Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families by performing quantitative analysis of cell culture supernatants by (i) electron microscopy for the quantification of virus particles, (ii) quantitative real time PCR for the quantification of genomes, and (iii) determination of focus forming units by coating fluorescent antibodies to infected cell monolayers for the quantification of virus infectivity.Information on the replication of viral haemorrhagic fever viruses is not readily available and has never been analysed in a comparative approach. Here, we compared the cell culture growth characteristics of haemorrhagic fever viruses (HFV), of the The comparative analysis revealed that filovirus and RVFV replication results in a surplus of genomes but varying degrees of packaging efficiency and infectious particles. More efficient replication and packaging was observed for Lassa virus, and Dengue virus resulting in a better yield of infectious particles while, YFV turned out to be most efficient with only 4 particles inducing one FFU. For Crimean-Congo haemorrhagic fever virus (CCHFV) a surplus of empty shells was observed with only one in 24 particles equipped with a genome. The complete particles turned out to be extraordinarily infectious. Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families. Only few laboratories specialize in the research on these agents. Basic virological information on these viruses is scant and described exclusively in the frame of case reports and pathological animal models. Although some progress has been achieved concerning the interaction of these viruses with mechanisms of innate immunity [Viral haemorrhagic fevers (VHF) are caused by various haemorrhagic fever viruses (HFV), of the immunity -9 and niimmunity  concise ®BRL, Invitrogen, Life Technologies, Paisley, U.K.), at 37°C. The supernatants with progeny virus were collected and stored at -80°C until use. All handling of live virus was carried out in biosafety (BSL) level 3 or 4 facilities.Viral stocks were prepared from Lassa virus (LASV) strain Josiah, Ebola Zaire virus (EBOVZ) strain Mayinga, Ebola Sudan virus (EBOSV) strains Maridi and Boniface, Marburg virus (MARV) strains Ravn, Ozolin and Musoke, Crimean Congo Haemorrhagic fever virus (CCHFV) strain IbAr 10200, Rift Valley fever virus (RVFV) strain ZH 548, Dengue 1 virus (DENV 1) strain 293, Yellow fever virus (YFV) strain Asibi. Confluent Vero cells (ATCC CCL-81) were inoculated with the respective virus and grown between 1 and 10 days in minimal essential medium (MEM) supplemented with penicillin/streptomycin solution, hepes and 2% heat inactivated foetal calf serum . Ten grid squares were counted in each case. One particle per square equals a concentration of 1.5 × 105 particles per ml. Grids were stained by 2% tungstophosphoric acid  at pH 6.The electron microscopy studies were performed using a Philips CM100 electron microscope  as previously described [10 in MEM and 100 μl of each dilution was transferred to confluent Vero cells in 96 well tissue culture plates, followed by incubation for 24-48 hours at 37°C and 5%CO2.The viruses were titrated 10-fold from 1:10 to 1: 10Subsequently the supernatants were removed, and the infected cells were washed 3 times with PBS, before fixation by cold acetone (80%) in distilled water for 60 minutes at -20°C. After fixation, fluorescent focus-forming assays were performed by incubation of specific antibodies on the infected cells for 30 minutes at 37°C, followed by incubation with secondary fluorescein-conjugated antibodies for additionally 30 minutes at 37°C table . End poiRNA was isolated in a BSL3/4 laboratory from the viral stocks by treatment with TRIZOL Reagent (ratio 1+3) (Invitrogen) for a minimum of 5 minutes, before decontamination of the tubes and transport to a BSL2 laboratory following the safety instructions. Quantitative real time RT-PCR (qPCR) was performed using published primers and probes and RNA standards for RVFV, YFV, EBOZV, EBOSV, MARV and DENV -15 and p-1/slope-1) of 0.9, 1.3 and 1.5 respectively.The new qPCR assays for the CCHFV 10200 M-segment, for LASV Josiah-N and DENV 3'NTR showed an analytical sensitivity of 100, 10 and 100 detected RNA molecules and efficiencies (E = 10The virus titers were determined from 1 ml virus tissue culture supernatant by (i) electron microscopy (EM), (ii) quantitative real time PCR (qPCR) of RNA genomes extracted from the culture supernatant and (iii) determination of focus forming units (FFU) on cell monolayers infected with the supernatant using fluorescent antibodies.10-steps) higher than the particle counts  of the FFU count to the genome count.YFV was the only virus were particle and FFU count equalled each other topped only by a +1 log10-step). In comparison to YFV a +1 log10-step increased genome count over the particle therefore leads to a -2 log10-step reduction of the FFU count to the genome count.Apart from YFV, CCHFV showed the lowest level of difference between FFU and particle count (-1 log2) are associated with one FFU, whereas for all other viruses much higher amounts (103-105) were counted .To interpret the data obtained, ratios were formed between all three quantitative data sets. The observed ratios of particles/FFU  for MARV and 4 to 5 log10-steps down for EBOZV and EBOSV. Due to the overproduction the resulting amounts of FFU are still veritable but it appears that efficient genome packing by MARV (1 genome in 3-5 particles) by producing more shells than genomes does not lead to a high rate of infectious particles, as about 1000 particles are needed to induce one FFU. For EBOZV and EBOSV a 100-fold overproduction of genomes only yields an even lower amount of infectious particles, as 103-105 are needed to induce one FFU. It seems fair to say that at least as observed in cell culture particle and genome production seem to run out of sync resulting in a proportionally low amount of infectious particles.In the group of the filoviruses there seems to be a clear separation between the growth characteristics of EBOZV and EBOSV on the one hand, and MARV strains on the other hand. While Ebola viruses appear to produce a 2 logIn experimental animal models for EBOZV doses as low as one plaque-forming unit suffice to cause infection . The amo8) and genomes (1011) leading to 1 genome in 119 being packed into a particle of which 103 are needed to induce one FFU. In recent experiments sheep were successfully inoculated with RVFV at a dose of 1 × 105 TCID50 [50 and FFU are not easy to relate but it seems that there is much scope to determine a much lower infectious dose of RVFV.Cellular growth of RVFV seems comparable to that of EBOZV Mayinga with very high yields of particles (105 TCID50 . TCID50 The difference between particle count and genome count for LASV is low and reflected in a genome to particle ratio of 22 indicating a good correlation between virus particles and genomes produced. The efficiency of the virus particles and their infectiousness (100/FFU) observed in cell culture is reflected by the fact that animal models can be highly susceptible to lethal infection as for example to 2 PFU for inbred strain 13 guinea pig . A similThe best yield of correctly packaged particles is obtained by YFV and CCHFV indicating efficient synchronisation of shell and genome production (table 3 are needed to induce one FFU.DENV packs genomes even more efficiently than LASV and YFV (1 in 15 as compared to 1 in 20). The resulting DENV particles are however much less infectious than the YFV particles and about 1010 - steps up from the particle count) is conducive to a genome packaging ratio of 1 in 21 of which only 4 are needed to induce one FFU. Rhesus monkeys have been successfully infected with 7 × 102 plaque forming units (PFU) determined on vero E6 cells [YFV presents itself as the most efficient packer, leading to a very high ratio of infectious particles since it is the only virus for which particle count and FFU count are the same. From the ratios formed it appears that a slight overproduction of genomes (1 logE6 cells . Our resCCHFV does not induce a CPE in vero cells and therefore additional viral RNA genomes released by cell lysis do not contribute to the genome count. Therefore the assembly modalities of the infectious CCHFV particles appear reverse to those of the other viruses, as only every 24th particle will actually contain a genome leading to a surplus of empty shells. The correctly assembled particles however are highly infectious as each of them induces a FFU. In the EM analysis malformed CCHFV particles were noticed (see Figure -/- mice lacking the IFN receptor. These mice do not develop an innate immune response and therefore develop acute disease which makes them a good model for CCHFV infection and disease [A recent study showed that 10 FFU sufficed to kill IFNAR  disease . These rThe analysed quantitative virus titers indicate that YFV and CCHFV virus are the most efficient in producing infectious particles. These viruses manage to synchronise genome and particle production in an optimal fashion as opposed for example to the filoviruses with an apparent overflowing and inefficient production of genomes and particles. YFV and CCHFV represent the optimal synchronisation of the replication strategies observed i.e. overproduction of genomes and overproduction of shells respectively.The authors declare that they have no competing interests.MW: performed qPCR drafted manuscript, AS: provided strains, involved in drafting and revising the manuscript, has given final approval of the version to be published.JCM: involved in drafting and revising the manuscript, has given final approval of the version to be published, LK: revised the manuscript, has given final approval of the version to be published, AA: revised the manuscript, has given final approval of the version to be published, FFT: revised the manuscript, has given final approval of the version to be published, KOH: performed EM, has given final approval of the version to be published, GL: performed FFU assay, has given final approval of the version to be published, AM: involved in drafting and revising the manuscript, has given final approval of the version to be published.


Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym. Vaccinia virus (VACV) is the virus used in the live vaccine against smallpox. Smallpox was declared eradicated by the World Health Organization in 1980  Bureau of Laboratories reported a suspected orthopoxvirus infection to the Centers for Disease Control and Prevention (CDC). In the affected person, a 26-year-old male resident of Maryland , multiple pustules had developed on the arm, chin, and back of the knee on June 16 . He sought treatment after a fever and headache developed on June 19 and was advised to go to an emergency room if his fever worsened . His conLesion samples were collected on June 24 and forwarded by the Maryland hospital to a virology reference laboratory in Michigan for testing. The samples were negative by PCR for varicella, adenovirus, and herpes simplex virus. Cytopathic effect suggestive of an orthopoxvirus was noted in MRC-5, A549, and primary rhesus monkey kidney cells, and the samples were forwarded to the MDCH laboratory for further testing. On July 4, the MDCH laboratory confirmed the presence of orthopoxvirus DNA in the lesion sample using an orthopoxvirus (nonvariola) and an orthopoxvirus generic real-time PCR .The Maryland Department of Health and Mental Hygiene was contacted on July 3 and, in collaboration with the Montgomery County Department of Health and Human Services, began an investigation. The patient was asked whether he recently received smallpox vaccination or had history suggestive of exposure to orthopoxviruses such as monkeypox virus . He reported having neither; however, his wife and child had returned from a trip to Brazil 2 weeks before his illness. Human VACV infections caused by contact with infected dairy cattle occur in regions of Brazil , was contacted and described having a 4-day rash on his right forearm in mid to late June with no systemic symptoms . SamplesIn the absence of an explanation for these 2 VACV (ACAM2000) infections, Maryland public health officials launched an investigation at the gym to identify additional cases and pinpoint the source of infection. Approximately 400 surveys were distributed to gym members through email and by hand at the gym. Members were asked whether they had any recent skin lesions similar to those shown in an attached photo. They were asked whether they had recently received a smallpox vaccination or had contact with someone recently vaccinated.Staphylococcus aureus (MRSA) by health care providers in late June and early July. Both attended the gym on the same day as case-patient 1  were clinically diagnosed with methicillin-resistant atient 1 . Serum satient 1 .Maryland public health officials reviewed cleaning protocols at the gym. They determined that equipment and pads were cleaned at least twice daily (stemming from a concern about MRSA transmission) and that appropriate cleaning products were being used.CDC identified 5 civilian clinics that had received ACAM2000 vaccine since late February in the Maryland area. These clinics reported having vaccinated 65 persons; none were members of the martial arts gym. The Military Vaccine Agency (Milvax) cross-checked its list of recent military vaccinees against the gym member list since late February. Although several of those identified as being vaccinated had an association with the gym, they were either not currently gym members or were not at the gym during this period. The source of virus introduction into the martial arts gym remains unknown. No further infections have been identified among gym members or health care workers exposed to case-patients.This cluster of community-acquired VACV infection was possibly the result of sequential person-to-person spread of virus through direct physical contact, although transmission through fomites cannot be ruled out. The ultimate source-person responsible for introducing the virus into the gym was not identified, but given the limited time that ACAM2000 had been available to providers in the region (late February 2008), the most likely source was a recent vaccinee. None of the current gym members were known to have been vaccinated within the 4 weeks before illness onset of the first case-patient. Unrecognized transmission of VACV among gym members may have been ongoing over several months.–Multiple cases of VACV infection caused by secondary transmission have been noted recently (


To the Editor: Malignant catarrhal fever (MCF) is an often lethal viral disease of susceptible biungulates from the Bovidae, Cervidae, and Suidae subfamilies. MCF in pigs has been associated with direct or indirect contact with sheep, which are the main reservoir of ovine herpesvirus 2 (OvHV-2) , and anorexia. After the onset of clinical signs, neurologic symptoms developed such as ataxia, tremors, convulsions, and aggressive behavior. Animals that survived longer showed forelimb paralysis, stood in a dog-sit position, and gnawed with abundant salivation on pen bars.,Specimens from randomly selected dead sows and gilts from the outbreaks during 2004–2008 were obtained for histopathologic examination, immunofluorescence testing for rabies virus, viral and bacterial isolation, and PCR. No bacterial or viral growth was detected, and direct immunofluorescence for rabies virus was negative. Microscopic examination showed high-grade nonpurulent meningoencephalitis characterized by lymphocytic cuffings with vasculitis in the brain hemisphere, the brainstem, the spinal cord, and, to a lesser extent, the cerebellum. Multifocal areas of edema, fibrinoid necrosis, and lymphocytic infiltration also were observed . OvHV-2 ,To find possible carriers of the virus, blood samples were collected from 9 pregnant sows, 10 nonpregnant sows, and 30 breeding boars and analyzed for OvHV-2 DNA. Samples from 3 boars were positive. Nasal swabs and semen samples were collected from these infected boars to investigate the potential mode of OvHV-2 transmission, and OvHV-2 DNA was detected only in semen samples. Two of the 3 semen samples had >350 copies/2 µg of total DNA, suggesting that these animals shed virus (In Brazil, porcine MCF has been found primarily in pregnant sows and gilts. Our findings of OvHV-2 DNA in the semen of asymptomatic boars suggest that the OvHV-2 in the sows and gilts originated from asymptomatic boars that were responsible for maintaining the virus in the herd. Whether virus shedding in the semen was temporary or lasted for a long period is not known.Emergence of OvHV-2 in boars that had no known contact with sheep was surprising, especially given the possibility of venereal transmission through contaminated semen. The occurrence of OvHV-2 infection in other specific pathogen–free farms is unknown, and it is not possible to suggest a strategy to guarantee OvHV-2-free herds.


Rangifer tarandus tarandus) industry in Alaska began with animals imported from Siberia (Russia) in the 1890's. Cervid herpes virus 2 (CvHV2) is endemic in reindeer in Scandinavia. We sought to determine if the same virus, or similar herpesviruses, were circulating in Alaskan reindeer and caribou (Rangifer tarandus granti). Serum samples from 292 reindeer were collected during annual reindeer handlings (1988-2005) near Nome, Alaska. In 2005, swab samples were collected from 40 calves from this herd, near Nome, Alaska. In 2007, ocular and nasal swab samples were collected from 30 apparently healthy reindeer calves near Wales, Alaska. Samples of plasma and white blood cells were collected from three Alaskan caribou herds, Mulchatna (n = 24), Teshekpuk (n = 34) and the Western Arctic (n = 87) in 2009.The reindeer , seroprevalence was 47% (136/292) and adult reindeer had higher seroprevalence than yearlings. The overall seroprevalence for caribou was 60% (87/145), with no significant differences among caribou herds. A virus neutralization test of 20 samples from both reindeer and caribou showed that ELISA positive samples always neutralized CvHV2 to a greater extent than BoHV1 or elk herpesvirus (ElkHV), indicating that CvHv2 is the most likely virus circulating. PCR of nasal and ocular swabs sampled from 30 reindeer calves in Wales, Alaska (2007) yielded four CvHV2 positive samples. PCR amplicons of the expected size (294 bp) were obtained from 2 of the 36 buffy coats samples from caribou, and the amplicon sequences were consistent with CvHV2.This study shows that Alaskan reindeer and Caribou are infected with an alphaherpesvirus. Based on sequence similarity, CvHV-2 is the most likely virus. Further studies should be conducted to determine the impact of this infection on the health of these animals. Rangifer tarandus tarandus) were introduced to Alaska in 1891 when 16 Siberian reindeer were introduced to Unalaska and Amaknak Islands [Indigenous Rangifer migrated to Alaska more than 13,000 years ago [ Islands . Over th Islands . In the  Islands .Rangifer tarandus granti) and increasing predation by wolves. The WAH is the largest caribou herd in Alaska, with a population of 377,000 in 2007 [There are a variety of possible reasons for this population's decline including commingling and emigration into the migratory Western Arctic caribou herd  have a broad host range, replicate quickly, lyse infected cells and establish latency in sensory neuron ganglia. In a range of ruminant species, clinical signs may be present in the eyes, respiratory tract, genital organs, and mammary glands. Systemic spread can result in abortions and enteritis, usually achieved by invasion of the reticuloendothelial system [Members of the subfamily, l system .Alphaherpesviruses antigenically similar to BoHV1 have been found to be endemic in reindeer in Norway , Sweden A recent serosurvey, based on a commercial ELISA kit for cattle and BoHV1, complemented by a virus neutralization test (VNT) showed that CvHV2 is endemic in Norwegian reindeer, with a mean seroprevalence across northern Norway of 48% (1502/3062) . The preMoraxella sp., Streptococcus spp., Staphylococcus spp. and Archanobacterium spp. were also isolated, it was concluded that CvHV2 was the primary agent during this outbreak, facilitating secondary bacterial infection. This conclusion was supported by the fact that virus could be isolated from eye swab samples more frequently in animals with a mild stage of the disease, whereas in severe cases of IKC, only bacteria were recovered [The clinical impact of CvHV2 infections in reindeer is unclear. Recently, CvHV2 was associated with an outbreak of infectious keratoconjunctivitis (IKC) in Norwegian semi-domesticated reindeer . Of the ecovered . Howeverecovered .CvHV2 has been isolated from respiratory and genital swab samples as well as fetal and ocular tissues of reindeer in Norway ,15,17. CRangifer tarandus caribou) to have antibodies to a virus related to BoHV1 [A study in Saskatchewan, Canada, found 55% of 42 woodland caribou  describe the level of exposure to herpesviruses in Alaskan reindeer and caribou and to identify risk factors for exposure, 2) determine which herpesvirus is circulating among Alaskan reindeer and caribou and 3) characterize the virus through sequencing of PCR amplicons obtained from nasal and ocular swabs.The overall frequency of seropositivity (ELISA) for reindeer was 47% 136/292), showing no significant difference between years of sampling. Two samples having inconclusive results were classified as negative prior to the statistical analysis. Adults had a higher likelihood of exposure (being seropositive) than calves , whereas gender and year of sampling were not significantly associated with seroprevalence (P > 0.05). The area under the ROC curve was acceptable at 0.687. The lowess curve .PCR amplicons of the expected size (294 bp) were obtained from 5 of the 50 swabs collected from apparently healthy reindeer calves . Sequences were found to be identical to the CvHV2 Salla82 strain (AF078727.2), except for the Davis reindeer sample, which was similar to the Norwegian reindeer isolates of CvHV2 , having 50, , a range considered to be narrow enough to compare viruses' neutralizations results.From the subpanel of samples chosen for the virus neutralization test (VNT), all samples considered negative by ELISA were negative for all viruses in the VNT (2 caribou and 2 reindeer). All samples considered positive by ELISA, except one weak positive (60.8% competition), were positive for at least one virus in the VNT. All samples classified as positive in the VNT neutralized CvHV2 to a higher titer than any other viruses. For reindeer, the highest serum dilution that neutralized CvHV2 was 1:362 followed by ElkHV with 1:181, then CvHV1 with 1:128 and finally BoHV1 with 1:91  with similar levels in Mulchatna (58%), Teshekpuk (67%), and Western Arctic (57%). The logistic regression model showed that differences in seroprevalence between genders and herds were not statistically significant (P > 0.05). As most animals were calves (140 out of 145) it was not possible to assess the effect of age on seroprevalence.JF951969 and BankIt1453414 Seq7 JF951970-Caribou 109754).PCR amplicons of the expected size (294 bp) were obtained from 2 of the 36 buffy coats samples. The amplicon sequences were identical to corresponding sequences of the CvHV2 Finnish strain . Sequences submitted to GenBank  [The higher frequency in older animals is consistent with previous studies. In Saskatchewan, it was found that the prevalence of herpesvirus antibody titers increased with age, with most seropositive animals being over three years old . In Norw2) . An incrThe single nucleotide difference between our PCR amplicons sequences and that of the Finnish CvHV2 strain  is identical to the substitution previously described in isolates obtained from Norwegian reindeer . This leThe impact of these viruses on the health of caribou and reindeer in Alaska is unknown. In Norway, CvHV2 has been shown to cause respiratory disease, abortions and keratoconjunctivitis ,17. It iFurther research, including virus isolation from both reindeer and caribou is necessary to determine if the alphaherpesvirus circulating in reindeer and caribou in Alaska represent only one virus or strain and if it is indeed identical to the Scandinavian CvHV2. Studies on Siberian reindeer or reindeer imported from Siberia, having had less or no contact with caribou, may shed light on the origin of the virus in Alaska. Alphaherpesvirus infections in reindeer and caribou do not appear to induce high mortality or severe and obvious disease syndromes, but this is difficult to evaluate due to large and fluctuating herd sizes, remote areas and limited access to animals. However, being endemic and establishing latent infections, and having the potential to cause abortion, weak offspring, mucosal lesions as well as playing a part in the etiology of IKC, such infections may have greater impact on reindeer health and fitness than previously thought. This may be especially relevant to stress situations, reactivation of latent infections and shedding and transmission of virus within the herd, i.e. to calves and young animals.This study shows that Alaskan reindeer and Caribou are infected with an alphaherpesvirus. Based on sequence similarity, CvHV-2 is the most likely virus. Further studies should be conducted to determine the impact of this infection on the health of these animals.All methods were approved by the appropriate ethical committees .Serum samples from 292 reindeer were collected during the summers of 1988 through 2005 from the reindeer herd of the Davis family on the Seward Peninsula of Alaska , which is based on glycoprotein B (gB) from BoHV1 as antigen, a highly conserved glycoprotein among ruminant alphaherpesviruses which enables the kit to detect antigenically related alphaherpesviruses . Reindee50 values for each of the viruses.A VNT including the viruses CvHV2, Cervid herpesvirus 1 (CvHV1), Elk herpesvirus (ElkHV) and BoHV1, was performed to determine which virus the animals were most likely exposed to and had produced antibodies against. This was done as previously described , choosinDNA was extracted  from swab samples collected from 30 apparently healthy calves in Wales, Alaska, 20 apparently healthy calves from the Davis reindeer herd and from buffy coats from 36 seropositive caribou from the WAH herd collected during Fall 2009. The buffy coat samples were extracted in pairs .Extracted DNA was amplified by a nested pan-alphaherpesvirus PCR as described previously ,21, whic® for Windows, further statistical analyses were done in Stata 10/SE for Windows . A biweight kernel density estimation was done to evaluate the distribution of ELISA titers and to determine if the cut-off value of 50% was appropriate. A lowess  was used to graph the effect of age on seroprevalence. Exposure frequencies per herd were calculated using the proportion command in Stata. A final logistic regression model was fitted to assess the adjusted associations of sex, age class , herd and year of sampling on serological status, seropositive or seronegative. The model was established using a backward selection procedure as previously described [After establishing the database in Excelescribed . Model fCvHV2: Cervid herpes virus 2; BoHV1: Bovine herpesvirus 1; ElkHV: Elk herpesvirus; WAH: Western Arctic caribou herd; PCR: Polymerase chain reaction; CvHV1: Cervid herpes virus 1; VNT: Virus neutralization test; CPE: Cytopathic effect; ED50: Effective dose 50%; ELISA: Enzyme-linked immunosorbent assay.AE participated in study design, carried out the field sampling and data collection for Alaskan reindeer, performed laboratory analysis, analyzed data and drafted the manuscript. CN participated in design of the study, conducted and coordinated laboratory analysis, cooperated in the statistical analysis and participated in drafting the manuscript. GF participated in study design, coordinated field logistics, and contributed historical samples from reindeer. KB conceived of and facilitated inclusion of caribou in the study by participating in study design and coordinating and conducting field sampling of caribou. IN performed the ELISAs for Alaskan reindeer. ES was responsible for the statistical analysis. MT participated in study design and coordination and helped to draft the manuscript. All authors have critically revised the manuscript and read and approved the final manuscript.


Enterovirus 71 (EV71) is an important human pathogen which may cause severe neurological complications and death in children. The virus caused several outbreaks in the Asia-Pacific region during the past two decades and has been considered a significant public health problem in the post-poliovirus eradication era. Unlike poliovirus, there is no effective vaccine or approved antivirals against EV71. To explore anti-EV71 agents therefore is of vital importance. Several strategies have been employed to develop antivirals based on the molecular characteristics of the virus. Among these, some small molecules that were developed against human rhinoviruses and poliovirus are under evaluation. In this review, we discuss the recent development of such small molecules against EV71, known drug resistance and possible solutions to it, and animal models for evaluating the efficacy of these antivirals. Although further investigation is required for clinical applications of the existing candidates, the molecular mechanisms revealed for the inhibition of EV71 replication can be used for designing new molecules against this virus in the future. Enterovirus of the family Picornaviridae. Since the virus was first isolated in California in 1969 [Enterovirus 71 (EV71) belongs to the genus  in 1969 -5. Most  in 1969 . More re in 1969 .Similar to other human enteroviruses, such as poliovirus, the transmission of EV71 occurs through the fecal-oral route. The primary replication sites for the virus are presumed to be in the tonsils and intestinal lymphoid tissue. Therefore, the virus can be spread from the gastrointestinal tract of infected people for weeks. This may also explain why most EV71 isolates were recovered from throat swabs and stool specimens. However, clinical features have suggested that the virus can reach the central nervous system (CNS) of people suffering from EV71 infection ,9. The tIn past decades, in an effort to eradicate polio, a dramatic reduction of epidemics has occurred through effective vaccines and improvement in public hygiene. However, the emergence of EV71 infection has recently developed into a new threat to children, especially because there are no specific treatments or vaccines to combat this ailment. Supportive therapy is still the primary management for severe cases of EV71 infection. Other than symptomatic treatment, intravenous immunoglobulin (IVIG) is clinically used to neutralize the virus and to nonspecifically suppress inflammation. Nevertheless, although this treatment has been routinely applied to severe cases of EV71 infection, its efficacy requires further evaluation. Therefore, the development of specific antiviral strategies against EV71 has become an urgent issue for the protection of children from the hazards of EV71 infection.Picornaviridae, EV71 is a small non-enveloped virus that encloses a positive-sense, single-stranded RNA molecule of approximately 7.4 kilobases [pro and 3Cpro[Like poliovirus, the prototype virus in the family ilobases . After iilobases ,13. Onceilobases . A singland 3Cpro.pro and 3Cpro proteins and lyses the infected cell [The viral RNA genome is not only the mRNA for viral protein translation, but it can also be the template for replication by the virus-encoded RNA-dependent RNA polymerase (RdRP), designated 3D. In the infected cell, viral RNA replication occurs within a vesicular membrane structure in the cytoplasm ,17. Otheted cell ,19. NewlBlocking virus entry is an ideal antiviral strategy. For example, to a certain extent, treating severe cases of EV71 infection with IVIG was partly dependent on its nonspecific neutralization of the virus . CorrespThe VP1 protein of poliovirus or rhinovirus has also been shown to form a canyon structure, which is important for receptor binding. In addition, conformational change of the VP1 protein is the crucial step for viral particle disassembly and release of viral RNA into the host cell. Therefore, designing small molecules that target VP1 may interfere with EV71 infection. Two molecules, pleconaril  . In addiEnviroxime was found as an anti-viral compound against the replication of rhinovirus and poliovirus . By analRNA interference is a cellular post-transcriptional process in which gene expression is silenced in a sequence-specific manner. Based on this concept, artificially generated small, interfering RNAs (siRNAs) are widely applied to study gene function. Because siRNAs can effectively downregulate gene expression, virus sequence-specific siRNAs have been considered to be potential therapeutic agents. Several studies have shown that virus-specific siRNAs can successfully suppress the replication of human viruses, such as poliovirus, HIV-1, and HCV -59. Thispro inhibitor, rupintrivir, for EV71 treatment was considered as a strategy to combat the IFN signaling inhibition [pro could be an IFN antagonist, because it reduces the expression level of the type I IFN receptor [Innate immunity is the host’s natural defense system against virus invasion. Production of type I interferons (IFNs), IFN-α/β, is the initial response of innate immunity and results in activation of IFN-stimulated gene expression to block viral replication. IFN-α has been used to treat HCV infection, but its application for enterovirus infection has not been established. To evaluate whether type I IFN has a therapeutic effect against EV71 infection, a recombinant murine IFN-α was administered to EV71-infected newborn mice, resulting in an increased survival rate . Similarhibition . By the hibition . Accordireceptor , making Because the EV71 RNA genome is synthesized by its RdRP, which does not have proofreading activity for faithfully replicating viral RNA, mutations in the newly synthesized viral genome are frequently generated during replication. Thus, EV71 variants that present antiviral resistance phenotypes could often be selected during antiviral treatment. For example, the elucidation of the mechanism of action of DTriP-22 was based on the appearance of drug-resistant mutants and whole viral genome sequencing . To addrTo verify the effectiveness of antivirals against EV71, animal studies are urgently required, in addition to in vitro experiments. Since Chumakov et al. established an experimental animal infection model, neonatal mice have been the most widely used for in vivo studies . For exaBased on the experience in the control of poliovirus epidemic, vaccination should be a primary strategy to prevent EV71 infection in children. Although there is no commercialized effective vaccine against EV71 available until now, several researches have disclosed the availability for the vaccine against EV71. For example, the viral capsid protein VP1 was initially designed as an immunogenic candidate for subunit vaccine -75. In aOver the past 2 decades, eradication efforts have substantially reduced the number of poliomyelitis cases worldwide. Unfortunately, reemerging EV71 infection has become another challenge for public health, especially in the Asia-Pacific region. Unlike poliomyelitis, an effective EV71 vaccine is still not available to provide immunity in children. Therefore, development of anti-EV71 agents has become an urgent issue to relive distress in epidemic areas. Based on the replication characteristics of picornaviruses, several strategies have been developed for designing antivirals against EV71, as summarized in Figure The authors declare that they have no competing interests.RLK wrote the manuscript. SRS participated in its organization and coordination and helped to draft the manuscript. Both authors read and approved the final manuscript.


The involvement of molecular mechanisms in a particular process such as embryonic mammary gland development, can be revealed by modulation of one or several factors that purportedly act in that process. If those factors or their inhibitors are soluble, their function can be tested by loading them onto small inert beads, which are then implanted in cultured explants of the tissue of interest, in this case embryonic flanks. We here describe a protocol for such experiments. Nowadays, genetic modification is a widely used technique to identify the role of a particular gene in a specific process. But if genetically modified animals are not available, too costly, or don’t survive until the developmental stage of interest; or if the molecule of interest is not encoded by genes (as is the case for e.g. retinoic acid), alternative methods are available to test the potential roles of these molecules. For example, if the molecule of interest is soluble, it can be loaded onto inert beads, which can be applied to, or implanted in, explant cultures of the tissue or organ of interest. Compared to diluting the soluble factor in the culture medium, this method has the advantage of localized application and reduced consumption of the factor.Wnt10b positive mammary streaks on the flanks, in the axillae and in the inguinae. On each lateral side of the body, these streaks soon fuse into one continuous line coincident with the asynchronous formation of five mammary disc-shaped placodes, one in the axilla, one in the inguen, and the other three residing on the flank in between forelimb and hindlimb [This technique can be applied to the study of various stages of embryonic mammary gland development. Mammary gland development in mice starts at around embryonic day (E) 10.5, as inferred from the formation of hindlimb .Klaus Kratochwil developed a culture method in which mammary rudiments (MRs) from E12.5 mouse embryos onwards can undergo normal morphogenesis, albeit it with a delay of about 1 day. For these cultures, Kratochwil dissected individual mammary buds with a few layers of contiguous mesenchyme. He placed these on a filter resting on a metal grid which itself was hanging over a central depression in a special glass culture dish (Grobstein-design), filled with less than 1 ml medium to just touch the filter . This cuThis protocol describes the culture of E10.5 and E11.5 flank explants with application or implantation of beads soaked in soluble molecules, to monitor the effect of these molecules on mammary development. In short, beads are loaded with the molecule of interest. Embryos are harvested at ages ranging between E10.5 and E12, and their flanks are dissected for culture as explants. A loaded bead is then grafted underneath the ectoderm . These e2 may negatively affect tissue viability. It is practical to use a mouse strain that carries a transgenic marker for the mammary line and rudiments, e.g. TOPGAL-F [Pregnant female mouse. Sacrifice her preferably by cervical dislocation, as COTOPGAL-F  or s-SHITOPGAL-F  for easyA (styrofoam) support and needles to pin down and stabilize the sacrificed pregnant female mouse for embryo dissection.70 % EtOH in squirt- or spray bottle, to spray the female’s belly before opening.Large scissors and blunt serrated forceps to open the mother’s belly skinSmaller scissors and serrated forceps to open the peritoneumForceps (e.g. Dumont #5) to lift and hold the uterus, and small scissors or Vannas spring scissors to dissect the uterus out of the body2 watchmaker forceps (e.g. Dumont #5), Vannas spring scissors, 2 Graefe knifes or Tungsten needles, Moria (mini) perforated spoon to transfer embryosSeveral sets of sterile dissection instruments (e.g. from Fine Science Tools):Sterile DPBS .100 mm petri dishes.35 mm petri dishes or 6-well culture plates .Stereoscope, preferably set up in a clean room reserved for organ culture experiments.Home-made metal support grids, cut from corrosion-resistant stainless steel or aluminium patio screen at 0.7 mm mesh size, in triangles or circles of approximately 30 mm diameter. Bend a 3 mm edge, on which the grids can stand in the dish. Punch holes (e.g. with paper hole-puncher) in the grid for photography of the explants. Alternatively, metal grids without bent edge can be hung over the well of commercially available organ culture dishes . Wash and sterilize the grids after each experiment by soaking them in 70 % EtOH, drying and autoclaving, and store under sterile conditions. Optionally, metal grids can be replaced by commercially available membrane inserts  for 35 mm dishes/6-well culture plates.2O, at 120 °C (not 140 °C as it will clog the filter pores and lead to tissue death) or bring to a boil on a hot plate. Pick the filters one by one out of the solution while it is still hot and let them dry on the edge  of a grid. If the filter is curled when it comes out of the gelatin solution, attach it convex to the grid. Let dry several hours and collect in 20–30 % serum  in DPBS. Seal the container and store at 4 °C .Nuclepore® Track-Etch membrane PC MB, 13 mm diameter, pore size 0.1 μm . Autoclave the filters  in 0.1 % Gelatin  in H10 % Fetal Bovine Serum (e.g. Hyclone cat# SV30160.03)9 % E9 chick extract 2 mM Glutamine/GlutaMAX™ Culture medium: DMEM (Dulbecco’s Modified Eagle Medium) (e.g. Gibco/Invitrogen) to which the following supplements are added2, 37 °C.Humidified incubator, set at 5 % COWear gloves and wash with 70 % EtOH.Set up a mating pair of mice and check the female daily for presence of a vaginal plug. With normal light (day)-dark (night) cycles, noon of the day a plug is found is considered embryonic day (E) 0.5, gestational day 1.Remove the uterus from female mouse at gestational day 11 or 12 under sterile conditions and transfer to sterile DPBS in 100 mm petri dish. Dissect E10.5 or E11.5 embryos under the stereoscope from the uterus, and remove parietal yolk sac, visceral yolk sac and amnion. Use a spoon to transfer liberated embryos to clean sterile DPBS, taking care not to damage the flanks.Optionally, somites are counted for more precise determination of the developmental stage  of the embryo. Under the stereoscope, place the embryo in a small drop of DPBS such that its tail can be placed stably along its forehead. Adjust the light and mirror of the stereoscope until the somite boundaries are visible by transparency. Approximately at the level of the anterior side of the hindlimb is the boundary between somites #24 and #25 in embryos at around E11.5  into the dish with embryonic flanks and push to the bottom of the dish. With closed forceps, gently push a flank onto the center of the filter, mesenchymal side down, ectodermal side facing up. Drag the filter towards the periphery of the dish, and decant the dish so that the explant is no longer submerged. Lift the filter with explant out of the dish and place it on the grid or membrane insert, above the hole and fill the dish with supplemented medium until it just touches the filter. The explant may not be submerged, but has to be situated at the medium/air interface. Avoid air bubbles in the medium and certainly underneath the filter.Place the lid on the dish and the dish in the incubator, and prepare flank explant cultures of the next embryo.In a separate dish, set up a similar Trowell-type culture of a heart, as a positive control for culture conditions. If the heart is not beating at the end of the experiment, culture conditions were not good.In separate dishes, set up similar Trowell-type cultures of an organ that can serve as a positive control for activity of the molecule that is loaded onto beads, e.g. several Fibroblast Growth Factors (most certainly FGF10) will enhance branching morphogenesis of the embryonic lung .Pasteur pipettes1 ml individually wrapped Stripettes (e.g. Corning® Costar® available from various vendors)2 or 10 μl pipetteMicrofuge (1.5 ml) and PCR (0.2 ml) reaction tubes, sterileDPBS, sterile60 mm petri dishStereoscope, preferably in a clean room reserved for organ cultureBeads: Heparin-Acrylic beads (Sigma) were the preferred choice for loading e.g. Fibroblast Growth Factors (FGFs)  but are Bovine Serum Albumin (BSA) , as a ‘vehicle’ for growth factors. Prepare a small stock solution of 1 mg/ml in DPBS and store at 4 °C for a few weeks.(recombinant) Growth factors (e.g. from R&D Systems) or other molecules of interest. For growth factors: Buy in lyophilized state and reconstitute according to manufacturer’s recommendations and 10× more concentrated than the final concentration in culture, usually in at least 0.1 % (1 mg/ml) BSA. Final concentrations for culture must be retrieved from the literature and by experimentation. Store small aliquots at −80 °C where they can be kept for at least several months. Dilute on ice to final concentration preferably just prior to the experiment, although single use aliquots (e.g. 10 μl) can be made of excess volume at final concentration and stored for at least several months at −80 °C.Using a 1 ml Stripette, transfer a small volume of bead suspension from the commercial stock to a 1.5 ml microfuge tube.Wash these beads several times with DPBS.Using a 1 ml Stripette, transfer beads to a 60 mm petri dish.Under the stereoscope, select beads with a diameter that is about similar to the width of a somite.Transfer sufficient beads for one experiment (add extra for unforeseen loss during experimentation) with a 2 or 10 μl pipette (or mouth pipette with capillary drawn Pasteur pipette) to a sterile 0.2 ml PCR reaction tube. Preferably keep the number (e.g. 40) of beads the same for similar experiments, to minimize loading variation among multiple experiments.Under the stereoscope, aspirate the DPBS entirely with 2 or 10 μl pipette or mouth pipette.Add 10 μl of growth factor/substance e.g. 100 ng/μl recombinant FGF10 in 100 ng/μl BSA in DPBS. Concentration and choice of vehicle may vary depending on the molecule of interest.Suspend an equal number of beads in 10 μl of 100 ng/μl BSA in DPBS  for negative control experiments.Incubate growth factors 1 h at 37 °C with occasional flicking of the tubes to promote equal loading.Store on ice until use. Excess beads in growth factor suspension may be stored up to 1 week at 4 °C.35 mm petri dishes with 3 ml DMEM without any supplements, to wash beads.Freshly prepared flank explant cultures, as described above.Loaded beads and vehicle/control beads, as described above.Sterile fine watchmakers forceps, e.g. Dumont #5.Photo-stereoscope, preferably in a clean room reserved for organ culture.2, 37 °C.Humidified incubator, set at 5 % COTransfer sufficient test and control beads for one experiment to separate 35 mm dishes with DMEM and place in incubator, 1 h before using them in an experiment.Place the explant culture under the stereoscope, take the lid off.When the experiment is intended to test the role of molecules active in the mesenchyme, use forceps to gently poke a small tunnel just below the surface ectoderm in parallel with the boundaries between the somites. Approach from the dorsal side and move ventrally towards the ventral/hypaxial tip of the somites. Be consistent in placing this tunnel at the same axial level, by counting the somites .Pick up a bead with the forceps and gently push in into the tunnel.Alternatively, place a bead on top of the explant at the desired position.Several beads can be placed at separate positions in/on one explant (see e.g. ).Perform negative control experiments with vehicle-loaded beads. Alternate the use of left and right flank explants for application of test-beads and vehicle/control beads, because the left and right flank differ in gene activity and mammogenic potential .Place test bead against the organ that is chosen as a positive control for the test molecule. Place vehicle/negative control bead in a separate culture of a positive control organ.Place the lids on the dishes and transfer dishes to incubator.Optionally, capture images of all explants at this stage, prior to culture.Optional: Loaded beads and vehicle/control beads, as described above; transfer to DMEM in 35 mm dish and place in incubator, 1 h before using them in an experiment.Supplemented medium as above under “Preparing Mouse Embryonic Flank Explant Cultures”; prewarmed at 37 °C.1 ml pipette with sterile tips.Sterile fine watchmakers forceps, e.g. Dumont #5.Photo-stereoscope, preferably in a clean room reserved for organ culture.2, 37 °C.Humidified incubator, set at 5 % COCulture for 1–3 days in the incubator. Replace supplemented medium  daily with pipette, avoiding air bubbles under the filter. When beads are loaded with growth factors or BSA as vehicle control, they need not be replaced. When beads are loaded with relatively unstable factors (e.g. retinoic acid) and placed on top of the explant, beads may be replaced daily.Capture images of all cultures at the end of the culture period Fig. .Wnt10b; or test any other gene of interest.Fixate and analyze explants by the method of choice, e.g. visualize the mammary line or MRs by LacZ staining if the embryos carry the TOPGAL transgene as a marker for mammary line and rudiments, or use a fluorescence stereoscope if they carry the s-SHIP-GFP transgene, or perform whole mount in situ hybridization for endogenous The use of beads loaded with molecules of interest in explant flank cultures facilitates the analysis of the role of those molecules in the earliest stages of mammary gland development. This technique can also be used to test epistatic cascades without having to crossbreed different mouse mutant strains with each other.


Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in  Aedes aegypti and Culex quinquefasciatus. Deletion of the NSm gene reduced infection of Ae. aegypti mosquitoes indicating a role for the NSm protein in mosquito infection. The virus with deletion of both NSs and NSm genes was the best vaccine candidate since it did not infect Ae. aegypti and showed reduced infection and transmission rates in Cx. quinquefasciatus.Rift Valley fever virus is transmitted mainly by mosquitoes and causes disease in humans and animals throughout Africa and the Arabian Peninsula. The impact of disease is large in terms of human illness and mortality, and economic impact on the livestock industry. For these reasons, and because there is a risk of this virus spreading to Europe and North America, it is important to develop a vaccine that is stable, safe and effective in preventing infection. Potential vaccine viruses have been developed through deletion of two genes (NSs and NSm) affecting virus virulence. Because this virus is normally transmitted by mosquitoes we must determine the effects of the deletions in these vaccine viruses on their ability to infect and be transmitted by mosquitoes. An optimal vaccine virus would not infect or be transmitted. The viruses were tested in two mosquito species:  Phlebovirus genus of the family Bunyaviridae, RVFV causes significant outbreaks of severe disease in livestock, including mortality in young animals, fetal deformities and abortion. RVFV infection in humans can result in a self-limiting febrile illness or more severe disease such as retinitis, hepatic necrosis, encephalitis, neurologic deficits or fatal hemorrhagic fever Aedes genus. Mosquitoes in the Culex genus are thought to be important in amplification of virus activity during outbreaks. The virus has also been detected in phlebotomine sand flies, Culicoides midges, and Amblyomma tick species although these infections are not thought to play an important role in the life cycle of the virus or in disease outbreak settings Aedes and Culex mosquito species have been shown to be competent vectors of the virus, indicating the potential for establishment of RVFV transmission cycles in North America Rift Valley fever virus (RVFV), a human and animal pathogen that is endemic in much of Africa, has in recent decades spread to Saudi Arabia, Madagascar and Yemen and has the potential to spread to other parts of the world via transport of infected livestock, humans or mosquitoes or by an act of bioterrorism Infection, replication and transmission of an arthropod-borne virus involve complex interactions between the virus and various cells/tissues/organs of the vector. Successful transmission requires that after being ingested in a viremic bloodmeal the virus must enter the epithelial cells of the midgut, replicate and escape from the midgut cells into the hemolymph. This is followed by infection of secondary organs, including the salivary glands, where the virus enters the saliva and can then be transmitted to a new host. Potential barriers in this process have been identified that can block infection, replication and/or transmission of a virus by the mosquito The RVFV genome is comprised of three segments of single-stranded, negative sense RNA. The small (S) segment codes for the structural nucleoprotein (NP) and the nonstructural NSs protein, the medium (M) segment encodes the two structural glycoproteins, Gn and Gc, as well as two nonstructural proteins (NSm and NSm-Gn) and the large (L) segment codes for the viral RNA-dependent RNA polymerase. The nonstructural NSs and NSm proteins have been shown to function as virulence factors. The NSs protein has multiple functions that suppress the mammalian host cell antiviral response by inhibiting IFN-β gene transcription, promoting degradation of protein kinase (PKR) and suppressing host transcription Historically, a number of different methods have been employed in development of RVFV vaccines, however due to drawbacks associated with currently available vaccines including the necessity for multiple inoculations, abortions/teratologic effects in some vaccinated animals or risk of reversion to virulent phenotype, none of the existing vaccines is approved for veterinary use in North America or Europe Aedes (Stegomyia) aegypti L. and Culex (Culex) quinquefasciatus (Say).The purpose of this study was to determine the effects of NSs and NSm gene deletions on infection, dissemination and transmission of these rRVF vaccine candidate viruses in mosquitoes. Results are presented for each of the three deletion mutant rRVF viruses and rRVF-wild type evaluated side-by-side in two mosquito species representing two different genera: Construction of the rRVF viruses has been previously described Aedes aegypti Rexville D mosquito strain used was an isofemale line derived from a population of Ae. aegypti collected as larvae in San Juan, Puerto Rico (Rexville) in 1991 Culex quinquefasciatus Sebring mosquito strain used was originally colonized in Florida in 1998 and has been in colony at the CDC in Fort Collins since 2004 Cx. quinquefasciatus colony was verified by examination of genetalia and by HotAce PCR Two mosquito species were used in this study. The Ae. aegypti mosquitoes fed a bloodmeal containing frozen rRVF-wt virus compared to 63% (n = 32) with freshly harvested virus.Multiple blood-feeding experiments were undertaken. Each experiment utilized freshly prepared virus due to an observed reduction in infection rates when frozen virus was used in bloodmeals: in a separate experiment we observed only a 10% (n = 19) rate of infection in For each experiment, adult 7- to 10-day old mosquitoes were placed in pint cartons (approx. 50–100 mosquitoes per carton) and starved for 24 hours prior to feeding. Artificial virus-laden bloodmeals were prepared using fresh virus grown in Vero E6 cells as described above. Virus was harvested 2–3 days after infection depending on growth curve results for each virus (data not shown). Infected cell culture supernatant was clarified by centrifugation at 10 K rpm, 4°C for 10 min. Defibrinated chicken blood  was washed 3 times with 1 volume of ice-cold phosphate buffered saline and centrifuged at 3 K rpm, 4°C for 3 min after each wash. Two parts clarified virus were mixed with 2 parts washed blood and 1 part FBS/10% sucrose. The bloodmeal was heated to 37°C and offered to mosquitoes using cotton balls that were soaked in the bloodmeal and applied to the mesh top of the mosquito cartons. Mosquitoes were allowed to feed for 30 min at 28°C/95% humidity after which the bloodmeal was removed. Fully engorged mosquitoes were collected, double-caged and held for 14 days at 28°C/95% humidity with 5% sugar water. Three engorged mosquitoes were immediately removed for each virus and titrated to determine the amount of virus ingested (input virus titer).Twenty-five to fifty mosquitoes from each virus group were tested for virus transmission at 14 days post exposure by collection of saliva as previously described Virus was isolated and sequenced from selected mosquitoes at 14 days post-exposure as follows. Viral RNA was extracted either from triturated mosquito supernatant or from a Vero cell amplification of mosquito supernatant (25 µL mosquito supernatant grown 3 days in a T25 flask of Vero cells) using the QIAamp viral RNA kit (Qiagen). RT-PCR was performed using the Titan One-Step RT-PCR kit . Products were agarose gel purified and sequenced using the BigDye Terminator v3.1 Ready Reaction Cycle Sequencing mix . Reactions were purified using the BigDye Xterminator Purification kit (Applied Biosystems) and analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems).10-transformed) titers among the virus constructs, while logistic regression was used to compare their infection, dissemination, and transmission rates. Wald 95% confidence intervals were computed for parameters of interest, and likelihood ratio tests were used to compare models. Because the data have cases for which all individuals in a virus test group were either negative or positive, we use Firth's penalized likelihood adjustment to the estimating score equations, as detailed in Heinze and Schemper (2002) and Heinze and Puhr (2010) and implemented in R (www.r-project.org) in the package logistfwww.r-project.org). Confidence intervals for the differences of virus effects were adjusted for multiple comparisons in both normal and logistic models using the methods described in Hothorn et al. (2008) Linear regression methods were used to compare  for details related to analysis of missing data and incorporation of imputation error Due to the necessity of using freshly grown, and therefore untitrated, virus in the oral mosquito feeds, the standard regression methods for the body titers and infection rates were augmented to adjust for the unknown amount of virus taken up during the feedings by using the information collected from the mosquitoes fed concurrently and stored just after feeding (input virus titer). This was necessary because the titers for the different viruses varied between each virus stock preparation. Although significant results were found, we cannot rule out that this variation in the virus titers may have affected the results in a manner that cannot be accounted for by the statistical analysis. To summarize the approach, we treated the unknown amount of virus taken up by the test mosquitoes as missing data, represented in the linear models as a simple, continuous random effect and in the logistic models as a continuous, random offset. We then used the estimated, predictive normal distributions of the concurrently fed individuals' input virus logFor the dissemination and transmission rates, Fisher's exact tests were used to test for an overall difference, and pairwise comparisons among viruses were made using score confidence intervals for the differences. The Bonferroni adjustment was used to account for the multiple comparisons.Ae. aegypti and Cx. quinquefasciatus experiments are listed in e = number positive divided by number exposed to virus and 2) Di = number positive divided by number infected. Similarly, transmission rates are shown as 1) Te = number positive divided by number exposed to virus and 2) Td = number positive divided by number disseminated. Where data from two separate experiments are presented in Viral RNA from 25 randomly selected infected mosquitoes representing both species and all four viruses was sequenced and compared to the nucleotide sequence of the bloodmeal viruses; no differences were observed. Results from Ae. aegypti mosquitoes is summarized in 10 PFU/mL) was significantly lower than for rRVF-wt (5.4 log10 PFU/mL) . Dissemination rates for the rRVF-ΔNSs virus  were significantly lower than for rRVF-wt , while transmission rates for rRVF-ΔNSs  and rRVF-wt  did not differ significantly. RVFV antigen was found to be similarly distributed throughout head tissues by IFA testing of individuals with disseminated rRVF-wt and rRVF-ΔNSs infections (data not shown).Replication of the rRVF-wt and deletion mutant viruses in Ae. aegypti infection rate for the rRVF-ΔNSm virus  was significantly less than for rRVF-wt . The rRVF-ΔNSm infection rate in experiment 1  was higher than that of experiment 2 , most likely due to the higher experiment 1 bloodmeal titer, although when calculated individually both rates were significantly less than that of rRVF-wt. The average body titer of rRVF-ΔNSm-infected mosquitoes (1.9 log10 PFU/mL) at 14 days post-exposure was significantly less than that of mosquitoes infected with rRVF-wt (5.4 log10 PFU/mL) . The dissemination rates for rRVF-ΔNSm  were significantly less than rRVF-wt . The transmission rate for rRVF-ΔNSm  was significantly less than that of rRVF-wt  when calculated as Te (number positive/number exposed); when calculated as Ti (number positive/number disseminated) the transmission rate did not differ significantly from rRVF-wt. Out of 129 Ae. aegypti mosquitoes that fed on a bloodmeal containing the rRVF-ΔNSm virus, five became infected and one developed a disseminated infection; this individual was also found to be transmission-positive. The distribution of RVFV antigen in head tissues of this individual did not appear to differ from that of rRVF-wt (data not shown). Full length sequencing of virus isolated from this individual revealed no genetic differences compared to the virus in the blood meal.The Ae. aegypti mosquitoes exposed to rRVF-ΔNSs-ΔNSm were found to be infection-, dissemination- or transmission-positive.None of the 75 Cx. quinquefasciatus mosquitoes is summarized in e (number positive/number exposed), the dissemination rate for the rRVF-ΔNSm virus  was significantly less than that of rRVF-wt , however, the dissemination rate calculated as Di (number positive/number infected) was not significantly different and there were no significant differences in transmission rates between these viruses. IFA testing demonstrated the presence of RVFV antigen distributed throughout head tissues from Cx. quinquefasciatus individuals with a disseminated infection; no qualitative differences were observed between tissues infected with the deletion mutant viruses and rRVF-wt (data not shown). Average body titers of individuals with disseminated infections ranged from 4.4–6.1 log10 PFU/mL, while titers in individuals with undisseminated infections ranged from 1.0–4.3 log10PFU/mL. These values are similar to those reported for Cx. pipiens mosquitoes by Turell et al. Replication of the rRVF-wt and deletion mutant viruses in in vivo infection, dissemination and transmission characteristics of several recombinant RVF viruses lacking the entire coding regions of the NSs and/or the NSm genes and demonstrate the critical role of the NSm gene for infection and transmission in two mosquito species that exhibit different capacities for transmitting RVFV. Ae. aegypti has been shown to be a moderately competent vector of RVFV, although it has not been shown to be a vector in nature Cx. quinquefasciatus is a potential vector of RVFV in nature, although laboratory studies have shown it to be a less efficient vector than Ae. aegypti.We report the Ae. aegypti were much greater than those in Cx. quinquefasciatus mosquitoes in our study. At 14 days post-exposure, 90% of rRVF-wt-infected Ae. aegypti individuals had titers greater than the average input virus titer. This is in marked contrast to the Cx. quinquefasciatus mosquitoes, where at 14 days post-exposure only 10.5% had a rise in body titer that was greater than the input virus titer and 35% of individuals with detectable virus at 14 days post-exposure had body titers ≤2.0 log10 PFU/mL. Additionally, dissemination rates for rRVF-wt in Cx. quinquefasciatus were low (≤16.7%) (Aedes species for RVFV Observed rates of dissemination of rRVF-wt in (≤16.7%) . These oin vivo testing demonstrated it to be highly attenuated, immunogenic and protective against challenge with wild type virus making it a potential vaccine candidate Cx. quinquefasciatus mosquitoes compared to wild type RVFV ZH548, while no difference was observed in Ae. vexans mosquito infection rates Ae. aegypti or Cx. quinquefasciatus mosquitoes although the dissemination rate for rRVF-ΔNSs was significantly lower than rRVF-wt in Ae. aegypti.The recombinant viruses tested comprised three groups: those with a deletion of the NSs gene from the S segment of the virus, those with a deletion of the NSm gene from the M segment, and those with both the NSs and NSm genes deleted. Recombinant RVFV lacking the NSs gene has been shown to maintain the virulence and growth characteristics of the wild type virus in mammalian cell culture, and in vivo studies have demonstrated this mutant virus to be highly immunogenic it is only partially attenuated relative to the wild type virus, retaining the ability to cause lethal hepatic or neurologic disease in a minority of infected animals Ae. aegypti mosquitoes, dissemination and transmission rates were also significantly reduced suggesting an important role for the NSm proteins in this species.Recombinant RVFV lacking the NSm gene exhibits efficient replication in cell culture and although in vitro and in vivo studies characterizing the double NSs and NSm deleted recombinant virus suggest that this hypothesis is correct Given the characteristics of RVFV mutant viruses individually lacking the NSs or NSm genes, Bird, et al., have hypothesized that combining these deletions in a single mutant virus would generate a stable, attenuated, immunogenic vaccine virus Ae. aegypti mosquitoes; none of the Ae. aegypti that ingested a bloodmeal containing the rRVF-ΔNSs-ΔNSm virus became infected (n = 75). In contrast, infection with rRVF-ΔNSs-ΔNSm of the less competent vector, Cx. quinquefasciatus, was significantly reduced compared to rRVF-wt, although to a lesser degree than in Ae. aegypti, and no significant differences in dissemination or transmission rates were observed. It was apparent, however, that in Cx. quinquefasciatus the additional deletion of the NSm gene reduced the infection rate of the double deletion virus, rRVF-ΔNSs-ΔNSm,  compared to the rRVF-ΔNSs single deletion virus   . The Cx.In vivo studies by Bird, et al., showed no detectable post-vaccination viremia in n = 20 rats Ae. aegypti vector a bloodmeal titer of 7.0 log10 PFU/mL did not result in infection of mosquitoes with this virus, suggest an extremely low likelihood that mosquitoes could acquire an infectious dose of virus from feeding on a vaccinated animal. This is important since, due to the segmented nature of the RVFV genome, the potential exists for reassortment and therefore reversion of mutations in a mosquito that has acquired both vaccine and wild type strains of the virus from feeding on multiple hosts Ae. aegypti. The combined deletion of both gene regions resulted in the greatest attenuation of RVFV replication in these mosquitoes, suggesting that the rRVF-ΔNSs-ΔNsm virus is an acceptable vaccine candidate with little possibility of environmental contamination due to the lack of efficient infection and transmission in mosquitoes.The results of this study demonstrate that deletion of the RVFV NSm gene alone or in combination with the NSs gene significantly affected the replication kinetics of the virus in the mosquito species tested, particularly in Ae. aegypti and Cx. quinquefasciatus species and the diminished whole body virus titers of infected Ae. aegypti mosquitoes suggest a possible role for NSm in modulation of a mosquito midgut infection barrier. Additionally, the reduction in rates of dissemination and transmission in Ae. aegypti indicate that NSm may also function as a suppressor of a midgut escape barrier. Although the mechanisms of these barriers are not understood, it is apparent that the genetic traits of the virus as well as those of the mosquito host species influence the infection, dissemination and transmission of arboviruses The RVFV NSm has been demonstrated to function as a suppressor of virus-induced apoptosis in mammalian cells in culture although a similar role has not been demonstrated in arthropod cells Table S1Aedes aegypti statistical analysis results for data in .(XLS)Click here for additional data file.Table S2Culex quinquefasciatus statistical analysis results for data in .(XLS)Click here for additional data file.


In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region , from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses.Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses “hot spots”, obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments.From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses.Obtained data highlighting the possible role of  West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses of the Japanese encephalitis antigenic complex Culex, wild birds are principal reservoirs of WNV and they are suspected of being also of USUV, both viruses can be pathogenic for these animals Even if USUV ecology is less known than the WNV one, the two viruses seem to show biological cycle similarities: the principal vectors of WNV and USUV are largely ornithophilic mosquitoes, mainly of the genus Praomys rat and from man WNV can be transmitted occasionally by mosquitoes to vertebrates other than birds; these are considered dead-end host or seem to have only a minor role in the environmental maintenance of the virus. USUV was rarely isolated from mammals; only reported isolations were from The first detection of USUV in Europe dates back to 2001 in Vienna WNV is widely distributed through Africa, Asia, Europe, and Australia, and it is also present in America, since 1999. In Europe, epidemics with tens to hundreds of human cases have been observed in southern Russia, south France, Spain, Romania and Hungary since the 1960s Detections of different human diseases due to mosquito-borne viruses in Emilia-Romagna in recent years  strongly suggested the need for an investigation to determine the presence and geographic distribution of arboviruses in this region. For this purpose, a mosquito based survey was started in late summer 2007; this plan was implemented to cover the whole season in 2008, sampling and analyzing 47,453 specimens belonging to nine mosquito species from Emilia-Romagna A total of 158 traps worked, with different frequency and in different periods  collectiCulex pipiens , followed by Aedes caspius (15.4%), Ae. vexans (2.4%) Ae. albopictus (0.6%) and Cx. modestus (0.1%). Less abundant species analyzed (under 0.1%) were Ae. dorsalis, Ae. detritus, Ae. geniculatus, Anopheles maculipennis s.l., An. plumbeus  on the August 26. The isolated WNV strain grew with an evident cytopathic effect, until first passage, on Vero cell culture; isolation was confirmed by described ELISA test and by specific reverse transcription-PCR. No isolation was obtained from C6/C36 cell culture.Biomolecular analyses were performed on aliquots of 1,789 mosquito pools  giving tNS5 gene) but not arising from the presence of WNV or USUV. These sequences were detected in 4 mosquito species: 20 in Ae. albopictus, 4 in Ae. caspius, 1 in An. maculipennis s.l. and 1 in Cx. pipiens. The 20 sequences obtained from Ae. albopictus mosquitoes  showed an almost complete identity between themselves as well as the 3 sequences from Ae. caspius  and that from Cx. pipiens (HQ441842); these sequences were very similar with the sequences obtained during the 2007–2008 survey performed in the same area An. maculipennis s.l. (HQ441867) and a sequence from Ae. caspius (HQ441866) showed an affinity for arthropod-borne flaviviruses, as highlighted in the phylogenetic tree obtained from these sequences and homologous sequences belonging to Flaviviridae family available in Genbank database  2 traps, obtaining 2,579 mosquitoes (grouped in 16 pools) with 4 WNV-positive pools and 9 USUV-positive pools. Collections near WND cases in horse were activated from 1 to 3 weeks after the reporting of case, and captured a total of 8 WNV-positive mosquito pools.The largest part of mosquitoes was caught by modified CDC traps baited by COCx. pipiens, while two USUV-positive pools belonged to Ae. albopictus  USUV-positive pools belonged to species bopictus . These tE gene . The alignment of these sequences, eliminating every gap and missing data, showed an identity ranging from 100 to 99.2% between them, with a number of differences ranging from 0 to 3 on a total of 353 positions considered. Generally, these differences didn't cause modification in consensus deduced aminoacid sequence. In only one sequence a transition (from C to T) in position 545 of retrotranscribed WNV genome (M12294) can lead to a mutation (from threonine to isoleucine) in position 182 of derived translated protein sequence.WNV-positive mosquito pools produced 24 very similar amplicon sequences, part of The consensus sequence obtained by mosquito pools had a 100% identity with the consensus sequence produced by 21 bird sequences obtained in Emilia-Romagna in 2009 NS5 gene USUV sequences, obtained by the flavivirus-genus PCR , showed a good identity, ranged from 100 to 98.3% on 233 positions which were compared, all these differences were silent and did not produce variations in translated amino acid sequence. The highest scores obtained by the BLAST of consensus sequence were with USUV isolated in Budapest (EF206350) and USUV strain Vienna 2001 (AY453411), both showed a 98% identity with the consensus sequence used.Also the 12 partial The first pool tested positive for WNV was sampled in Bologna province on July 21 and the last two in Modena Province on September 9 . The higThe first 4 pools which tested positive for USUV were sampled in Bologna province (in different sites) on July 8, two weeks before the collection of the first WNV-positive pool, the last one in Forlì-Cesena province on October 3 . The higThe dissemination surfaces of WNV and USUV represented by kernel density estimation (KDE) are represented in The land use classification showed that 62 traps (39.2%) were placed in urban areas, 31 in green areas (19.6%), 45 in rural areas (28.5%), 10 in both woodlands and wetlands (6.3%).When Cx. pipiens mosquito pools were classified according to the environment of the sampling stations, most of the pools sampled after the week from July 6 (date of capture of first USUV-positive pool) were from urban areas , 163 pools (21%) were from rural areas, 120 pools (15.4%) were from green area .Generally, the number of USUV-positive pools in different monitored environments reflected the abundance of Cx. pipiens pools in the same environment. Concerning WNV, the proportion of positive pools captured in rural areas was significantly higher (p<0.01), in fact on a total of 778 pools of Cx. pipiens, 163 (21%) were sampled in this environments where 14, out of a total of 27 (51.9%), tested positive for WNV. On the contrary WNV-positive pools were scarce in urban areas (p<0.05), on a total of 414 (53.2%) pools from this environment only 8 (29.6%) were WNV positive. Furthermore WNV-positive pools were frequently observed in wetland , even ifThe inquiry conducted on the 190,516 sampled mosquitoes pointed out important epidemiological data on temporal and geographic diffusion of the detected viruses in Emilia-Romagna and their spreads in mosquitoes.Both WNV and USUV biomolecular data showed a high homology between detected sequences and suggested that a single strain of both WNV and USUV circulated in 2009 in the surveyed area. This hypothesis was confirmed by the high identity also observed between WNV-sequences obtained by mosquito pools and sequences obtained by birds in the same area and available in GenBank. These sequences were also very similar to those obtained the year before in mosquitoes, birds and human, suggesting the virus overwintering between 2008 and 2009. The high identity between sequences obtained and 2008 Israeli sequences suggest a common origin of WNV strains circulating in the two countries in last two years, but further data are necessary to support this observation.Results of several studies imply a silent circulation of WNV in Europe The USUV sequences published on GenBank and containing homologous part of obtained USUV sequences are scarce. Among them the USUV strains originating from Vienna and Budapest showed the highest nucleotide identity rates with the Italian viruses, implying that - following a single introduction of USUV to central Europe - the same USUV strain is now circulating in central Europe.Twenty-four sequences obtained from positive flavivirus-PCR not referred to WNV or USUV, are due to the presence of RNA of two viruses, also detected during the 2007–2008 survey and probably resulting from the presence of mosquito-only flaviviruses Cx. pipiens pools only, confirming the evidence obtained in 2008 Ae. vexans and Ae. caspius, which leads us to hypothesize the poor involvement of these mosquito species in the virus circulation in Emilia-Romagna. Cx. modestus mosquito, which is consider a highly competent species for WNV During 2009, WNV was detected in Cx. pipiens mosquito (54 pools), suggesting this species is also the main vector of this virus. Interestingly, USUV was detected in two pools of Ae. albopictus, indicating this species as possible USUV vector. Further experimental studies are needed to confirm the vector competence and the role of the Tiger mosquito in the spread of this virus. If this hypothesis will be proved the epidemicity of USUV should be reconsidered, taking into account the uninterrupted spread of Tiger mosquito in the world and the abundance of this invasive species in man-made environments, moreover the health risk connected to the presence of this exotic mosquito will be reconfirmed Ae. albopictus mosquito to serve as a bridge vector, capable of mediating the spillover of a virus from rural-cycle to urban-cycle, role already suggested for this species in regard to other viruses, including WNV All but two positive USUV pools were composed of The highest number of mosquitoes analyzed was sampled in regularly working fixed stations . The useCx. pipiens mosquito USUV-positive pools sampled in Lombardia region (Ticino River Park) in August-September 2009 (unpublished data).The simultaneous circulation of USUV and WNV in Emilia-Romagna was demonstrated by the presence of positive mosquito pools in the same area and in the same period and also by the presence of mosquito positive pools for both viruses. Furthermore, the KDE analysis produced a largely overlaying area of the two viruses “hot spots” , but thiDespite the simultaneous circulation and the reported ecological affinity between the two viruses, the data obtained didn't point out any obvious interactions between them. The PCR detections showed a stronger and longer USUV circulation in mosquitoes with respect to WNV, as displayed by the higher number of USUV-positive pools detected and by the presence of USUV-positive pools two weeks earlier than WNV. Indeed the land use data highlighted differences between the ecology of the two viruses. While the USUV-positive pools were uniformly captured in different environments, the number of WNV-positive pools was significantly higher in rural areas, highlighting a greater circulation of this virus in this environment . FurtherBesides the detection in surveyed area of WNV/USUV PCR-positive birds, two European Magpies and one Gull The earliest evidence of 2009 summer WNV re-activation in Emilia-Romagna region was detected in a mosquito pool sampled in July 21, about a week before the first reported positive bird (July 30) and about three weeks before the six diagnosed WN neuroinvasive human cases reported in second half of August The spread of the detected viruses in sampled mosquitoes and in different sampled areas were evaluated using MLE, this index could also yield assessment of viral activity under survey epidemiological and ecological conditions. WN MLE data seem to indicate that the virus has resumed the 2008 expansion from east to west , along tCx. pipiens mosquitoes, but the possibility of virus isolation by the mosquito pool with a PCR positive aliquot can be increased by a better sample management, for instance by limiting freezing and thawing cycles of samples. The introduction of other PCRs in the described monitoring system could allow the detection of other arboviruses, as achieved for Tahyna virus by orthobunyavirus-genus PCR in 2008 The effectiveness of the described survey was demonstrated by the isolation of a WNV strains from a pool of The results obtained by the described survey are encouraging and show how this mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses.The survey area was the Emilia-Romagna portion of the Pianura Padana, near the Po River, the most important Italian plain, characterized by an intensive agriculture and animal husbandry. The eastern part of the area is located on the Adriatic Sea and it is characterized by the presence of large natural wetland areas . The climate condition is typically sub-continental and it becomes gradually Mediterranean toward the costal part of the region. The Pianura Padana environment is strongly influenced by human activities, characterized by intensive agricultural with few hedges, rare scattered trees and a dense irrigation network, non-cultivated zones are rare . Most common crops are cereals and maize, vineyards or orchards are locally abundant and poplar cultivation is widespread in floodable areas near rivers. The costal part of the region is characterized by pinewood and typical Mediterranean thicket vegetation. All the monitored territories are densely populated and characterized by the abundant presence of villages, city and industrial areas.Sampling stations utilized in this survey worked with different frequency of sampling. Stations could be clustered by geographic origin and period of sampling into 9 homogenous groups, as shown in Some sampling stations (Bassa Bolognese and Lidi Ferraresi samples) were active since 1991 to monitor mosquito abundance in mosquito control plans, these stations worked weekly for the large part of the mosquito season.Other stations were specifically fixed to monitor the 2008 presumptive WNV circulation area in Ferrara and Bologna provinces; these traps worked monthly and their disposition was set to evenly cover the entire area. Twelve more traps were placed in Modena provinces, and work weekly. Other traps were positioned in Reggio Emilia, Parma, Piacenza and Forlì-Cesena provinces. All these stations were monitored by modified CDC traps baited by CO2 (CO2 traps) 2 traps. These traps were activated in proximity of particular epidemiological events, like detection of WNV-positive mosquito pool or following the detection of WND in horses.Moreover extraordinary stations were sampled only once on the regional territory by gravid traps or COAll traps were georeferenced and worked at night from roughly 5:00 pm to 9:00 am.Ochlerotatus taxon was considered an Aedes sub-genus. Mosquitoes were pooled according to date, location and species, with a maximum number of 200 individuals per pool g for 3 minutes, finally aliquots were collected from grinded samples and submitted to biomolecular analysis. A 200 µl aliquot was collected by the 101–200 mosquitoes homogenates, to optimize time and costs aliquots from other pools were combined in superpools, with an approach similar to that described in Chisenhall et al. Mosquitoes were identified to species level using morphological characteristics according to three classification keys RNAs present in aliquots were extracted using Trizol®LS Reagent ; cDNA synthesis was achieved using random hexamer  and SuperScript® II Reverse transcriptase  according to the manufacturer's instructions.NS5 gene fragment, for the detection of flavivirus-genus according to Scaramozzino et al. E gene of WNV according to the method of Tang et al. Pools were analyzed using 3 different polymerase chain reactions (PCRs): 1) traditional PCR, targeted to E-gene sequences of detected WNV a traditional PCR protocol was performed on positive samples with the same primers as the real time-PCR protocol.Fragments obtained by flavivirus-genus PCR were sequenced by an automated fluorescence-based technique following the manufacturer's instructions . In order to obtain These sequences were employed to perform basic local alignment search tool (BLAST) in the GenBank library to confirm the specificity of positive reaction and to estimate the degree of identity of detected strains. The sequences obtained were aligned with available GenBank sequences by ClustalW of the freeware program MEGA 4 Virus isolation was attempted starting from the remaining part of PCR positive mosquito homogenates using Vero cell line , incubated at 37°C, and C6/36 cell line For WNV and USUV identification, supernatant fluids of inoculated cell cultures were tested through two different sandwich enzyme-linked immunosorbent assay (ELISA). The first ELISA, specific for WNV, was performed by using a monoclonal antibody reactive against WNV envelope protein domain III (EDIII) while the other sandwich-ELISA, was performed using monoclonal antibodies cross reactive between members of Japanese encephalitis antigenic complex Cx. pipiens samples maximal likelihood estimation (MLE) was calculated Cx. pipiens pools sampled in two weeks in homogeneous groups of sites, with at least 1,500 sampled specimens throughout the season.To estimate the proportion of infected mosquitoes in Land use characteristics of the sampling stations were obtained by overlaying georeferenced traps to Emilia-Romagna Region Land Use 2003 layer in ArcView 3.x GIS. According to obtained data, stations were ranked in five categories: urban area , rural area , green area , woodland , wetland .2 test (p<0.05).Abundance of positive PCR pools and total number of Cx. pipiens pools sampled in different environment were compared by using χKernel density estimation (KDE) was applied on two datasets composed by the mosquito pools positive for WNV and USUV. KDE is a geospatial technique based on the kernel function, a bivariate probably density function Figure S1Aedes albopictus in 2008 (GQ477006), FE-Aa1: sequence detected in Ae. albopictus in 2009 (HQ441847), AeveFV2: sequence detected in Ae. vexans in 2008 (GQ477001), OccaFV3: sequence detected in Ae. caspius in 2007 (GQ476995), OccaFV4: sequence detected in Ae. caspius in 2008 (GQ476991), FE-Cp: sequence detected in Culex pipiens in 2009 (HQ441842), FE-Ac3: sequence detected in Ae. caspius in 2009 (HQ441845); GenBank sequences: AeFV: Aedes flavivirus (AB488408), CxFV: Culex flavivirus , CFA: cell fusing agent , KRV: Kamiti River virus , PoMoFlavA153: sequence detected in Ae. caspius sampled in Portugal in 2007 (EU716422), YFV: Yellow fever virus (U54798).Phylogenetic trees of the sequences of detected mosquito-only flaviviruses, other mosquito-only flaviviruses and yellow fever virus. The Neighbor-Joining Phylogeny tree of a portion of NS5 gene, amplified by flavivirus-PCR, was constructed by the informatics program MEGA4 (model p-distance) with the sequences of amplified fragments and homologous fragment of flaviviruses obtained in GenBank library. Only the percentages over 70% in the bootstrap test (1000 replicates) are shown next to the branches. ABBREVIATIONS (GenBank accession number); study area detected sequences: AealFV2: sequence detected in (0.46 MB TIF)Click here for additional data file.Figure S2Flaviviridae. The Neighbor-Joining Phylogeny tree of a portion of NS5 gene, amplified by flavivirus-PCR, were constructed by the informatics program MEGA4 (model p-distance) with the sequences of amplified fragments and homologous fragment of Flaviviridae obtained in GenBank library. Flavivirus genus branch was colored in black, Pestivirus genus was in azure and Hepacivirus genus was in blue. Flavivirus genus was further divided into insect-only viruses in green, non-know vector in orange, tick-borne in yellow and mosquito-borne in red. ABBREVIATIONS (GenBank accession number); study area detected sequences: MO-Ac: sequence detected in Aedes caspius in 2009 (HQ441866), PV-Am: sequence detected in Anopheles maculipennis in 2009 (HQ441867), OccaFV3: sequence detected in Ae. caspius in 2007 (GQ476995). GenBank sequences: AeFV: Aedes flavivirus (AB488408), APOIV: Apoi virus (NC003676), BORV: Border disease virus (NC003679), BVDV: Bovine viral diarrhea virus (NC001461), CFA: cell fusing agent (NC001564), CSFV: Classical swine fever virus (NC002657), CxFV: Culex flavivirus (AB262759), DENV: Dengue virus , HCV: Hepatitis C virus , JEV: Japanese encephalitis virus GQ902063, KRV: Kamiti River virus (AY149905), MODV: Modoc virus (NC003635), OHFV: Omsk hemorragic fever virus (NC005062), POWV: Powassan virus (NC003687), RBV: Rio bravo virus (NC003675), TBEV: Tick-borne encephalitis virus (DQ401140), USUV: Usutu virus (HM138707), WNV: West Nile virus (GU047875), YFV: Yellow fever virus (U54798).Phylogenetic trees of the sequences of presumptive detected flaviviruses and other viruses of family (1.86 MB TIF)Click here for additional data file.


We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface. We calculate inter-glycoprotein distances and the fractional volume occupied by glycoproteins. The models suggest that for typical HA densities on virus, Fabs can bind to epitopes on the HA stem domain. The models also show how membrane curvature may influence the number of glycoproteins that can simultaneously interact with a target surface of receptors. The latter strain maintains a filamentous phenotype in the laboratory and displays a structural regularity that may be exploited for structural study 21HGE) into spike density perpendicular to the surface. Intermolecular distances were calculated between the centers-of-mass of the HA models (78 Å from membrane). For studies of FI6 Fab binding 3ZTJ) with different numbers of Fabs bound was examined for overlap with other HA models. To measure the relative distance of receptor binding sites, the O2 position of the sialic acid in the receptor-binding site was determined for all HA coordinates built on the virus surface.Growth, purification, and cryotomography of A/Udorn/72 and A/Aichi/68 X-31 virus were done as previously described 33.1Cryotomography was used to study the three-dimensional structure of frozen-hydrated influenza virions (H3N2). Udorn virions typically show a capsular  or filamentous morphology. We identify glycoproteins as strong densities with distinct features at the highest radius of the particles beyond the membrane. The HA glycoproteins are 13 nm long spikes with a density profile similar to the X-ray crystal structure of the trimeric ectodomain. The NA is 14 nm long and has density concentrated in the tetrameric head domain similar in size and shape to the crystal structure, located at the membrane distal end of a thin stalk. Clusters of NAs 3.2We build a structural model for the virus envelope by placing the X-ray model for the HA ectodomain at peak density positions on the virus membrane. Because of the anisotropic resolution of the tomograms due to the missing data wedge, the images of the virus surface are blurred along the direction of the membrane at the sides of the particles, which cannot be tilted toward the electron beam. For this reason, we only build models for the glycoproteins on the top and bottom cylindrical surfaces of the virus and restrict our analysis to these surfaces. These positions are indicated for a Udorn virion in We measure the distance between each glycoprotein position and its five nearest neighbors on both X-31 and Udorn virions and plot these as separate histograms in 3.3ϕ about the cylindrical axis of the particle from the point of closest approach. Note that for the typical spacings described above, the ∼100 Å distance between spikes corresponds to a relative angle of 12̊. Assuming at least one HA can engage receptors on a surface, then the binding sites of the next closest HA are on average 12 Å further away from the surface. For a spherical-shaped particle, different directions of curvature are identical. In the case of capsular or filamentous particles, HAs along the axis maintain the same distance and could simultaneously engage receptors.A striking feature of the virus particles is the curvature of the membrane. For capsule or filament-shaped viruses of the most typical dimension in our preparations, the virus has a small radius of curvature perpendicular to the long axis of the capsule . One con4Cryotomography of the influenza virus X-31 The images in Earlier studies of the surface glycoprotein density have relied upon bulk scattering methods such as neutron diffraction The structural models of the envelope glycoprotein on the virus surface suggest geometric constraints on receptor binding determined by the glycoprotein spacing and radius of curvature of the virus membrane. In vitro experiments indicate a weak millimolar binding constant of the HA glycoprotein for sialic acid receptors. Furthermore, influenza host specificity is dependent on very small affinity differences for sialic acid receptors with different glycosidic linkages The three-dimensional structural models of the glycoprotein on the surface of influenza virions describe important structural parameters that govern antibody recognition of the HA including the density and accessibility of epitopes. The average glycoprotein spacing observed (∼100 Å) is short enough for bivalent IgGs, which possess flexibly linked antigen binding sites that can extend 150 Å apart, to cross-link adjacent HAs 5The three-dimensional maps of influenza virus determined by electron cryotomography show the packaging of the genomic segments in the virus interior and the envelope structure including a dense matrix layer inside the bilayer and glycoproteins outside. We have used X-ray structures of the HA to build three-dimensional models for the surface glycoprotein distribution that show large scale structural features that are likely to be important for understanding of the virus life-cycle. Electron cryotomography can also be applied to visualize neutralizing antibodies in complex with virus and viruses interacting with target membranes.