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A One Health cross-sectoral surveillance approach was implemented to screen biological samples from bats, pigs, and humans at high-risk interfaces for zoonotic viral spillover for five viral families with zoonotic potential in Viet Nam. Over 1600 animal and human samples from bat guano harvesting sites, natural bat roosts, and pig farming operations were tested for coronaviruses (CoVs), paramyxoviruses, influenza viruses, filoviruses and flaviviruses using consensus PCR assays. Human samples were also tested using immunoassays to detect antibodies against eight virus groups. Significant viral diversity, including CoVs closely related to ancestors of pig pathogens, was detected in bats roosting at the human–animal interfaces, illustrating the high risk for CoV spillover from bats to pigs in Viet Nam, where pig density is very high. Season and reproductive period were significantly associated with the detection of bat CoVs, with site-specific effects. Phylogeographic analysis indicated localized viral transmission among pig farms. Our limited human sampling did not detect any known zoonotic bat viruses in human communities living close to the bat cave and harvesting bat guano, but our serological assays showed possible previous exposure to Marburg virus-like (Filoviridae), Crimean–Congo hemorrhagic fever virus-like (Bunyaviridae) viruses and flaviviruses. Targeted and coordinated One Health surveillance helped uncover this viral pathogen emergence hotspot.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-angstrom-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.

The trade-off hypothesis, the current paradigm of arbovirus evolution, proposes that cycling between vertebrate and invertebrate hosts presents significant constraints on genetic change of arboviruses. Studying these constraints in mosquito-borne viruses has led to a new understanding of epizootics. The trade-off hypothesis is assumed to be applicable to tick-borne viruses too, although studies are lacking. Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the family Bunyaviridae, is a major cause of severe human disease worldwide and shows an extraordinary amount of genetic diversity compared to other arboviruses, which has been linked to increased virulence and emergence in new environments. Using a transmission model for CCHFV, utilizing the main vector tick species and mice plus next generation sequencing, we detected a substantial number of consensus-level mutations in CCHFV recovered from ticks after only a single transstadial transmission, whereas none were detected in CCHFV obtained from the mammalian host. Furthermore, greater viral intra-host diversity was detected in the tick compared to the vertebrate host. Long-term association of CCHFV with its tick host for 1 year demonstrated mutations in the viral genome become fixed over time. These findings suggest that the trade-off hypothesis may not be accurate for all arboviruses.

SummaryViral noncoding RNAs have been shown to play an important role in virus-host interplay to facilitate virus replication. We report that members of the genus Flavivirus, a large group of medically important encephalitic RNA viruses, produce a unique and highly structured noncoding RNA of 0.3–0.5 kb derived from the 3′ untranslated region of the viral genome. Using West Nile virus as a model, we show that this subgenomic RNA is a product of incomplete degradation of viral genomic RNA by cellular ribonucleases. Highly conserved RNA structures located at the beginning of the 3′ untranslated region render this RNA resistant to nucleases, and the resulting subgenomic RNA product is essential for virus-induced cytopathicity and pathogenicity. Thus, flaviviruses evolved a unique strategy to generate a noncoding RNA product that allows them to kill the host more efficiently.

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 x 10(7) Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 degrees C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 x g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 degrees C before examination for evidence of C. abortus using polymerase chain reaction. C abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The. persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C abortus would eliminate the bacteria from the ZP. (C) 2016 Elsevier Inc. All rights reserved.

Background The family Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. Goal We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. Results Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. Conclusion Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses., Author Summary The number of international travelers has increased dramatically in recent decades. This has contributed to the increase in infectious diseases in travelers which are not present in their countries of origin and so may cause a threat to the public health. Viruses transmitted by biting insects (vector-borne viruses) are an important group within these travel-related diseases. They are found across the world and can cause debilitating and life-threatening symptoms, like inflammation of the brain or excessive bleeding. Many of these diseases are difficult to distinguish from each other. They cause comparable symptoms and are genetically closely related. Testing for long lists of diseases is time consuming and expensive. Here we develop a novel testing tool that allows doctors and researchers to test for multiple viruses with just one test. The method, which uses a specific part of the virus that makes distinguishing between infections with these closely related viruses possible, requires only one drop of blood. This allows us to test for multiple viruses simultaneously with the same amount of blood previously used to test for only one virus, while distinguishing between genetically closely related viruses.

Background: Vectors derived from alphaviruses are gaining interest for their high transfection potency and strong immunogenicity. Objectives: After a brief introduction on alphaviruses and their vectors, an overview is given on current preclinical immunotherapy studies using vector systems based on alphaviruses. The efficacy of alphavirus vectors in inducing immune responses will be illustrated by a more detailed description of immunization studies using recombinant Semliki Forest virus for the treatment of human papilloma virus-induced cervical cancer. Results: Immunization with recombinant alphavirus results in the induction of humoral and cellular immune responses against microbes, infected cells and cancer cells. Preclinical studies demonstrate that infectious diseases and cancer can be treated prophylactically as well as therapeutically. Conclusions: Alphavirus-based genetic immunization stratagies are highly effective in animal model systems, comparing quite favourably with any other approach. Therefore, we hope and expect to see an efficient induction Of tumour-or microbial immunity and a positive outcome in future clinical efficacy studies. (c) 2005 Elsevier B.V. All rights reserved.

Background: Vectors derived from alphaviruses are gaining interest for their high transfection potency and strong immunogenicity.Objectives: After a brief introduction on alphaviruses and their vectors, an overview is given on current preclinical immunotherapy studies using vector systems based on alphaviruses. The efficacy of alphavirus vectors in inducing immune responses will be illustrated by a more detailed description of immunization studies using recombinant Semliki Forest virus for the treatment of human papilloma virus-induced cervical cancer.Results: Immunization with recombinant alphavirus results in the induction of humoral and cellular immune responses against microbes, infected cells and cancer cells. Preclinical studies demonstrate that infectious diseases and cancer can be treated prophylactically as well as therapeutically.Conclusions: Alphavirus-based genetic immunization stratagies are highly effective in animal model systems, comparing quite favourably with any other approach. Therefore, we hope and expect to see an efficient induction Of tumour-or microbial immunity and a positive outcome in future clinical efficacy studies. (c) 2005 Elsevier B.V. All rights reserved.

International audience; Slave trading of Africans to the Americas, during the 16th to the 19th century was responsible for the first recorded emergence in the New World of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. Many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. Here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. We cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. Using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. Finally, we present recommendations for development by an international panel of experts reporting directly to World Health Organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. Success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment.

6 páginas.-- Mansfield, K. L. (et al.)., In 2011, a neurological disease was reported in a herd of goats (Capra hircus) in Asturias, Spain. Initial sequencing identified the causative agent as louping ill virus (LIV). Subsequently, with the application of whole genome sequencing and phylogenetic analysis, empirical data demonstrates that the LIV-like virus detected is significantly divergent from LIV and Spanish sheep encephalitis virus (SSEV). This virus encoded an amino acid sequence motif at the site of a previously identified marker for differentiating tick-borne flaviviruses that was shared with a virus previously isolated in Ireland in 1968. The significance of these observations reflects the diversity of tick-borne flaviviruses in Europe. These data also contribute to our knowledge of the evolution of tick-borne flaviviruses and could reflect the movement of viruses throughout Europe. Based on these observations, the proposed name for this virus is Spanish goat encephalitis virus (SGEV), to distinguish it from SSEV., C. G. acknowledges support from CYTEMA. This work was jointly funded from the European Commission Seventh Framework Programme under ANTIGONE (project 278976), the UK Department for Environment, Food and Rural Affairs (Defra) project SE4112 and the INIA grant E-RTA2013-00013-C04-04 (FEDER co-funded, Spain).

The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques (Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.

Society responds to changes in climate and land use via mitigation measures, including rainwater retention and storage in rewetted and newly constructed wetlands. Humans living close to these wetlands express concerns about future mosquito nuisance situations, and request the necessary distance between human occupation and wetlands to avoid such problems. Wetland managers need to know the distance required, as well as the type of management needed for such buffer or barrier zones. Here we performed an extensive literature survey to collect quantitative information on mosquito flight distance and the relevant environmental conditions. Mosquitoes have an average maximum flight distance of between 50 m and 50 km, depending on the species. Long-distance or migratory flights are strongly related to species ecological preferences and physiology, are survived by few specimens, and do not relate to nuisance situations. Nuisance-related or non-oriented flights are also species-specific and cover much shorter distances-between 25 m and 6 km for the 23 species analyzed. Based on these results, we made regression-based estimations of the percentages of the population that cross certain distances. A 90% reduction in breeding site population density would require minimal distances of 56 m for Anopheles saperoi and 8.6 km for Anopheles sinensis, and much greater distances for Aedes vexans, Culex quinquefasciatus, and Culiseta morsitans. Little useful information was available regarding the environmental conditions under which non-oriented flights took place. Qualitatively, the review showed that flight capacity was influenced by landscape structure, meteorological conditions (temperature, humidity, and illumination), and species physiology (energy available for flight). Overall, our findings suggest that predictions regarding the construction of barrier zones around breeding sites can be made based on mosquito and host density and human nuisance perception, and that barrier zone usefulness strongly depends on the mosquito species involved. Additional quantitative research is needed to better document the non-oriented dispersal patterns of the mosquitoes that populate rewetted and newly constructed wetlands, and the effects of vegetation types in barrier zones on mosquito densities. (C) 2013 Elsevier GmbH. All rights reserved.

Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.

To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses. ispartof: PLoS One vol:10 issue:2 ispartof: location:United States status: published

West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.