Limited susceptibility of mice to Usutu virus (USUV) infection and induction of flavivirus cross-protective immunity

© 2015 Elsevier Inc. Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. In Europe, West Nile virus (WNV) outbreaks have dramatically increased in number and severity in recent years, with dozens of human and horse deaths and a high avian mortality across the continent. Besides WNV, the only clinically relevant mosquito-borne flavivirus detected so far in Europe has been the Usutu virus (USUV), which after being reported for the first time in Austria in 2001, quickly spread across Europe, causing a considerable number of bird deaths and neurological disorders in a few immunocompromised patients. Even though USUV infects multiple avian species that develop antibodies, there is little information about USUV susceptibility, pathogenicity and cross-reactive immunity. Here, the susceptibility of suckling and adult mice to USUV infection and the induction of cross-protective immunity against WNV challenge have been addressed.Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), PLATESA (P2013/ABI-2906) from the Comunidad Autónoma de Madrid, and by the Network of Animal Disease Infectiology and Research-European Union (NADIR-EU- 228394)Peer Reviewe

Widespread distribution of hepatitis E virus in Spanish pig herds

<p>Abstract</p> <p>Background</p> <p>Hepatitis E virus (HEV) infection is a serious health problem in developing countries and is also increasingly reported in industrialized regions. HEV is considered a zoonotic agent and strains isolated from swine and human sources are genetically similar. Thus, HEV is of increasing importance to both public and animal health. The aim of the present study was to evaluate the distribution of HEV in a large population of pigs from herds located in different autonomous regions throughout Spain.</p> <p>Results</p> <p>The presence of anti-HEV IgG antibodies was analyzed in 1141 swine serum samples (corresponding to 381 pigs younger than 6 months and 760 pigs older than 6 months) collected from 85 herds. Herds were located in 6 provinces in 4 autonomous regions throughout Spain. At least one pig tested positive for anti-HEV IgG in over 80% of herds. Of individual pigs, 20.4% (233/1141) were positive for anti-HEV IgG, with the prevalence being higher in adult pigs than in those under 6 months (30.2% <it>vs. </it>15.5%). A subset of serum samples taken at 2- to 5-week intervals showed that seroprevalence dropped between 3 and 11 weeks of age, and then rose significantly by the 15th week. Pigs were also examined for the presence of HEV-RNA by RT-PCR. Of pigs tested for the presence of HEV-RNA 18.8% (64/341) were positive, with at least one pig in almost half of the herds testing positive. HEV-RNA amplicons from several positive pigs were sequenced and all were of genotype 3.</p> <p>Conclusions</p> <p>HEV was found to be widely distributed among swine farms across Spain, with the prevalence being highest among animals older than 6 months. These results indicate that HEV infection either is or is likely to become endemic in the Spanish swine population.</p

A vaccine based on a modified vaccinia virus Ankara vector expressing Zika virus structural proteins controls Zika virus replication in mice

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8+ T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKVassembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8+ T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIK

Identification of West Nile virus RNA-dependent RNA polymerase non-nucleoside inhibitors by real-time high throughput fluorescence screening

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 μM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1–4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 μM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors

Evaluation of an enzyme-linked immunosorbent assay for detection of West Nile virus infection based on a recombinant envelope protein produced in Trichoplusia ni larvae

West Nile virus (WNV), a Flavivirus distributed most widely, is presenting lately variable epidemiological and ecological patterns, including an increasing virulence that has already caused over 1000 human deaths in USA. Currently, diagnosis of WNV is achieved mainly by enzyme-linked immunoassays (ELISAs) based on the use of inactivated whole WNV (iWNV) as antigen, although results have to be confirmed by plaque reduction neutralization tests (PRNTs). Expression of WNV envelope recombinant E (rE) protein and its usefulness as ELISA antigen are described. Production of rE was achieved upon infection of Trichoplusia ni insect larvae with a recombinant baculovirus. Once optimized, the rE-based ELISA was validated with a battery of mouse and equine sera characterized previously. Concordance with the iWNV-based ELISA used routinely was good (95%), as it was with the reference PRNT (90%), with specificity of 94.4% and sensitivity of 88.1%. Production of rE protein in insect larvae allows for an easy, low cost and quite large-scale yield of partially purified antigen which is suitable for serological diagnosis of WNV, without the need for manipulation of large quantities of infective virus. © 2010 Elsevier B.V

Infection with Usutu Virus Induces an Autophagic Response in Mammalian Cells

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection. © 2013 Blázquez et al.Instituto Nacional de Investigación Agraria y Alimentaria (INIA); Network of Animal Disease Infectiology and Research-European Union (NADIR-EU-228394); Spanish Research Council (CSIC)Peer Reviewe