When designing live-attenuated respiratory syncytial virus (RSV) vaccine candidates, attenuating mutations can be developed through biologic selection or reverse-genetic manipulation and may include point mutations, codon and gene deletions, and genome rearrangements. Attenuation typically involves the reduction in virus replication, due to direct effects on viral structural and replicative machinery or viral factors that antagonize host defense or cause disease. However, attenuation must balance reduced replication and immunogenic antigen expression. In the present study, we explored a new approach in order to discover attenuating mutations. Specifically, we used protein structure modeling and computational methods to identify amino acid substitutions in the RSV nonstructural protein 1 (NS1) predicted to cause various levels of structural perturbation. Twelve different mutations predicted to alter the NS1 protein structure were introduced into infectious virus and analyzed in cell culture for effects on viral mRNA and protein expression, interferon and cytokine expression, and caspase activation. We found the use of structure-based machine learning to predict amino acid substitutions that reduce the thermodynamic stability of NS1 resulted in various levels of loss of NS1 function, exemplified by effects including reduced multi-cycle viral replication in cells competent for type I interferon, reduced expression of viral mRNAs and proteins, and increased interferon and apoptosis responses.

Insects constitute the largest proportion of animals on Earth and act as significant reservoirs and vectors in disease transmission. Rice thrips (Haplothrips aculeatus, family Phlaeothripidae) are one of the most common pests in agriculture. In this study, the full genome sequence of a novel Ollusvirus, provisionally named \"Rice thrips ollusvirus 1\" (RTOV1), was elucidated using transcriptome sequencing and the rapid amplification of cDNA ends (RACE). A homology search and phylogenetic tree analysis revealed that the newly identified virus is a member of the family Aliusviridae (order Jingchuvirales). The genome of RTOV1 contains four predicted open reading frames (ORFs), including a polymerase protein (L, 7590 nt), a glycoprotein (G, 4206 nt), a nucleocapsid protein (N, 2415 nt) and a small protein of unknown function (291 nt). All of the ORFs are encoded by the complementary genome, suggesting that the virus is a negative-stranded RNA virus. Phylogenetic analysis using polymerase sequences suggested that RTOV1 was closely related to ollusvirus 1. Deep small RNA sequencing analysis reveals a significant accumulation of small RNAs derived from RTOV1, indicating that the virus replicated in the insect. According to our understanding, this is the first report of an Ollusvirus identified in a member of the insect family Phlaeothripidae. The characterisation and discovery of RTOV1 is a significant contribution to the understanding of Ollusvirus diversity in insects.

The presence of viruses that spread to both plant and fungal populations in nature has posed intriguingly scientific question. We found a negative-strand RNA virus related to members of the family Phenuiviridae, named Valsa mali negative-strand RNA virus 1 (VmNSRV1), which induced strong hypovirulence and was prevalent in a population of the phytopathogenic fungus of apple Valsa canker (Valsa mali) infecting apple orchards in the Shaanxi Province of China. Intriguingly, VmNSRV1 encodes a protein with a viral cell-to-cell movement function in plant tissue. Mechanical leaf inoculation showed that VmNSRV1 could systemically infect plants. Moreover, VmNSRV1 was detected in 24 out of 139 apple trees tested in orchards in Shaanxi Province. Fungal inoculation experiments showed that VmNSRV1 could be bidirectionally transmitted between apple plants and V. mali, and VmNSRV1 infection in plants reduced the development of fungal lesions on leaves. Additionally, the nucleocapsid protein encoded by VmNSRV1 is associated with and rearranged lipid droplets in both fungal and plant cells. VmNSRV1 represents a virus that has adapted and spread to both plant and fungal hosts and shuttles between these two organisms in nature (phyto-mycovirus) and is potential to be utilized for the biocontrol method against plant fungal diseases. This finding presents further insights into the virus evolution and adaptation encompassing both plant and fungal hosts.

The genetic variation and population structure of gene N (nucleocapsid) and part of gene L (replicase) from 13 eggplant mottle dwarf virus (EMDV) isolates from Spain were evaluated and compared with sequences of EMDV isolates from other countries retrieved from GenBank. Phylogenetic inference of part of gene L showed three main clades, one containing an EMDV isolate from Australia and the other two containing isolates from Iran and Europe, as well as four subclades. EMDV isolates from Spain were genetically very similar and grouped in a subclade together with one isolate from Germany and one from the UK. No new recombination events were detected in addition to one recombination previously reported, suggesting that recombination is rare for EMDV. The comparison of synonymous and non-synonymous rates showed that negative selection played an important role, and only two codons were under positive selection. Genetic differentiation (Fst test), phylogenetic and nucleotide diversity analyses suggest a unique introduction of EMDV to Spain and low gene flow with other countries. In contrast, Greece and Italy showed diverse populations with high gene flow between both.

The phylogenetic relationships of ninety-five rose rosette virus (RRV) isolates with full-length genomic sequences were analyzed. These isolates were recovered mostly from commercial roses that are vegetatively propagated rather than grown from seed. First, the genome segments were concatenated, and the maximum likelihood (ML) tree shows that the branches arrange independent of their geographic origination. There were six major groups of isolates, with 54 isolates in group 6 and distributed in two subgroups. An analysis of nucleotide diversity across the concatenated isolates showed lower genetic differences among RNAs encoding the core proteins required for encapsidation than the latter genome segments. Recombination breakpoints were identified near the junctions of several genome segments, suggesting that the genetic exchange of segments contributes to differences among isolates. The ML analysis of individual RNA segments revealed different relationship patterns among isolates, which supports the notion of genome reassortment. We tracked the branch positions of two newly sequenced isolates to highlight how genome segments relate to segments of other isolates. RNA6 has an interesting pattern of single-nucleotide mutations that appear to influence amino acid changes in the protein products derived from ORF6a and ORF6b. The P6a proteins were typically 61 residues, although three isolates encoded P6a proteins truncated to 29 residues, and four proteins extended 76-94 residues. Homologous P5 and P7 proteins appear to be evolving independently. These results suggest greater diversity among RRV isolates than previously recognized.

Severe fever with thrombocytopenia syndrome virus (SFTSV), which has been reported in China, Korea, Japan, Vietnam, and Taiwan, is a causative agent of severe fever thrombocytopenia syndrome. This virus has a high mortality and induces thrombocytopenia and leukocytopenia in humans, cats, and aged ferrets, whereas immunocompetent adult mice infected with SFTSV never show symptoms. Anti-SFTSV antibodies have been detected in several animals-including goats, sheep, cattle, and pigs. However, there are no reports of severe fever thrombocytopenia syndrome in these animals. Previous studies have reported that the nonstructural protein NSs of SFTSV inhibits the type I interferon (IFN-I) response through the sequestration of human signal transducer and activator of transcription (STAT) proteins. In this study, comparative analysis of the function of NSs as IFN antagonists in human, cat, dog, ferret, mouse, and pig cells revealed a correlation between pathogenicity of SFTSV and the function of NSs in each animal. Furthermore, we found that the inhibition of IFN-I signaling and phosphorylation of STAT1 and STAT2 by NSs depended on the binding ability of NSs to STAT1 and STAT2. Our results imply that the function of NSs in antagonizing STAT2 determines the species-specific pathogenicity of SFTSV.

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions.

Mammalian arenavirus (mammarenavirus) mRNAs are characterized by 5\'-capped and 3\'-nonpolyadenylated untranslated regions (UTRs). We previously reported that the nonpolyadenylated 3\'-UTR of viral mRNA (vmRNA), which is derived from the noncoding intergenic region (IGR), regulates viral protein levels at the posttranscriptional level. This finding provided the basis for the development of novel live-attenuated vaccines (LAVs) against human pathogenic mammarenaviruses. Detailed information about the roles of specific vmRNA 3\'-UTR sequences in controlling translation efficiency will help in understanding the mechanism underlying attenuation by IGR manipulations. Here, we characterize the roles of cis-acting mRNA regulatory sequences of a prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in modulating translational efficiency. Using in vitro transcribed RNA mimics encoding a reporter gene, we demonstrate that the 3\'-UTR of nucleoprotein (NP) mRNA without a poly(A) tail promotes translation in a poly(A)-binding protein-independent manner. Comparison with the 3\'-UTR of glycoprotein precursor mRNA, which is translated less efficiently, revealed that a 10-nucleotide sequence proximal to the NP open reading frame is essential for promoting translation. Modification of this 10-nucleotide sequence also impacted reporter gene expression in recombinant LCMV. Our findings will enable rational design of the 10-nucleotide sequence to further improve our mammarenavirus LAV candidates and to develop a novel LCMV vector capable of controlling foreign gene expression.

Viruses with negative-strand RNA genomes (NSVs) include many highly pathogenic and economically devastating disease-causing agents of humans, livestock, and plants-highlighted by recent Ebola and measles virus epidemics, and continuously circulating influenza virus. Because of their protein-coding orientation, NSVs face unique challenges for efficient gene expression and genome replication. To overcome these barriers, NSVs deliver a large and multifunctional RNA-dependent RNA polymerase into infected host cells. NSV-encoded polymerases contain all the enzymatic activities required for transcription and replication of their genome-including RNA synthesis and mRNA capping. Here, we review the structures and functions of NSV polymerases with a focus on key domains responsible for viral replication and gene expression. We highlight shared and unique features among polymerases of NSVs from the Mononegavirales, Bunyavirales, and Articulavirales orders.

RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.