Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

Potent neutralizing antibodies in humans infected with zoonotic simian foamy viruses target conserved epitopes located in the dimorphic domain of the surface envelope protein.

Human diseases of zoonotic origin are a major public health problem. Simian foamy viruses (SFVs) are complex retroviruses which are currently spilling over to humans. Replication-competent SFVs persist over the lifetime of their human hosts, without spreading to secondary hosts, suggesting the presence of efficient immune control. Accordingly, we aimed to perform an in-depth characterization of neutralizing antibodies raised by humans infected with a zoonotic SFV. We quantified the neutralizing capacity of plasma samples from 58 SFV-infected hunters against primary zoonotic gorilla and chimpanzee SFV strains, and laboratory-adapted chimpanzee SFV. The genotype of the strain infecting each hunter was identified by direct sequencing of the env gene amplified from the buffy coat with genotype-specific primers. Foamy virus vector particles (FVV) enveloped by wild-type and chimeric gorilla SFV were used to map the envelope region targeted by antibodies. Here, we showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain. Epitopes recognized by neutralizing antibodies have been conserved during the cospeciation of SFV with their nonhuman primate host. Greater neutralization breadth in plasma samples of SFV-infected humans was statistically associated with smaller SFV-related hematological changes. The neutralization patterns provide evidence for persistent expression of viral proteins and a high prevalence of coinfection. In conclusion, neutralizing antibodies raised against zoonotic SFV target immunodominant and conserved epitopes located in the receptor binding domain. These properties support their potential role in restricting the spread of SFV in the human population.

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats.

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE: Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

Evidence of excretion of Schmallenberg virus in bull semen.

Schmallenberg virus (SBV) is a novel orthobunyavirus, discovered in Germany in late 2011. It mainly infects cattle, sheep and goats and could lead to congenital infection, causing abortion and fetal abnormalities. SBV is transmitted by biting midges from the Culicoides genus and there is no evidence that natural infection occurs directly between ruminants. Here, we could detect SBV RNA in infected bull semen using qRT-PCR (three bulls out of seven tested positive; 29 positive semen batches out of 136). We also found that highly positive semen batches from SBV infected bulls can provoke an acute infection in IFNAR-/- mice, suggesting the potential presence of infectious virus in the semen of SBV infected bulls.