Mariana Varela

Endogenous retroviruses regulate periimplantation placental growth and differentiation

Endogenous retroviruses (ERVs) are fixed and abundant in the genomes of vertebrates. Circumstantial evidence suggests that ERVs play a role in mammalian reproduction, particularly placental morphogenesis, because intact ERV envelope genes were found to be expressed in the syncytiotrophoblasts of human and mouse placenta and to elicit fusion of cells in vitro. We report here in vivo and in vitro experiments finding that the envelope of a particular class of ERVs of sheep, endogenous Jaagsiekte sheep retroviruses (enJSRVs), regulates trophectoderm growth and differentiation in the periimplantation conceptus (embryo/fetus and associated extraembryonic membranes). The enJSRV envelope gene is expressed in the trophectoderm of the elongating ovine conceptus after day 12 of pregnancy. Loss-of-function experiments were conducted in utero by injecting morpholino antisense oligonucleotides on day 8 of pregnancy that blocked enJSRV envelope protein production in the conceptus trophectoderm. This approach retarded trophectoderm outgrowth during conceptus elongation and inhibited trophoblast giant binucleate cell differentiation as observed on day 16. Pregnancy loss was observed by day 20 in sheep receiving morpholino antisense oligonucleotides. In vitro inhibition of the enJSRV envelope reduced the proliferation of mononuclear trophectoderm cells isolated from day 15 conceptuses. Consequently, these results demonstrate that the enJSRV envelope regulates trophectoderm growth and differentiation in the periimplantation ovine conceptus. This work supports the hypothesis that ERVs play fundamental roles in placental morphogenesis and mammalian reproduction.

Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

RNA Interference Targets Arbovirus Replication in Culicoides Cells

ABSTRACT Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Coevolution of endogenous betaretroviruses of sheep and their host.

Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. Jaagsiekte sheep retrovirus (JSRV) is a pathogenic exogenous retrovirus and the causative agent of ovine pulmonary adenocarcinoma. The sheep genome contains at least 27 copies of endogenous retroviruses (enJSRVs) highly related to JSRV. enJSRVs have played several roles in the evolution of the domestic sheep as they are able to block the JSRV replication cycle and play a critical role in sheep conceptus development and placental morphogenesis. Available data strongly suggest that some dominant negative enJSRV proviruses (i.e. able to block JSRV replication) have been positively selected during evolution. Interestingly, viruses escaping the transdominant enJSRV loci have recently emerged (less than 200 years ago). Thus, endogenization of these retroviruses may still be occurring today. Therefore, sheep provide an exciting and unique system to study retrovirus-host coevolution. (Part of a multi-author review).

Friendly viruses the special relationship between endogenous retroviruses and their host

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and have coevolved with their hosts for millions of years. Some ERVs play a critical role in placental development, contribute to genome plasticity, and protect the host against infection of related pathogenic and exogenous retroviruses, thus some ERVs have been positively selected and maintained in the host genome. The sheep genome contains 27 endogenous retroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. enJSRVs are able to protect their host against JSRV infection by blocking different steps of the viral replication cycle. In addition, enJSRVs are absolutely required for sheep placental development. Thus, enJSRVs‐JSRV provides a unique and interesting model to study the symbiotic relationship and interplay between host ERVs and evolution. This review will provide some examples of the biological functions of ERVs. In particular, the role of ERVs in reproductive biology and in protecting the host against pathogenic retrovirus infections will be emphasized using enJSRVs/JSRV and the sheep as a model.

Immunophenotyping of Inflammatory Cells Associated with Schmallenberg Virus Infection of the Central Nervous System of Ruminants

Schmallenberg virus (SBV) is a recently discovered Bunyavirus associated mainly with abortions, stillbirths and malformations of the skeletal and central nervous system (CNS) in newborn ruminants. In this study, a detailed immunophenotyping of the inflammatory cells of the CNS of affected animals was carried out in order to increase our understanding of SBV pathogenesis. A total of 82 SBV-polymerase chain reaction (PCR) positive neonatal ruminants (46 sheep lambs, 34 calves and 2 goat kids) were investigated for the presence of inflammation in the brain and spinal cord. The study focused on 15 out of 82 animals (18.3%) showing inflammation in the CNS. All 15 neonates displayed lymphohistiocytic meningoencephalomyelitis affecting most frequently the mesencephalon and the parietal and temporal lobes. The majority of infiltrating cells were CD3-positive T cells, followed by CD79α-positive B cells and CD68-positive microglia/macrophages. Malformations like por- and hydranencephaly, frequently found in the temporal lobe, showed associated demyelination and axonal loss. SBV antigen was detected in 37 out of 82 (45.1%) neonatal brains by immunohistochemistry. In particular, SBV antigen was found in 93.3% (14 out of 15 ruminants) and 32.8% (22 out of 67 ruminants) of animals with and without encephalitis, respectively. Highest amounts of virus-protein expression levels were found in the temporal lobe. Our findings suggest that: (i) different brain regions display differential susceptibility to SBV infection; (ii) inflammatory cells in the CNS are found only in a minority of virus infected animals; (iii) malformations occur in association with and without inflammation in the CNS; and (iv) viral antigen is strongly associated with the presence of inflammation in naturally infected animals. Further studies are required to explore the cell tropism and pathogenesis of SBV infection in ruminants.

Molecular Evolution Analysis of the Human Immunodeficiency Virus Type 1 Envelope in Simian/Human Immunodeficiency Virus-Infected Macaques: Implications for Challenge Dose Selection

ABSTRACT Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIVsf162p4. We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.

Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses

The host innate immune response mediated by type I interferon (IFN) and the resulting up-regulation of hundreds of interferon-stimulated genes (ISGs) provide an immediate barrier to virus infection. Studies of the type I ‘interferome’ have mainly been carried out at a single species level, often lacking the power necessary to understand key evolutionary features of this pathway. Here, using a single experimental platform, we determined the properties of the interferomes of multiple vertebrate species and developed a webserver to mine the dataset. This approach revealed a conserved ‘core’ of 62 ISGs, including genes not previously associated with IFN, underscoring the ancestral functions associated with this antiviral host response. We show that gene expansion contributes to the evolution of the IFN system and that interferomes are shaped by lineage-specific pressures. Consequently, each mammal possesses a unique repertoire of ISGs, including genes common to all mammals and others unique to their specific species or phylogenetic lineages. An analysis of genes commonly down-regulated by IFN suggests that epigenetic regulation of transcription is a fundamental aspect of the IFN response. Our study provides a resource for the scientific community highlighting key paradigms of the type I IFN response.

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence

ABSTRACT Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.