Bunyaviridae has more than 530 members that are distributed among five genera or remain to be classified. The genus Orthobunyavirus is the most diverse bunyaviral genus with more than 220 viruses that have been assigned to more than 18 serogroups based on serological cross-reactions and limited molecular-biological characterization. Sequence information for all three orthobunyaviral genome segments is only available for viruses belonging to the Bunyamwera, Bwamba/Pongola, California encephalitis, Gamboa, Group C, Mapputta, Nyando, and Simbu serogroups. Here we present coding-complete sequences for all three genome segments of 15 orthobunyaviruses belonging to the Anopheles A, Capim, Guamá, Kongool, Tete, and Turlock serogroups, and of two unclassified bunyaviruses previously not known to be orthobunyaviruses (Tataguine and Witwatersrand viruses). Using those sequence data, we established the most comprehensive phylogeny of the Orthobunyavirus genus to date, now covering 15 serogroups. Our results emphasize the high genetic diversity of orthobunyaviruses and reveal that the presence of the small nonstructural protein (NSs)-encoding open reading frame is not as common in orthobunyavirus genomes as previously thought.The family  Bunyaviridae ranks among the largest families of RNA viruses. The family has more than 530 named members that are either assigned to the five included genera Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, or Tospovirus, or remain to be classified [i.e., Herbert virus, Kibale virus, and Taï virus, form a deeply-rooted sister taxon to the genus Orthobunyavirus [The family assified ,2,3. Manassified . In addiassified ,3. In adnyavirus .Bunyaviruses have tripartite, negative-sense, single-stranded RNA genomes ,8. The tOrthobunyavirus is the most complex genus of the family Bunyaviridae and currently contains approximately 220 named viruses. The majority of these viruses were assigned to more than 18 different serogroups based on presence, lack, or extent of serological cross reactions using various assays, such as complement fixation (CF), hemagglutination inhibition (HI), and neutralization test (NT) [The genus est (NT) ,12,13,14est (NT) ,22,23. Rest (NT) ,26,27,28Here we report the first coding-complete genomic sequences of 15 distinct orthobunyaviruses from the Anopheles A, Capim, Guamá, Koongol, Mapputta, Tete, and Turlock serogroups .Aedes and Anopheles mosquitoes mostly collected in South American and Caribbean countries. Eleven different viruses of this group have been assigned to the two species Anopheles A orthobunyavirus  and Tacaiuma orthobunyavirus  [Aedes mosquitoes collected in 1955 in Trinidad and Brazil [Viruses of the Anopheles A serogroup have been isolated from viruses) ,30,31,32d Brazil , but hasAcara orthobunyavirus , Benevides orthobunyavirus (Benevides virus), Bushbush orthobunyavirus , Capim orthobunyavirus (Capim virus), and Guajará orthobunyavirus (Guajará virus). These viruses are endemic to South and Northern America, where they are transmitted by mosquitoes among small vertebrates [Caluromys philander) in 1958 in Pará State, Brazil, and was also recovered from sentinel spiny rats (Proechimys spp.) and Culex mosquitoes. Antibodies to the virus were detected in spiny rat sera, but not in human sera in Pará State. Guajará virus was first isolated from a sentinel Swiss laboratory mouse in Pará State, Brazil. Additional isolates were obtained in South America from wild rodents and mosquitoes of different species, and antibodies against the virus were detected repeatedly in spiny rats [Capim serogroup viruses are classified into five species: tebrates ,2,32. Weiny rats ,34,35.Bertioga orthobunyavirus , Bimiti orthobunyavirus (Bimiti virus), Catú orthobunyavirus (Catú virus), Guamá orthobunyavirus , and Timboteua orthobunyavirus (Timboteua virus) [The Guamá serogroup viruses are distributed among five established species: a virus) ,2,32. Gua virus) ,38,39,47Koongol orthobunyavirus [Culex mosquitoes [Ficalbia mosquitoes in New Guinea [The Koongol serogroup currently consists of Koongol virus and Wongal virus, both of which are assigned to the species nyavirus ,2,32. Bosquitoes . Koongolw Guinea . HI testw Guinea ,49,50.Anopheles mosquitoes collected in Northern Territory and Griffith, Australia, respectively. Mapputta and Trubanaman viruses, which we have sequenced during this study, were initially isolated from Anopheles mosquitoes, respectively, collected in Queensland, Australia in the 1960s [The Mapputta serogroup currently has seven members  that have not yet been assigned to species ,24,51. Bhe 1960s ,41. Antihe 1960s .Batama orthobunyavirus (Batama virus) and Tete orthobunyavirus  comprise the Tete serogroup [Ploceus cucullatus) collected in South Africa in 1959 [Viruses of the species erogroup ,2,32. Toerogroup , have beerogroup ,55,56. T in 1959 .M’Poko orthobunyavirus  and Turlock orthobunyavirus  [Culex bitaeniorhynchus mosquitoes collected in Poona (today Pune), India in 1955 [Culex mosquitoes and birds in India and Malaysia. Anti-Umbre virus antibodies could be detected in sera collected from wild birds and sentinel chickens from Malaysia [Finally, we sequenced Umbre virus, a virus of the Turlock serogroup that is represented by viruses belonging to the two species e virus) . Turlock in 1955 . AdditioMalaysia .Bunyaviridae, Tataguine virus and Witwatersrand virus . Open reading frame (ORF) analysis and general work with assembled contigs were performed using the Lasergene 11.0.0  software package. After de novo assembly of trimmed reads, BLASTx  analysis was performed against orthobunyaviral sequences, and matching contigs were extracted. All resulting contigs contained parts of non-coding terminal regions and full-length ORFs corresponding to their matches in the BLASTx search. Identified ORFs were translated, and the resulting amino acid (aa) sequences were used in further analyses. Deduced aa sequences of the proteins encoded by sequenced orthobunyaviral genome segments and corresponding sequences of selected representatives of already characterized orthobunyaviruses were aligned using all available multiple sequence alignment methods implemented on M-Coffee server [Primary analysis of sequencing data and e server  and onlye server .Phylogenetic trees were inferred using MrBayes 3.2.4  under LGN-glycosylation sites were determined with the open-source NetNGlyc 1.0 Server [Signal peptide cleavage sites of orthobunyaviral glycoprotein precursors were determined from deduced aa sequences using SignalP 4.1 Server . Transme0 Server .Orthobunyavirus, including previously and newly characterized viruses, are presented in Segment-specific ORF-based phylogenies of the genus Genomic data of the Anopheles A serogroup orthobunyaviruses were thus far limited to S segment sequences of Anopheles A and Tacaiuma viruses . Here, wCaraparu orthobunyavirus , Madrid orthobunyavirus (Madrid virus), Marituba orthobunyavirus , and Oriboca orthobunyavirus  [All Guamá serogroup members studied in the present work  clustered together in all three phylogenetic trees , and forviruses) ,2. Groupviruses) .The measured divergence of Guamá serogroup viruses is somewhat inconsistent among phylogenies inferred for different viral proteins . For insThe most obvious example of this observation is the relationship between Catú BeH 151 and Guamá BeAn 277 viruses, which were found to be almost identical when compared by their N protein sequences, differing only by two aa (99.2% sequence identity). However, their glycoprotein precursor polyprotein and L protein sequences are more divergent . These data are in agreement with original studies that distinguished these two viruses in NT, but not in CF tests . The obsCapim and Guajará viruses fall basal to viruses of the Guamá group phylogenies inferred for N and L proteins, but are placed inside the Guamá serogroup in the glycoprotein precursor polyprotein phylogeny . Capim aThe obtained genomic sequences of Mapputta virus MRM186 is more than 99% identical to the sequences of the same isolate reported previously , and all158–S164) found in all other N protein sequences belonging to Tete group viruses, but not in the Tete virus SAAn 3518 sequence reported earlier. Our analyses reveal Bahig and Matruh viruses to be closely related, with 98.8%, 99.1%, and 87.2% aa identities among their N, L, and glycoprotein precursor polyprotein sequences. This observed relationship is in agreement with the results of CF tests, which showed that these viruses were practically indistinguishable, and HI tests, which proved them to be easily distinguishable [Sequence information for this serogroup was limited to the S segment of Tete and Batama viruses  and a paOrthobunyavirus.Two unclassified bunyaviruses, I612045 virus (GenBank HM627179-81) and Oyo virus (GenBank HM639778-80) form a monophyletic group with Tete serogroup viruses, with Oyo virus falling basal to the other viruses in this group. While the measured nt and aa identities of I612045 virus with other orthobunyaviruses clearly indicate its taxonomic status as a member of Tete serogroup, Oyo virus is indeed distinct and may represent a distinct species in the genus Here we report coding-complete sequences of all three genomic segments of Koongol and Umbre viruses. The sequence of the Umbre virus IG1424 M segment is more than 99% identical to a previously published M segment of the same isolate . PhylogeGenomic sequence information for the Turlock serogroup was thus far limited to partial Umbre virus M segment sequences. We expanded the sequence space of this serogroup by determining the coding-complete Umbre virus genome sequence and confirmed the relationship of Umbre virus to Kongool and Witwatersrand viruses .Orthobunyavirus.We present sequence information for Tataguine virus, the closest relative of which appears to be Lukuni virus, with 47.5% aa identity for N, 35.5% aa identity for glycoprotein precursor polyprotein, and 53.5% aa identity for L. Anopheles A and Anopheles B group viruses, along with Tataguine virus, form a monophyletic group with Tataguine virus at its base. Our data indicate that Tataguine virus may have to be assigned to a new species in the genus As mentioned above, based on the obtained phylogenies, Witwatersrand virus clusters together with Umbre (Turlock serogroup) and Koongol (Koongol serogroup) viruses in the same branching order independent of the analyzed protein. The closest relative of Witwatersrand virus is Koongol virus (48.5% to 59.6% aa identities for the three proteins), indicating that Witwatersrand virus should be classified as an orthobunyavirus.Orthobunyavirus with unique extensions predominantly located at the amino termini [The N proteins of bunyaviruses encapsidate viral RNA and are major CF determinants . The len termini .Forty-six aa of the N protein are conserved among the previously determined 51 sequences of orthobunyaviruses from the Bunyamwera, California, Group C, and Simbu serogroups . Our anaA number of researchers have evaluated whether NSs is involved in the immune response to orthobunyavirus infections. Despite not expressing NSs, Tacaiuma virus (Anopheles A serogroup) antagonizes host interferon (IFN) production through a yet unrecognized mechanism  and is aEach orthobunyavirus M segment contains a single continuous ORF encoding a glycoprotein precursor polyprotein that is cotranslationally cleaved into glycoprotein Gn, the nonstructural protein NSm, and the glycoprotein Gc . Among tRegions highly similar to the fusion peptide identified in La Crosse virus glycoprotein precursor polyprotein (positions 1066–1087) are present in the predicted polyproteins of the sequenced orthobunyaviruses of all serogroups. Ten out of twenty-two La Crosse virus fusion peptide aa are strictly conserved across the genus. This finding indicates that all orthobunyavirus Gc glycoproteins have analogous functions and thereby act as class II fusion proteins . SupportN-glycosylation of viral membrane proteins plays a crucial role in correct protein folding and functioning, including receptor binding, membrane fusion, and cell-penetration processes [N-glycosylation sites of the membrane glycoproteins of Bunyamwera virus (Bunyamwera serogroup) are indeed glycosylated. Glycosylation of Bunyamwera virus Gn’s N60 site is essential for correct protein folding of both Gn and Gc [Orthobunyavirus. Finally, with the exception of Koongol virus, transmembrane prediction using hidden Markov models (TMHHM) 2.0 generally predicted the same distribution and type of transmembrane regions for the glycoprotein precursor polyprotein sequences of the analyzed viruses, which included two transmembrane domains in Gn, three in NSm, and one in Gc. The Koongol glycoprotein precursor polyprotein has four predicted transmembrane regions, lacking two domains usually located at the C-terminal half of NSm.rocesses . All thrn and Gc . Interesn and Gc . NetNGlyThe L sequence aa lengths of the studied orthobunyaviruses are comparable to those of previously studied viruses, ranging from 2241 aa in the case of Lukuni virus to 2293 aa in the case of Umbre virus . Tete grBunyaviridae was originally established to group viruses that produce morphologically similar and often serologically cross-reactive virions [i.e., the currently accepted genera Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus [i.e., arenaviruses, emaraviruses, tenuiviruses, and Mourilyan virus [Bunyaviridae Study Group has therefore recently initiated discussions on a taxonomic re-evaluation of the entire bunyavirus-like supergroup with the ultimate goal to establish a novel taxonomy that adequately reflects the phylogenetic relationships of all these viruses. Unfortunately, the genomic “sequence space” of the supergroup is still very limited, thereby impeding those efforts.The family  virions . Largely virions ,12,13,14 virions ,88, whicspovirus . Recent an virus ,108,109.Bunyaviridae family possess segmented genomes, the phenomenon of segment reassortment plays a significant role in their evolution [Orthobunyavirus are intra-group reassortants. Our data show that all examined viruses of the Guamá serogroup are in all likelihood genome segment reassortants. Furthermore, viruses of the Guamá and Capim serogroups form a monophyletic lineage on the tree inferred for the glycoprotein polyprotein precursor protein (M segment), suggesting inter-group reassortment of M segments in their natural history. Another important observation that could be made is the classification of Witwatersrand virus and Tataguine virus as likely members of two new novel species in the genus Orthobunyavirus.Within the classic orthobunyavirus group, genomic sequence information was limited to viruses of 10 of 18 serogroups ,26,27,28volution . EarlierOrthobunyavirus, but may be important for differentiating pathogenic from nonpathogenic viruses.Finally, our data show that presence of an ORF encoding an NSs protein is not a universal feature for orthobunyaviruses. Among the viruses of 15 orthobunyavirus serogroups for which genomic data are now available, only viruses of eight serogroups along with Witwatersrand virus encode NSs proteins. Therefore, the presence or absence of NSs protein should not be considered as a taxonomic characteristic of the genus Orthobunyavirus.Our analyses advance the overall resolution of orthobunyavirus phylogeny. Although sequence information for the 3′ and 5′ genomic segment termini could not obtained in this study, we are confident that the determined phylogenetic placement on the phylogenetic tree of all viruses studied here will hold. Complementation of our results by genomic sequences of viruses from the remaining unsequenced orthobunyavirus serogroups should allow official taxonomic re-organization of the genus


Southern rice black-streaked dwarf virus (SRBSDV) in the white-backed planthopper (WBPH) and in rice plants both altered the vector’s feeding behavior. When viruliferous WBPH (carrying SRBSDV) were fed on uninfected plants, they spent more time in salivation and phloem sap ingestion than non-viruliferous insects. In comparison with uninfected plants, infected plants showed an arrestant effect on non-viruliferous WBPH for phloem sap ingestion. Differential feeding behaviors were also detected between the WBPH that inoculated or acquired SRBSDV and those that failed to. The WBPH that inoculated SRBSDV exhibited more probing bouts, salivation events and phloem sap ingestion events and longer salivation than those that failed to. The WBPH that acquired SRBSDV were quicker to reach phloem and spent more time in phloem sap ingestion than those that failed to. These behavior alterations in the vector may have adaptive advantages for SRBSDV transmission and spread success because greater salivation by viruliferous vectors on uninfected hosts will promote virus inoculation, whereas more sap ingestion by non-viruliferous vectors on infected hosts will promote virus acquisition.Plant viruses are primarily transmitted by insect vectors and virus infection may influence on the vectors’ feeding behaviors. Using an electrical penetration graph, we detected that infection with the  Pathogenic and parasite organisms may have interacted evolutionarily with their hosts and vectors to influence vectors’ performance and behavior favoring their spread1Southern rice black-streaked dwarf virus (SRBSDV), a newly reported Fijivirus of the family Reoviridae910Sogatella furcifera (Hovath) (Hemiptera: Delphacidae), is the only confirmed vector of SRBSDVNilaparvata lugens (Stål) (Hemiptera: Delphacidae), or the small brown planthopper, Laodelphax striatellus Fallen (Hemiptera: Delphacidae)The In the interactions between plant-virus-vector, a plant virus can exert an influence on its vector’s developmental fitness. Like other persistent propagative viruses13et al.Barley yellow dwarf virus (BYDV) and its aphid vector, Rhopalosiphum padi L. (Hemiptera: Aphididae)Myzus persicae (Sulzer)-Potato leaf roll virus (PLRV) pathosystemet al.Tomato yellow leaf curl virus (TYLCV) induced an arrestant behavior in the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) because viruliferous whitefly adults remained motionless for a longer time and moved more slowly than non-viruliferous whiteflies (not carrying the virus) after their first contact with eggplants, a non-host plant of this virus. Headspace volatiles from Luteovirus PLRV-infected mature leaves of Solanum tuberosum L. attracted the aphid M. persicae (Hemiptera: Aphididae)21A plant virus can also alter its vector’s behavior to influence the virus transmission and spread. One such behavior alteration concerns the vector’s orientation preference to virus-infected host plants. Wang Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) infected with the Bunyavirus Tomato spotted wilt virus (TSWV) exhibited an increased feeding time and frequency of all feeding behaviors versus uninfected maleset al.B. tabaci than in non-viruliferous whiteflies. Alvarez et al.M. persicae between PLRV-infected and uninfected potato plants but only after the infected plants showed visual symptoms of infection. More recently, Liu et al.B. tabaci in TYLCV-infected tomato plants. These studies, based on detailed recordings of specific probing and feeding behaviors, imply that infection with a plant virus can directly (through the infected vectors) or indirectly (through infected host plants) alter the vector’s probing and feeding behavior in a way that increases the transmission efficiency of the virus.Another manipulative behavior involves the vector’s probing and feeding behavior in response to its virus infection status. Male thrips R. padi, the duration of the E waveform (phloem sap sucking) generated by aphids that successfully acquired BYDV was longer than in those that failed to ref. Cicadulina spp. that acquired the Maize stripe virus (MSV) successfully generated an L2 waveform (similar to the E waveform of aphids) more frequentlyCucumber mosaic virus (CMV) acquisition by Aphis gossypii Glover (Hemiptera: Aphididae) and the generation of potential drop waveform (intracellular punctures)B. tabaci to tomato plants depends on the time duration spent in salivation into the phloem sieve elementsSuccessful virus inoculation and acquisition is associated with the vector’s specific feeding behavior. For example, in the acquisition of BYDV by In this study, the probing and feeding behaviors of the WBPH in response to SRBSDV infection status and virus transmission success were recorded using an electrical penetration graph (EPG) technique. These EPG recordings were compared between viruliferous and non-viruliferous WBPH on uninfected rice seedlings, non-viruliferous WBPH on infected and uninfected rice seedlings, viruliferous WBPH that inoculated the virus to uninfected plants and those that failed to, and non-viruliferous WBPH that acquired the virus from infected plants and those that failed to. The transmission and spread of plant viruses depend on the feeding behavior of their vectors. Hence, according to the VMH, we hypothesize that SRBSDV can alter the behavior of its vector to enhance its transmission.PDI, probing duration per insect) during the 6 h of the EPG recording period. When compared with the non-viruliferous WBPH, the viruliferous WBPH showed longer PDI on uninfected rice seedlings  of salivation and stylet movement  and N4-a . Likewise, the WDE in N4-a was shorter in virus-acquired insects  and WDI  and phloem sap ingestion (N4-b) compared with non-viruliferous insects. Similar results have been reported in the thrips F. occidentalis, where TSWV-infected male thrips spent more time feeding than uninfected malesB. tabaci, where TYLCV-viruliferous insects sustained a longer duration of the salivation phase in phloem sieve elements than non-viruliferous whiteflieset al.ucumber mosaic virus (CMV) and Potyvirus potato virus Y (PVY), which are transmitted by both Aphis gossypii Glover and Myzus persicae Sulzer, showed that the transmission of plant viruses from viruliferous insect vectors to uninfected plants was tightly related to the production of II-1 waveforms . In these studies, the plants are uninfected; the differences in probing behavior between the viruliferous and non-viruliferous vectors can result from the virus infection status of the vector, i.e. the viruses have directly altered the probing behavior of their vectors. These behavioral alterations may promote virus spread, since a long salivation duration preceding the phloem sap ingestion by the viruliferous vectors can help inoculate the viruses to uninfected plants.When viruliferous and non-viruliferous DFnp) and probed longer  in phloem sap ingestion (N4-b) and exhibited fewer probing events , more salivation (N2) waveform number and longer N2 waveform duration and greater stylet activity near and in the phloem region  by the beet leafhopper, Circulifer tenellus, depended only on the production of the D1 waveform  instead of on the duration of the D1 waveform. The difference may lie in the use of a different strategy by leafhoppers than by aphids and whiteflies for phloem ingestion; the phloem sap ingestion by the leafhopper is not necessarily preceded by salivation into the phloemIn contrast to the WBPH that failed to inoculate SRBSDV, those that succeeded in inoculation showed a shorter probing duration per probe PDP; , more prDFnp, WDI) in salivation (N2) and less event time (WDE) and total time (WDI) in stylet activities in the phloem region (N4-a) but more event time and total time in phloem sap ingestion (N4-b) than the WBPH that failed to acquire the virus Interestingly, the WBPH that acquired SRBSDV settled on rice plants without piercing activities for a shorter time . The virus stock culture was maintained using WBPH as vector and three-leaf to tillering stage rice seedlings  as host plants within 80 mesh insect-proof cages (50 × 50 × 50 cm) in a climate chamber at 27 ± 1 °C, a relative humidity (RH) of 75–80% and a photoperiod of 16L:8D. Stock cultures of WBPH exposed and not exposed to SRBSDV were also maintained under the same conditions in separate insect-proof mesh cages.th instar nymphs were transferred from a stock culture into glass tubes (2.5 × 15 cm) each with a rice seedling and 10 ml of Kimura solution. Newly emerged macropterous WBPH females were transferred to a new glass tube with a rice seedling within a 12 h period and then used in the experiments.Macropterous WBPH females emerged within 24 h from the stock cultures were used as the test insects in the EPG recordings. To obtain the females, 5th instar WBPH nymphs for 24 h. Ten days after the exposure, a leaf was cut from each of the seedlings and tested for the SRBSDV infection status using RT-PCR (SRBSDV positive or not). The infected and uninfected rice plants were maintained separately in insect-proof cages inside climate chambers.Potted rice seedlings (TN1) cultured in plastic pots (5 cm diameter × 7 cm height) were used in the EPG recordings. To obtain infected rice seedlings, the virus-free seedlings (approximately 20 d old) were individually confined with 3 viruliferous 4The SRBSDV-infection status of rice seedlings and WBPH were detected by one-step RT-PCR9-Ω input resistance in a Faraday cage . For the IAP, viruliferous WBPH feeding on uninfected rice seedlings were electrically monitored to obtain an EPG; and for the AAP, non-viruliferous WBPH feeding on infected rice seedlings were monitored. A control, i.e., non-viruliferous WBPH feeding on uninfected rice seedlings, was included in the EPG recordings.The feeding behavior of the WBPH during the SRBSDV inoculation access period (IAP) and acquisition access period (AAP) were recorded using a Giga-8 direct current electrical penetration graph (DC-EPG) amplifier with a 10et al.The obtained macropterous WBPH females were starved for 2 h (supplied with water only on filter paper in a flask) before the EPG recordings. To facilitate the attachment of thin gold wires to the planthoppers, each WBPH female was immobilized using a micro-vacuum pump  by suctioning on its abdomen. Then, one end of a gold wire (18 μm diameter × 3–4 cm length) was attached to the dorsal thorax of the insect using a drop of water-soluble silver glue and allowed to dry for approximately 10 s. The wired insect was then connected to the amplifier through a copper nail that was inserted into the EPG probe and placed on a rice leaf sheath of a potted plant. This treatment did not hamper the feeding of the planthopper. A copper wire (2 mm diameter × 10 cm length) connected to the amplifier and vertically inserted into the pot soil was used as the plant electrode. The EPG signals were digitized with a converter , and the gain of the amplifier was acquired and stored with the PROBE 3.4 software . The substrate voltage was adjusted so that the EPG signals fit into the +5 V to −5 V window provided by the PROBE software. A 1 h acclimation period was set between the wiring time and the beginning of the EPG recording as described by Moreno-Delafuente et al.Similar to previous reports38et al.et al.et al.DFnp, min); probing duration per insect , which is the sum of the durations of each event of all the waveforms made by an insect; probing duration per probe , which is the sum of the probing duration of an insect divided by the number of probes made by the insect; number of waveform events per insect (NWEI), which is the sum of the number of events of a particular waveform; waveform duration per event , which is the sum of the durations of each event for a particular waveform divided by the number of events of that particular waveform; and waveform duration per insect , which is the sum of the durations of each event of all the waveforms made by an insect. All the parameters were averaged across the number of insects, probes or events  transformationHow to cite this article: Lei, W. et al. EPG Recordings Reveal Differential Feeding Behaviors in Sogatella furcifera in Response to Plant Virus Infection and Transmission Success. Sci. Rep.6, 30240; doi: 10.1038/srep30240 (2016).


Despite accounting for approximately one fifth of all acute gastroenteritis illnesses, norovirus has received comparatively less attention than other infectious pathogens. With several candidate vaccines under development, characterizing the global economic burden of norovirus could help funders, policy makers, public health officials, and product developers determine how much attention and resources to allocate to advancing these technologies to prevent and control norovirus.We developed a computational simulation model to estimate the economic burden of norovirus in every country/area  stratified by WHO region and globally, from the health system and societal perspectives. We considered direct costs of illness  and productivity losses.Globally, norovirus resulted in a total of $4.2 billion (95% UI: $3.2–5.7 billion) in direct health system costs and $60.3 billion (95% UI: $44.4–83.4 billion) in societal costs per year. Disease amongst children <5 years cost society $39.8 billion, compared to $20.4 billion for all other age groups combined. Costs per norovirus illness varied by both region and age and was highest among adults ≥55 years. Productivity losses represented 84–99% of total costs varying by region. While low and middle income countries and high income countries had similar disease incidence , high income countries generated 62% of global health system costs. In sensitivity analysis, the probability of hospitalization had the largest impact on health system cost estimates , while the probability of missing productive days had the largest impact on societal cost estimates .The total economic burden is greatest in young children but the highest cost per illness is among older age groups in some regions. These large costs overwhelmingly are from productivity losses resulting from acute illness. Low, middle, and high income countries all have a considerable economic burden, suggesting that norovirus gastroenteritis is a truly global economic problem. Our findings can help identify which age group(s) and/or geographic regions may benefit the most from interventions. Diarrheal disease remains the fourth most common cause of mortality and second most common cause of morbidity worldwide in children under the age of 5 years. Despite There are several possible reasons for comparative lack of attention towards norovirus. First, disease burden has been challenging to estimate, in part because diagnostics of adequate sensitivity have not been widely available. Second, some associate norovirus primarily with outbreaks in cruise ships  and healthcare facilities; howeverWith a number of norovirus prevention and control measures currently under development , undersWe constructed a computational simulation model in Microsoft Excel  with the Crystal Ball add-in  to estimate the economic burden of norovirus for any country, from the third party payer, health system, and societal perspectives. The model first determines the number of norovirus illnesses and norovirus-associated deaths in each of four age-groups  in the specified country/area. Each norovirus case had probabilities of seeking medical care  and hospitalization. Additionally, each case had a probability of missing productive days .Third party payer or health system costs included all direct medical costs of illness . Societal costs included direct and indirect  costs. The cost per hospital bed day and duration of hospitalization were used to estimate hospitalization costs. Daily income served as a proxy for productivity losses associated with absenteeism and lost productive days due to norovirus illness and were accrued by all norovirus cases. A norovirus-specific premature death resulted in accruing the net present value of that person’s lifetime earnings, based on person’s age of death and his/her remaining years of life based on life expectancy. Costs are presented in 2013 US dollars, with past costs converted using country-specific Consumer Price Index (CPI) ratios, and future costs (future lifetime earnings) discounted at 3%, annually.All inputs were age- and country-specific when available; For young children in LMICs, the probability of seeking medical care for diarrhea  came froGross national income (GNI) per capita (Atlas Method), used to estimate daily income , came from the World Bank when notIn our baseline scenario we assumed that everyone with norovirus illness accrued lost productive days, used the healthcare seeking ratios found in th and 97.5th percentiles across the 10,000 Monte Carlo trials.For every simulation, the number of norovirus illnesses, deaths, and their associated costs were estimated for each country/area from each perspective and summed to give the total for all WHO regions, income strata, and worldwide. Simulation runs consisted of 10,000 probabilistic  trials, varying each parameter throughout their ranges to estimate uncertainty. We report the median and 95% uncertainty interval (95% UI), based on the 2.5Globally, norovirus was estimated to cause a median number of 699 million illnesses  and 219,000 deaths  across all ages per year. These cases and deaths resulted in a median $4.2 billion (95% UI: $3.2–$5.7 billion) in direct health system costs  and $56.2 billion (95% UI: $40.9–$78.3 billion) in productivity losses; thus costing society a total of $60.3 billion (95% UI: $44.4–$83.4 billion) annually. Approximately half of all productivity losses were due to mortality. When excluding hospitalization, norovirus resulted in a median $2.8 billion (95% UI: $2.1–$3.9 billion) in direct health system costs, costing society a total of $59.1 billion (95% UI: $43.4 to $81.8 billion).Globally, LMICs have a greater cumulative norovirus disease burden  compared to high income countries, but norovirus-associated costs were consistently higher in high income countries . HoweverFurther exploring the impact of missing productive days, global societal costs were $51.8 billion (95% UI: $39.5 –$69.8 billion), $43.9 billion (95% UI: $34.6 –$57.2 billion) and $35.9 billion (95% UI: $29.7 –$44.2 billion) when the probability of missing productive days was 75%, 50%, and 25%, respectively. While societal costs decreased (as a result of lower productivity losses), productivity losses still represented a substantial portion of the total burden as pre-mature mortality resulted in $17.8 billion globally. Even when only 25% of illnesses missed productivity days, productivity losses represented 86% and 91% of the total burden in high income and LMICs, respectively, as they are largely driven by pre-mature mortality.Worldwide, we estimate that the approximately 699 million norovirus illness and 219,000 deaths result in $4.2 billion (95% CI: $3.2–$5.7 billion) in health system costs and $60.3 billion (95% CI: $44.4–$83.4 billion) in societal costs annually. Productivity losses represent the largest portion of this economic burden in all regions–of which, half of losses were due to norovirus deaths. As most persons with AGE do not seek medical care and therefore do not incur healthcare costs, overlooking productivity losses would severely underestimate the true cost of norovirus illness. Total cost was most sensitive to hospitalization rates, probability of missing productive days, and care seeking rates. Lacking better data on how norovirus affects productivity, our baseline scenario assumed that everyone would lose productivity time each day ill (1 to 3 days). However, it may be unlikely that everyone with norovirus have a productivity decrement, thus additional scenarios evaluated the impact of this parameter. These scenarios also serve as a proxy for uncertainty in the labor force, especially in LMICs where productivity losses may be less of an issue if only one parent works. Total societal costs were still considerable, $35.9 billion (95% UI: $29.7 –$44.2 billion), when only 25% of illnesses missed productive days, as half of all productivity losses are due to norovirus mortality.Our results suggest that the economic burden of norovirus gastroenteritis may exceed that of other diseases that have received more attention. For example, the annual societal cost of rotavirus in LMICs is an estimated $423 million ($262 million to $590 million) and direct medical treatment costs results in an estimated $325 million ($202 to $453 million) in the absence of vaccination . GloballOur results also demonstrate the considerable economic burden of norovirus in both high income countries and LMICs, highlighting that norovirus is a truly global problem. When determining global and LMIC priorities, focusing on mortality and major chronic outcomes can overlook diseases such as norovirus AGE, for which severe outcomes are uncommon. Such self-limiting but high incidence diseases still result in productivity losses that cumulatively can lead to considerable burden to society. Tracking productivity losses can be challenging, but ultimately are important to consider. LMICs with fewer material resources and human capital may be particularly susceptible to productivity losses. As our sThese results can inform funding agencies and public heath bodies regarding where to best allocate limited resources and gauge investments and potential returns for interventions and control measures. Without additional investment and attention, some efforts may not be realized. This is especially important with vaccines for norovirus, which are in the development pipeline. DifferenAll models, by definition, are simplifications of reality and therOur results present an economic argument for greater consideration of norovirus. The large cost of norovirus overwhelmingly are from productivity losses resulting from acute illness. Thus, these results point to the importance of taking a broader view of economic impact that includes productivity losses. Productivity losses tend to go unrecognized, but make up 94% of the global economic burden of norovirus. Focusing only on medically-attended outcomes substantially underestimates the total economic impact of norovirus illness. Additionally, low, middle and high income countries all have a considerable economic burden, suggesting that norovirus gastroenteritis is a truly global economic problem.Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention, or the US Department of Health and Human Services.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Streptococcus pneumoniae is a common cause of child death. However, the economic burden of pneumococcal disease in low-income countries is poorly described. We aimed to estimate from a societal perspective, the costs incurred by health providers and families of children with pneumococcal diseases.We recruited children less than 5 years of age with outpatient pneumonia, inpatient pneumonia, pneumococcal sepsis and bacterial meningitis at facilities in rural and urban Gambia. We collected provider costs, out of pocket costs and productivity loss for the families of children. For each disease diagnostic category, costs were collected before, during, and for 1 week after discharge from hospital or outpatient visit.A total of 340 children were enrolled; 100 outpatient pneumonia, 175 inpatient pneumonia 36 pneumococcal sepsis, and 29 bacterial meningitis cases. The mean provider costs per patient for treating outpatient pneumonia, inpatient pneumonia, pneumococcal sepsis and meningitis were US$8, US$64, US$87 and US$124 respectively and the mean out of pocket costs per patient were US$6, US$31, US$44 and US$34 respectively. The economic burden of outpatient pneumonia, inpatient pneumonia, pneumococcal sepsis and meningitis increased to US$15, US$109, US$144 and US$170 respectively when family members’ time loss from work was taken into account.The economic burden of pneumococcal disease in The Gambia is substantial, costs to families was approximately one-third to a half of the provider costs, and accounted for up to 30 % of total societal costs. The introduction of pneumococcal conjugate vaccine has the potential to significantly reduce this economic burden in this society.The online version of this article (doi:10.1186/s12962-016-0053-4) contains supplementary material, which is available to authorized users. There was an estimated 826,000 deaths due to pneumococcal diseases. In the year 2000, representing more than 11 % of all childhood deaths with 90 % of deaths from pneumococcal pneumonia occurring in Africa . In addiIn spite of the extensive disease burden, data describing the economic burden of pneumococcal disease in sub-Saharan Africa are limited. A study in Kenya estimated the treatment cost for episodes of pneumonia in children to vary from US$54 to US$99 in regional and district hospitals and up to US$177 in the national hospital. For meningitis, the estimated costs were higher, US$189 to US$201 and up to US$284 in the national hospital . AnotherMost studies of the economic burden of disease or vaccine cost-effectiveness do not collect primary data on costs but instead rely upon other sources such as estimates from neighbouring countries often with a number assumptions , 10 or tEffective PCVs are now available and with the help of Gavi, the Vaccine Alliance, these vaccines are becoming part of routine immunisation in many developing countries . In 2009A study to determine the impact of PCV in The Gambia has been underway since 2008 . Our ecoThe study was undertaken in 2011 and 2012 at two sites, Basse Health Centre (Basse), in a rural town in upper river region (URR), The Gambia and Royal Victoria Teaching Hospital (RVTH) recently renamed Edward Francis Small Teaching Hospital in Banjul, the capital city. The health centre at Basse serves as a referral centre for all minor health centres in URR and RVTH serves as the main tertiary hospital for all regions.Treatment of children aged under 14 years in The Gambia is provided free of charge. Case management of pneumonia follows WHO’s Integrated Management of Childhood Illness strategy. Even though the costs directly related to treatment at health facilities are covered by the government, families incur out-of-pocket expenses and loss of income before presentation to hospital, while taking care of a sick child at hospital and after discharge. Additionally, sequelae following pneumococcal meningitis are common and result in substantial economic burden to families.Streptococcus pneumoniae was isolated from blood, and (4) an illness in which pathogenic bacteria was isolated from cerebrospinal fluid.Children were potentially eligible for the study if they were aged less than 5 years and attended Basse or RVTH. Patients attending RVTH were assessed for eligibility by study staff on a daily basis between 9am and 2pm. Children were given treatment at the outpatient department (OPD) or admitted to the ward according to the discretion of the attending clinician. Patients in Basse were enrolled in a surveillance project to monitor individuals with pneumonia, sepsis and meningitis . SurveilAfter confirmation of eligibility and informed consent, study staff interviewed the child’s caregiver. For hospitalised patients, data were collected for the day of admission and any days of illness prior to this first interview. Subsequently, caregivers were interviewed on a daily basis to obtain retrospective data during the previous 24 h, until the day of discharge. Caregivers of children treated at the OPD were interviewed on the day of presentation and after the discharge of patients. Staff conducted a home visit 7 days after discharge or outpatient visit and collected data on costs incurred during the days following discharge/visit until the day of the home visit. All data were recorded on standardised questionnaires.We collected data in order to determine costs per case. Data included staff cost, clinical consumables, drugs consumed by each patient, diagnostic tests, the number of days on intravenous fluid, number of days on oxygen therapy, and the length of hospital stay. We used the unit costs of each medication provided by the national pharmaceutical service which procures and distributes drugs to all government facilities , as did the PATH Research Ethics Committee (HS 560).We aimed to collect cost data from a sample of 100 pneumonia cases at each centre, 50 inpatient and 50 outpatient. Assuming a mean cost of U$100 per case with a standard deviation of US$33 , enrolmeAll costs were presented in total, mean, median, and inter quartile range (IQR) by facility and by diagnosis. To determine the total provider costs, the estimated average treatment cost for each diagnostic category was added to the estimated bed day cost multiplied by the average duration of admission for each diagnostic category. Estimation of costs to the caregiver and family associated with sequelae following bacterial meningitis was calculated in a similar manner. We placed a value on the time loss based on the daily wage or how much the caregiver would pay for the work to be carried out, whichever was provided in the questionnaire, both options were available . The totTwo different scenarios were analysed. One scenario assumed lower levels of access to diagnostic tests. Given the use of X-ray and microbiology investigations in Basse was greater than at the RVTH and other facilities, a sensitivity analysis was conducted based on utilization of these investigations at RVTH. In a second scenario, we compared costs for diagnostic test from private laboratories to costs at the MRC which were used in the base case analysis.A total of 340 patients were recruited, 197 (57.9 %) from Basse and 143 (42.1 %) from RVTH. There were more males at both sites. Overall 57.4 % of the children were male. The median age (IQR) in months was 9 (4–18) and 10 (4–24) in Basse and RVTH respectively. The age distribution was not different between the two sites . Table The average inpatient stay was 5.8 days; 4.6 days in Basse and 7.6 days at RVTH. The maximum stay was 35 days. Thirty-six (10.6 %) children died and one absconded from hospital. Ten (4.2 %) children died on the same day they were admitted. The children who died had an average length of stay of 3.9 days compared to 6.1 days for children who survived . Deaths were more common among the meningitis cases 16/29 (55.2 %) compared to sepsis (10/36) and pneumonia (10/175) cases . No patient who was treated for pneumonia as an outpatient died during the study period.The mother was the caregiver for 90.6 % of cases and the father for 6.8 %. On average, there were 12 (IQR 6–15) adults and 15 (IQR 7–20) children aged under 18 years per household in Basse and 5 (IQR 3–7) adults and 6 (IQR 3–7) children per household in urban areas. The average daily household expenses were greater in Basse at US$9.05,  than RVTH at US$5.40, . The per capita expense per day based on the size of the household however was US$ 0.49 and US$ 0.33 at Basse and RVTH respectively.Thirteen percent of households  reported that there were one or more days in the previous month when there was no food in the house. The source of income to cover medical expenses was exclusively from current income in 25.7 % of households, savings 21.0 %, cutting down other expenses 10.1 %, and borrowing or donations from relatives and friends 6.2 and 18.5 % respectively. Selling assets was not common (1.1 %) and 17.4 % had a combination of financing strategies.The paediatric bed occupancy rate at RVTH and Basse were 0.88 and 0.95 respectively. Aggregating the estimated costs per patient day for food, laundry, and personnel, the estimated cost per paediatric patient day at Basse was US$5.29 and US$4.37 in RVTH.The distributions of costs were generally right-skewed. The average total provider treatment cost for outpatient pneumonia, inpatient pneumonia, pneumococcal sepsis and bacterial meningitis were US$5.50, US$60.90, US$90.90 and US$131.00 in Basse and US$9.80, US$67.90, US$51.60 and US$104.20 at RVTH respectively per patient  and pharmacies (16.5 %) were common. In the urban area, the average cost for treatment at private clinics was US$19.00. In the rural area, the average cost of treatment from a traditional healer prior to admission was US$8.80  of these children were traced, eleven had died (23 % CFR) two had travelled out of the country, and two did not consent. In total, twenty-five children, 18 from Basse and 9 from RVTH were followed up.The mean duration between the onset of meningitis episodes and the interview was 27 months (includes time on admission and maximum 24 months after discharge). The average age of the children was 34 months, . The average cost to the families for taking care of the children during the acute phase was US$35.6; (US$21.30 in Basse and US$61.00 at RVTH). The average costs due to disability over the first 6 months period after discharge was US$26.0 and the average cost per patient (acute phase and disability) was US$62.5 (Table Fifteen out of the twenty-seven (55.6 %) children examined had at least one form of neurological deficit (data not shown). On average, a child could not perform any of their usual activities for a period of 10 days (range 1-90) after the onset of the episode of meningitis, and for 28 days (range 0–480 days) children could not perform some activities. Only three households had a family member that was unable to work because of the need to care of the disabled child. The estimated mean loss of earnings per household was US$2.79 over 6 months which translates to an average of US$174 throughout the life of the child. On average, each household spent less than 1 day per month at health facilities providing care due to the child’s disability.A sensitivity analysis was conducted based on varied utilization of diagnostic tests at the two facilities. The first scenario was based on the rates of diagnostic testing at RVTH where only 2 % (2/93) of inpatients had a blood culture. We thus assumed that 2 % of inpatients in Basse had a blood culture. Under this scenario, the total cost for blood culture in Basse declined from US$2250 to US$50 Table . There wIn this study, we have filled an important evidence gap by estimating the costs of a range of pneumococcal diseases, from a societal perspective, in a typical sub-Saharan Africa setting. We have included the costs to the health care provider, out-of-pocket costs incurred by families, productivity loss and also assessed the economic burden associated with sequelae following bacterial meningitis.The provider costs associated with the treatment of pneumococcal diseases in the Gambia are substantial, ranging from US$5 for outpatient pneumonia to a peak of US$130 for hospitalized pneumonia patients in urban settings. Out-of-pocket costs to families are also high, with expenditure between $2 for children treated for pneumonia as outpatients and US$60 for children with meningitis.The average provider costs for one case of outpatient pneumonia was US$8. Outpatient pneumonia is the most common of the disease categories and therefore despite the cost per case being relatively small, the overall cost to the provider is substantial. At RVTH, there are approximately 30 cases of outpatient pneumonia for each case of meningitis (median cost US$85), consequently the economic burden due to outpatient pneumonia was more than five times that of meningitis. In the case of meningitis, the average cost for treating one case was higher than that for inpatient pneumonia nevertheless, given that the number of pneumonia admissions is 15 times that of meningitis admission, the overall economic burden for pneumonia admissions was about ten times greater.Similar to other African studies, meningitis was the most expensive condition to treat , 18. In The treatment protocol for pneumonia and meningitis was similar across all surveyed sites; outpatient pneumonia was treated with cotrimoxazole or amoxicillin, while meningitis was treated with benzyl penicillin and chloramphenicol. The availability of X-ray and microbiology tests however varied between locations as did the provision of food for patients. The health facility survey showed however that national treatment guidelines for pneumonia and meningitis are used throughout the country, therefore our estimates for drug costs can be generalized within the country.Studies in Colombia and India have shown differences in treatment costs at tertiary facilities compared to the secondary and primary level facilities , 20. ThiWe showed that provider costs represent 50 to 70 % of the societal costs. Out-of-pocket costs ranged from one to ten times of the average daily household expenses (US$5 to US$9). Daily household expenses were higher in Basse where the household size was larger.The findings of this report have a number of limitations. We enrolled few cases of pneumococcal sepsis and bacterial meningitis. Sepsis cases were particularly low at RVTH reflecting the lack of reliable microbiology services. We identified a total of 36 cases of pneumococcal sepsis and the median costs for diagnostic tests were US$49 and US$12 at Basse and RVTH. Most of the pneumococcal sepsis cases occurred in children admitted with pneumonia, so it was not unexpected that the costs of these two conditions, inpatient pneumonia and sepsis were similar. We included all cases of bacterial meningitis identified during the study period. Of the 29 bacterial meningitis cases, the mean provider treatment costs ranged from US$104 to US$131. In general, antibiotic treatment duration for pneumococcal meningitis is longer than for other bacteria . The samThe economic burden of pneumococcal diseases may be underestimated since we know that not all cases present to health facilities . A healtThe study excluded some cost components, such as costs of buildings, equipment, maintenance, and utilities from the main analysis. We did not collect burial costs at Basse however, if we extrapolate the average burial cost for children at RVTH to Basse, the family out-of-pocket cost increases by another US$650.Our difficulty in tracing children for follow-up after pneumococcal meningitis is likely to reflect the high mortality associated with this condition. Even though we confirmed 11 deaths, it is possible that more of the children not traced (23/70) had died. For those children that were followed up, the estimated monthly costs of providing care for the child was around US$3. For one in ten families, one family member was not able to work due to the need to provide special levels of care for the child.Clearly, pneumococcal meningitis continues to be associated with substantial economic and social burdens for families, even when children survive the acute illness. Meningitis is such a dramatic event that mothers would remember details of their child’s illness but we cannot rule out recall bias. Mothers were to give costs at the time of the illness but it is possible that they stated current costs rather than actual costs incurred at the time of the incidence resulting in overestimation of costs due to inflation over time.Our findings demonstrate that pneumococcal disease in The Gambia is associated with substantial economic costs of more than a US$100 for admitted cases. The health system meets 50 to 80 % of these costs. Out-of-pocket costs were one to ten times the average daily household expenditure. Compared to Kenya and Pakistan, fewer costs were passed on to caregivers by the health system , 18. TheThe ultimate aim of economic analysis is to improve and support decisions around allocation of health resources. Understanding the costs associated with treating pneumococcal disease highlights the magnitude of the costs that can be averted when these diseases are prevented. The economic burden numbers presented in this analysis are, in aggregate, smaller than are reported in similar analyses in other larger countries. However, the economic burden of childhood pneumococcal disease is significant both for households and for the country as a whole. From the household perspective, out-of-pocket costs ranged from one to ten times daily household expenditures. These costs can be dramatic for the economically disadvantaged. Likewise, the economic burden of one case of inpatient pneumonia (US$109) is much larger than The Gambia’s health expenditure per capita of US$29 . These nThe economic burden of pneumococcal diseases in The Gambia is substantial. Apart from the significant costs to the health care providers, families of children with pneumococcal diseases incur considerable expenses during the course of treating the sick child. The introduction of pneumococcal conjugate vaccine has the potential to significantly reduce this economic burden.


As reported: “A health care worker at Texas Health Presbyterian Hospital, who cared for dying Ebola patient has been tested positive for the virus after a preliminary test, officials said early Sunday. If confirmed, it would be the first known person-to-person transmission of the disease in the United States” .Every day people hear bad news around the distribution of Ebola in Africa and also the possibility of its changing to other continents. Coming up a successful remedy for this horrible disease of the century is expected but inventing and producing adequate medicines to be usable for the increasing number of patients may have challenges. The only available drug “Zmapp” has been produced by the biotech firm “Mapp Biopharmaceutical Inc.”, which is based in San Diego. “Mapp Biopharmaceutical” is working with the “National Institutes of Health and the Defense Threat Reduction Agency” that has followed to discover and develop an Ebola medicine for several years . NeverthDespite the enormous attraction of research on the Ebola drug discovery and development, running on this virus is not simply accessed, because it requires biosafety level 4-equivalent containment. Unfortunately, despite high standards of security in such labs, research laboratory workers are still at risk of contracting Ebola hemorrhagic fever especially during animal experimentation. Until 2011, three laboratory accidents with Ebola virus were documented in the literature and one of them led to mortality . That exAs of 1976 (the date of Ebola emerged), Ebola hemorrhagic fever affected poor countries and such a neglected diseases have not been a research priority for pharmaceutical companies  but now Undoubtedly, Ebola epidemic in 2014 is the biggest epidemic of this virus, so far, since multiple countries in the West-Africa have been feigned. Established on the present information, a few events have been reported in Nigeria and a single case reported in Senegal but fortunately not further spread in those lands. Nevertheless, the first “travel-associated” case of Ebola was diagnosed in the United States on September 30, 2014. As of the date of writing this article, it is described that the US Center for Disease Control and Prevention (CDC) and collaborators are taking precautions to prevent the spread of Ebola within the US in association with other governmental agencies, the World Health Organization (WHO), as well as other domestic and international collaborators. Furthermore, CDC has outspread teams of health care professionals to West-Africa and other affected countries .Historically for the first time, Ebola (referred to the figure of “Ebola River” in Zaire) was struck in Sudan and Zaire 1976). Bibliography shows that the first outbreak of Ebola (Ebola-Sudan) infected over 284 people , while the second Ebola virus appeared in Yambuku, Zaire, Ebola-Zaire (EBOZ) some months later with the highest mortality rate (88%), infected 318 people. Notwithstanding the great efforts of dedicated researchers so far, its natural reservoir has never been set up. The third form of Ebola , was found in 1989 in infected monkeys that were imported into Virginia from the Philippines. All the same, a few individuals infected and never developed Ebola hemorrhagic fever (EHF). The last known strain of Ebola  was emerged in 1994 during a necropsy on a dead chimpanzee from the Tai Forest, Cote d’Ivoire. The etiologist who did necropsy accidentally infected herself [. BibliogAn overview of chronology of Ebola, published by US CDC , expressBats are known the most probably natural reservoirs of Ebola virus, followed by plants, arthropods, and birds. In the first epidemic, bats were found in the cotton manufactory, in which the first cases of infection were observed 1976, 1979). In reality, bats are the only infected animals with no clinical signs among 24 plants and 19 vertebrate species that experimentally inoculated with Ebola virus 6, 1979. . MoreoveThe Ebola virus was thought to be a new strain of the Marburg virus, but it was renamed “Ebola virus” in 2010 referred to the name of “Ebola River” in its original country, Congo (formerly named Zaire). In fact, this virus is just a single member of the species “Zaire Ebolavirus” in the genus Ebolavirus, family Filoviridae. The Zaire species of the Ebola virus are the most dangerous, causing almost 90% mortality rate . The genLiterature reveals filoviruses have a broad spectrum cells to target in susceptible host species, of which monocytes, macrophages, dendritic cells (DCs), hepatocytes, adrenal cortical cells, fibroblasts and endothelial cells. The replication of virus is centered around the cells of the mononuclear phagocyte system resulting in quick circulation and widespread transferring throughout the host body . The EboAs literature shows, filoviruses are able to enter the target cells through several uptake mechanisms such as lipid raft-dependent and receptor-mediated endocytosis, as well as macropinocytosis. When viruses fuse into cellular membrane, nucleocapsids are released into the cytoplasm and play a role of template for transcription and replication. Concisely, the replicated RNAs are encapsidated by the nucleocapsid proteins while nucleocapsids are transported to the locations of viral release, where budding is ongoing. Viral assembling and budding might happen either at intracellular membranes (multivesicular budies) or at the plasma membrane ,17. As aInvestigations of filovirus showed that they exploit the infected cells to replicate successfully that potentially may lead to cell destruction. Intriguingly, viruses exhibited mechanisms to avoid cell death, because destroying the infected cells inhibits the production of progeny viruses. The mentioned mechanisms have been developed through a complicated interaction between viruses and cell death signaling pathways . HoweverThere is evidence that Ebola virus surface protein (GP) plays a role in cytotoxicity and cell damage, inducing cell rounding and detachment as well as membrane permeabilization. Furthermore, GP expression can down-modulate the cell surface proteins such as adhesion molecules, MHC class I proteins, and EGF receptor. Literature demonstrated that the GP-induced cytotoxicity may happen due to interaction of GP with the GTPase dynamin, resulting in noise with the intracellular trafficking of cell surface proteins . On the It is easily-documented that small interfering RNAs (siRNAs) and phosphorodiamidate morpholino oligomers (PMOs) could target Ebola virus RNA polymerase L protein to prevent disease in nonhuman primates ,24. ThesAlthough there is no specific approved medicine, early supportive care, rehydration and symptomatic cures such as using RBC, platelets or fresh frozen plasma, as well as heparin to prevent disseminated intravascular coagulation, and clotting agents to decrease bleeding can be helpful . Maintaiin vitro on Ebola infected mice. The most recent news show that this drug has been successful in the treatment of a French woman got Ebola in Liberia. The woman received three experimental drugs for the treatment that one of them was favipiravir [Presently, there is no ground for using alternative therapies or traditional medicine for prevention or treatment of Ebola hemorrhagic fever. US FDA advises people to be careful of advertisements for the benefits of different anti-Ebola products . Neverthipiravir ,30.BCX4430 , brincidofovir  and lamivudine  have recently been reported to show positive results in treatment of Ebola-infected patients . In factin vitro-effective known drugs that are currently being used for other indications including ion channel blockers (for treatment of heart arrhythmias) like amiodarone, dronedarone and verapamil, which could block up the entry of Ebola virus into cells [in vitro and in vivo [Alongside other antiviral drugs that are under investigations, scientists could find a number of to cells . In sum, in vivo . Since tin vitro and in vivo experiments. Successfulness of natural proteins like “Scytovirin” that is a cyanobacterial based (95-amino acid) antiviral candidate leads scientists to another group of compounds which could be produced through biotechnological approaches.It seems that patients, who have rescued from Ebola after infection, should possess strong immunity system that acted faster than Ebola virus and restricted its destroying activity. Therefore, synthetic or naturally immunostimulats may help patients in combination therapy with anti-Ebola medicines. Furthermore as mentioned above, if estrogen receptor modulators (like clomiphene) would probably be effective in clinical trials, other phyto-estrogens from different classes of secondary metabolites like isoflavonoids and phytosterls could be candidates for Although there is some information about the Ebola virus and its interaction with infected or non-infected cells, its target cells, stages of the replication cycle, and its cytopathic effects, until now we are far from having an efficient medication to control the virus outbreak. To be prosperous in the founding of a medicine for Ebola, the signaling pathways in the infected cells that contribute in replication and fate must be better elucidated. There are promising candidates in clinical trials for prevention of the disease like DNA vaccines or vaccines derived from adenoviruses, vesicular stomatitis Indiana virus (VSIV) or filovirus-like particles (VLPs). These vaccines could protect nonhuman primates from Ebola and hopefully can be engaged in human -40.


Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels.Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big BlueTM transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores.As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis infection increases host mutation frequency are warranted.These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which  Microsporidiosis in the immunocompromised first became evident during the Acquired Immunodeficiency Syndrome (AIDS) pandemic, but remains a concern in Human Immunodeficiency Virus (HIV)-infected individuals without access to therapy, as well as those immunosuppressed due to cancer therapy, diabetes, organ transplants, and autoimmune disease . Howeverreatment . As chroreatment , these dreatment . Thus, mChlamydia trachomatis which, like many microsporidia species, replicates in a cytoplasmic parasitophorous vacuole (PV), increases host cellular chromosomal instability in culture [in vivo [An estimated 2 million infectious disease-linked cancer cases occurred worldwide in 2008, many attributable to intracellular pathogens like Human Papilloma Virus . Infecti culture  and is a[in vivo . Like ot[in vivo ,10. Mult[in vivo . Microsp[in vivo  - an imp[in vivo . Though [in vivo . Thus, win vivo mutational frequency in response to genotoxic insults and contains ~40 chromosomally-integrated copies of the λ-phage genome as a mutation target [cII locus will not plaque at 24°C in G1250 E. coli, but mutational inactivation of the cII gene allows lytic replication at 24°C. In contrast, both cII mutant and cII-wild type phage produce plaques at 37°C [cII-mutant plaques obtained at 24°C by the total number of plaques at 37°C. We obtained heterozygous Big Blue™ murine embryonic fibroblasts (BB-MEFs) by crossing homozygous male Big Blue™ mice with female BALB/c mice. The tissue, minus head and peritoneal cavity contents, from 2–2.5-week-old embryos was minced and trypsinized. Single cell suspensions were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and frozen in liquid nitrogen until use.The Big Blue™ transgenic mouse  measures n target . After m at 37°C . Thus, BE. intestinalis spores were propagated in Vero African Green Monkey kidney cells (ATCC CCL-81) cultured in DMEM with 2% FBS. After 10–12 days in culture, spores were harvested by centrifugation, washed with 0.25% sodium dodecyl sulfate and sterile water, counted and stored as described [E. intestinalis spores  were inactivated by 1.14 J/cm2 of ultraviolet (UV) light in a Spectroline XL-1500 cross linker and did not produce PVs in subsequent infections . In preliminary time course studies, 42 hours post-infection (hpi) was the latest time at which no difference in host cell death between mock- and E. intestinalis BB-MEFs was observed. Genomic DNA, purified from replicate cultures, was packaged into λ phage capsids in vitro. G1250 E. coli suspensions were infected with phage populations, plated in soft agar and incubated either under non-selective (37°C) conditions for 16 h or selective (24°C) conditions for 48 h [cII mutation frequency was determined as described above. Interestingly, the cII mutation frequency was increased by 2.5-fold in E. intestinalis productively-infected versus mock-infected cells . In contrast, the mutation frequency in BB-MEFs exposed to E. intestinalisUV was similar to that in mock-infected cells . These data indicate that: i) microsporidia infection increases host nuclear mutation frequency 2–3 fold; and ii) the observed mutation frequency increase depends upon infection with viable spores.To ascertain infected cell mutation frequency, BB-MEFs were mock-infected (exposed to medium without spores), for 48 h . All putE. intestinalis-infected BB-MEF cells were stained with Uvitex 2B  and DAPI (to label host cell nuclei), and intracellular microsporidia PVs and host nuclei were counted. Approximately 8% infection was achieved in E. intestinalis-infected BB-MEFs at 42 hpi  this increase requires some post-attachment step(s) in microsporidia development. Because the Big Blue™ assay does not detect mutations: i) in other parts of the phage/host genome; or ii) that do not alter cII gene function, these data likely underestimate MEF nuclear mutation frequency. However, this assay determines whether mutation frequency changes under different experimental conditions – such as infection with various pathogens. For example, gastric infection with the bacterium Helicobacter pylori and liver infection with the fluke Fasciola hepatica increase Big Blue™ mouse mutation frequencies 5-fold and 2-fold in vivo, respectively [E. intestinalis increases mutation frequency is within the range observed for other pathogens [These data demonstrate that: i) ectively ,20. Althathogens ,20 and tathogens . It is lH. hepaticus and other Gram-negative pathogens secrete cytolethal distending toxins that damage host DNA and increase host cell genomic instability [E. intestinalis PV may interfere with host DNA segregation, as does that of C. trachomatis[Microsporidia infection could increase host cellular mutation frequency by several mechanisms. First, microsporidia-infected patients have elevated levels of reactive chemical species , suggesttability . Notablytability , indicatachomatis. As the Microsporidia can establish chronic infections , even afEncephalitozoon intestinalis; AIDS: Acquired immunodeficiency syndrome; HIV: Human immunodeficiency virus; PV: Parasitophorous vacuole; BB-MEF: Big Blue™ embryonic fibroblast; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; UV: Ultraviolet; E. intestinalisUV: UV-inactivated E. intestinalis spores; h: Hours; hpi: Hours post-infection; H. hepaticus: Helicobacter hepaticus; C. trachomatis: Chlamydia trachomatis; DAPI: 4’, 6-Diamidino-2-Phenylindole, Dichloride.E. intestinalis: The authors declare that they have no competing interests.CL participated in the experimental design and performed microsporidia infections, percent infectivity and cell death assays, statistical analyses, drafted the figures, and assisted in manuscript preparation. MS performed animal husbandry tasks, BB-MEF isolation/culture, and quantified mutation frequencies. RVS designed the overall study, performed data analysis/trouble shooting, drafted part of the manuscript, and provided critical reagents. JRH designed experiments, analyzed data, drafted part of the manuscript, reviewed the manuscript and figures, and provided critical reagents. All authors reviewed the manuscript before submission. All authors read and approved the final manuscript.E. intestinalis (E. int.)-infected; or iii) UV inactivated, E. intestinalis (E. int.UV)-infected BB-MEF monolayers. At 42 hpi, BB-MEF cultures on glass coverslips were fixed with 1:1 acetone: methanol and DAPI stained. Stained cells were mounted in Vectashield  and the number of host cell nuclei were counted in 20 random 630x fields per coverslip using an Olympus BH-2 fluorescent microscope. Host nuclei/reticle was then calculated for each group and plotted. The results shown are averaged from 3 replicates in 3 independent experiments. Means were compared with the unpaired Student’s t test using Microsoft Excel; p values below 0.05 were considered significant. NS = not significant.Microsporidia infection does not induce host cell loss at 42 hpi. Host nuclei counts were performed on: i) replicate mock (mock)-infected; ii) Click here for file


PNPLA3 I148M sequence variant favors hepatic lipid accumulation and confers susceptibility to hepatic fibrosis and hepatocellular carcinoma. The aim of this study was to estimate the effect size of homozygosity for the PNPLA3 I148M variant (148M/M) on the fibrosis progression rate (FPR) and the interaction with age at infection in chronic hepatitis C (CHC).The FPR was estimated in a prospective cohort of 247 CHC patients without alcohol intake and diabetes, with careful estimation of age at infection and determination of fibrosis stage by Ishak score.PNPLA3 148M/M was associated with faster FPR in individuals infected at older age , but not in those infected at younger age (p = ns). The negative impact of PNPLA3 148M/M on fibrosis progression was more marked in subjects at risk of altered hepatic lipid metabolism . At multivariate analysis, PNPLA3 148M/M was associated with FPR , independently of several confounders, and there was a significant interaction between 148M/M and older age at infection (p = 0.025). The association between 148M/M and FPR remained significant even after adjustment for steatosis severity (p = 0.032).Older age at infection was the strongest determinant of FPR (p<0.0001). PNPLA3 I148M variant and age at infection in determining fibrosis progression in CHC patients.We observed an interaction between homozygosity for the  Hepatitis C virus (HCV) infection affects roughly 130–170 million people worldwide. Individuals with chronic HCV-related hepatitis (CHC) are at high risk of liver fibrosis, an in about 20% of cases CHC progresses to cirrhosis. Therefore, HCV is a leading cause of end-stage liver disease and hepatocellular carcinoma However, even after taking into account all known viral, host related, and environmental factors, the rate of progression to advanced fibrosis is highly variable among individuals, thereby suggesting a role for inherited variants. Indeed, single-nucleotide polymorphisms at different genetic loci have been associated with fibrosis progression in candidate gene studies Patatin-like phospholipase domain-containing 3 (PNPLA3), encoding for the I148M protein sequence variant, is the major common genetic determinant of hepatic fat content. The I148M variant influences the metabolism of triglycerides, lipid droplets remodeling, and lipoprotein secretion in hepatocytes PNPLA3 I148M is the major risk factor for both alcoholic and nonalcoholic steatohepatitis, showing an age-modulated effect on liver damage susceptibility and fibrogenesis PNPLA3 148M/M) also influences the risk of advanced fibrosis and hepatocellular carcinoma in CHC The rs738409 C>G polymorphism of PNPLA3 148M/M on fibrosis progression rate (FPR) and the interaction with age at infection in a unique validated cohort of 247 CHC subjects without significant alcohol intake and diabetes, characterized by a careful estimation of age at infection and with histological determination of liver damage Therefore, the aim of this study was to estimate the effect size of We analyzed a previous published cohort of 247 CHC patients followed-up at the Migliavacca Liver Disease Center at the Fondazione IRCCS Ca' Granda Policlinico of Milan PNPLA3 148M/M status are presented in The baseline features of the study cohort stratified by Liver histology was evaluated by a single expert pathologist. Mean biopsy length was 26.3 mm. The severity of hepatic inflammation was evaluated by the Ishak score PNPLA3 rs738409 C>G single nucleotide polymorphism, encoding for the I148M protein sequence variant, was genotyped by a 5′-nuclease Taqman assay  at the Metabolic Liver Diseases lab at the Fondazione Ca' Granda IRCCS. Success rate was 100% and perfect concordance between duplicates and internal controls was observed.Genomic DNA was obtained from peripheral whole blood as previously described 10 transformation of FPR was employed to obtain linearity. FPR estimation assumes that no significant liver fibrosis was present at the time of infection, and that liver fibrosis progression is constant during time PNPLA3 148M/M at risk genotype PNPLA3 148M/M was fitted into the model to test the interaction between PNPLA3 genotype and age at infection on FPR. To account for other potential confounders, a second model was tested, with further adjustement for the route of HCV transmission (intra-venous drug use: IVDU or other), body-mass (BMI < or ≥25 Kg/m2), and age at liver biopsy. Finally, to check whether the relationship between PNPLA3 genotype and FPR was independent of steatosis, a final model further adjusted for the histological severity of steatosis (grade 2–3 vs. 0–1) was evaluated. Models were checked through the regression diagnostic plots to verify normality, linearity of the data, and constant variance. The effect of the explanatory variables was considered significant if p<0.05 (two-tailed). Analyses were performed by the SPSS 21.0  statistical analysis software.For descriptive statistics, variables were shown as mean ± SD and frequencies, and compared by Student's t-test or chi-square test according to data distribution. P values were considered significant when <0.05 (two-tailed). FPR was calculated by taking the ratio between the staging value of Ishak score and the disease duration (years), and it was treated as a continuous variable. Mean disease duration was 25±10 years. LogPNPLA3 I148M variant did not violate Hardy-Weinberg equilibrium (p>0.8), and was in line with that expected unselected subjects: 120 patients were PNPLA3 148I/I (48%), 106 148I/M (43%), and 21 had the unfavorable 148M/M genotype (9%).Detailed characterization of the study cohort has previously been reported PNPLA3 148M/M status are presented in PNPLA3 148M/M status (p = ns). There was no statistically significant difference in the prevalence of grade 2–3 steatosis according to PNPLA3 148M/M status (p = ns), although a nonsignificant trend was observed in the whole cohort including subjects with ultrasonographic estimation of steatosis severity .Clinical features of patients stratified by 10 FPR −1.1±0.3 vs. −0.9±0.3 in subjects younger or older than 21 years, median value, respectively; p<0.0001). The FPR stratified by age at infection  and PNPLA3 148M/M and is presented in PNPLA3 148M/M was associated with faster FPR in older (p = 0.001), but not in younger patients (p = ns). The positive association between PNPLA3 148M/M and FPR in older patients was confirmed in sensitivity analyses using different age cut-offs (th centile (p = 0.038), 18 years (p = 0.045), and 27 years, the 75th centile (p = 0.01).Older age at infection was strongly associated with FPR . The association between steatosis grade 2–3 and FPR was confirmed in the whole cohort including subjects with ultrasonographic estimation of steatosis severity . Since PNPLA3 I148M interferes with hepatocellular lipid metabolism, we next evaluated whether the effect of PNPLA3 148M/M on FPR was influenced by steatosis severity and risk factors for derangement of hepatic lipid metabolism. Results are shown in PNPLA3 148M/M with FPR was more consistent in the presence of grade 2–3 steatosis (p = 0.024), or in the presence of other steatogenic factors: namely infection with genotype 3 (p = 0.005), or overweight (p = 0.05). The association between PNPLA3 148M/M and FPR was nearly significant in patients with steatosis grade 2–3 including subjects with ultrasonographic estimation of steatosis severity (p = 0.056).Moderate-severe steatosis was associated with faster FPR (logPNPLA3 148M/M was an independent predictor of FPR (p = 0.022). The model estimated an incremental progression of 0.08±0.03 log10 unit of fibrosis per year conferred by 148M/M status. Furthermore, there was a significant interaction between PNPLA3 148M/M and older age at infection in determining FPR . In exploratory analyses, there was no significant interaction between PNPLA3 148M/M and other determinants of FPR.At the multivariate generalized linear model considering known determinants of FPR , as well as the interaction with older age at infection  remained unaffected.To test the robustness of the association between model 2; . In thisPNPLA3 148M/M  and the interaction with older age at infection  remained significantly associated with FPR independently of the steatosis severity . In the overall series of patients including those with ultrasonographic estimation of steatosis, PNPLA3 148M/M  and the interaction with older age at infection  remained significantly associated with FPR.Finally, to test whether the effect of 148M/M status on fibrosis progression was independent of the histological severity of steatosis, we further adjusted the extended model for the presence of grade 2–3 steatosis model 3; . Again, PNPLA3 I148M sequence variant influences fibrosis progression in CHC.In this paper, we showed that in a prospective cohort with careful estimation of the date of infection and absence of major confounders the Homozygosity for the I148M variant, which in line with the prevalence in the Italian population PNPLA3 I148M on fibrosis progression in CHC is modulated by the age at infection. The negative impact of 148M/M on FPR in individuals infected at older age was confirmed in sensitivity analyses with lower age cut-offs, and was prominent in individuals infected in adulthood. Previous studies suggested that PNPLA3 I148M modulates liver damage progression in an age-dependent fashion. However, despite older age at onset was associated with faster progression of liver damage in alcoholic liver disease, the effect size of 148M/M was larger in younger patients PNPLA3 I148M variant on liver damage in nonalcoholic fatty liver disease was larger in pediatric case series Our study is the first to show that the effect of PNPLA3 I148M on fibrosis progression in CHC is presently unknown. It could be speculated that the pattern of activation of the immune system towards HCV is different depending on the age at infection. According to this hypothesis, HCV infection after the developmental age would lead to immune-mediated hepatic damage, exerting a permissive role on PNPLA3 I148M-related fibrogenesis PNPLA3 I148M in determining hepatic steatosis, which would favor viral replication during the acute infection The reason underlying the opposite direction of modulation by age of the effect of PNPLA3 I148M synergizes with host features and environmental triggers in determining liver damage associated with altered hepatic lipid metabolism PNPLA3 148M/M on fibrogenesis was more evident in subjects with grade 2–3 steatosis, and in those with other conditions favoring hepatocellular lipid accumulation, such as infection with genotype 3 and overweight.Indeed, evidence is accumulating that PNPLA3 and steatosis in this subgroup PNPLA3 I148M may promote fibrogenesis by directly altering hepatic stellate cells lipid metabolism and trans-activation Of note, we reported for the first time an effect of 148M/M on fibrosis progression in genotype 3 patients, even if previous studies did not detect an association between PNPLA3 I148M variant with fibrosis progression and hepatocellular carcinoma risk PNPLA3 I148M genotyping may help refining treatment prioritization to novel therapeutic regimens based on direct antiviral agents Therefore, given the independent association of the PNPLA3 148M/M with fibrogenesis in CHC Limitations of this study include the assumption of the absence of liver fibrosis at the time of infection, which is likely given the absence of cofactors of liver damage, the young age at infection and low risk profile. Furthermore, the model tested assumes the consistency and linearity of FPR during time, and of fibrosis progression across Ishak score stages, which are approximations of a more complex reality PNPLA3 I148M variant is associated with FPR in CHC. There is a significant interaction between age at infection and PNPLA3 genotype in determining fibrosis progression, but the association between PNPLA3 and FPR is independent of the histological severity of steatosis. These findings further establish the PNPLA3 I148M variant as a moderator of CHC natural history, and have possible relevance for treatment prioritization and follow-up of CHC patients.In conclusion, homozygosity for the Data S1Study database.(XLSX)Click here for additional data file.