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.

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5\' hammerhead and 3\' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

Autophagy is a homoeostatic process by which cytoplasmic material is targeted for degradation by the cell. Viruses have learned to manipulate the autophagic pathway to ensure their own replication and survival. Although much progress has been achieved in dissecting the interplay between viruses and cellular autophagic machinery, it is not well understood how the cellular autophagic pathway is utilized by viruses and manipulated to their own advantage. In this review, we briefly introduce autophagy, viral xenophagy and the interaction among autophagy, virus and immune response, then focus on the interplay between NS-RNA viruses and autophagy during virus infection. We have selected some exemplary NS-RNA viruses and will describe how these NS-RNA viruses regulate autophagy and the role of autophagy in NS-RNA viral replication and in immune responses to virus infection. We also review recent advances in understanding how NS-RNA viral proteins perturb autophagy and how autophagy-related proteins contribute to NS-RNA virus replication, pathogenesis and antiviral immunity.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

The nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) plays key roles in assembling genomic RNA into ribonucleoprotein (RNP), which serves as a template for both viral gene transcription and genome replication. However, little is known about the molecular mechanism of how TSWV N interacts with genomic RNA. In this study, we demonstrated that TSWV N protein forms a range of higher ordered oligomers. Analysis of the RNA binding behavior of N protein revealed that no specific oligomer binds to RNA preferentially, instead each type of N oligomer is able to bind RNA. To better characterize the structure and function of N protein interacting with RNA, we constructed homology models of TSWV N and N-RNA complexes. Based on these homology models, we demonstrated that the positively charged and polar amino acids in its predicted surface cleft of TSWV N are critical for RNA binding. Moreover, by N-RNA homology modeling, we found that the RNA component is deeply embedded in the predicted protein cleft; consistently, TSWV N-RNA complexes are relatively resistant to digestion by RNase. Collectively, using homology modeling, we determined the RNA binding sites on N and found a new protective feature for N protein. Our findings also provide novel insights into the molecular details of the interaction of TSWV N with RNA components.

The transcriptional strategy of orchid fleck virus (OFV), which has a two-segmented negative-strand RNA genome and resembles plant nucleorhabdoviruses, remains unexplored. In this study, the transcripts of six genes encoded by OFV RNA1 and RNA2 in the poly(A)-enriched RNA fraction from infected plants were molecularly characterized. All of the OFV mRNAs were initiated at a start sequence 3\'-UU-5\' with one to three non-viral adenine nucleotides which were added at the 5\' end of each mRNA, whereas their 3\' termini ended with a 5\'-AUUUAAA(U/G)AAAA(A)n-3\' sequence. We also identified the presence of polyadenylated short transcripts derived from the 3\'-terminal leader regions of both genomic and antigenomic strands, providing the first example of plus- and minus-strand leader RNAs in a segmented minus-strand RNA virus. The similarity in the transcriptional strategy between this bipartite OFV and monopartite rhabdoviruses, especially nucleorhabdoviruses (family Rhabdoviridae) is additional support for their close relationship.