Viral suspensions were exposed to temperatures ranging from 70 to 130°C for 0 to 90 min depending on the virus, and then the recovered viral preparations were tittered. Inactivation rates were calculated from curves that were analysed according to the first order kinetics model. Full inactivation was obtained for MVM in 90 min at 80°C and in 2 s at 130°C, for H1N1 in 14 s at 70°C and in 1 s at 110°C, for CVB4 and HSV-1 in 5 s and 7 s respectively at 70°C and in 1 s at 100°C. Clearly, MVM was more resistant than H1N1 that was more resistant than HSV-1 and CVB4, which was reflected by increasing inactivation rates. The impact of short time exposure to heat onto the infectivity of viruses contained in a small volume of suspension has been determined. For the first time, the inactivation of viral particles contained in drops exposed to temperatures higher than 100°C has been investigated. It appears that heating can have an unexpected faster virucidal effect than previously described.Heat inactivation of viruses was reported, however, the thermal resistance of viruses in droplets has not been studied. The aim of this study was to evaluate the pattern of heat resistance of minute virus of mice (MVM), coxsackievirus B4 (CVB4), influenza A virus (H1N1), and herpes simplex virus type 1 (HSV1) contained in droplets. Four μL droplets containing viruses (> 10 Viruses can be transmitted through aerosol or droplet. Therefore airfilters are used to collect viruses . However6 TCID50 mL−1 can be obtained in vitro.In the present study, the virucidal effect of warmed surface on viruses contained in droplets has been investigated. A system has been developed to evaluate the resistance to heating of non-enveloped viruses; minute mouse virus (MVM), coxsackievirus B4 (CVB4), and two enveloped viruses, influenza virus type A (H1N1) and herpes simplex virus type 1 (HSV-1). These viruses are relevant models to assess the virucidal effect of heat. MVM is a parvovirus, which is known to be the most resistant to thermal inactivation , and theCVB4 E2 is a strain provided by Ji-Won Yoon, Julia McFarlane Diabetes Research Center  . The vir−1 to 10−8 in MEM supplemented with 2% FBS, 1% non-essential amino acids and 1% L-glutamine for HSV-1 and CVB4; DMEM supplemented with 2% FBS and 1% L-glutamine for MVM; and MEM for H1N1. Then the plates were incubated for 5 d (H1N1 and CVB4), 7 d (HSV-1), 3 weeks (MVM) in a 5% CO2 atmosphere at 37°C. Afterwards the plates were examined using an inverted microscope to evaluate the extent of the virus-induced cytopathic effect in the cell culture. Calculation of estimated virus concentration was carried out by the Spearman-Kärber method and expressed as log10 TCID50 4 μL−1 . An aluminium cone was plunged into hot glycerol. After a few seconds, 4 μL of virus suspension were put on aluminium surface during desired time. 200 μL of cool titration media were then quickly added and withdrawn, and then mixed with 800 μL of cool titration media.CVB4E2 positive strand RNA was quantitated by two-step quantitative RT-PCR as described previously . BrieflyThe first-order kinetic model assumes a linear relationship between the decreases in logarithmic reduction of the number of survivors over treatment time according to the formulai.e., N(t) (TCID50 4 μL−1), and the initial infectious titer N0 (TCID50 4 μL−1). K is the inactivation rate in log10 TCID50 4 μL−1 s−1, and t is the treatment time (seconds).S(t) is the ratio between the infectious titer after an exposure time t, P < 0.05. Statistical analysis was performed using Graphpad Prism version 5.00 .Statistical analysis of the results was performed by Mann–Whitney when appropriate. Curves were compared using an analysis of covariance (ANCOVA) when appropriate. Differences were considered to be statistically significant when Virus inocula (4 μL) were applied in cone shaped aluminium foils that were set in a glycerol bath at various temperatures . 1 s to 90 min after inoculation, 200 μL of cool media were added and afterwards harvested to recover the virus inocula. Harvested fluids were spiked to 800 μL of cool media and then titration was carried out.10 TCID50 4 μL−1 in 6 s, while, in these conditions, the titer of H1N1 was 2.42 log10 TCID50 4 μL−1 (P=0.017)  . A 4 log10 TCID50 4 μL−1 s−1 (P<0.0001) at 80°C, −1.44 vs −1.90 log10 TCID50 4 μL−1 s−1 (P<0.0001) at 90°C, −2.00 vs −1.77 log10 TCID50 4 μL−1 s−1 (P<0.0001) at 100°C.The inactivation rates of H1N1 vs HSV1 were, respectively, −0.81 vs −1.28 log10 TCID50 4 μL−1, at 70°C in 4 s whereas in similar conditions the inactivation of H1N1 was only −1.13 log10 TCID50 4 μL−1 (P=0.028). Inactivation rates of CVB4 vs H1N1, at 80°C, 90°C, and 100°C were, respectively, −1.59 vs −0.81 (P<0.0001), −2.15 vs −1.44 (P<0.0001), and, −4.27 vs −2.00 log10 TCID50 4 μL−1 s−1 (P<0.0001).Drops containing CVB4 a non-enveloped virus, were inactivated with kinetics roughly similar to those of HSV1. Compared with H1N1, CVB4 appeared to be less resistant to inactivation by heating. Indeed CVB4 was dramatically inactivated, −4.23 log10) was obtained only when drops were exposed to 80°C for 30 min, 90°C for 60 s (inactivation rate: −0.06 log10 TCID50 4 μL−1 s−1) and 100°C for 30 s. Temperatures higher than 100°C, 110°C, 120°C and 130°C for 9, 3 and 2 s respectively resulted in a dramatic inactivation (> 4 log10).The impact of temperatures less than or equal to 100°C on to the infectious level of MVM contained in 4 μL of viral suspension was moderate, since an inactivation (up to 4 log10 TCID50 mL−1). The mixtures were incubated, at room temperature, for 30 min afterwards the level of infectious particles were determined. The level of infectious particles was not reduced when the viral suspension was incubated in medium containing aluminium (data not shown).In so far as it was reported that metals, like silver or copper, are able to inactivate viruses , 14, 17 10 reduction)  whereas the infectious titer values were markedly different (6.21 logduction) . These d10 reduction)  in PBS, in PBS supplemented with 2% FBS and in conditioned medium enriched with cellular components, obtained from mock-infected Hep-2 cell cultures. When CVB4 stock and CVB4 spiked in various media was heated at 70°C for 4 s, the level of infectious particles was dramatically lower than in controls in every case (reduction ranging from 4.7 to 4.9 logduction) . Thus th10 TCID50 4 μL−1 of infectious virus without the need to concentrate viral preparations. The individual variation of thermal resistance from one virus to a another one was unlikely due to pH of virus suspension since pH values were closely similar ranging from 7.5 to 8 (data not shown). The fast thermal inactivation in our experiments was not related to the presence of proteins or cellular components in virus suspension as illustrated in the case of purified CBV4 selected as the testing agent spiked in various media.This study is different in many respects from those of other investigators. Several considerations for the system used in the present report are noteworthy. For the first time, the level of a small volume (4 μL) of virus suspension applied on a surface subjected to heat for a short time (as short as 1 s) has been investigated. It was decided to work with 4 μL volumes for two reasons: this volume is small enough for rapid thermic transfer and large enough to contain up to 4.5 logA critical issue was the ability to use the system at high temperature for a short time. Our system opens up the possibility to test the virucidal effect of heat under dried conditions from room temperature to 171°C which is the temperature of glycerol decomposition. One advantage of this system is that inocula immediately reached for setting temperature for the desired time and is instantly cooled at 4°C. Moreover, the system can be easily settled into a class II biological safety cabinet to prevent contamination.Metals, like silver, iron, copper, have proven to exert antiviral activity against viruses , 13, 16.The thermal inactivation of viruses was obtained in a very short time in most or our experiments and the inactivation curves were roughly linear, therefore, they were analyzed according to the first order kinetics model which enabled a comparison of inactivation rates obtained in our study with those obtained by other teams . However10 TCID50 s−1 in our study, whereas the inactivation rates were −0.0092 and −0.023 log10 TCID50 s−1 at 70 and 80°C respectively in a previous study  , 23.10 TCID50 4 μL−1 s−1, and in the case of MVM at 110, 120, and 130°C the values were −0.66, −1.54 and −2.54 log10 TCID50 4 μL−1 s−1 respectively.In the present study, for the first time, the inactivation of viral particles contained in drops exposed to temperatures higher than 100°C has been investigated. Thermal inactivation rates at such temperatures were determined with H1N1 and MVM only since HSV1 and CVB4 were fully inactivated in 1 s at 100°C. The inactivation rate of H1N1 at 110°C was −3.54 loget al. no inactivation of MVM was observed after dry heat exposure at 90°C for 1 min while in our conditions MVM was fully inactivated (10 reduction in the case of HSV-1), whereas no reduction of MVM infectious titers was observed (data not shown). Thus it may be suggested that drying contribute to some extent to thermal inactivation of HSV-1, H1N1 and CVB4 but not of MVM; however drying was obtained in two hours whereas the impact of heating was obtained in a few seconds in our experiments. Nevertheless, it cannot be ruled out that the combination of water evaporation and heat intervene in thermal inactivation of viruses. Whether drying combined with heating play a role, remains an open issue, future studies will be directed along this line in our laboratory.A full inactivation of MVM was obtained in our experiments when viral particles were exposed to 90°C for 1 min, however in a previous study 10 min at 90°C (wet-heat) were needed to inactivate the MVM , suspensIn conclusion, the thermal resistance of viruses contained in a small volume of suspension, has been investigated and, particularly noteworthy, the impact of short time exposure to temperatures ranging from 70°C to 130°C onto virus infectivity has been determined. This is the first report of inactivation of viral particles contained in drops exposed to temperatures higher than 100°C. The thermal resistance of H1N1, compared to HSV1 another enveloped virus, and to CVB4 a non-enveloped virus has been observed. Furthermore, it has been displayed that heating can have an unexpected faster virucidal effect than previously described.


Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.A novel lyssavirus was isolated from brains of Indian flying foxes ( Lyssavirus: rabies virus (RABV), Lagos bat virus, Mokola virus (MOKV), Duvenhage virus, European bat lyssavirus types 1 and 2, Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Khujand virus, Irkut virus, Shimoni bat virus, Bokeloh bat lyssavirus, West Caucasian bat virus, and Ikoma lyssavirus (IKOV)  , is a large frugivorous and nectarivorous bat that lives in forest, urban, and rural areas and is one of the most persecuted  bats in southern Asia (<150 km) to forage and have a wide distribution . We report identification of a lyssavirus in Indian flying foxes in Sri Lanka.In Sri Lanka, lyssavirus surveillance has focused on canine RABV as the primary public health concern. The Indian flying fox  and the Animal and Plant Health Agency . Specimens were collected under permit no. WL/3/2/62/14 from the Sri Lanka Department of Wildlife Conservation.During January 1, 2014–October 31, 2015, a total of 62 grounded bats were collected in an area inhabited by a long-established roost of ≈20,000 Indian flying foxes in Gannoruwa, Peradeniya, Sri Lanka , which is located 600 m above sea level. Most bats were found dead. One bat (AK-42), which had clinical signs of illness, died shortly after capture .The first bat collected (AK-15) was a fresh carcass of a mature male that weighed 1.5 kg. A detailed necropsy showed that the animal had been healthy and had well-developed pectoral muscles. Except for a few multifocal hemorrhages in the lungs and mild, diffuse hyperemia and edema in the brain, gross pathologic findings were unremarkable. However, Negri bodies of various sizes were identified in the brain , panel AVirus was isolated by using N2A cells (Complete genome sequences (GenBank accession nos. KU244266–9) were obtained from brain RNA samples by using next-generation sequencing according to previous methods . This noLyssavirus but is distinct from viruses circulating in nonvolant mammals in Sri Lanka.Representative canine and golden palm civet RABV sequences from Sri Lanka were included in the dataset, but those sequences clustered with other RABVs, distinct from the GBLV sequence (nucleotide identity 78%). Nucleotide identity across the complete genome ranged from 61% (IKOV) to 76.5% (ABLV), which showed that GBLV is a member of the genus We report isolation of a novel non-RABV lyssavirus (GBLV) that is most closely related to RABV and ABLV. GBLV is pathogenic; it caused fatal disease in 4 Indian flying foxes, and clinical signs for these flying foxes were similar to those observed in other bat lyssavirus infections . DiagnosAlthough rabies is prevalent in Sri Lanka, and a number of wildlife species have been confirmed as being rabid, most of the RABVs involved have not been genetically typed. Furthermore, over a 12-year period, only 1 bat tested for RABV was shown to be uninfected (We report a novel non-RABV lyssavirus identified in Sri Lanka, which indicates that Indian flying foxes are a reservoir for lyssaviruses on the Indian subcontinent and nearby regions. Indian flying foxes are widespread in urban and rural areas and occasionally come in contact with humans and domestic dogs, which provides opportunities for virus spillover. Indian flying foxes are also reservoirs for Nipah virus in Bangladesh and India, where transmission to humans has resulted in outbreaks and human deaths. Other bat species, including insectivorous bats, might also be reservoir hosts for lyssaviruses in the study region. Thus, further surveillance is required to understand the role that bats play in the epidemiology of lyssaviruses in Asia.Continued and extended surveillance of bats and other mammalian species is necessary to determine the distribution and prevalence of GBLV. Detailed phylogenetic analysis and monoclonal typing and antigenic mapping will help clarify the evolutionary relationship between GBLV and other lyssaviruses, in particular RABV and ABLV. In vitro and in vivo cross-neutralization and protection studies will elucidate properties of GBLV and provide information on protection from this virus by available prophylaxis.


Background. Previous studies on the role of inflammation in the pathophysiology of sickle cell disease (SCD) suggested that the CCR5Δ32 allele, which is responsible for the production of truncated C-C chemokine receptor type 5 (CCR5), could confer a selective advantage on patients with SCD because it leads to a less efficient Th1 response. We determined the frequency of the CCR5Δ32 polymorphism in 795 Afro-Brazilian SCD patients followed up at the Pernambuco Hematology and Hemotherapy Center, in Northeastern Brazil, divided into a pediatric group  and an adult group . The adult patients were also compared to a healthy control group . Methods. The CCR5/CCR5Δ32 polymorphism was determined by allele-specific PCR. Results. No homozygous patient for the CCR5Δ32 allele was detected. The frequency of heterozygotes in the study population (patients and controls) was 5.8%, in the total SCD patients 5.1%, in the children 5.4%, in the adults with SCD 4.8%, and in the adult controls 8.1%. These differences did not reach statistical significance. Conclusions. Our findings failed to demonstrate an important role of the CCR5Δ32 allele in the population sample studied here. Sickle cell disease (SCD) is caused by either homozygosity for the hemoglobin S (HbS) gene  or compound heterozygosity for HbS and another structural hemoglobin variant or beta-thalassemia , 2.AG → GTG) at the sixth codon of the β-globin gene (HBB), which causes glutamic acid to be replaced by valine at the sixth position of the polypeptide chain [α, and adhesive molecules [ CCR5 gene, which encodes CCR5, a Th1-cell-associated chemokine receptor, has been associated with chronic inflammatory states [ CCR5Δ32, which leads to truncation and loss of the receptor on the cell surface [ CCR5Δ32 allele could confer a selective advantage on patients with SCD because it induces a less efficient Th1 response [ CCR5Δ32 allele would increase with advancing patient age. Thus, in order to investigate if the CCR5Δ32 polymorphism could confer a selective advantage on its carriers, we compared the frequencies of the CCR5Δ32 allele between two groups of SCD patients (pediatric and adult), seen at the Pernambuco Hematology and Hemotherapy Center, HEMOPE, in Northeastern Brazil, as well as the SCD adult group and a normal control group formed by blood donors.HbS results from a single nucleotide substitution  followed up regularly at HEMOPE were analyzed. The HEMOPE Foundation Ethics Committee approved this study (n° 017/06), and informed consent was obtained from all participants or those legally responsible for them.A total of 795 DNA samples from Afro-Brazilian SCD patients between 3 months and 70 years of age  with 483 individuals and an adult group (18 to 70 years old) with 312 individuals. An adult control group of 247 DNA samples from healthy blood donors  from the same geographical region and with ethnic background similar to those of the patients was analyzed for the CCR5 polymorphism, genomic DNA was extracted from leukocytes using a commercially available kit according to the manufacturer's instructions . The CCR5Δ32 deletion was detected by polymerase chain reaction (PCR) adapted from Chies and Hutz [To analyze theand Hutz , using tμL containing 0.05 U Taq DNA polymerase; 0.1 mM dNTPs; 100 nM of each primer; 3.0 mM of MgCl2; 1x Taq buffer; 200 ng of DNA and deionized water for 30 cycles . The amplified products were run on a 3% agarose gel stained with ethidium bromide and visualized under UV light. The amplification products are shown in  CCR5 allele produced a 206 bp fragment, while amplification of the mutant allele (CCR5Δ32) produced a 174 bp fragment.The PCR reactions were carried out in a Veriti Thermal Cycler (Life Technologies) in a final volume of 30,0 χ2) was used to determine whether the gene distribution in the individuals studied was in Hardy-Weinberg equilibrium. To compare proportions, the chi-square test and Fisher exact test were used. A significance level of 5% was used for all the statistical tests.The statistical analysis was carried out with SAS 9.2 for Windows. The chi-square test . None of the patients or controls was homozygous for the CCR5Δ32 allele. The frequency of heterozygotes in the study population (patients and controls) was 5.8% , corresponding to an allelic frequency of 2.9%. Of the 795 SCD patients, 41 (5.1%) were heterozygous , with 26 (5.4%) being in the pediatric group and 15 (4.8%) in the adult group. In the control group, 20 individuals (8.1%) had the CCR5Δ32 polymorphism, corresponding to an allelic frequency of 4.05%.TheP = 0.72) and of the adult group and the respective controls  failed to identify any statistically significant differences.Statistical comparisons of the pediatric and adult groups , found a relatively high prevalence of the CCR5Δ32 allele (5.1%) compared with healthy controls from the same ethnic group (1.3%) [ CCR5Δ32 heterozygotes in SCA patients from the Southern Brazil compared with only 2% in normal controls. Differently, our results did not show any significant difference between the frequencies of CCR5Δ32 heterozygotes of the adult and the pediatric populations , as well as adult patients and adult controls . However, the sample size analyzed here (795 SCD patients), besides a less heterogeneous ethnic origin , confers consistency on these results.Regarding SCD, there are few studies investigating if the mutantp (1.3%) . Vargas p (1.3%)  reported CCR5Δ32 allele varies in the Brazilian population, reflecting the history of immigration from very varied ethnic backgrounds. However, after studying 795 patients, from the Northeastern region of the country, we failed to find any significant result which could suggest that the presence of the CCR5Δ32 mutant allele contributes to the development of a less aggressive spectrum of SCD and confers an important selective advantage on its carriers in this population.In conclusion, our data show once more that the frequency of the


The Kokobera group of flaviviruses circulates in Australia and Papua, New Guinea, and has been associated with occasional human polyarticular disease. To facilitate future studies to identify virulence determinants, the complete coding regions of the Stratford virus, and isolates of the Bainyik virus and Torres virus were obtained. Flaviviridae; genus, Flavivirus) currently includes 5 candidate species: Kokobera (KOKV), Stratford (STRV), Bainyik (previously strain MK7979), Torres (previously strain TS5273), and New Mapoon (NMV) viruses  as a scaffold. Any discrepancies in the sequence were resolved by construction of primer sets which spanned the region in question, followed by amplification and Sanger sequencing.Sequencing was as described in detail previously . BrieflyThe nucleotide sequence length obtained varied  mainly due to inefficiencies in the ability of the method to amplify the RNA genome termini. Nucleotide alignments with the complete 10874 nt KOKV genome sequence indicated that the Bainyik virus was more closely related to it than STRV or the Torres virus, as expected . This was consistent with previous reports based on analyses of the untranslated regions (UTR) and envelope sequences (KM225263), Bainyik virus (KM225264), and Torres virus (KM225265).The GenBank accession numbers for the virus sequences are STRV (


To the Editor: Coronaviruses (CoVs) in bats are genetically diverse, andevidence suggests they are ancestors of Middle East respiratory virus CoV (MERS-CoV),severe acute respiratory syndrome CoV, and human CoVs 229E and NL63 ; and released,except for 72 bats that died or were euthanized upon capture. Lungs and livers ofeuthanized bats were harvested and homogenized. Caves were in proximity tohuman-inhabited area but not in proximity to camels.Taphozousperforatus) tested negative for CoV. We also sampled 31 desert pipistrellebats (Pipistrellus deserti) and detected an HKU9-like betacoronavirus(β-CoV) in the liver of 1 bat . From 257 specimens from Egyptianfruit bats (Rousettus aegyptiacus), we detected β-CoV in 18samples from 18 different bats . A murine hepatitis virus–like CoVwas detected in the lung of 1 bat. HKU9-like viruses were detected in 5 oral, 2 lung, 5liver, and 5 rectal samples. Overall, 5.1% of the bats tested positive.In Egypt, we sampled 3 bat species . Eighty-Rhinolophus hipposideros batsand 6 Miniopterus schribersii bats tested negative. One of 3Rhinolophus ferrumequinum bats sampled was positive. We sampled 438Rousettus aegyptiacus bats from 10 different locations and detectedHKU9-like viruses in 24 rectal swab specimens . Overall, 5.5% of thebats tested positive.In Lebanon, we sampled 4 bat species. Four A subset of the samples  were tested forMERS-CoV by using the specific upstream of E quantitative reverse transcription PCR; alltested negative. Serum samples from 814 bats tested negative for MERS-CoVantibodies.RdRp) genes of viruses detected in R. aegyptiacusbats in Lebanon and Egypt were closely related to the RdRp gene of HKU9CoV (RdRp fragments sequenced had<90% nt similarity among groups A, B, and C. Within-group nucleotide similarity was>90%, and amino acid variability was 2%–4% (Phylogenetic analysis revealed that the RNA-dependent RNA polymerase(HKU9CoV . Our viras 2%–4% . The phyas 2%–4% . VirusesRdRp fragments to suggest the circulation of atleast 3 diverse groups comprising 3 different CoV species.Most of the positive samples were detected in Egyptian fruit bats. These arecave-dwelling species that inhabit regions of East Africa, Egypt, the EasternMediterranean, Cyprus, and Turkey (Our detection of CoVs in oral, rectal, lung, and liver samples suggests that CoVinfection in those bats was systemic, although the bats were apparently healthy. One bathad a murine hepatitis virus–like infection. This bat was captured from a broodthat inhabited the windowsills of a historic building in urban Cairo. This infectionmight have been a cross-species infection from mice to bats in the same habitat.Although bats rarely come in direct contact with humans, humans can come into morefrequent contact with bat urine and feces and, in the case of fruit bats, bat salivathrough partially eaten fruits. Bats in the Middle East are not eaten for food but areoccasionally hunted. In this study, HKU9-related viruses were detected in apparentlyhealthy fruit bat species from Egypt and Lebanon and appear to cause systemic infection.HKU9-related viruses are not known to cause human disease. MERS-CoV was not detected inbats sampled in this study. More surveillance for bat CoVs in the Middle East is needed,and the zoonotic potential for bat-CoVs requires further study.Laboratory methods.Nucleotide and amino acid pairwise distances of coronaviruses found in bats,Egypt and Lebanon, 2013–2015.


Wolbachia are widely distributed in arthropods. There is growing empirical evidence that Wolbachia directly interacts with viruses and other parasites inside the arthropod host, sometimes resulting in low or no pathogen replication. Previous theoretical studies showed that this direct effect of Wolbachia can result in a reduced virus prevalence (within the population), suggesting that Wolbachia could be used in the biological control of vector-borne diseases . However, Wolbachia might also indirectly affect virus dynamics because Wolbachia-induced reproductive phenotypes  increase the larval mortality of hosts and thus alter the age structure of populations. We investigated this indirect effect using mathematical models with overlapping generations, and found the results to depend strongly on the host's life history. In general, the indirect effect can result in two different outcomes: (1) reduced virus prevalence and virus invasion ability, and (2) increased virus prevalence and virus invasion ability. The former occurs for host species with larval competition and undercompensation, the latter for hosts with either adult competition or larval competition and overcompensation. These findings suggest that the effect of Wolbachia on a specific virus is sensitive to the host's life history. We discuss the results with respect to biocontrol programs using Wolbachia.Intracellular bacteria of the genus  Wolbachia, which impedes replication of dengue virus in the yellow fever mosquito Aedes aegypti . In order to evaluate opportunities and potential risks of this new approach, mathematical modeling is useful because it allows investigating factors where pest control is most effective, but also circumstance in which unexpected or counterintuitive effects occur.The study of mutualistic and parasitic interactions is a major theme in ecology and evolution  and male killing (MK). CI-Wolbachia induce a sperm-egg incompatibility in matings between infected males and uninfected females, resulting in no or a reduced number of offspring. This allows Wolbachia to invade rapidly in host populations as demonstrated by theoretical and empirical studies  and related species lay eggs in small breeding containers, in which larvae compete for resources. This results in scramble competition amongst larvae. However, resource competition between adults is generally thought to be negligible or absent in this system .An important feature of both models is that they allow for overlapping generations and distinguish between larval and adult stage. This is in contrast to previous theoretical work on N at the beginning of each time step. Let X denote the virus frequency of adults at this point. In order to calculate the virus frequency in the next time step (X′) each phase of the life cycle is modeled separately.The adult competition model (ACM) describes the temporal change in the infection frequency of the virus with a deterministic non-linear recursion equation. During each time step, the following events occur in order: (1) virus transmission and virus-induced mortality, (2) mating and reproduction, (3) natural death of larvae and adults, and (4) density dependent population regulation . Note that individuals, who were already infected, do not suffer from the pathogen and have the same survival probability as uninfected individuals. As a result, the population consists of i = [X + tV(1 − s)X(1 − X)]N infected and u = [(1 − X) − tVX(1 − X)]N uninfected individuals.First, virus transmission takes place. We assumed that each individual has random contact to one other individual. If an infected and an uninfected individual meet, transmission occurs with probability dA of adults and dL of larvae die. All surviving larvae develop into adults. The resulting population consists of (1 − dA) i infected and (1 − dA) u + ε(1 − dL)(i + u) uninfected adults.Next, mating and reproduction happen. We assumed that each individual lays ε eggs that all develop into larvae. Adults and larvae face natural mortality, i.e., the fraction Finally, density dependent regulation occurs. Mathematically, this is realized by normalization. The infection frequency in the next time step is calculated as the number of infected individuals divided by the total number of individuals. This yields the following recursion equation:I) and uninfected (U) adults in a population. The temporal change of I and U is described by two non-linear recursion equations. Within each time step, the following events occur in order: (1) mating and reproduction, (2) natural death of larvae and adults, (3) virus transmission between adults and virus-induced mortality, and (4) density dependent regulation of larvae and emerging of new adults  keeps track of the absolute numbers of virus-infected (I + U). Further, a fraction dA of adults dies, resulting in dA) I infected and dA) U uninfected individuals.First, adults mate and produce on average ε eggs per individual. Second, a fraction c that is linear in the number of infected individuals, i.e., c = K. The rate c describes the average number of contacts with infected individuals per uninfected individual. The interpretation of K is as follows. If K, each uninfected individual has exactly one contact to one infected individual. For K there are several contacts, and for K not every uninfected individual has contact. Next, we calculate the number of newly infected adults. For this, we first calculate the probability of an uninfected individual to stay uninfected within this time step as (1 − tV)c. Here, c stands for the average number of contacts and (1 − tV) for the probability to not get infected despite having contact with an infected individual. Accordingly, the total number of newly infected adults computes to (1 − (1 − tV)c)s) in comparison to uninfected individuals or individuals with “old” infections. As a result, there are s)(1 − (1 − tV)c)tV)cThird, virus transmission occurs between adults. In this model, virus transmission depends explicitly on the number of adult individuals in the population (and not on the infection frequency as in the ACM). We first introduce a contact rate L larvae develop into L = exp(−αLβ)adults. The parameters  describe the density dependent mortalities of the larvae. Over- and undercompensation occurs depending on the choice of the parameters and the actual value of the variables .The fourth and last step is the density dependent regulation during larval development. We followed a standard model for mosquito population dynamics developed by Christopher Dye , and assL = ε(1 − dL)(I + U) and c = (1 − dA) I/K.We are now able to write down the full recursion equations. It holds that:Python 2.7.1 scripts and visualized with Gnu R. The ACM was analyzed by numerically iterating equation (1) . The LCM was analyzed by iterating Equations (2) and (3). For each parameter constellation tested, the system was first iterated 1000 time steps without the virus to avoid population growth effects. Then, one single individual was infected. Temporal dynamics and virus equilibria were calculated in the same way and with the same accuracy as the ACM  was increased to mimic the effect of Wolbachia. The system was again allowed to reach equilibrium, and the equilibrium values before and after the change in Ld were compared. For all parameters tested, an increase in Ld resulted in increased viral frequencies. For example, a Ld of 0.4 led to a virus frequency of 42%. However, if Ld was increased to 0.45, then the equilibrium frequency was at 59%, and an increase to 0.55 resulted in the high virus frequency of 83%  and low cost of infection (s). We screened the -space for regions, in which the virus can invade the host population. Figure Ld.Next, we investigated how changes in larval mortality affect virus invasibility. In general, virus spread and persistence is promoted by high horizontal transmission .In summary, increasing larval mortality has two effects: (1) virus frequencies are increased Figures , 3A, andLd of 0.3, 0.6, and 0.9 lead to virus equilibrium frequencies of 58, 69, and 81%  reduce larval density in infected populations. The mathematical analysis revealed that the outcome of the indirect effect is sensitive to the host's life history. The findings suggest that the spread of Wolbachia results (1) in reduced virus prevalence for host populations with density dependent regulation at the larval stage and undercompensation, and (2) in increased virus prevalence for host populations with density dependent regulation at the adult stage or larval competition and overcompensation.In this study, we investigated the effect of Wolbachia as a reduction in larval mortality without taking into account the infection dynamics of Wolbachia itself. This approach was chosen to emphasize that MK and CI phenotypes have a similar effect on the virus dynamics. However, we also investigated an extended version of the ACM that describes explicitly the coinfection dynamics of the virus with Wolbachia (either MK or CI). The following general conclusions can be made (see supplement for details). First, the results revealed that, as MK frequency increases, the virus frequency increases, and achieves its maximum for the highest MK frequency plCI, where lCI denotes the CI-level . Accordingly, a parameter constellation of lCI = 0.5 and p = 27.5%, which is realistic for T. urticae  or the population size (LCM) of the host. The indirect effect was not observed by Fenton et al. (Previous theoretical analysis demonstrated a n et al. . It woulWolbachia, e.g., if some host individuals loose the infection during their life span. An important implication of this study is that hybrid zones involving uni- and bidirectional CI can stably persist, which would not be possible according to standard models (cf. Engelstädter and Telschow, Wolbachia during the host life cycle. It may be an interesting direction for future research to study how such a Wolbachia loss affects the infection dynamics of both Wolbachia and the virus.We have pointed out that the host life-cycle A previous theoretical study by Martinez-Rodriguez et al.  pointed Wolbachia affect the infection dynamics of viruses even if Wolbachia does not interfere with the virus directly in coinfected hosts. This indirect effect of Wolbachia can result in increased or decreased virus frequencies depending on the host's life cycle. These findings point out that the usability of Wolbachia in biocontrol programs depends crucially on the specific host-virus system.In summary, our results show that 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.


Bacteroidales family S24-7 is a prominent example of one of these groups. Marker gene surveys indicate that members of this family are highly localized to the gastrointestinal tracts of homeothermic animals and are increasingly being recognized as a numerically predominant member of the gut microbiota; however, little is known about the nature of their interactions with the host.Our view of host-associated microbiota remains incomplete due to the presence of as yet uncultured constituents. The Candidatus Homeothermaceae,” using 30 population genomes extracted from fecal samples of four different animal hosts: human, mouse, koala, and guinea pig. We infer the core metabolism of “Ca. Homeothermaceae” to be that of fermentative or nanaerobic bacteria, resembling that of related Bacteroidales families. In addition, we describe three trophic guilds within the family, plant glycan (hemicellulose and pectin), host glycan, and α-glucan, each broadly defined by increased abundance of enzymes involved in the degradation of particular carbohydrates.Here, we provide the first whole genome exploration of this family, for which we propose the name “Ca. Homeothermaceae” representatives constitute a substantial component of the murine gut microbiota, as well as being present within the human gut, and this study provides important first insights into the nature of their residency. The presence of trophic guilds within the family indicates the potential for niche partitioning and specific roles for each guild in gut health and dysbiosis.“The online version of this article (doi:10.1186/s40168-016-0181-2) contains supplementary material, which is available to authorized users. Firmicutes and Bacteroidetes, and the divergent nature of gut-associated genera in comparison to other phylum members not associated with this environment indicates host selection and evolution occurring over a long period [The host microbiome has been firmly established as critical to host physiology. Evidence now supports the microbiome as influential in diverse processes ranging from infection susceptibility  to behavg period . The relg period , 6). DesBacteroides group” first recognized in 2002 [Bacteroidales, named after one of the earliest environmental clones belonging to the lineage, S24-7  and performed a comparative genomics analysis. The recovered genomes define a family that is most closely related to, but distinct from, the genera Barnesiella and Coprobacter. Analysis of 16S rRNA gene databases suggests a strong habitat preference for the homeothermic gut. Metabolically, we infer S24-7 are fermentative or nanaerobic [To further our understanding of the naerobic  species,S24-7 family. Notably, 98 % of these sequences originate from homeothermic animals, 99 % of which are associated with the gastrointestinal system  are herbivores or omnivores, with many of the omnivores likely to consume a mostly herbivorous diet . The only two carnivorous hosts within the database are the dhole , one of only four canids classified as hypercarnivores [Ca. Homeothermaceae” hosts potentially reflects the metabolic capacity of the family.The Silva database , 14 from mouse , four from guinea pig , and two from koala . Average genome size was 2.69 Mb, with a notable outlier from the koala gut of 4.46 Mb , maximum 28 % (K1), Additional file We obtained 30 near complete “Ca. Homeothermaceae” population genomes were phylogenetically placed within the Bacteriodales order by generating a genome tree based on 120 single-copy marker genes within 300 Bacteroidales reference genomes obtained from NCBI . Two ofCa. Homeothermaceae” cell envelope is that of a diderm (Gram-negative), as demonstrated by the absence of typical monoderm protein domains and the presence of the majority of typical diderm domains, consistent with the Bacteroidetes phylum . All “Ciewed in ).Fig. 2I1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenate), B7 (biotin), and B9 (folate), a range which is consistent with other Bacteroidetes [6 production, was identified in any of the genomes, despite the remainder of the pathway being present. The lack of PdxH has also been noted in some Bacteroides species [12  production pathway is absent from all “Ca. Homeothermaceae,” however, a subset encode a partial pathway originating from adenosyl cobyrinic acid. Vitamin B12 transporters were identified within 28 of the 30 genomes , one of which is 97 % complete, suggesting that not all “Ca. Homeothermaceae” have this capacity. We were unable to confirm the operons’ absence through genomic context due to a lack of synteny in the surrounding regions despite close relatives in some instances . If truly absent, as with complex I in H5, this would indicate relatively recent independent loss of this operon from multiple “Ca. Homeothermaceae” lineages. The variable presence of respiratory complexes in “Ca. Homeothermaceae” suggests a level of energetic flexibility within the family and potentially relatively recent purging of non-essential respiratory elements in the gastrointestinal environment.In addition to complex I, complex II (fumarate reductase/succinate dehydrogenase) is present in 28 genomes and is composed of three subunits: a flavoprotein, an iron-sulfur protein, and a single transmembrane protein, indicating a type B structure . Both geronments . Four ge+ translocating NADH:ubiquinone oxidoreductase Nqr complex was identified in 27 “Ca. Homeothermaceae” genomes  was identified in 24 of the genomes, and eight of these also encode a catalase (H2O2 to H2O + O2) protein  thioredoxins within each genome and a single copy of the thioredoxin reductase TrxB in all but three genomes: GP1 and M13 lack TrxB, while M12 contains two copies. In addition, 26 genomes contain a non-heme ferritin protein permitting the storage of excess iron. Overall, “Ca. Homeothermaceae” members appear well equipped to deal with oxidative stress, supporting potential microaerobic growth.To support a prediction of “Ca. Homeothermaceae” proteins carry the general secretory pathway signal peptide, as predicted by SignalP [Ca. Homeothermaceae” or neighboring populations. Several immune-related peptidases are also secreted: 20 of the 30 population genomes contain a metalloprotease belonging to peptidase family M6 (immune inhibitor A family)  were present in 22 genomes (other genomes contained incomplete gene sets), in addition to the regulatory two-component sensor system, PorX (response regulator) and PorY (histidine kinase), responsible for the co-regulation of a subset of T9SS genes  that dictates the use of the system and is cleaved during translocation [Ca. Homeothermaceae” genomes, ~75 % of which also carried a general secretory pathway signal peptide, supporting their movement to the periplasm and subsequent secretion by the T9SS. The majority of proteins within this group are annotated as hypothetical (60 %); however, there is a homolog of a characterized immune-related peptidase, streptopain (SpeB). SpeB, encoded by Streptococcus pyogenes, contains the peptidase C10 domain and is capable of degrading multiple components of the immune system  was also identified in the majority of the “location , 39. We iewed in ). StreptCa. Homeothermaceae” coding sequences, a level similar to that of other Bacteroidales families  enriched in “Ca. Homeothermaceae” in comparison to related Bacteroidales families, as was the starch binding module CBM26  and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology annotation  profiles for a selection of microbial isolates from the gut and compared their glycoside hydrolase distribution with that of the “ion Fig. . In suppdegrader , was foued bread . Functio the gut . TherefoBacteroides thetaiotaomicron [Bacteroidetes [susCD-like gene pairs are found within all “Ca. Homeothermaceae” genomes, and most also contain the gene pair in association with carbohydrate-active enzymes  and susD  suggesting that equivalent sequence diversity exists within each group and therefore potentially a similar, although not necessarily overlapping, diversity of substrates available to this system.Carbohydrate degrading enzymes may be clustered together in polysaccharide utilization loci (PUL), first described in aomicron  and now roidetes . Such loroidetes , 46. Mul species , 50, 51.aomicron . We consy scores  for all To determine whether the metabolic guilds extend to broader genome content, we annotated each genome using both KEGG and COG orthology detection. Ordination plots generated from both annotation systems clustered the metabolic guilds discretely, although with low levels of separation Fig. . Only a Ca. Homeothermaceae” to other Bacteroidales families. “Ca. Homeothermaceae,” Bacteroidaceae, and Prevotellaceae separated clearly based on COG annotations, while Porphyromonadaceae members were intermingled with other families, potentially reflecting the phenotypic diversity of this family  as suggested by genomic analysis of other oxalate degrading species [Ca. Homeothermaceae” have oxalate degrading genes, whereas only five of the 13 α-glucan-focused guild have them. As with urease, gene trees confirm that this function is rare within the Bacteroidales  than in lean (10 %), suggesting that a higher energy diet may better support the family, although there was no dietary composition information available for this dataset. We also identified a substantial increase in the prevalence of “Ca. Homeothermaceae” within the Hadza hunter-gatherer population; 70 % of individuals were found to carry at least one population. Only two species were identified within this group of individuals: the typically prevalent H8/H9 and species H4, also a member of the α-glucan guild. The Hadza diet is heavily plant based, composed of tubers, meat, honey, foliage, and berries, with tubers being particularly important due to their constant availability [Ca. Homeothermaceae” within the Hadza suggests this diet is particularly amenable to the maintenance of the identified species, although does not result in any increase in their overall abundance.The most prevalent “lability . Tubers lability . The incBacteroidales family S24-7 is encountered frequently in culture-independent studies and is gaining recognition due to both its prevalence, particularly in murine-based datasets, and its fluctuating abundance in cross-sectional and intervention type studies. Increased abundance has been described in mice fed a low-fat diet and, in association with increased exercise [S24-7 is also the dominant family during hibernation of arctic ground squirrels [S24-7 is currently unknown, as they remain uncultured and no genomic studies have been undertaken. Here, we recover a set of S24-7 population genomes from metagenomic samples, enabling inference of their core metabolism, and propose the name “Ca. Homeothermaceae” reflecting an ecological distribution limited to the guts of homeothermic animals , further reports confirming the roles of “Ca. Homeothermaceae” in vivo are likely to appear in the future.Overall, this study provides the first genomic insights into the uncultured gut-inhabiting “Homeothermus . Inferred to be Gram-negative, non-motile, nanaerobic, and able to ferment a wide range of carbohydrates including arabinose, cellobiose, fructan, fructose, glycerol, lactose, maltose, raffinose, sucrose, xylan, and xylose, with a focus on arabinan and xylan based on enzyme abundance.Homeothermus arabinoxylanisolvens . Description is the same as that for genus “Ca. Homeothermus.” Represented by population genome M4 (acc. no. LUJO00000000) obtained from metagenomic sequencing of Mus musculus fecal sample.Ca. Homeothermus” with the following additions; -aceae ending to denote a family. Additional fermentation substrates include pectin, rhamnose, and trehalose. Three trophic guilds are proposed within the family based on the relative abundance of carbohydrate-active enzymes with different substrates: α-glucans, complex plant cell wall components, and host glycans. Type genus: “Ca. Homeothermus.” Order: Bacteroidales.The description is the same as for the genus “Barnesiella, Butyricimonas, Coprobacter, Dysgonomonas, Odoribacter, Paludibacter, Parabacteroides, Proteiniphilum, “Ca. Sanguibacteroides,” and Tannerella have been removed as they do not form a monophyletic group with the type genus, Porphyromonas.The description is the same as that given by Krieg et al.  with theBarnesiella (type genus) and Coprobacter. Description is drawn from that of Barnesiella given by Sakamoto et al. [Coprobacter given by Shkoporov et al. [-aceae ending to denote a family. Cells are Gram-negative, obligately anaerobic, non-spore-forming, and non-motile. Saccharolytic. Type genus: Barnesiella. Order: Bacteroidales.Includes the genera o et al.  and Coprv et al. : -aceae Dysgonomonas (type genus) and Proteiniphilum. Description is drawn from that of Dysgonomonas given by Hofstad et al. [Proteiniphilum given by Chen et al. [-aceae ending to denote a family. Cells are Gram-negative, fermentative, and facultatively (Dysgonomonas) or obligately (Proteiniphilum) anaerobic. Type genus: Dysgonomonas. Order: Bacteroidales.Includes the genera d et al.  and Protn et al. : -aceae Marinifilaceae given by Iino et al. [Marinifilaceae contains the genera Butyricimonas, Marinifilum (type genus), Odoribacter, and “Ca. Sanguibacteroides.” Cells are Gram-negative, non-spore-forming, non-motile, and facultatively (Marinifilum) or obligately  anaerobic.The description is drawn from that of o et al.  with thePaludibacter (type genus). Description is the same as for the genus Paludibacter given by Ueki et al. [-aceae ending to denote a family. Type genus: Paludibacter. Order: Bacteroidales.Includes the genus i et al. : -aceae Parabacteroides and Tannerella (type genus). Description is drawn from that of Tannerella given by Sakamoto et al. [Parabacteroides given by Sakamoto et al. [-aceae ending to denote a family. Cells are Gram-negative, non-motile, and obligately anaerobic. Type genus: Tannerella. Order: Bacteroidales.Includes the genera o et al.  and Parao et al. : -aceae Fecal samples were obtained from six 12-week-old female C57BL/6 mice housed in accordance with the University of Newcastle Animal Care and Ethics Committee; reference number A-2013-303. DNA was extracted from feces using the PowerSoil DNA Isolation Kit  according to the manufacturer’s instructions. Library preparation was performed using the Nextera DNA Library Preparation Kit . Libraries were sequenced at the Diamantina Institute, The University of Queensland, using the Illumina HiSeq 2500 platform generating ~9 Gbp of 100 bp paired-end reads per sample.Koala fecal samples originated from a 12-year-old male as previously described . Public https://github.com/jstjohn/SeqPrep) then remaining pairs were quality trimmed using Nesoni v0.128 (https://github.com/Victorian-Bioinformatics-Consortium/nesoni) with a minimum Phred quality threshold of 20. Assembly of pooled reads was performed using CLC Genomics Workbench v7.0.4  using a word size of 30 and bubble size of 1500. Scaffolding was performed during assembly, and reads were mapped back to contigs with default settings. Minimum contig length was 300. Mapping of reads to final assemblies was performed using BWA v0.7.10 [Reads from mouse fecal samples were adapter trimmed and merged using SeqPrep ( v0.7.10  with defhttps://sourceforge.net/projects/bbmap/). Assembly was performed either per individual run (human) or pooled (guinea pig) using CLC Genomics Workbench v8.5.1  either using default settings (human) or using a word size of 30 and a bubble size of 1000 (guinea pig). Minimum contig length was 500. Scaffolding was performed during assembly, and reads were mapped back to contigs with default settings. Gap filling was performed on assemblies from human datasets using Abyss-sealer [http://ecogenomics.github.io/BamM/) with default settings.SRA data from guinea pig and human datasets was quality and adapter trimmed using Trimmomatic v0.3.2  with defs-sealer  with defCa. Homeothermaceae” genomes were refined by removing contigs with incongruent coverage profiles as identified by RefineM v0.0.3 (https://github.com/dparks1134/RefineM). Gap filling was performed on these assemblies using FinishM v0.0.6 (https://github.com/wwood/finishm). Reads mapping to each genome were extracted from a complete assembly mapping  using BamM v1.5.1 (http://ecogenomics.github.io/BamM/) then remapped to each individual genome using CLC Genomics Workbench v8.5.1  with default settings. These mappings were used for manual investigation of specific genomic features.Population genomes were obtained either from previous studies  or de noCa. Homeothermaceae” population genomes in the context of the order Bacteroidales was generated based on an alignment of 120 concatenated single-copy, bacterial-specific, marker genes implemented within an in-house pipeline. Marker genes from 300 Bacteroidales and 37 Sphingobacteriales (outgroup) genomes were obtained from publicly available genomes within the NCBI database and aligned using Mafft v7.221 [A maximum-likelihood tree of “t v7.221 . A maximt v7.221  under thhttps://github.com/Ecogenomics/mingle) utilizing the BLAST [susC and susD gene trees were calculated using the picante R package [Individual gene trees were generated using FastTree v2.1.7  implemenhe BLAST  workflowhe BLAST . Default package . All tre package .https://github.com/widdowquinn/scripts/blob/master/bioinformatics/calculate_ani.py). Average amino acid identity between each genome pair was calculated using CompareM v0.0.5 (https://github.com/dparks1134/CompareM). Protein families were identified using pfam_scan.pl against the Pfam database release 28 [ftp://ftp.jcvi.org/pub/data/TIGRFAMs/). Antibiotic resistance genes were predicted using the Resfams database [Genomes were annotated using Prokka v1.11 . All sublease 28  employinlease 28 . Archetylease 28  were uselease 28  against database .http://kbase.us/), and Pathway Tools v19.0 [Ca. Homeothermaceae.” Clusters of Orthologous Groups (COG) profiles were identified using BLASTP v2.2.30+ [http://www.ncbi.nlm.nih.gov/COG/, last accessed July 2015) with e-value cutoff of 1e-6.Kyoto Encyclopedia of Genes and Genomes (KEGG) term annotation was performed using KAAS  and KEGGls v19.0 , and curls v19.0  were usev2.2.30+  against Carbohydrate-active enzymes (CAZy) were identified using hmmscan from HMMER v3.1b2  against Differential abundance of annotated features  was analyzed using DESeq2 R package  based onCa. Homeothermaceae” were assessed by mapping public human and mouse gut metagenome datasets downloaded from the SRA against all genomes using BWA v0.7.12 [Ca. Homeothermaceae” population bins and thus represents a conservative estimate. Relative abundance of each population was based on the percentage of total reads attributed to each genome exceeding the minimum cutoff percentage, normalized for genome size. Diet-related datasets were PRJEB7759 (mouse), PRJNA278393 (Hadza), and PRJEB2054 (lean vs. obese). Additional human samples originated from projects PRJEB1220, PRJEB4410, PRJEB6456, and PRJEB7774Prevalence of each population genome and overall abundance of “ v0.7.12 . Genome ANI, average nucleotide identity; AR, antibiotic resistance; CAZy, carbohydrate-active enzyme; COG, Clusters of Orthologous Groups; KEGG, Kyoto Encyclopedia of Genes and Genomes; PUL, polysaccharide utilization loci; Sus, starch utilization system.


Cydia pomonella (L.) (Lepidoptera: Tortricidae) is a major fruit feeding pest of apples. Understanding susceptibility differences of various apple cultivars to CM oviposition is an important step in developing resistant varieties as well as monitoring and management strategies for this pest in apple orchards planted with mixed-cultivars. In this context, oviposition preferences of CM for the fruits of different apple cultivars were studied in laboratory bioassays using a series of no-choice and multiple-choice tests in 2006, 2007, and 2008. In 2006 and 2007, 10 apple cultivars, viz., Arlet, Fuji, Gala, Golden Delicious, Honeycrisp, Pristine, Delicious, Stayman, Sunrise, and York Imperial were evaluated, while in the 2008 tests, Golden Delicious, Honeycrisp, and York Imperial were evaluated. During the 2006 tests, preferred apple cultivars for CM oviposition were Golden Delicious and Fuji, while the least preferred were Arlet, Pristine, Sunrise, and Honeycrisp. Similarly, during the 2007 tests, Golden Delicious, Fuji and Stayman remained the preferred cultivars, while Arlet, Honeycrisp, Pristine, and Sunrise remained the least preferred cultivars. In the 2008 tests, Golden Delicious and Honeycrisp were the most and least preferred cultivars, respectively. Based on the oviposition preferences from these bioassays, a susceptibility index for each cultivar was developed. This index may be used as a standard measure in cultivar evaluations in breeding programs, and may assist fruit growers and crop consultants to select the most appropriate cultivar(s) for monitoring and detecting the initial signs of fruit injury from CM in an apple orchard planted with mixed-cultivars.Codling moth (CM),  Cydia pomonella (L.) (Lepidoptera: Tortricidae), probably originating in Europe , however, other species belonging to various plant families, such as pears (Pyrus sp.), quinces (Cydoniaoblonga Mill.), peaches (Prunus persica) (L.), wild haws (Crataegus sp.), English walnuts (Juglans regia L.) (Prunus sp.), nectarines (Prunus sp.), and sweet cherry (Prunus avium L.) are also reported as host plants (The codling moth (CM), n Europe , is a sen Europe , and cauegia L.) , plum , which are naturally occurring sesquiterpene compounds  for field oviposition/infestation by CM and other insects. Fruits of approximately similar size were vertically suspended in oviposition chambers by tying the stem to the top of the oviposition chamber using aluminum wire. Fruits damaged while being placed in oviposition chambers were discarded and replaced by new fruits from the same lot.Fruits of all cultivars were collected from unsprayed (without insecticide application) trees (10–33 years old) in apple orchards established in south facing slopes with typical well-drained soils of the Appalachian region. Fruits were stored in small cardboard boxes in a cold room (0°C). Fruits were removed from the cold room approximately 4–5 h before the start of each experiment. All fruits were washed three times with clean cold water and were carefully inspected via a 10X Opti-VisorCodling moth adults used in this study were obtained from a laboratory colony established from adults or larvae collected from a block of apples located at The Pennsylvania State University, Fruit Research and Extension Center (FREC), Biglerville, PA, USA. Green thinning apples of various cultivars were used to maintain the laboratory colony/insect culture throughout the year during this study year. Pupae were collected from rearing containers in cardboard strips, and kept in environmentally controlled chambers (18–20°C) till their use. CM pupae of similar age were selected and sexed, and placed into the oviposition chambers. The adult moths were allowed to emerge, mate, and freely oviposit on fruits. Pupae were regularly monitored for adult emergence. If there was no adult emergence from a pupa within 3 days of release, then it was replaced by an adult (2–3 days old) of the same sex from the same pupal lot.Multiple-choice oviposition preference tests and no-choice preference tests were conducted for both fruit maturity sets. In the no-choice tests, the oviposition chamber consisted of transparent plastic cups (1.0 L) internally lined with charcoal-colored fiberglass screen. In the multiple-choice tests, a cylindrical chamber  made of transparent fiberglass internally lined with fine aluminum mesh screening served as the oviposition chamber. In the multiple-choice tests, fruits of each cultivar were allocated to one of several locations at random in the oviposition chamber. During the study period, insects were maintained under laboratory conditions . The year-wise description of these bioassays is as follows:Nine cultivars  were evaluated in multiple-choice and no-choice tests during the first set (July) of experiments. In the second set of experiments (August), all cultivars  were again evaluated. Each treatment (cultivar) was replicated at least eight times in the multiple-choice tests and 10 times in the no-choice tests. All fruits were collected during 14–17 July and 12–15 August for the first set (19 July) and second set (25 August) of experiments, respectively. Fruits of all cultivars  were collected from non-insecticide sprayed trees at FREC, Biglerville. Fruits of ‘Arlet,’ ‘Pristine,’ and ‘Sunrise’ cultivars  were received from the USDA Appalachian Fruit Research Station, Kearneysville, WV, USA. In both no-choice experiments (early and late), one pair of unmated male and female adults was placed per cup, and the number of deposited eggs was counted after 8 days. In multiple-choice tests, seven and six pairs of unmated adults were utilized in the early and late experiments, respectively. Total numbers of eggs were counted after 15 days (early), and 10 days (late). The position of each egg on fruit  was recorded.All 10 cultivars were evaluated in multiple-choice and no-choice tests conducted during the months of July and August. Each treatment (cultivar) had 8 and 10 replicates in the no-choice and multiple-choice tests, respectively. Fruits were collected during 12–15 July (early) 11–14 August (late). Fruits of all cultivars  were collected from non-insecticide sprayed trees at FREC, Biglerville. Fruits of ‘Arlet,’ ‘Pristine,’ and ‘Sunrise’ cultivars  were received from the USDA Appalachian Fruit Research Station, Kearneysville, WV, USA for the early set of experiments, and for the late set of experiments from The Russell E. Larson Agricultural Research farm, Rock Springs, PA, USA. The early set of multiple-choice and no-choice tests were conducted on 17 July, while the late set of experiments were conducted on 16 August. In the no-choice tests, two pairs of unmated male and female adults were used in both sets of no-choice tests. In multiple-choice tests, three pairs of unmated male and female adults were used in both sets of multiple-choice tests. In all tests, the total numbers of deposited eggs on fruits were counted after 10 days. The position of eggs on the fruits was recorded as per the procedure used in the 2006 bioassays.Based on the results of bioassays conducted during the first two years, only three cultivars  were further evaluated in the third year. Fruits of similar size were collected from non-insecticide sprayed trees at FREC, Biglerville, and utilized the same day for both the no-choice and multiple-choice experiments. The study was replicated 15 and 8 times in the no-choice and multiple-choice tests, respectively. In the multiple-choice tests, two fruits of each treatment/cultivar were used in each replication. In the early experiment, fruits were collected on 22 July, and used in both types of tests on the same day, and observations on eggs were taken after 10 days. In the late set, fruits were collected on 28 August, and observations were recorded after 11 days in both no-choice and multiple-choice tests. Similar to previous years, the position of eggs on the fruits was recorded in 2008.Table 2) was used to determine: (a) the oviposition preference of CM among apple cultivars; (b) differences in oviposition preferences during the early and late season ; and (c) differences in oviposition preferences in the multiple-choice and no-choice tests. The second model, which includes the egg counts by position on the fruit, was used to determine CM oviposition-site preferences across different cultivars (Table 3). The mixed-model ANOVA analysis was performed using R software  was used to analyze the data. In the analysis, two similar statistical models were used to address the study objectives. The first model (post hoc test (P < 0.05) when ANOVA was significant (Oviposition preference based on the mean number of eggs (per pair of CM per fruit) was determined for each cultivar. The data sets were transformed  by taking the natural log of the “eggs per pair” variable. Pairwise comparisons were done among all cultivars, and means were separated using Tukey’s honest significant differences nificant .The CM oviposition susceptibility index (based on oviposition preferences of CM) for each cultivar was characterized as:SI = Susceptibility index; SEPP = Standardized mean eggs per pair of moths for an apple cultivar [i]; j = Time of fruit collection (early or late); k = year of observation; and t = type of tests .Where, Standardized mean eggs per pair of moths for an apple cultivar were determined as following:SEPP = Standardized mean eggs per pair of moths for an apple cultivar [i]; EPP [i] = Mean number of eggs per pair of moths on an apple cultivar [i]; and EPPmax [i] = Maximum number of eggs per pair of moths on an apple cultivar [i].Where, Mean total number of eggs per pair of moths (EPP) on a cultivar was calculated by the following equation:EPP [i] = Mean number of eggs per pair of moths on an apple cultivar [i]; EPPC[i] = Mean number of eggs per pair of moths on calyx side of an apple cultivar [i]; EPPS[i] = Mean number of eggs per pair of moths on stem side of an apple cultivar [i]; and EPPL[i] = Mean number of eggs per pair of moths on lateral side of an apple cultivar [i].Where post hoc test (P < 0.05) when ANOVA was significant , CM females laid significantly higher numbers of eggs on ‘York Imperial,’ ‘Golden Delicious,’ and ‘Delicious’ than other cultivars . In contrast, the lowest numbers of eggs were laid on the ‘Honeycrisp’ cultivar . On the stem site of fruits, CM females laid significantly more eggs on ‘Stayman,’ ‘York Imperial,’ ‘Golden Delicious,’ and ‘Delicious’ than all other cultivars . On the lateral site of fruits, CM females preferred ‘York Imperial,’ ‘Golden Delicious,’ and ‘Delicious’ than the cultivars ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ .In the multiple-choice test, on the calyx site of fruits (Table 4), the female moths significantly preferred to oviposit on ‘Golden Delicious’ (P = 0.009), ‘Fuji’ (P = 0.005), and ‘Delicious’ (P = 0.008), compared to ‘Pristine’ . On the stem site, ‘Golden Delicious’ was significantly more preferred than ‘Delicious’ (P = 0.014), ‘Pristine’ (P = 0.013), ‘Honeycrisp’ (P < 0.001), ‘Arlet’ (P = 0.047), and ‘Sunrise’ . On the lateral site, ‘Golden Delicious’ was significantly more preferred than ‘Honeycrisp’ (P = 0.049), ‘Arlet’ (P = 0.004), and ‘Sunrise’ ; however, the total number of eggs on ‘Golden Delicious’ was not significantly different from that of all other cultivars .In the no-choice test, on the calyx site (Table 4), the oviposition preference of CM was not significantly different for all cultivars (P > 0.05), except for ‘Fuji’, when compared to ‘Delicious’ (P = 0.042), and ‘Sunrise’ . On the stem  and lateral  sites of fruits, ‘Stayman,’ ‘Golden Delicious,’ ‘Fuji,’ ‘Honeycrisp,’ and ‘Arlet’ rather than ‘Delicious,’ ‘Gala,’ and ‘Sunrise’ were the significantly preferred cultivars (P < 0.05).In the multiple-choice test, on the calyx site (Table 4), CM deposited more eggs on ‘Golden Delicious’ than on ‘Gala’ (P = 0.011), otherwise, there was no significant differences between ‘Golden Delicious’ and all other cultivars . On the stem  and lateral  sites of fruits, CM deposited more eggs on ‘Golden Delicious’ and ‘Fuji’ than on ‘Gala,’ ‘Honeycrisp,’ and ‘Sunrise’ (P < 0.05).In the no-choice test, on the calyx site (Table 4), CM significantly preferred ‘Golden Delicious’ for oviposition over ‘Pristine’ (P = 0.006), ‘Arlet’ (P = 0.032), ‘Sunrise’ (P = 0.006), and ‘Gala’ . On the stem site of fruits, ‘Golden Delicious’ was again the significantly preferred cultivar for oviposition over other cultivars, viz., ‘Pristine’ (P < 0.001), ‘Honeycrisp’ (P < 0.001), ‘Arlet’ (P < 0.001), ‘Sunrise’ (P < 0.001), and ‘Gala’ . However, the preference for ‘Golden Delicious’ was similar to ‘Stayman’ (P = 0.092), ‘York Imperial’ (P = 0.457), ‘Fuji’ (P = 0.777), and ‘Delicious’ . On the lateral site, ‘Golden Delicious’ was significantly more preferred than all other cultivars (P < 0.05), except ‘Stayman’ (P = 0.996), ‘York Imperial’ (P = 0.777), and ‘Delicious’ . In contrast, ‘Arlet’ was the least preferred cultivar for oviposition on the lateral site of fruits .In the multiple-choice test of early season 2007, on the calyx site (Table 4), CM deposited higher numbers of eggs on ‘Golden Delicious’ and ‘Gala’ than on ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ . On the stem  and lateral  sites of fruits, ‘Golden Delicious’ and ‘Fuji’ received the highest number of eggs over ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ (P < 0.05).In the no-choice test (July 2007), on the calyx site of fruits (Table 4), ‘Golden Delicious’ was more preferred for oviposition than ‘York Imperial’ (P = 0.036) and ‘Sunrise’ . On the stem site of fruits, the moths again preferred ‘Golden Delicious’ for oviposition over all other cultivars (P < 0.05), except ‘Stayman’ (P = 0.978) and ‘Fuji’ . In contrast, ‘Pristine’ was the least preferred cultivar . On the lateral site, ‘Stayman,’ ‘Golden Delicious,’ and ‘Fuji’ were the most preferred cultivars for oviposition (P < 0.05), except for ‘York Imperial’ and ‘Delicious’ .In the multiple-choice test conducted during the late season study of 2007, on the calyx site (Table 4), CM deposited less eggs on ‘Pristine’ than ‘Gala’ (P = 0.047), however, such lower preference for ‘Pristine’ was not significantly different from all other cultivars . On the stem  and lateral  sites of fruits, CM showed less preference for ‘Pristine’ (P < 0.05) than all other cultivars, except for ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ (P > 0.05).In the no-choice test, on the calyx site (Table 4), on the calyx  and stem  sites of fruits, ‘Golden Delicious’ was the most preferred cultivar for oviposition over the other two cultivars (P < 0.05). On the lateral site, ‘Honeycrisp’ was less preferred for oviposition than ‘Golden Delicious’ .In the multiple-choice test (Table 4), the oviposition preference of CM did not differ significantly across all the cultivars . On stem  and lateral sites of fruits , CM deposited more eggs on ‘Golden Delicious’ over ‘Honeycrisp’ and ‘York Imperial’ (P < 0.05).In the no-choice test (July 2008), on the calyx site (Table 4), on the calyx site, ‘Golden Delicious’ was the most preferred cultivar over ‘Honeycrisp’ (P = 0.004), but it was not more preferred over ‘York Imperial’ . On stem  and lateral  sites of fruits, ‘Honeycrisp’ was the least preferred cultivar when compared to ‘Golden Delicious’ and ‘York Imperial’ (P < 0.05).In the multiple-choice test , stem , and lateral  sites of fruits, ‘Golden Delicious’ was the most preferred cultivar for oviposition over that of ‘Honeycrisp’ and ‘York Imperial’ (P < 0.05).In the no-choice test (August 2008), on calyx . All types of interactions presented in Table 2 had a significant impact on the oviposition preference of CM (P < 0.05). Oviposition sites  on fruits had a highly significant influence on CM oviposition . All the interactions of oviposition sites with other covariates, except Site:Season (P = 0.458), Site:Cultivar:Season (P = 0.115) and Site:Cultivar:Year:Season (P = 0.615), displayed a significant interactive impact on the oviposition of CM .All covariates  had a significant influence on the oviposition of CM on different apple cultivars (P = 0.002), ‘York Imperial’ (P = 0.002), ‘Fuji’ (P = 0.011), ‘Delicious’ (P < 0.001), ‘Pristine’ (P < 0.001), ‘Honeycrisp’ (P < 0.001), ‘Arlet’(P < 0.001), ‘Sunrise’ (P < 0.001), and ‘Gala’ . In contrast, ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ were noticeably less susceptible to oviposition by CM .In terms of the CM oviposition susceptibility index , ‘Golden Delicious’ had a significantly higher susceptibility index than ‘Stayman’ ] for these different apple cultivars are also reported , study year and oviposition sites  on fruits had a significant influence on the oviposition of CM on different apple cultivars. Covariate interactions  were also significant. Oviposition-sites on fruits may vary from one cultivar to another. In the multiple-choice and no-choice tests, CM deposited more eggs on lateral and stem sites than on the calyx site of fruits. Such patterns of egg deposition could be due to the physical characteristics of the apple fruit surface as discussed earlier or due to the ‘vertical’ placement of fruits, as in all these tests, fruits were vertically placed in oviposition chambers. In contrast, oriental fruit moth adult females preferred to oviposit on the calyx and stem sites of apple fruit, and their oviposition site preferences are also reported to vary between different apple cultivars .Susceptibility to various pest infestations may vary among cultivated varieties as well as wild varieties . In the past, susceptibility of apple cultivars/germplasms to different arthropod pests has been studied using several methods, such as their impact on pest developmental rate and pest survival rate , damage viz., ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ ‘Sunrise,’ and ‘Gala’ (less preferred cultivars) in the majority of the multiple-choice tests. In the no-choice tests, CM deposited more eggs on these preferred cultivars than the less preferred cultivars. Regardless of choice test type and season, CM deposited significantly more eggs on ‘Golden Delicious’ over other cultivars, viz., ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise.’ In both types of tests, more eggs were laid on lateral and stem sites than the calyx site of fruits across different cultivars. In terms of a CM oviposition susceptibility index, ‘Golden Delicious’ was the most susceptible cultivar to oviposition, while ‘Pristine,’ ‘Honeycrisp,’ ‘Arlet,’ and ‘Sunrise’ were least susceptible. From an integrated pest management perspective, the newly developed susceptibility index can assist fruit growers and consultants select the most appropriate cultivar(s) for monitoring and detecting the initial signs of fruit injury from this pest. For instance, ‘Golden Delicious’ is the most preferred cultivar for oviposition, therefore it should be the cultivar of choice for monitoring CM injury in mixed-cultivar planted orchards. If it is not present in a block/orchard, then the next preferred cultivar for oviposition should be selected for examining CM injury or oviposition. In addition, results of these studies would be helpful in breeding programs, particularly in developing CM resistant apple varieties. As previously discussed, oviposition by CM is likely stimulated by fruit volatiles, and variations in the production and release of these volatiles from different apple cultivars may result in different oviposition preferences. Further investigations are needed to understand the biochemical as well as physical aspects of fruits and other factors involved in determining apple cultivar susceptibility for CM oviposition.To summarize, CM preferred to oviposit on later maturing cultivars ‘Golden Delicious,’ ‘Stayman,’ ‘York Imperial,’ ‘Fuji,’ and ‘Delicious’ (preferred cultivars) than early maturing cultivars, 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.


Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated. Bunyavirus infections cause febrile illnesses of varying severity worldwide; however, despite their public health importance most remain relatively unstudied. In order to clarify the genetic relationships among African orthobunyaviruses associated with human infection, we have sequenced multiple strains of Bwamba (BWAV), Pongola (PGAV) and Nyando virus (NDV). Based on genetic analysis we showed that, while different BWAV and PGAV virus strains are closely related, NDV strains were highly variable and warrant classification as two distinct virus species. In addition, sequencing of the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi (KKV) viruses showed that both are closely related to NDV. This was unexpected considering that these viruses were isolated in South America and Southeast Asia, respectively, and are mainly associated with bats. Further, our experiments also indicated that BWAV and PGAV, as well as NDV, MDCV and KKV, are able to infect both bat and mosquito cell lines, suggesting that ecological studies focusing on these potential host and vector species are warranted. In the future, the availability of complete genetic information for these viruses, together with an understanding of their genetic relationships, will aid in better defining the distribution and public health impact of these viruses. Bunyaviridae are a large, diverse group of more than 350 viruses divided into 5 genera, of which more than 150 belong to the genus OrthobunyavirusThe Phlebovirus) and Crimean-Congo Hemorrhagic fever virus , there are a number of viruses in the genus Orthobunyavirus that are also agents of human disease, with many of them being highly prevalent within their endemic areas. Among these viruses, by far the most prevalent appears to be Bwamba virus (BWAV), which has been reported to be among the most common arthropod-borne diseases in Africa In addition to the well-known agents of human bunyavirus disease in Africa, such as Rift Valley Fever virus  in South Africa Interestingly, a closely related virus, Pongola virus (PGAV), has been isolated from mosquitoes  is also capable of causing moderate-severe febrile disease in humans. Although to date only a single human case with multiphasic fever, myalgia and vomiting has been reported from the Central African Republic Orthobunyavirus is presently divided into 18 serogroups Tadarida plicata and Taphozous theobaldi) in Thailand and Cambodia Stricticimex parvus and Cimex insuetus) Until now, bunyavirus classification has relied almost exclusively on serological testing, which may include complement fixation, hemagglutination inhibition, immunofluorescence assay and/or viral neutralization assays, and on the basis of these assays, the genus de novo sequencing efforts While genome sequencing is known to be both a rapid and accurate means of identifying viruses, and is indeed the standard for the identification of most other virus families, it is dependent on the availability of sufficient pre-existing sequence data, something that is presently lacking for bunyaviruses. Indeed, where genetic analysis of these viruses has been performed it is often extremely limited and focuses almost exclusively on the S-segment. However, this issue has been increasingly recognized within the field and is beginning to be addressed, particularly as a result of large-scale In order to improve our understanding of the evolutionary relationships and genetic diversity among orthobunyaviruses causing human disease in Africa, and particularly the viruses of the Bwamba/Pongola virus and Nyando virus serogroups, we have undertaken the complete genome sequencing of multiple strains of each of these viruses. In addition, we have determined the first complete sequences of MDCV and KKV, which has allowed their definitive identification, and based on the genetic relationships identified in our analyses, we have begun to explore the possibility that additional host and vector species may be involved in the ecology of these viruses.ATCC CRL-1586), Tb 1 Lu  and RE05  were grown in Eagle's Minimum Essential Medium  supplemented with 10% fetal calf serum , 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin (Life Technologies) at 28°C in the presence of 5% CO2.Vero E6 , Division of Vector-Borne Diseases (DVBD) arbovirus reference collection and the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) arthropod-borne virus reference collection. Information regarding their origin is summarized in Virus stocks were grown in Vero E6 cells in DMEM supplemented with 2% FCS, 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and 10 µg/ml Mycokill AB . Virus growth was monitored based on the appearance and progression of cytopathic effect (CPE) in cells. When advanced CPE was observed (50–75% of cells detached), the culture supernatants were harvested for RNA isolation.g for 5 min at 4°C to pellet cell debris. For Sanger sequencing, RNA was then extracted using the QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer's directions. Alternatively, for Next Generation sequencing (NGS), samples were further purified and concentrated through a centrifugal filtration device (Millipore) prior to RNA extraction, as previously described Cell culture supernatants from infected cells were spun twice at 1,000× et al.5′-GTT TCC CAG TAG GTC TCN NNN NNN N-3′). RNA was then hydrolyzed in NaOH and the single-stranded cDNA (ss-cDNA) products purified using the QIAquick system (Qiagen). The resulting ss-cDNAs were randomly amplified using a 1∶9 mixture of the arbitrary 17-octamer primer and a primer targeting a specific 17-mer sequence (5′- CGC CGT TTC CCA GTA GGT CTC-3′). The resulting ss-cDNA templates were used as template for PCR using Platinum Taq polymerase. PCR products were purified using the QIAquick kit following the manufacturer's protocol (Qiagen) and used as template for sequencing on the 454 Titanium FLX sequencer (454/Roche Life Sciences).cDNA for NGS was synthesized using a previously described modification cDNA samples were quantitated using Picogreen reagent (Life Technologies) and prepared according to the Rapid Library Preparation Method Manual – GS FLX Titanium Series October 2009 (454 Life Sciences). A multiplex was titrated in medium volume emulsion (MVE) format to determine the optimal copy-per-bead ratio (CPB) which produced the best sequencing quality. A 454 Titanium sequencing run was then performed using 1 CPB.de novo assembled using GS De Novo Assembler v2.6 (454 Life Sciences) and CLC Genomics Workbench 4.0 (CLC Bio). Translated BLAST (blastx) was performed to remove non-viral contaminants and the initial assembly was performed using Sequencher v5.0 (Gene Codes). Assembled contigs were then verified, refined or corrected by mapping the 454 reads using GS Reference Mapper v2.6 (454 Life Sciences). Where needed, several rounds of manual assembly and trimming were performed in Sequencher with verification done using GS Reference Mapper to eliminate discrepancies or errors discovered during the prior reference mapping procedure.Genomic viral sequences were produced on the 454 FLX sequencer and Based on the assembled data obtained from NGS, primers for reverse transcription PCR (RT-PCR) and Sanger sequencing were designed (primer sequences available upon request). RT-PCR was performed with Superscript III reverse transcriptase (Life Technologies) and iProof DNA polymerase (Bio-Rad). The 3′ and 5′ termini of each genome RNA segment were amplified using both a 3′ and 5′ RACE approach based on ligation-anchored PCR, as previously described Orthobunyavirus from GenBank , NDV (ERET147), MDCV and KKV) or 5 d (BWAV and PGAV) prior to fixation overnight in 10% formalin containing 0.1% crystal violet (Sigma-Aldrich).The genome sequences determined in this study were deposited in GenBank under the following accession numbers : KJ867176, KJ867177 and KJ867178 ; KJ867179, KJ867180 and KJ867181 ; KJ867182, KJ867183 and KJ867184 ; KJ867185, KJ867186 and KJ867187 ; KJ867188, KJ867189 and KJ867190 ; KJ867191, KJ867192 and KJ867193 ; KJ867194, KJ867195 and KJ867196 ; KJ867197, KJ867198 and KJ867199 ; KJ867200, KJ867201, and KJ867202 ; KJ867203, KJ867204 and KJ867205 .In order to better understand orthobunyavirus evolution as it relates to the relationships between and degree of diversity among BWAVs, PGAVs and NDVs, we have determined the complete genome sequences for all 3 viral RNA segments from multiple strains of each of these viruses. In addition, we have determined the complete sequences of a single strain of each of the “uncharacterized” bunyaviruses MDCV and KKV  indicated a possible, but weakly supported, evolutionary relationship to the NDV group (data not shown). On this basis we additionally undertook full-length sequencing of a single strain each of MDCV and KKV using equenced . Howeverequenced –S4.Analysis of the genome structures for all of these viruses indicates that they are consistent with what is known for previously analyzed orthobunyavirus genomes . The S sIn order to apply rational criteria to the taxonomic assignments within these virus groups, we next analyzed the levels of sequence divergence between the different strains of BWAV, PGAV and NDV , S6, S7.Among the NDVs, the situation observed was rather different. While the NDV clade is clearly highly divergent from all other recognized virus groups, it also demonstrated much more divergence between strains. Analysis showed that the MP401 and UgAr1712 strains exhibit 92%–99% identity at the nucleotide level and 96%–100% identity at the amino acid level, consistent with the levels of conservation observed between BWAV and PGAV strains. Similarly, the ERET147 and YM176-66 strains showed the expected high levels of sequence conservation, with 80%–98% identity at the nucleotide level and 90%–98% at the amino acid level. However, between these groups much more substantial differences in sequence were observed and identity levels dropped to 61–89% at the nucleotide level and 57%–86% at the amino acid level, respectively. These levels are then very similar to those seen when comparing BWAV and PGAV, members of two different virus species, and suggest that these two NDV clades should also be recognized as distinct orthobunyavirus species.3′-AGTAGTGTAC…GCACACTACT-5′). In addition we found that the downstream 5 nt fit well to the sequences determined for Jamestown Canyon virus (JTCV), a member of the closely related California Encephalitis virus group. Further, where deviations from this prototype sequence were observed, compensatory mutations are found in the other UTR, which would then maintain base pairing at these positions. Such deviations from the established sequences of CEV group members were seen for the MDCV and KKV S segment UTRs and the BWAV and PGAV M segment UTRs  . Based oent UTRs . This obBeyond these well conserved terminal sequences we found that the UTR sequences exhibit a general lack of conservation in both sequence and length between different virus species. The 3′ UTRs varied in length from 24–85 nt , with the PGAV M segment 3′UTR being uncommonly long in comparison to other 3′ UTR sequences. Compared to the 3′ UTRs, the 5′ UTR sequences were generally much longer, ranging from 85–310 nt, and with sequences in excess of 200 nt determined for the BWAV, MDCV and KKV S segment 5′ UTRs and the KKV M segment 5′ UTR. Interestingly, despite the marked variability of these sequences between virus species, within a single species these sequences are in fact highly conserved. This can be clearly seen when examining the NDV(MP401) and NDV(UgAr1712) UTR sequences in comparison to those of NDV(ERET147) and NDV (YM176-66) . As suchThe genetic assignment of MDCV and KKV to the NDV clade was surprising, since neither of these viruses has been previously associated with transmission from mosquitos, which are the sole established vector for the transmission of BWAV, PGAV and NDV. In contrast, MDCV and KKV were both originally isolated from bats, which is rather unusual for orthobunyaviruses and has not been reported for BWAV, PGAV or NDV. However, it raises the possibility that these viruses might have a broader host/vector range than is currently appreciated.Rousettus aegyptiacus) and South American (Tadarida brasiliensis) bat species, as well as an Aedes albopictus mosquito cell line. Our data clearly indicate that all of these viruses have the ability to grow in both bat and mosquito cell types with titre increases in infected cells of between 1.5–4 logs between 0 and 72 hours post-infection  In our study, very little genetic divergence was noted among different strains of BWAV and PGAV, despite substantial differences in the location and/or time at which they were collected, indicating that molecular detection methods based on information from one or a few virus strains may be adequate to detect all of the genetic diversity present for BWAV and PGAV. However, given the small number of isolates available for each of these virus species, we cannot exclude the possibility that other strains showing much more substantial sequence divergence also exists. In contrast to the situation for BWAV and PGAV, for NDV a large degree of genetic divergence was noted among the strains analyzed. Indeed, this genetic divergence was to such an extent that, based on a cut-off value of 10% amino acid divergence, a division of the existing NDV strains into two distinct virus species would be warranted. Given the prevailing naming conventions among bunyaviruses  we would, therefore, propose that the prototype NDV (MP401), isolated from the Nyando river valley in vitro data also indicate that bats should be more closely considered in future ecological and epidemiological investigations looking at BWAV, PGAV and NDV, as all of these viruses display at least a fundamental ability to infect cells derived from various bat species. Further, we cannot currently exclude that productive infection of bat cells may in fact be a feature of a wide range of other orthobunyavirus species as well, opening up the possibility that a broader host range than is currently appreciated might generally exist for orthobunyaviruses, and specifically that consideration of bats as a potential host for other orthobunyaviruses may also be warranted.The placement of MDCV within a larger NDV clade is also supported by early serological data which indicated reactivity by immunofluorescence assay (IFA) and complement fixation (CF), but not neutralization (NT), to both NDV and CEV group viruses in vitro, and while these data only show that infection of mosquitos is fundamentally possible at a cellular level, when taken together with the close genetic relationship of these viruses to a number of well-documented mosquito-borne viruses, this clearly indicates that the possibility of mosquito-borne transmission should be considered in future field studies. Such studies will be particularly important given that evidence exists supporting the relevance of KKV for human infection. In particular, high levels of seroprevalence among Guano miners working in caves known to have infected bats and/or bedbugs have been reported While for MDCV no arthropod vector has yet been established, for KKV, until now only bedbugs have been identified as a potential vector species The inclusion of MDCV and KKV in the NDV clade was also surprising given their geographical distribution, having been isolated exclusively from South America and Southeast Asia, respectively. The existence of such a highly genetically diverse clade, which spans three continents, suggests on the one hand that additional related but unrecognized virus species likely exist, and also that transmission between these non-contiguous geographical locations has most likely been facilitated by an unknown host species common to all of these virus groups. In particular, transmission of KKV and MDCV via migratory bird routes through various flyways would appear possible (i.e. via the American Flyways<>Black Sea and Mediterranean flyways<>Asia Flyways<>American Flyways), and indeed infection of bird species has been shown to be possible for several other orthobunyaviruses, including Turlock virus and Mermet virus Orthobunyavirus genus. Further, our in vitro data, informed by the genetic relationships established as part of our sequencing efforts, have identified the possibility of infection with these viruses in an expanded range of host and vector species. The availability of complete genetic information for these viruses, as well as a better understanding of their genetic relationships, will be instrumental in assisting future surveillance efforts aimed at determining the distribution and public health impact of these viruses, as well as efforts in identifying the contributions of various potential host and vector species.Overall the data contained in this study have not only led to the genetic identification of two previously uncharacterized viruses, but in doing so, has considerably expanded our knowledge of virus diversity along the BWAV/PGAV and NDV genetic lineages. On this basis we have also presented arguments for a more refined and evidence based approach to the taxonomic classification of the viruses in these groups, something that is increasingly appreciated as being sorely needed within the Figure S1Phylogenetic relationships among the BWAV/PGAV and NDV/MDCV/KKV group viruses as inferred using the Neighbor-Joining method. Neighbor-Joining trees were constructed using the Tamura-3 parameter model on the nucleotide sequences of the S segment, M segment and L segment, as indicated. Bootstrap values based on 1,000 replicates are indicated. Viruses lineages added based on sequences determined as a part of this study are indicated in color: Bwamba virus (orange), Pongola virus (red), Nyando virus (blue), Mojuí dos Campos virus (purple), Kaeng Khoi virus (pink).(TIF)Click here for additional data file.Figure S2Comparison of 3′ and 5′ untranslated region (UTR) sequences in BWAV/PGAV and NDV/MDCV/KKV group viruses. The 3′ and 5′ UTR sequences are shown for the indicated viruses. For comparison, prototype members of the Bunyamwera virus group (Bunyamwera virus) and California Encephalitis virus group (Jamestown Canyon) are also shown. Previously identified highly conserved terminal sequences are highlighted in blue, while start/stop codons are indicated in green. Sequences that are predicted to form base-pairing interactions between the two UTRs (3′- 3 nt/8 nt/4 nt…4 nt/8 nt/3 nt- 5′) are delineated with black lines, and those nucleotides that differ from the CEV prototype sequence are indicated in red. Abbreviations: BUNV: Bunyamwera virus, BWAV: Bwamba virus, JTCV: Jamestown Canyon Virus, KKV: Kaeng Khoi virus, MDCV: Mojuí dos Campos virus, NDV: Nyando virus, PGAV: Pongola virus.(TIF)Click here for additional data file.Table S1GenBank accession numbers for viruses used in the phylogenetic analyses.(DOCX)Click here for additional data file.Table S2Homology among N and NSs open reading frame sequences within the NDV clade.(DOCX)Click here for additional data file.Table S3Homology among GPC open reading frame sequences within the NDV clade.(DOCX)Click here for additional data file.Table S4Homology among L open reading frame sequences within the NDV clade.(DOCX)Click here for additional data file.Table S5Homology among N and NSs open reading frame sequences within the BWAV/PGAV clade.(DOCX)Click here for additional data file.Table S6Homology among GPC open reading frame sequences within the BWAV/PGAV clade.(DOCX)Click here for additional data file.Table S7Homology among L open reading frame sequences within the BWAV/PGAV clade.(DOCX)Click here for additional data file.