixed vs. non-mixed). ANOVA was used to determine whether there were statistically significant differences between the residual chlorine concentrations of the three chlorine solutions following addition to excreta matrices. An Independent Samples T-test was used to compare pH and residual chlorine differences between the chlorine-based and lime-based approaches.The efficacy of the various approaches was evaluated by recording the final concentration of microorganisms present and calculating the log reduction of each microorganism for each approach . Additionally, calculation of log reduction was performed for the various approaches  and also for each excreta matrix. The Kruskal-Wallis test (rank-based non-parametric one-way ANOVA) was used to determine whether there were statistically significant differences between efficacies for the various solutions and suspensions tested. Furthermore, a Dunn-Bonferroni 4 (1.7 x 106) CFU mL-1; IE = 3.5 x 104 (1.1 x 106) CFU mL-1; SOMPH = 4.9 x 104 (7.1 x 105) PFU mL-1; F+PH = 1.3 x 105 (5.4 x 105) PFU mL-1; and GB124PH = 4.6 x 105 PFU mL-1 (2.2 x 106). There was no statistical difference (p >0.05) between the average initial levels and the average final levels of microorganisms in the “control” buckets (without chlorine or lime). Average levels (median and geometric mean) of surviving microorganisms following chlorine and lime-based treatment are provided in the supporting information ) initial levels of indicator organisms (prior to chlorine or lime addition) were as follows: FC = 7.7 x 10ormation . OverallOverall, the hydrated lime suspensions demonstrated greater efficacy compared with the chlorine solutions. Statistically, for FC, HL 20% and HL30% demonstrated greater log reductions than achieved using the three chlorine-based approaches. For IE, HL 30% demonstrated greater log reduction (p<0.05) than all chlorine–based approaches.For SOMPH, there were no significant differences in log reduction (p>0.05) between all lime- and chlorine-based approaches. For the ‘spiked’ F+PH and GB124PH, all lime suspensions demonstrated statistically greater log reduction (p<0.05) than the chlorine solutions. There was no significant statistical difference in log reduction (p>0.05) between any of the chlorine solutions .As displayed in Figs Efficacy of the various approaches decreased as the organic content and suspended solids increased within the matrices (EM 10% and EM 20%). This trend was more pronounced and significant for the chlorine-based approaches.For EM 10%, log reduction ranged from 0.48 to 2.58 for chlorine-based approaches and from 0.92 to 5.43 for lime-based approaches. Faecal coliform log reduction was statistically higher for all lime-based approaches (max. = 4.89) compared with chlorine-based approaches (max. = 2.58). For IE, HL 30% demonstrated statistically a higher log reduction (4.28) than all chlorine-based approaches (max. = 1.96) and HL 20% demonstrated a statistically higher log reduction than ‘domestic bleach’. Interestingly, the only statistical difference reported for SOMPH reduction was that the log reduction for ‘NaDCC’ (2.43) was higher than for ‘HL 10%’ (0.92). For F+PH and GB-124 phages all lime-based approaches demonstrated statistically higher log reductions  than chlorine-based approaches  .post hoc Dunn-Bonferroni pairwise comparison test did not demonstrate any adjusted significance value below 0.05. For SOMPH, no statistical difference was observed between any of the approaches and log reductions ranged from 1.05 to 1.98. For F+PH, the HL 20% (M = 5.29) and HL 30% (M = 5.29) suspensions demonstrated statistically higher log reductions than the chlorine-based approaches (max. = 1.48). For GB-124 phages, all log reductions for lime-based approaches (5.40–5.41) were also statistically higher than for chlorine-based approaches (0.56–0.59)  was compared with ‘HTH’ (1.53) and ‘NaDCC’ (2.00) chlorine solutions. For IE, the HL 20% (M = 3.64) and HL 30% (M = 3.71) suspensions demonstrated statistically higher log reductions than chlorine-based approaches (max. = 1.36), but the .59) See .A summary of the physico-chemical excreta matrix characteristics is presented in -1. As solutions were diluted at a ratio of 1:10 with the excreta matrices, it was assumed that initial levels of free chlorine within the buckets would be 454.54 mg L-1 . The concentration of chlorine solutions was consistently 0.5%, or 5,000 mg L-1 to residual free chlorine levels ranging from 187.5 to 241.3 mg L-1 in EM 0%; 1.11 to 2.31 mg L-1 in EM 10% and 0.76 to 0.98 mg L-1 in EM 20%.It is clear that as the levels of organic matter and suspended solids increase in the excreta matrix, the higher becomes the chlorine demand exerted. Initial levels of free chlorine declined from 454.54 mg LThe chlorine solutions and lime suspensions exhibited mean pH values of 7.90 (HTH); 6.17 (NADCC); 11.81 (bleach); 12.90 (HL 10%); 12.91 (HL 20%) and 12.93 (HL 30%). Mean pH values of excreta-treatment mixes did not vary significantly with respect to contact time (p> 0.05). There were a few statistically significant differences between pH values of mixed and non-mixed tests (p< 0.05). Mean pH levels of excreta-treatment mixes according to excreta matrix, treatment and mixing method are presented in Generally, pH values for the lime-excreta combinations were always higher when mixing was employed, although this assumption was only statistically supported for the EM 20%—HL 20% combination .This laboratory-based study aimed to assess the performance and suitability of on-site approaches for the safe handling and containment of human excreta using current WASH guidelines recommended for use in emergency settings. In addition to elucidating the suitability of existing chlorine-based approaches –12  upon performance. These higher concentrations demonstrated higher log reductions, especially HL 30% ; which There was no statistical difference in terms of performance between any of the chlorine-based approaches . Their highest log reduction levels were for NaDCC, were as follow: FC = 2.90, IE = 2.36, SOMPH = 3.01, F+PH = 2.36 and GB124PH = 0.74  and from 0.8 to 0.83 mg L-1 (EM 20%) , their performance being significantly lower than the various lime-based approaches in the EM 10% and 20% . This wa(EM 20%) . It is a(EM 20%)  may alsoWASH treatment protocols  and 20 sGiven the increased risks to healthcare workers associated with any potential disturbance of buckets contents, the observation that mixing had no statistically significant effect on the efficacy of chlorine-based approaches is useful to note. Mixing did appear to slightly improve the performance of the lime-based approaches , which m+PH and GB124PH showed considerable resistance to chlorination, also in accordance with the results of previous studies [Myoviridae, Siphoviridae, Podoviridae and Microviridae. In this study, the authors noted that the lime and chlorine tolerant SOMPH phages were all similar in terms of their plaque size and shape, possibly suggesting that they may belong to the same virus family. However, further analysis of the morphology and classification of these resistant phages using electron microscopy was considered beyond the remit of this study.Of the bacterial indicators assessed in this study, IE appeared to be more resistant to chlorine and lime-based approaches than FC under all conditions, a finding that agrees with those of previous studies . Of the  studies ,54. SOMP- ions present in lime suspensions saponify lipids in the enveloping membrane [According to the extant literature, the observed differences in resistance among the various bacterial and viral indicators can be attributed to their mechanisms of removal and inactivation. For instance, the main mechanisms of bacterial disinfection by chlorine and lime-based treatments are chemical reactions that lead to the lysing of microorganism cell walls. OHmembrane , while tmembrane . In contmembrane , 58.Pseudomonas syringae phage Phi6 has been proposed as a potential surrogate for the Ebola virus in a recent study [+PH for assessing removal is an essential precautionary measure.However, whilst studies have shown that the Ebola virus is highly sensitive to chlorine-based approaches, demonstrating a 6.6 log reduction when exposed to 0.5% chlorine on surfaces , its behnt study , as it hnt study , adoptinResearch has also shown that laboratory-grown microorganisms may be more susceptible to treatment processes than indigenous microorganisms , 62. ThiThis research has demonstrated that although chlorination (at 0.5% concentration) using a range of chlorine-based products appears to be effective when handling raw wastewater, these products are considerably less effective at ensuring the safe handling, containment and removal of more concentrated forms of human excreta (containing greater concentrations of organic matter). Furthermore, chlorination of human excreta also raises other safety concerns, such as the potential re-growth of resistant pathogenic microorganisms . HoweverHowever, production of lime suspensions requires larger volumes of hydrated lime compared with the equivalent volume of chlorine compounds required to produce the solutions used in this study. Transportation of large quantities of hydrated lime powder may therefore be considered a logistical problem in emergency settings. Nevertheless, lime powder is a very common building material, which is available in most parts of the world. Preparing and handling lime suspensions is relatively easy, but, like chlorine-based approaches requires the use of basic PPE. Furthermore, lime suspensions appear to demonstrate a relatively long shelf-life and maintain their high pH levels for weeks. The disposal of highly caustic hydrated lime wastes may of course cause an adverse effect on the environment. Therefore, hydrated lime sludge should be disposed in landfills sites or waste stabilization ponds. However, hydrated lime is commonly used as a soil conditioner where soil pH is low and is extensively used to raise soil pH in agricultural settings. Consequently, these effects are unlikely to be too damaging to the environment in the longer term.Supporting our study, recent research  has highSeveral groups are known to be currently researching alternative approaches for the safe handling, containment, disposal and treatment of human excreta. However, the results from this study are very encouraging, but further research is still required  without constituting a public health hazard; and to establish optimum ratios and contact times between solutions/suspensions and excreta matrices). Therefore, it is important that the international scientific community continues to explore alternative options with which to develop a ‘toolbox’ of methods to ensure that risks to human health are minimized across all potential disease transmission pathways within emergency settings.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Acanthamoeba polyphaga mimivirus, in 2003, and several others thereafter, posed intriguing questions regarding virus origins, evolution, classification, and their place in the “tree of life.” Second, the discoveries of “Lokiarchaeota” and several other closely-related archaeal members that encode several eukaryote-specific proteins challenged the three-domain canonical structure of the tree of life. These discoveries have reopened debates on central questions in evolutionary biology research such as the origin of viruses, the origin of eukaryotes, evolutionary relationship between Archaea and Eukarya, and the structure and topology of the tree of life. In this Research Topic, we received a broad range of contributions addressing these and other related questions.We live in exciting times for microbiology research. The significant technological and scientific advancements in the past decades have now enabled scientists to pursue discovery and description of novel microbial and viral linages from previously uncharted Earth habitats. Two such discoveries are particularly exciting and noteworthy in this regard. First, the discovery of the first “giant virus,” Moelling and Broecker discussed the “virus first” model for the evolution of life on Earth. They provided several examples of virus diversity and abundance in a range of Earth environments and in the mammalian genomes. According to their view, ribozymes and viroids could have started early evolution albeit they also acknowledged competing alternatives such as the “proteins first” and the “metabolism first” scenarios of origins of life. In a separate contribution from the same authors, they examined the possibility that complex antiviral defense strategies and immune systems in cellular organisms could have evolved from viruses and transposable elements. The authors thus highlighted the crucial roles viruses could have played during the evolution of cells.Ramisetty and Sudhakari discussed why the selection of “grounded prophages” may be favored by bacterial cells. Grounded prophages are unable to excise from bacterial genomes due to mutations in either recombinase gene or genes encoding attachment sites. Lysogens with grounded prophages are protected from specific phage infections and the future activation of lytic cycles. These grounded prophages can also serve as hotspots or buffer zones where genes encoding antibiotic resistance and virulence may integrate. Their work thus highlights another important contribution of viruses to the evolution of cells that contradicts the view of viruses as mere cellular pathogens.Legendre et al. highlighted an often-ignored feature of viral genomes, the existence of many protein-coding genes with no detectable homologs. These virus-encoded ORFans constitute the large majority of genes of giant viruses but are mostly ignored when discussing models of virus evolution, while their origin remains a mystery. The authors provided evidence that ORFans in pandoraviruses, the largest viruses known to date, can be unique even among closely related members of the same virus family. These ORFans likely originate from intergenic regions and suggest that the pandoravirus pan-genome is open. These findings indicate that the genomes of viruses are incredibly dynamic in their gene creation capabilities, a notion hardly acknowledged in standard virus evolutionary scenarios where all genes are bound to have an ancestor.Hendrickson and Poole presented several analogies of nucleus-like compartmentation outside eukaryotes and discussed three possible explanations for the emergence of compartmentation in cells: physical protection, crosstalk avoidance, and non-adaptive origins.The presence of nucleus is a defining distinction between eukaryotes and prokaryotes. While nucleus-like compartmentation has been described or proposed in planctomycetes and jumbophage-infected bacteria, it is unclear how these compartmentations are linked to the origin of nucleus in eukaryotes. Harrison et al. explored the diversity of ribonucleotide reductases (RNRs) from marine viroplankton, ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and are prominent in viral genomes. They found cyanophage Class II RNR enzymes were misannotated and were actually part of the large oxygen-dependent Class I clade. Since the marine Synechococcus and Prochlorococcus hosts carry only Class II enzymes, the finding confirms that the viroplankton RNRs are not host derived nor dependent on the adenosylcobalamin (B12) cofactor of class II enzymes. This is an important clarification for connecting genomic information and phenotypic traits in the context of viral ecology.Wang et al. analyzed the genomes of 21 Coxsackievirus A4 isolates from hand, foot and mouth disease cases in children from the Shandong province of China. Phylogenetic analysis of the VP1 gene, when benchmarked to a large Coxsackievirus collection, revealed that genotypes C, D1, and D2 identified in the early 2000s have been taken over by the D2 genotype in China. Calculation of substitution rates revealed ongoing virus evolution and several dynamic fluctuations in the history of the virus isolates. The study is important for the molecular epidemiological characterization of A4, which has been understudied because of the paucity of genomic data.Flynn and Moreau engaged in a comparative host-centered exploration of endogenous viral elements (EVE) in ants. Using a comprehensive bioinformatic pipeline, they screened all 19 published ant genomes for EVEs and assessed their phylogenetic relationships to closely related exogenous viruses. EVEs similar to proteins from single stranded RNA viruses, viral glycoproteins, and retro-virus-derived proteins were widely present in ant genomes, suggesting tendencies to endogenize. EVEs therefore mimic the diversity of viral lineages.Ongrádi et al. isolated an adenovirus from a domestic cat and found it was related to human adenovirus. Their careful molecular, biological, and phylogenetic characterizations highlight important information with the potential to re-define the adenovirus research field. It also prompts a broader analysis of adenoviruses of many different types. Further epidemiological and pathomechanistic studies can help understand the molecular mechanisms of virus host jumps, which could be engineered to combat both feline and human AIDS.Flodman et al. performed an extensive analysis of interaction between commercially available restriction enzymes and bacteriophages containing modified nucleotides. They evaluated restriction resistance of phages M6, Vil, and phi W-14 against 200 commercially available enzymes. This work has enormous value for genetic engineering and understanding interaction of proteins with virus-modified DNA.Sun et al. showed evidence that polysaccharides isolated from the leaves of the Aloe vera plant, known to mitigate virus infection, inhibit the replication of a H1N1 subtype of influenza virus at the time of virus adsorption. The anti-influenza activities were for the first time explored in vitro in cell cultures and in a mouse model. This study opens the door to the development of novel anti-influenza drugs.Zhou et al. presented a novel mismatch-tolerant loop-mediated isothermal amplification (LAMP) method with an efficiency high enough to be applied to the detection of viruses. The methodology tolerates mismatches in primers and templates. They used this new methodology to detect variants of antigenically-distinct serotypes of the dengue virus.Finally, The Research Topic is thus a collection of ideas ranging from our basic understanding of virus origins and evolution to new approaches of virus identification and treatment. We hope this resource will be a useful reference for future studies in basic and applied virology.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zika virus (ZIKV) became a global human health concern owing to its rapid spread worldwide and its association with congenital and neurological disorders. The current epidemiological profile of arboviruses in Brazil is characterized by widespread co-circulation of Dengue virus, Chikungunya virus, and ZIKV throughout the country. These viruses cause acute diseases frequently with overlapping symptoms, which could result in an inaccurate diagnosis based solely on clinical and epidemiological grounds. Here we conducted a screening for ZIKV RNA in serum samples from patients across Brazil with suspected ZIKV infection.Using RT-qPCR, we investigated ZIKV RNA in 3001 serum samples. Samples were passively acquired through a private laboratory network, between December 2015 and August 2016, from 27 Brazilian Federative Units. We performed descriptive statistics on demographic variables including sex, age, and geographic location.ZIKV was detected in 11.4% (95%CI = 10.3–12.6%) of the sera. ZIKV RNA was detected in sera collected throughout the country, but during the analyzed period, RNA was more frequently detected in samples from the Southeast, Midwest, and North regions (3.9 to 5.8 times higher) when compared to the Northeast and South regions.These data reinforce the importance of laboratory diagnosis, surveillance systems, and further epidemiological studies to understand the dynamics of outbreaks and diseases associated with ZIKV and other arboviruses.The online version of this article (10.1186/s12985-018-1070-z) contains supplementary material, which is available to authorized users. Flavivirus of the family Flaviviridae contains several well-known mosquito-borne viruses of medical importance, including Zika virus (ZIKV), Dengue virus (DENV), West Nile virus, Japanese encephalitis virus, and Yellow fever virus  (Table p ≥ 0.07)  cut-off = 36), representing a positivity rate of 11.4% . All samples were positive for the internal control (Ct cutoff = 32). The detection of ZIKV RNA in the screening was 1.59 times higher for women than for men  or between the South and Northeast regions (p = 0.83) , 87.8% and 21.7% of the suspected cases of CHIKV and DENV infections, respectively, were reported in the Northeast region . The co-The preponderance of young adults, especially women, sampled in this study could stem from a greater likelihood for women to seek health care, even with mild symptoms, and especially due to the concerns regarding sexual transmission of ZIKV and the association of ZIKV infection during pregnancy with microcephaly and other neurological disorders , 3, 7. AAlthough some studies highlight the role of urine and whole blood as a source of sample for ZIKV diagnostics since the virus has been detected in higher titers than in serum for prolonged times , we wereIt is also important to note that the overlapping ZIKV infection in clinical presentation with other arboviruses may artificially conflate diagnoses made solely on clinical/epidemiological criteria. In 2016, widespread dengue and chikungunya outbreaks took place in Brazil with more than 1.6 million suspected cases . AlthougGiven the epidemiological context, laboratory differential diagnosis, distinguishing ZIKV from other arboviruses is imperative, given the widespread co-circulation of these viruses in Brazil and the fact that syndromic surveillance is not enough to accurately define the etiology of many acute exanthematous illnesses. ZIKV constitutes a public health burden, and future studies are crucial to the understanding of outbreak dynamics and disease associated with ZIKV infection, as well to the establishment of control and prevention measures, including any future vaccination programs.Additional file 1:Figure S1. Geopolitical map of Brazil. The Rio Grande do Norte and Bahia states, where Zika virus was first reported, is highlighted. Brazilian Federative Units are indicated as follows: DF: Distrito Federal; GO: Goiás; MS: Mato Grosso do Sul; MT: Mato Grosso; AC: Acre; AM: Amazonas; AP: Amapá; PA: Pará; RO: Rondônia; RR: Roraima; TO: Tocantins; AL: Alagoas; BA: Bahia; CE: Ceará; MA: Maranhão; PB: Paraíba; PE: Pernambuco; PI: Piauí; RN: Rio Grande do Norte; SE: Sergipe; PR: Paraná; RS: Rio Grande do Sul; SC: Santa Catarina; ES: Espírito Santo; MG: Minas Gerais; RJ: Rio de Janeiro; SP: São Paulo. Brazilian states in each region are as follows: South: RS, PR, and SC; North: AC, AP, AM, PA, RO, RR, and TO; Southeast: MG, SP, RJ and ES; Midwest: MT, MS, GO and DF; Northeast: AL, BA, CE, MA, PB, PE, PI, RN and SE. (TIFF 261 kb)Additional file 2:Table S1. Reported cases and incidence of dengue, chikungunya, and Zika, in Brazil, in 2016. Incidence rates are indicated per 100,000 inhabitants. Data regarding the total reported cases (absolute numbers) and the incidence rates of Zika, chikungunya and dengue in different regions of Brazil were retrieved from official bulletins and tabulated. The bulletin published by Brazilian Ministry of Health, containing the data above is available at: http://combateaedes.saude.gov.br/images/boletins-epidemiologicos/2016-Dengue_Zika_Chikungunya-SE52.pdf (Accessed 26 Jan 2018). The percentages of reported cases of Zika, chikungunya and dengue were estimated per region , and are shown within brackets. The data presented here refer to the epidemiological weeks 1 to 52 of 2016. An epidemiological week (EW) is a standardized method of counting weeks to allow for the comparison of data year after year. By international convention EWs are counted from Sunday to Saturday. The first EW of the year ends, by definition, on the first Saturday of January. The epidemiological calendar of 2016, used by Brazilian Ministry of Health, is available at http://portalsinan.saude.gov.br/calendario-epidemiologico-2016. According to guides of Brazilian Ministry of Health, in areas with previous history of dengue virus, chikungunya virus or Zika virus transmission, during the epidemics periods, the confirmation of the majority of the cases should be performed by the clinical-epidemiological criteria, after the confirmation of viral circulation and epidemiological investigation of first cases in a given area. In general, blood collection and testing has been recommended for one to every 10 patients with suspected dengue fever, applying the same proportion to Zika and chikungunya (http://bvsms.saude.gov.br/bvs/publicacoes/guia_vigilancia_saude_volume_2.pdf). (DOC 39 kb)Additional file 3:Table S2. Decrease in the incidence of Zika virus infection in Brazil, from 2016 to 2017. Incidence values are indicated per 100,000 inhabitants. *Data from epidemiological week 1 to 52/2016. **Data from epidemiological week 1 to 52/2017. Data regarding Zika virus infection incidence in different regions of Brazil were retrieved from official bulletins and tabulated. The ratio incidence of Zika virus infection cases 2016/ incidence of Zika virus infection 2017 was estimated and presented. The incidence rates presented here refer to epidemiological weeks 1 to 52 of each year: 2016 and 2017. The bulletins, published by Brazilian Ministry of Health, containing the incidence rates are available at http://combateaedes.saude.gov.br/images/boletins-epidemiologicos/2016-Dengue_Zika_Chikungunya-SE52.pdf (Accessed 26 Jan 2018), and at http://portalarquivos2.saude.gov.br/images/pdf/2018/janeiro/23 /Boletim-2018-001-Dengue.pdf (Accessed 26 Jan 2018). An epidemiological week (EW) is a standardized method of counting weeks to allow for the comparison of data year after year. By international convention EWs are counted from Sunday to Saturday. The first EW of the year ends, by definition, on the first Saturday of January. The epidemiological calendar of 2016 and 2017, used by Brazilian Ministry of Health, are available at http://portalsinan.saude.gov.br/calendario-epidemiologico-2016 and at http://portalsinan.saude.gov.br/calendario-epidemiologico?layout=edit&id=161, respectively. (DOC 33 kb)Additional file 4:Table S3. Information of each participant were taken from patient’s file. * Geographic regions and state initials as follows: NORTH REGION: AC: Acre; AP: Amapá; AM: Amazonas; PA: Pará; RR: Roraima; RO: Rondônia; and TO: Tocantins. MIDWEST REGION: DF: Distrito Federal; GO: Goiás; MT: Mato Grosso; and MS: Mato Grosso do Sul. SOUTHEAST REGION: ES: Espírito Santo; MG: Minas Gerais; RJ: Rio de Janeiro; and SP: São Paulo. SOUTH REGION: PR: Paraná; RS: Rio Grande do Sul; and SC: Santa Catarina. NORTHEAST REGION: AL: Alagoas; BA: Bahia; CE: Ceará; MA: Maranhão; PB: Paraíba; PE: Pernambuco; PI: Piauí; RN: Rio Grande do Norte; and SE: Sergipe. (XLSX 154 kb)Additional file 5:Table S4. Analysis of the difference of Zika virus detection in all Brazilian regions during 2015–2016. The difference of Zika virus detection is shown by comparing all regions each other . Southeast, Midwest, and North were more likely for the detection of Zika virus than Northeast and South regions. No differences were observed in Zika virus detection between Southeast, Midwest, and North regions, or between the South and Northeast regions. Statistical analysis was carried out using the Chi-squared test. . (DOC 33 kb)


PHKA2.Glycogen storage disease (GSD) type IX, characterized by liver enlargement and elevated aminotransferase levels, is the most frequent type of GSD. The global incidence of GSD type IXa is only about 1/100,000 individuals. Case reports of GSD type IX are rare in China. We present the first case report of GSD type IXa in Northeast China caused by mutation of An 11-year-old boy was referred to our hospital because of liver enlargement with consistently elevated transaminase levels over 6 months.PHAK2.Histopathological results following an ultrasound-guided liver biopsy confirmed a diagnosis of GSD. Further genetic testing showed that the patient had GSD type IXa caused by the c.133C>T mutation in We placed the patient on a high-protein and high-starch diet and provided hepatoprotective and supportive therapy.The patient's transaminase levels decreased significantly and were nearly normal at 10-month follow-up.This is the first reported case of GSD type IXa in Northeast China. We hope that the detailed and complete report of this case will provide a reference for the diagnosis of liver enlargement of unknown etiology in future clinical practice. Case reports of GSD type IXa are rare in China. Until now, there were no reports of GSD type IXa occurring in Northeast China. We present the first case diagnosed in our hospital of a child with GSD type IXa, which was caused by a mutation in PHKA.Glycogen storage disease (GSD) types I, III, IV, VI, and IX are generally related to liver dysfunction. The most frequent subtype, GSD type IX, is caused by phosphorylase kinase (PhK) deficiency and accounts for 25% of all GSD.2A 11-year-old boy was referred to our hospital for liver enlargement with significantly elevated transaminase levels.2.1The patient went to a local hospital after experiencing nausea for no obvious cause for a period of 6 months. Examinations revealed liver enlargement and elevated transaminase levels. The patient subsequently took Chinese medicinal herbs (uncertain dose and composition) for 2 months, and his symptoms improved. A follow-up examination showed that the liver was still enlarged. The patient's chief complaint at that time was slight upper abdominal discomfort on the right side. The patient was then received by our hospital for further diagnosis and treatment. The patient was healthy before experiencing his current symptoms, and factors that might cause liver injury, including infection, autoimmune disease, alcohol consumption, or ingestion of other chemical drugs and poisons, were excluded.2.2The patient was 130 cm tall, weighed 31 kg, and displayed dysplasia of the teeth and abnormal relaxation of the ligament of the metacarpophalangeal joint. The superficial abdominal veins could be exposed by palpitation, and the liver edge could be felt 8 cm below the costal margin and 7 cm below the xiphoid , resulting in a change from arginine (Arg) to tryptophan (Trp) at amino acid position 45 in the PhK protein. The patient's mother was also found to carry the c.133C>T mutation in PHKA2, indicating that the patient inherited the mutation from his mother . An abdominal noncontrast computed tomography (CT) scan was performed, which showed only a marked enlargement of the liver Figs. . We test2.4We placed the patient on a high-protein, high-starch diet and provided hepatoprotective and supportive therapy. The patient attended regular follow-up visits with careful re-examinations from June 2018 until March 2019.3In order to roughly evaluate the morbidity of GSD type IXa, we did a literature search in May 2019. We systematically identified all potentially relevant case reports in the past 20 years (2000–2019) in Asia from 3 electronic databases: MEDLINE, PubMed, and Web of Science. Search terms such as “glycogen storage disease type IXa,” “glycogen storage disease,” “PHKA2 gene,” “case report,” and “Asia” were used in various combinations and permutations across the databases. Finally we made screening for 36 cases with detailed history information.–18). The onset of GSD type IXa can be in young age groups and the average age is 7.2 years. In the total 36 cases, 23 cases from China, accounting for 63.8% of the total 36 cases, followed by the Korea (22.2%).The proportion of cases from Japan (13.9%) is the lowest. It is noteworthy that the most patients in the table we mentioned were mainly manifested as liver enlargement (91.60%) and elevated transaminase (94.40%) in the early stage. However, less than half of the 36 patients showed signs of hypoglycemia (47.20%), hyperlipidemia (30.50%), and short stature (38.80%).We individually analyzed data of the 36 patients including age, region, the site of mutation, and clinical manifestation  encodes the α subunit, which includes 33 exons. Mutation of PHKA2 results in GSD type IXa, also known as X-linked liver GSD, which accounts for approximately 75% of all cases of GSD type IX. About 134 cases of GSD type IXa have been reported at present, and 99 types of PHKA2 gene mutation have been documented in the Human Gene Mutation Database, including 47 missense mutations, 28 deletion mutations, 9 insertion mutations, 9 nonsense mutations, and 6 splice-site mutations. Most patients with GSD type IXa are characterized by an enlarged liver and increased aminotransferase levels. In addition, some patients exhibit delayed growth. Compared with other types of GSD, GSD type IXa are generally mild, with many patients being asymptomatic or only experiencing a mild increase in blood triglyceride level. Hypoglycemia and hyperlactacidemia are less frequent symptoms of GSD type IXa. Because of its generally mild, atypical symptoms, GSD type IXa difficult to diagnose and is rarely reported in China. The related biomarkers, such as blood glucose and aminotransferase levels, are often changed very little, so they may be ignored or given little concern. Although liver biopsy can provide an exact diagnosis of GSD, it is invasive and cannot provide information about the exact type of GSD, so it has little prognostic value. Furthermore, liver biopsy provides no information about other family members. For those reasons, liver biopsy is often unacceptable to patients and parents of children. On the contrary, genetic testing is the most objective, secure, and reliable method. Genetic testing provides the exact type of GSD and therefore also has prognostic value. There is still progress to be made with genetic testing for GSD, however, because the procedure is complex and relative expensive. It is our hope that with further advancements liver biopsy can be gradually replaced by genetic testing for GSD. GSD type IXa is commonly considered a kind of benign lesion. Long-term follow-up studies of patients with GSD type IXa have shown that the clinical symptoms, including liver enlargement and increased aminotransferase levels, can gradually disappear, and the growth, which is delayed early on, may achieve a standard state in the later stages and even tend to be normal in adulthood. Not all cases have such favorable outcomes, however, and some even progress to liver cirrhosis. Although some specialists view GSD type IXa as self-healing and asymptomatic, the frequent morning nausea and vomiting can still have a negative impact on children's social and academic development and quality of life. Furthermore, the psychological effects caused by growth delay and shortened stature during adolescence should not be ignored. In addition, studies have shown that treatment and management of the glycogen metabolic disorder caused by GSD can remarkably improve the clinical symptoms and biomarkers. Hence, early treatment with a high-starch and high-protein diet and involvement in long-term follow-up with ultrasound examinations to monitor liver function and relief of symptoms can prevent complications and improve patients’ quality of life.GSD is a group of genetic and metabolic disorders caused by various enzyme deficiencies. Deficiency of PhK causes GSD type IX. The first case report in the literature of PhK deficiency was in 1966.The patient in this case report was mainly characterized by liver enlargement and elevated transaminase levels. Because of the mild clinical symptoms, more detailed laboratory tests should be considered for such patients. In this case, laboratory tests revealed low blood glycogen and high triglyceride levels, which caused us to suspect GSD. This is the first reported case of GSD type IXa in the area of Northeast China. We hope that the detailed and complete report of this case will provide a reference for the diagnosis of liver enlargement of unknown cause in future clinical practice. There are some limitations to this single case study. For privacy reasons, only the patient and the patient's mother could be genetically tested. It would be useful to perform genetic screening of the patient's entire family and to establish a long-term follow-up to monitor the patient's progress.The authors thank their patient and his guardian for giving informed consent for publication of the case.Conceptualization: Jun-Qi Niu.Data curation: Qian Zhu.Formal analysis: Xiao-Yu Wen.Funding acquisition: Jun-Qi Niu.Investigation: Xiao-Yu Wen.Methodology: Xiao-Yu Wen.Resources: Ming-Yuan Zhang.Software: Ming-Yuan Zhang.Supervision: Qing-Long Jin.Validation: Qing-Long Jin.Writing – original draft: Qian Zhu.Writing – review and editing: Xiao-Yu Wen.


Nextstrain utilizes a growing database of more than 2,000 West Nile virus genomes and harnesses the power of phylogenetics for students, educators, public health workers, and researchers to visualize key aspects of virus spread and evolution. Using Nextstrain, we use virus genomics to investigate the emergence of West Nile virus in the U S, followed by its rapid spread, evolution in a new environment, establishment of endemic transmission, and subsequent international spread. For each figure, we include a link to Nextstrain to allow the readers to directly interact with and explore the underlying data in new ways. We also provide a brief online narrative that parallels this review to further explain the data and highlight key epidemiological and evolutionary features (nextstrain.org/narratives/twenty-years-of-WNV). Mirroring the dynamic nature of outbreaks, the Nextstrain links provided within this paper are constantly updated as new West Nile virus genomes are shared publicly, helping to stay current with the research. Overall, our review showcases how genomics can track West Nile virus spread and evolution, as well as potentially uncover novel targeted control measures to help alleviate its public health burden.It has been 20 years since West Nile virus first emerged in the Americas, and since then, little progress has been made to control outbreaks caused by this virus. After its first detection in New York in 1999, West Nile virus quickly spread across the continent, causing an epidemic of human disease and massive bird die-offs. Now the virus has become endemic to the United States, where an estimated 7 million human infections have occurred, making it the leading mosquito-borne virus infection and the most common cause of viral encephalitis in the country. To bring new attention to one of the most important mosquito-borne viruses in the Americas, we provide an interactive review using Nextstrain: a visualization tool for real-time tracking of pathogen evolution ( Flavivirus) is globally distributed and maintained by a complex transmission cycle involving multiple species of mosquitoes and birds )o border ,115. Moro border  and Puero border ) reveal  America –118. HowCulex mosquitoes [Culex vectors and avian hosts is endemic throughout the Americas [An important question is why large human WNV outbreaks have not been detected in Latin American countries ,122. Thesquitoes ,122. MorAmericas . ConsideAmericas  and seroAmericas ,104–106,Americas , the preFig 5), to better understand endemic WNV transmission using virus genomics approaches [westnile4k.org/) provides a template for future public health and academic lab partnerships. Platforms such as Nextstrain (nextstrain.org/WNV/NA) and Virological  allow these new data to be continually communicated to the public.Given how much WNV has thrived in the US, significant national investment will be required to control future outbreaks. Among this investment should include sustained and collaborative efforts to fill in the many gaps in WNV genomic sampling throughout the country, especially from the last 10 years  makproaches , Hepp anproaches , Bialosuproaches , and theGenerating a well-distributed temporal and spatial WNV genomic dataset can be used to determine which strains are causing outbreaks and, importantly, where those strains came from and when they became locally established. This work will help to identify if WNV outbreaks in Ames, Iowa, for example, are connected to outbreaks as far away as Des Moines, Iowa (approximately 60 km); Omaha, Nebraska (approximately 270 km); or even Chicago, Illinois (approximately 560 km). Systematically searching for connections among WNV outbreaks around the country may reveal endemic “transmission networks,” which could be exploited for control and forecasting purposes. WNV control is primarily based on local interventions to temporarily reduce the populations of mosquito vectors , but theS1 FigA) phylogeny , (B) spread , and (C) genetic diversity (amino acid diversity plotted as entropy and positioned by codon sequence). In the WNV genome, the protein abbreviations are as follows: C = capsid, prM = premembrane, E = envelope, NS1-5 = nonstructural protein 1–5. A live display, which can be used to select and zoom in on various features, can be found at nextstrain.org/WNV/NA?p=grid. WNV, West Nile virus.A depiction of the main interactive interface on Nextstrain to visualize the virus ((PDF)Click here for additional data file.S2 Fignextstrain.org/WNV/NA?l=unrooted&m=div&p=full. WNV, West Nile virus.An unrooted maximum likelihood WNV phylogenetic tree shows that there is little geographic structure  during the early spread phase, as best demonstrated by the Texas (gold) and New York (purple) genomes often clustering together. This suggests that environment was highly conducive for transmission and that the virus could freely spread. The minimal mixing between California and New York genomes, however, suggests that geographic structure is starting to form as the virus transitioned into its endemic phase. Additional WNV sequencing throughout the US from the last 10 years will help to better understand if structure is starting to form and at what scale. States are listed by two-letter abbreviations and countries are listed by three-letter abbreviations. This “unrooted” tree view can be visualized on Nextstrain by toggling between the “Tree Options: Layout”. A live display can be found at (PDF)Click here for additional data file.S3 Fig−4 substitutions/site/year. The tips are colored by increasing time. This “clock” view can be visualized on Nextstrain by toggling between the “Tree Options: Layout.” A live display can be found at nextstrain.org/WNV/NA?c=num_date&d=tree&l=clock. WNV, West Nile virus.A root-to-tip plot showing the divergence (substitutions per site) of sequenced WNV genomes (tips) from the inferred ancestral sequence (root) by the collection dates (shown in years) is used to estimate the evolutionary rate at approximately 4×10(PDF)Click here for additional data file.S4 FigA) nonstructural protein 4A (NS4A) A85T and (B) NS5-K314R. Branches  and tips (sequenced genome) are colored by amino acid at position (A) NS4A site 85 (nucleotide position 6721) and (B) NS5 site 314 (nucleotide position 8621). Both (A) NS4A-A85T and (B) NS5-K314R help to form a well-supported clade (yellow branches at the top) but also occur independently throughout the tree . All other alleles (nucleotide and amino acid changes) can be visualized using Nextstrain by entering the loci using the “Color By: genotype” function or by selecting a loci on the “Diversity” plot . Live displays can be found at (A) nextstrain.org/WNV/NA?c=gt-NS4A_85 and (B) nextstrain.org/WNV/NA?c=gt-NS5_314. WNV, West Nile virus.WNV genotype SW03 is defined by two amino acid substitutions, ((PDF)Click here for additional data file.S5 FigA) in Maricopa County, Arizona [B), throughout California [C) throughout New York [A) https://nextstrain.org/WNV/NA?c=lineage&f_authors=Hepp%20et%20al, (B) https://nextstrain.org/WNV/NA?c=lineage&f_authors=Duggal%20et%20al, and (C) https://nextstrain.org/WNV/NA?c=lineage&f_authors=Shabman%20et%20al. WNV, West Nile virus.Since the emergence of WN02 (blue) and SW03 (green) in 2001, the genotypes continue to cocirculate in locations throughout the US For example, both genotypes were detected during the same years and vector species ( Arizona  (B), thrlifornia , and (C)New York . Data fr(PDF)Click here for additional data file.S6 Fignextstrain.org/WNV/NA?f_state=NY&d=tree. WNV, West Nile virus.WNV is likely establishing persistent local transmission networks throughout the US, but this can be easily demonstrated from the 570 genomes available from New York (most generated by ). Multip(PDF)Click here for additional data file.


This manuscript aims to provide an overview of the unique considerations and best practice principles associated with the manufacture of human viral challenge agents.Considerations are discussed on the entire process from strain and viral source selection through manufacturing, safety and efficacy testing. The human viral challenge (HVC) model is an important tool to help accelerate the drug development process but producing viruses suitable for use in the model presents a unique set of challenges. There are many case by case decisions and risk assessments to consider and no clear international standard to produce viruses for this purpose. The authors present challenge virus manufacturing considerations from the current literature, regulatory guidance and their own direct experience in producing challenge viruses. The use of these viral stocks in clinical studies, as published in peer-reviewed journals, is also briefly described.The online version of this article (10.1186/s13104-018-3636-7) contains supplementary material, which is available to authorized users. Edward Jenner performed possibly the first documented Human Viral Challenge (HVC) study in 1796  under vaConsidering the lack of specific guidance on production of viral CAs, this publication aims to provide some guidance to others considering producing CAs by addressing the key points we considered in the production of our respiratory virus CAs. We incorporate reviews of the latest regulatory and GMP guidelines, the scientific literature and our own discussions with the FDA and MHRA. The authors have conducted multiple HVC studies with over 2500 volunteers using numerous different challenge viruses including influenza, respiratory syncytial virus (RSV) and human rhinovirus (HRV).Safety of challenge viruses is of paramount importance and when selecting a suitable virus, hence a thorough literature review of the naturally occurring pathogenicity of the virus and potential risks should be undertaken. As an example; (H1N1) A/California/7/2009-like (pdm09-like) influenza virus isolates may have two key mutations in the Haemagglutinin receptor binding site, D222G and D222N. Both lead to greater tropism for the lower respiratory tract and are associated with severe clinical outcome . Viral gDifferent viral strains can be used and, based on the level of pre-existing immunity in subjects, be expected to result in differing pathogenic profiles. For viruses where the key correlates of protection are known, tests can be conducted to assess pre-existing immunity ensuring the virus produces a suitable pathogenic profile and importantly, evaluable endpoints. Sero-suitability of subjects may include testing of serum IgG, mucosal secretory IgA and T cell immunity –9 dependA challenge virus must be clinically relevant and present a similar pathogenic profile to that observed in a natural infection or, where appropriate, attenuated pathogenicity or disease exacerbation. For instance, rhinovirus types, A and C have been associated with asthma exacerbations, while Rhinovirus type B frequently induce a subtly different cytokine pathway leading to moderate asthma attack . Wild-tyManufacturing substrates can introduce mutations and the number of passages of the virus during manufacture should always be minimised. However, sequencing of isolates obtained during active infection in the model has shown that such mutations are rapidly selected out and thus may be of limited consequence .Viral seed material can be obtained from several sources, including an existing good manufacturing practice (GMP)-produced virus inoculum, clinical isolates or generated via reverse genetics. Careful consideration should be taken as to the source of the material regarding documentation, e.g., isolate history.Isolates can be obtained either from subjects who acquire an infection naturally (field isolates) or from subjects inoculated experimentally  where the donor shows a suitable pathogenic profile. All clinical isolates must be acquired from a reputable source. The donor should be in good health with a full medical history, free from cancer or chronic illness, HIV, Hepatitis B, Hepatitis C and HTLV; nor have received blood products to mitigate the risk of any unknown pathogens and transmissible spongiform encephalopathies (TSEs).Field isolates would likely be sourced if an appropriate seed could not be obtained from a reputable provider of virus such as the World Health Organisation (WHO), Center for Disease Control (CDC) or European Centre for Disease Control (ECDC). An isolate from an experimentally infected volunteer has advantages, the health status and medical history are known in a great level of detail as well as when in the course of infection the isolate was obtained.The use of reverse genetics to manufacture a virus removes the need for adventitious agent testing of the donor. The appropriate pathogenicity factors can be included or eliminated from these Genetically Modified Microorganisms (GMMs). When reverse genetics is used to produce a wild-type-like virus the pathogenicity profile should be like that of the naturally occurring strain. The GMM can be deliberately attenuated; NIBRG-14 is an attenuated version of a highly pathogenic influenza (bird flu) strain with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04(H5N1) virus. The polybasic cleavage site of the HA, known to be related to high pathogenicity, is removed . Such GMViral seed material may require pre-production processing; such upstream processing would typically involve plaque purification, rate limiting dilution, filtration and possibly selective microbial treatment.Whenever possible, a pilot stock would be produced and screened for a range of adventitious agents as determined by the risk assessment, and as a minimum sequenced to confirm identity.The manufacturing substrate will vary depending on the virus and range from embryonated eggs, cell culture and other in vivo methodologies. Each method poses challenges and risks to be assessed. Some challenge agents, including viruses and bacteria, can be propagated in Specific Pathogen Free (SPF) embryonated eggs with the advantages of low cost, ease of production and pathogen filter effect .Most challenge viruses are propagated in cell culture. GMP cell lines must be fully qualified, their history and providence known; examples include Vero cells, MRC-5 cells and PerC6 cells –19. OtheManufacturing should be conducted in a GMP setting designed to minimise the risks involved in any production that cannot be brought to light through testing of the final product. As cited in the EU regulations , we consA GMP manufacturing facility should be audited both from a scientific and Quality Assurance perspective. The scientific audit determines knowledge of viral handling best practices and the Quality Assurance audit ensures appropriate regulatory requirements are met.Any materials to be released formally from the facility must be accompanied by a copy of the complete batch record, a certificate of GMP compliance and a certificate of analysis.There are different approaches to the downstream processing of a post-GMP production harvest, but all have the same final requirements. The virus must be as free as possible from all components used during the production that may affect its final aim, i.e. a suitable pathogenicity profile while ensuring that the safety of the volunteers inoculated are not put at risk. Any product produced in a mammalian cell line should have minimal residual host cell DNA content , 21.Adventitious agent testing forms a critical and substantial part of the safety data required for a challenge virus stock. The stock must be tested for any adventitious agents that could reasonably be expected to contaminate it and potentially cause a safety risk to study subjects. We do not propose a definitive specific list of adventitious agents that one should test for as the appropriate testing strategy used will depend on a range of factors as well as the current opinion of the regulator. We simply describe the best practice principles that we applied in the manufacturing of our challenge viruses and give some examples in the hope of giving some guidance to others (see Additional file A diluent is required to dilute the challenge stock to an appropriate inoculum titre, ensuring stability in storage before use (see Additional file The harvest should be diluted and filled into single-use vials under GMP conditions. We propose a minimum testing panel for the release of the product from the manufacturing facility before the full adventitious testing panel required before human use (see Additional file Risk assessment and literature reviews to consider potential adventitious agents that could contaminate the product during each stage of the process from seed virus collection, pre-production processing, production, through to final fill of the GMP manufactured product should be conducted. For our challenge viruses, we considered the potential risks from all of the raw materials and cell banks/eggs used and possible newly emerging agents at the time of manufacture. Testing included; sterility, microbial enumeration testing, Mycoplasma & Mycobacterium detection tests along with testing for non-specific viral contaminants by in vitro inoculation of indicator cells. Also, we conducted tests for the quantification of reverse transcriptase activity as well as extensive agent-specific molecular testing for a panel of contaminants including known human viruses of both chronic and acute infections as well as the list of other potential pathogen contaminants that were highlighted by the risk assessment.Consideration should be given to the use of an animal model to check pathogenicity. However, for ethical considerations, it should only be used when it is sure to have good correlation with human disease and provide clear guidance for subsequent human studies.We have characterised numerous new challenge viruses following manufacture via the HVC; these included respiratory syncytial virus , human rThe production and testing of an influenza GMP challenge virus are shown schematically in Fig. There are numerous decisions to be made in the complex and specialized process of manufacturing a new challenge virus with each virus production creating a unique set of circumstances to consider. This is made harder by the lack of specific regulatory and technical guidance. Therefore, we used our own direct experience in producing viral challenge agents to present an overview of the considerations and the best practice principles involved in virus manufacture so that this may serve as an initial guide to others embarking on challenge agent production. We propose that applying the methodical risk-based approach discussed above will enable new viral challenge agents to be produced, like our own, in a scientifically robust and safe manner that gives reliable results and generates clear evaluable endpoints in the HVC model. This will in turn further expand the utility of the HVC model in evaluating the efficacy of antiviral compounds and vaccines or human host interactions.The authors acknowledge that, due to the lack of documented regulatory guidance specific to challenge virus production, the considerations and manufacturing options discussed are mainly based on the direct experience of the authors.Additional file 1: Table S1. Minimum GMP facility Release Testing Panel, testing for egg/cell manufacturing substrates and diluent. Table S2. Examples of Adventitious Agent Testing panels for different scenarios. A. testing panel for 25% sucrose in PBS challenge virus diluent. B. testing panel for Allantoic fluid of control eggs. Figure S1. Stability of hVIVO’s A/Perth/16/2009 [H3N2] challenge virus. Each point on the graph represents a stability testing timepoint with the mean titre illustrated by the line, demonstrating ongoing infectious virus stability from the point of manufacture to latest available stability testing time point at the time of manuscript submission.


Influenza A(H1N1)pdm09 (pH1N1) virus has become established in swine in the United Kingdom and currently co-circulates with previously enzootic swine influenza A virus (IAV) strains, including avian-like H1N1 and human-like H1N2 viruses. During 2010, a swine influenza A reassortant virus, H1N2r, which caused mild clinical disease in pigs in the United Kingdom, was isolated. This reassortant virus has a novel gene constellation, incorporating the internal gene cassette of pH1N1-origin viruses and hemagglutinin and neuraminidase genes of swine IAV H1N2 origin. We investigated the pathogenesis and infection dynamics of the H1N2r isolate in pigs  and in ferrets, which represent a human model of infection. Clinical and virologic parameters were mild in both species and both intraspecies and interspecies transmission was observed when initiated from either infected pigs or infected ferrets. This novel reassortant virus has zoonotic and reverse zoonotic potential, but no apparent increased virulence or transmissibility, in comparison to pH1N1 viruses. The ability of swine to support replication of phylogenetically diverse influenza A virus (IAV) strains from avian and mammalian origin poses a public health risk because of the potential for viral antigenic change resulting in variants with zoonotic properties (avN1) lineage 1C , also designated lineage IB  lineage 1A   and assayed by using a real-time quantitative reverse transcription PCR (qRT-PCR) (6 50% egg infectious dose (EID50)/mL. REU measures the amount of virus RNA present and not infectivity. However, it can be inferred from the linear relationship with the dilution series that REU values are proportional to the amount of infectious virus present.Virus RNA was extracted by using a QIAmp Viral RNA Biorobot Kit  for statistical analyses. We determined the area under the curve of the shedding profile for each animal and compared between groups by using the Tuckey multiple comparisons test and 1-way analysis of variance. Positive-sense RNA encoding the virus M gene was quantified by using a positive strand-specific real-time qRT-PCR  Act of 1986 and Animal Research: Reporting of In Vivo Experiments guidelines.avN1, H3N2, and H1N2 viruses) appropriate for the pig population in the United Kingdom (http://destronfearing.com) for daily temperature monitoring, and a clinical scoring system was used for daily clinical monitoring  were bred in the United Kingdom. Before infection, all animals were confirmed free of influenza A virus RNA by performing real-time qRT-PCR analysis of nasal swab specimens (pigs) or nasal washes (ferrets)  to deliver droplets with a diameter of 30–100 μm. A total dose of 106 EID50 units diluted in phosphate-buffered saline to a final volume of 4 mL (2 mL/nostril) was administered. We anesthetized ferrets and infected them by intranasal droplet instillation of a total dose of 106 EID50 in 0.4 mL inoculum (0.2 mL/nostril).We intranasally infected animals by using protocols appropriate for the species and nasal structure. We infected pigs by using a MAD Nasal Intranasal Mucosal Atomization Device  and 5 dpi, as well as for 2 animals at 7 dpi. Gross pathology was assessed and tissue samples were collected in 10% [vol/vol] phosphate-buffered formalin for histopathologic analysis. Lesions were scored as described for ferrets  and study II (interspecies transmission). In study I, immunologically naive animals (n = 6) of the same species were placed in direct contact at 1 dpi. In study II, animals of 1 species (n = 6) were infected and immediately thereafter cohoused animals of the other species (n = 6) were reintroduced. Pigs were housed on the floor and immediately adjacent to ferret cages (minimum distance 30 cm), and the floor of the ferret cage was at the height of the heads of the pigs. Virus shedding was monitored daily from 1 dpi through 12 dpi in nasal swab specimens from pigs (1/nostril) or nasal wash samples from ferrets . Serum samples were obtained before infection and at the end of the study at 21 dpi. An overdose of barbiturates was used for euthanasia of all animals.l-glutamine supplemented with 100 U/mL penicillin, 1,000 μg/mL streptomycin, and 1% fetal bovine serum . Ferret nasal wash samples were obtained by using 1 mL phosphate-buffered saline (GIBCO). Tissues were analyzed by preparing a 10%–20% [wt/vol] homogenate in 1 mL supplemented Leibovitz-15 medium by using an Omni GLH Homogenizer .Daily nasal swab specimens, which were pooled for each pig, were eluted in 1 mL Leibovitz L-15 medium with Serum samples were analyzed by using an influenza A virus NP competitive ELISA (IDVet) according to the manufacturer’s specifications and dilutions of 1:40 for pig serum samples and 1:10 for ferret serum samples. Antibody titers to the H1N2r virus were measured by HI as described for pig  and salivary glands (7 dpi), as well as in the left cranial lung lobe in 2 directly infected ferrets at 3 dpi and 5 dpi.Histopathologic changes were consistent with mild inflammatory disease. For pigs , panel AInfluenza A virus NP labeling showed replicating virus in pig and ferret tissues, notably in pig respiratory turbinates, at 5 dpi , panel CIn study I , panel AWe monitored virus RNA shedding daily in nasal swab specimens (pigs) or washes (ferrets) and quantified results by using real-time qRT-PCR. Nasal shedding started in directly infected animals at <1 dpi and in uninfected contacts at 1–2 days after contact , panel B,,,In study II , panel ASerologic analysis by competitive ELISA for NP, which is present within virions, demonstrated that, in interspecies transmission study II, infected and contact ferrets seroconverted by 21 dpi/days after contact , panel A,Our study showed that pigs and ferrets were susceptible to infection with the H1N2r virus and showed clinical signs and virologic parameters indicating mild disease. Longitudinal postmortem analysis indicated that this virus could be more adapted to a swine host because infection disseminated more rapidly throughout the respiratory tract, and distribution of infection in the lung on all postmortem days was more evident in pigs than in ferrets. However, in contrast to pigs, ferrets shed a larger total quantity of virus for an extended duration, and seroconversion occurred in all infected and contact ferrets by 21 dpi. However, interpretation of data is limited by the different intranasal infection protocols and nasal sampling techniques for the 2 species. All animals had increased specific antibody titers postinfection. However, in comparison with ferrets, titers in pigs were lower and, in some contact pigs, were below the positive threshold. These findings are comparable with outcomes reported for pH1N1 virus infection of pigs and ferrets pdm09 and swine influenza A(H1N2) viruses.


Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation . In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in  Flaviviral vertebrate amplifying host(s), invertebrate vector(s), genetics, and environmental factors shape the viral geographical distribution and epidemic disease pattern. Newly emerging dengue virus genotypes, West Nile virus clades, or Zika virus strains exhibited an enhancement in mosquito vector competence. However, hosts and viral determinants responsible for the occurrence of JEV genotype replacement remains unclear. Here, we demonstrated that emerging GI viruses with enhanced transmission potential in amplifying hosts such as pigs and avian species was encoded by three critical GI-specific mutations in NS2B/NS3 proteins. This discovery provides insight into the viral genetic mechanism underlying the GI virus advantage and adaptation in the pig/avian species-mosquito cycle. Our results also emphasize the importance of monitoring viral evolution in amplifying vertebrate hosts to clarify the role of avian species in local transmission of GI virus in JE endemic and epidemic countries. Culex tritaeniorhynchus-amplifying host (pigs and avian species) transmission cycle, and humans and horses are accidental, dead-end hosts [Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a single-stranded, positive-sense RNA genome encoding three structural proteins  and seven non-structural proteins  [nd hosts . An estind hosts .Culex tritaeniorhynchus, stillborn piglets, and JE patients in Japan, Korea, Vietnam, Thailand, Taiwan, China, and India [Aedes albopictus mosquito-derived cells, birds, and ducklings than GIII virus [Culex tritaeniorhynchus mosquitoes, which play critical roles in local transmission of JEV. In addition, the experimental evidence associated with the difference in viral genomic factor(s) does not fully support the occurrence of GIII replacement during the past 20 years [GI virus emerged in the 1990s and has gradually replaced GIII virus as the most frequently isolated JEV genotype from nd India –15. We snd India , 16. EmeII virus –19; howe20 years , 18, 20.in vitro and in vivo replication characteristics of emerging GI virus and previously dominate GIII virus in Culex tritaeniorhynchus mosquito vector and amplifying hosts . We did not include dead-end hosts in this study because of the remote possibility of the involvement of dead-end hosts in the transmission of JEV. More importantly, we constructed, generated and applied various genotypic chimeras of recombinant JEVs from infectious cDNA clones to determine the viral genetic determinants that enhanced and increased the replication efficiency of GI virus over GIII virus. These genetic determinants would be critically important for selection of viral hosts and genes to monitor GI virus activity and evolution in a natural transmission cycle.In this study, we investigated the Aedes albopictus-derived C6/36 cells and Culex tritaeniorhynchus-derived CTR209 cells with GIII viruses (CH1392 and T1P1 strains) and GI viruses (YL2009-4 and TC2009-1 strains) at 28°C  of GIII or GI JEVs. We determined the infection rate by detection of viral titer in each mosquito 14 days post-infection (DPI). GIII CH1392 virus and GI TC2009-1 virus had higher infectious rates (58.33% and 66.67%) than GIII T1P1 virus and GI YL2009-4 virus (25.00% and 16.67%) whereas no genotypic differences were observed in infectivity or in viral titer among positive mosquitoes  and primate-derived control cells (VERO cells) at 37°C  . The virin vivo. JEV–infected pigs exhibited either an asymptomatic manifestation or developed fever with sufficiently high viremia to ensure the transmission of virus by engorged mosquitoes [5 ffu of GIII CH1392 virus, GI YL2009-4 virus, and or PBS into ten-week old, specific-pathogen free (SPF) pigs (three SPF pigs per group), and then monitored daily body temperature and viremia. At 3 DPI, three GI virus-infected pigs developed fever averaging 40.6°C while GIII virus-infected and PBS-inoculated pigs maintained normal body temperature until euthanasia at 8 DPI . However, viremia was undetectable at 4-DPI in all virus- and PBS-inoculated pigs , PK-15,  and DF-1 cells   . InteresConversely, we investigated the influence of the replication ability of GIII virus- derived NS1-5 and NS1-3 proteins on the rGI backbone for amplification in host-derived cells. The chimeric clones pCMV GI/GIII NS1-5 and pCMV GI/GIII NS1-3 were constructed to produce rGI/GIII NS1-5 and rGI/GIII NS1-3 viruses as described above . The recin vivo, we compared the replication ability of rGIII/GI NS1-3 virus to rGIII virus by subcutaneously inoculating 107 ffu and 104 ffu of the viruses into ten-week old SPF pigs and 1-day old chickens, respectively  of GI NS1-3 proteins involved in the enhancement of GI replication, we conducted a loss-of-function experiment by introduction of a single GIII virus-specific and highly consensus substitution for NS1-3 proteins of the rGIII/GI NS1-3 chimeric virus instead of the rGI virus . The infin vivo replication ability of rGIII/GI NS1-3 virus in 1-day old chickens. The rGIII and parental rGIII/GI NS1-3 viruses were inoculated as infection controls  in National Chung Hsing University (NCHU) (protocol number: 102–107) and National Pingtung University of Science and Technology (NPUST) (protocol number: 104–013). All experimental protocols followed the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.Gallus gallus domesticus) and 80 2 day-old minimum disease-free ducklings (Cairina moschata) were purchased from JD-SPF Biotech Co., Ltd and Livestock Research Institute (Council of Agriculture in Taiwan), respectively. These animals were kept in isolators at the avian holding facility in NCHU. Thirteen second-generation SPF pigs (Lee-Sung Strain) were purchased from the Agricultural Technology Research Institute in Taiwan, and housed in the negative air-pressure animal facility certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) in the Animal Disease Diagnostic Center of NPUST, Taiwan. Avian and pig euthanasia performed by the use of CO2 inhalation and electrical stunning were approved by IACUCs in NCHU and NPUST, respectively.A total of 196 1 day-old specific pathogen free (SPF) chickens  derived from embryos of Culex tritaeniorhynchus [2 at 28°C or 37°C. GIII subcluster II JE CH1392 and T1P1 viruses , and GI-b subcluster II JE YL2009-4 and subcluster I TC2009-1 viruses  were used in this study. JEV strains CH1392, YL2009-4, and TC2009-1 were isolated from pools of field-captured Culex tritaeniorhynchus in 1990 and 2009, and T1P1 was isolated from the pool of Armigeres subalbatus in 1997 [C6/36 cells  derived from the midgut of rhynchus  were grorhynchus  suppleme in 1997 , 56. AllJEVs or rJEVs were inoculated onto C6/36, CTR209, PK-15, DF-1, CER, DE, and VERO cells at an MOI of 0.5 at 28°C for mosquito cells and at 37°C for mammalian cells. Infected cells were washed three times with 1X PBS after a one-hour incubation, and then subsequently incubated at 28°C or 34°C for mosquito cells and at 37°C or 41°C for mammalian cells. The supernatants from infected cells were collected and stored at −80°C until used. All experiments were conducted in triplicate, and the viral titers in the supernatants were determined by the micro-antigen focus assay.4 cells/100 μL/well and incubated at 37°C, 5% CO2 overnight to allow a cell monolayer to form. The serially diluted samples were added into wells for one hour at 37°C. Infected VERO cells were overlayed with 1% methylcellulose mixed with DMEM medium supplemented with 2% FBS, and incubated at 37°C for 32 to 36 hours. The methylcellulose overlays were discarded by washing with 1X PBS, and the infected VERO monolayers were fixed with 75% acetone in PBS for 20 minutes. The fixed cells were air-dried and stained with mouse anti-JEV polyclonal antibody , followed by staining with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody . The foci were developed after the addition of Vector-VIP  into each well. The viral titer was calculated by the average number of foci-forming unit (ffu) per ml or per mosquito.The micro-antigen focus assay was used to determine viral titers in the supernatants of infected cells, in the plasma recovered from infected animals, or in the supernatants of homogenized mosquitoes. Briefly, VERO cells were seeded in to 96-well plates at a cell count of 2.25×10Culex tritaeniorhynchus mosquitoes were fed with 10% sucrose and maintained at 28°C. Mosquitoes were starved for 1 day prior to the blood-feeding experiment. The mosquitoes were fed per os with a JEV viremic blood meal, a mixture of pig blood cells and 8×106 ffu/ml of JEV according to the previous study [Laboratory-hatched female us study  and the 5 ffu of GIII CH1392 virus, GI YL2009-4 virus, or 107 ffu of rJEVs. Experimental pigs were monitored, the daily body temperatures were recorded, and clinical signs of infection were noted. Pig plasmas were recovered at different days post infection. All pigs were euthanized with electrical stunning at 8 DPI.Thirteen ten-week old, JEV-seronegative SPF pigs were used to determine the replication ability of field-isolated JEVs (nine pigs) and rJEV (four pigs). SPF pigs were anesthetized with stresnil  and subcutaneously inoculated with PBS or 104 ffu of GIII CH1392 virus, GIII T1P1 virus, GI YL2009-4 virus, or GI TC2009-1 virus. The daily activity and clinical signs of infection for chickens and ducks were monitored after JEV infection. We collected blood from four infected chickens or ducklings 1 day prior to infection and at 1, 2, 4, and 6 DPI. Plasma was mixed with anticoagulant at a final concentration of 0.33% sodium citrate (Sigma-Aldrich) in 0.85% sodium chloride (Sigma-Aldrich), and centrifuged at 3,000 rpm for 15 minutes. The ten-fold diluted plasma was recovered from the supernatant and stored at −80°C until used.Eighty 1-day old chickens  and eighty 2-day old ducklings  were subcutaneously inoculated with one dose of PBS or 104 ffu of rJEVs. The plasma was recovered from infected chickens at 48 or 60 HPI as described above, and stored at −80°C until used.One hundred sixteen 1 day-old chickens  in this study. JEV viral RNA was transcribed by CMV promoter and terminated by SV40 poly-A terminator. The precise JEV 3’ terminal sequence was generated by a ribozyme sequence of hepatitis delta virus (HDVr) incorporated right after the 3’UTR of JEV (5). To construct GIII and GI JEV chimeric infectious clones, we replaced five genetic fragments or the complete viral genome of pCMV GIII with the corresponding genes of GI JE YL2009-4 virus by blunt-end ligation with T4 DNA ligase (New England Biolabs) to generate six recombinant viruses . We replaced the pCMV GI infectious cDNA clone with the corresponding gene fragment of GIII virus using the protocol of Gibson assembly reaction (New England Biolabs) and generated two additional recombinant viruses, rGI/GIII NS1-5 and rGI/GIII NS1-3 . All fra4, 0.2 mM dNTPs, 0.4 mM mutagenesis primers  was added into a mixture of Lipofectamine 2000 (Life Technologies) and Opti-MEM, and then the final mixture was incubated at room temperature for 30 minutes. Next the mixture was added onto the 80% confluent cells for five hours at 37°C, and then replaced with the culture medium. After a 3- to 4-day incubation, the production of rJEVs was detected in the transfected cells by an immunofluorescence assay utilizing mouse anti-JEV NS1 antibody . The recombinant viruses secreted from transfected cells were harvested and subsequently amplified in C6/36 cells. Virus plaques were identified by plaque assay. The viral RNA was extracted from virus-infected C6/36 cells with RNeasy mini kit (Qiagen), and transcribed into cDNA with the JEV 3’UTR primer 5’-AGATCCTGTGTTCTTCCTCA-3’ using the Superscript III transcription reaction (Thermo Fisher Scientific). The complete genome of recombinant virus was confirmed by sequencing the PCR products amplified from the viral cDNA template.t-test was used to compare two data groups. The multiple-group comparison was calculated by One-way ANOVA, and the post test analysis performed by using Turkey’s Multiple or Dunnett’s Multiple Comparison Test. P<0.05, the significant difference in the two-group and multiple-group comparison.The statistical analysis was performed by GraphPad Prism v5.01. Student’s two-tailed S1 Fig(A-L) The cell lysates of JEV-infected C6/36 cells at 28°C (A and D), CTR cells at 28°C (B and E) or at 34°C (C and F), VERO (G and J), PK-15 (H and K), and DF-1 (I and L) cells at 41°C were collected at 48 HPI. The viral NS3 protein and cellular α-tubulin or β-actin were detected by Western blot using mouse anti-NS3 protein, anti-α-tubulin or anti-β-actin antibodies. (D-F and J-L) The ratio of band intensity (NS3/α-tubulin) or (NS3/β-actin) was estimated by ImageJ version 1.44 with Mean and SEM calculated from triplicates. The difference in relative intensity was calculated using one-way ANOVA followed by Turkey’s Multiple Comparison Test. A significant difference is indicated by an asterisk (P< 0.05).(TIF)Click here for additional data file.S2 FigGIII CH1392 virus ((TIF)Click here for additional data file.S3 FigGIII CH1392 virus ((TIF)Click here for additional data file.S4 FigGIII CH1392 virus ((TIF)Click here for additional data file.S5 Figt-test or one-way ANOVA followed by Turkey’s Multiple Comparison Test. The statistical difference is noted by an asterisk (P< 0.05).(A-C) The viral RNA in the plasma of JEV-infected pigs (A), chickens (B), and ducklings (C) was measured by real-time RT-PCR at 2 DPI. The detection limit is indicated as a dotted line. A dot plus a horizontal line represents an individual animal and a mean RNA-copy number of the group, respectively. The statistics comparing either two or four viruses were determined by a Student’s two-tailed (TIF)Click here for additional data file.S6 FigThe viral RNA sequence, beginning with 151 nucleotides from the 5’terminal followed by poly(A) linker and 118 nucleotides from 3’terminal, is used to predict the cyclization structure at 37°C and 41°C with mfold web server. The ΔG value of the cyclization structure is indicated.(TIF)Click here for additional data file.S7 Fig(A and B) The viral RNA in plasma collected from the infected pigs (A) at days 0 to 8, and from the infected chickens (B) at 60 HPI, respectively, were measured by real-time RT-PCR. Each dot represents an individual animal. A horizontal dotted line indicates the detection limit. Mean with SEM is showed. The RNAemia in chicken plasma was statistically analyzed with one-way ANOVA, followed by Turkey’s Multiple Comparison Test. A significant difference is indicated by an asterisk .(TIF)Click here for additional data file.S8 Fig(A and B) The viral RNA in collected plasma was detected at 60 HPI (A) or 48 HPI (B) and measured by real-time RT-PCR. A horizontal dot line represents an individual animal and mean of a group respectively. Error bars indicate SEM. A horizontal dot line indicates the detection limit. The statistical analysis was determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test utilizing rGIII/ GI NS1-3 virus (A) or rGIII virus (B) as a control. A significant difference is showed as a red dot (P< 0.05).(TIF)Click here for additional data file.S9 FigrJEVs were incubated with VERO, PK-15, and DF-1 cells at an MOI of 5 at 37°C for 1 hour. Then, the infected cells were serially diluted and used to infect VERO cells using a micro-antigen focus assay. The number of infected cells releasing infectious particles was estimated and shown as ffu. The infectivity rate was calculated as the number of ffu divided by number of infected cells.(TIF)Click here for additional data file.S10 Fig(A) The focus was viewed by light microscopy. (B) The size of focus was estimated by ImageJ version 1.44. Mean with SEM for four foci is showed. The difference in size was calculated using one-way ANOVA followed by Turkey’s Multiple Comparison Test.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.