A genome-wide CRISPR/Cas9 screen reveals the requirement of host sphingomyelin synthase 1 for infection with Pseudorabies virus mutant gD–Pass

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked

Identification of African swine fever virus-like elements in the soft tick genome provides insights into the virus’ evolution

BACKGROUND: African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus’ evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host’s genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery. - RESULTS: For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks. - CONCLUSION: Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector ‘arms race’, a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.Background Results - Evidence of ASFLI-elements in the O. moubata tick cell genome - Phylogenetic analysis shows ASFLI-elements are close relatives of ASFV sequences - ASFLI-elements are present in recently sampled O.moubata, O. porcinus and approx. 100-year-old O.moubata and O. porcinus field-collected ticks from Africa - Phylogenetic reconstruction using full-length mitochondrial genomes of soft ticks reveals a possible integration of an ASFLI-element might have occurred over 1.46–1.47 million years ago (mya) - Molecular clock analyses using ASFLI-elements from different Ornithodoros species provide an estimate for a time to the most recent common ancestor consistent with orthologous dating - Ornithodoros tick species and tick cell lines show differences in the infectability with various ASFV genotype isolates - RNA sequencing demonstrates ASFLI-element-specific mRNA—small-interfering and piwi-interacting RNAs in tick cells - The reconstructed ASFLI-A104R protein is highly similar to its ASFV homologue but is not expressed in tick cell lines Discussion Conclusion Methods - Virus strains - Tick rearing, tick infection and tick cell cultures - Nucleic acid extraction - Oligonucleotide design - PCR - qPCR - RT-qPCR - Sanger sequencing - Next-generation sequencing - Amplicon sequencing for assembly validation - Data analysis - Phylogenetic analysis - Clock rate estimates and Bayesian time-scaled trees - Protein expression and purification in E. coli and rabbit immunisation - Transfection - SDS-PAGE and immunoblotting - Statistical analysi

Widespread occurrence of Squirrel adenovirus 1 in red and grey squirrels in Scotland detected by a novel real-time PCR assay

The Eurasian Red Squirrel (Sciurus vulgaris) is distributed throughout large parts of Europe and Asia. However, its distribution in certain regions of Europe is endangered by the invasive, non-native Grey Squirrel (Sciurus carolinensis). Adenoviruses were already described in squirrels in Great Britain almost two decades ago. In 2013, a squirrel adenovirus (SqAdV-1) was additionally found in a red squirrel from Germany, which suffered from acute diffuse catarrhal enteritis, and the complete genome sequence was determined. Here, samples from dead red (n = 25) and grey (n = 12) squirrels collected in Scotland, UK, were analysed for the presence of this squirrel-associated virus. By using a newly developed real-time PCR targeting the adenoviral polymerase gene, viral DNA was detected in at least one of four tissue samples tested per animal in 64.0% of the red squirrels and 41.7% of the grey squirrels. Exceptionally high viral genome loads were detected in the intestine and liver, but SqAdV-1 DNA was also present in lung and kidney samples of affected animals. Almost complete genome sequence determination of a red squirrel-derived SqAdV-1 strain from Scotland indicated a very high degree of identity to the first German strain. Sequence analysis of the hexon gene, which encodes one of the major antigens of the virion, revealed an identity of 100% between viruses found in red and grey squirrels from Scotland. In conclusion, SqAdV-1 appears to be widespread in the Scottish red and grey squirrel population, which highlights the necessity for continuous wildlife surveillance. The novel real-time PCR assay offers a highly sensitive and robust method for SqAdV-1 surveillance

Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import

To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic