ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

Viral Metagenomics Revealed Sendai Virus and Coronavirus Infection of Malayan Pangolins (Manis javanica).

Pangolins are endangered animals in urgent need of protection. Identifying and cataloguing the viruses carried by pangolins is a logical approach to evaluate the range of potential pathogens and help with conservation. This study provides insight into viral communities of Malayan Pangolins (Manis javanica) as well as the molecular epidemiology of dominant pathogenic viruses between Malayan Pangolin and other hosts. A total of 62,508 de novo assembled contigs were constructed, and a BLAST search revealed 3600 ones (≥300 nt) were related to viral sequences, of which 68 contigs had a high level of sequence similarity to known viruses, while dominant viruses were the Sendai virus and Coronavirus. This is the first report on the viral diversity of pangolins, expanding our understanding of the virome in endangered species, and providing insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into other mammals.

Anticancer effect of inactivated Sendai virus strain Tianjin on human osteosarcoma HOS cells.

Ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) has been proved to have antitumor effects in many kinds of tumor cells. Here, we investigated the anticancer properties of UV-Tianjin on human osteosarcoma HOS cells and the underlying molecular mechanism. Apoptosis, intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential were determined by flow cytometry analysis. The expression levels of apoptosis-related proteins were tested by western blotting. The results showed that UV-Tianjin concentration-dependently induced apoptosis in HOS cells. UV-Tianjin-induced apoptosis was mediated by the mitochondrial pathway, which was confirmed by mitochondrial dysfunction, downregulation of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-xL (Bcl-xL) and myeloid cell leukemia-1 (Mcl-1), upregulation of B-cell lymphoma 2 associated X protein (Bax) and Bcl-2 Homologous Antagonist/Killer (Bak), as well as the cleavage of caspase-9 and -3. Further analysis showed that UV-Tianjin augmented the phosphorylation of c-Jun N-terminal kinase, the extracellular-regulated kinase and p38, the major components of mitogen-activated protein kinase (MAPK) pathways, as well as the generation of ROS. Moreover, UV-Tianjin-induced apoptosis was remarkably attenuated by MAPK inhibitors and ROS inhibitor. Taken together, our results indicated that UV-Tianjin exerts antitumor effects by inducing mitochondria-dependent apoptosis involving ROS generation and MAPK pathway in human osteosarcoma HOS cells.

Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

[Study of the susceptibility of mice to Sendai strain Tianjin].

To explore the infectivity characteristics and susceptibility of Sendai strain Tianjin in 129Sv, DBA/2, Kunming and BALB/c mice and determine the optimal small rodent animal model for strain Tianjin research, the Sendai strain Tianjin was propagated for 72h in 9-11 day-old chicken embryos, the allantoic fluids were then harvested and the virus titer (1:1280) was detected by hemagglutination assay. Four different kinds of mice were intranasally inoculated with 5 microl and the diluted 30 microl virus solution. The weight loss of mice was monitored for 12 consecutive days and the survival rate was observed. Kunming and BALB/c mice were sacrificed on the first day prior to infection and on the fourth and seventh days post infection of the diluted 30 microl Sendai strain Tianjin. Their left lobes of lung were fixed with 4% formalin for histopathologic examination. The maximum percentage of average weight loss of 129Sv, DBA/2 were 13.0%, 4.7% with 100% survival rate when 129Sv, DBA/2, Kunming and BALB/c were inoculated with 5 microl virus solution, while the mice were inoculated with diluted 30 microl virus solution, the maximum percentage of average weight loss reached 21.7%, 30.3%, 16.7% and 9.7% with the survival rate of 20%, 0%, 80% and 100%. Lung infections of mice Kunming on the fourth and seventh day post infection were more severe than that of BALB/c, showing a large number of inflammatory cell exudation and thickening of the submucosa. It suggested that DBA/2 was the most susceptible to the infection of strain Tianjin. The mice susceptibility ranked as DBA/2>129Sv>Kunming>BALB/c. Mice DBA/2 and 129Sv could be used as the optimal small rodent animal models in the research of pathogenicity and vaccine of Sendai strain Tianjin.

Contribution of the leader sequence to homologous viral interference among Sendai virus strains.

Sendai viruses (SeV) derived from persistent infection have a capacity to interfere with co-infected wild-type virus. Here we showed that interference was also caused by the laboratory strains Z and Nagoya. The leader mutations A(20)U and A(24)U related to viral adaptation from mice to chicken eggs significantly affected the capacity for viral interference, especially through genome amplification. Furthermore, recombinant SeV that possessed the mutations A(34)G and G(47)A, which are commonly found in the leader sequence of persistent infection-derived SeV strains, had an increased capacity for interference. Viral replication of human parainfluenza viruses 1, 2, and 3, but not the mumps virus or Newcastle disease virus, was suppressed by co-infection of a persistent infection-derived SeV strain, suggesting suppression of closely related human paramyxoviruses. These results indicate that homologous interference is partly dependent on the promoter sequence and further suggest involvement of promoter activity for genome amplification related to host factors in viral interference.