Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transcription start site mapping of geminiviruses using the in vitro cap-snatching of a tenuivirus.

Geminiviruses are a family of single-stranded DNA viruses that cause significant yield losses in crop production worldwide. Transcription start site (TSS) mapping is crucial in understanding the gene expression mechanisms of geminiviruses. However, this often requires costly and laborious experiments. Rice stripe virus (RSV) has a mechanism called cap-snatching, whereby it cleaves cellular mRNAs and uses the 5' cleavage product, a capped-RNA leader (CRL), as primers for transcription. Our previous work demonstrated that RSV snatches CRLs from geminiviral mRNAs in co-infected plants, providing a convenient and powerful approach to map the TSSs of geminiviruses. However, co-infections are not always feasible for all geminiviruses. In this study, we evaluated the use of in vitro cap-snatching of RSV for the same purpose, using tomato yellow leaf curl virus (TYLCV) as an example. We incubated RNA extracted from TYLCV-infected plants with purified RSV ribonucleoproteins in a reaction mixture that supports in vitro cap-snatching of RSV. The RSV mRNAs produced in the reaction were deep sequenced. The CRLs snatched by RSV allowed us to locate 28 TSSs in TYLCV. These results provide support for using RSV's in vitro cap-snatching to map geminiviral TSSs.

Bayesian Phylogeographic Inference Suggests Japan as the Center for the Origin and Dissemination of Rice Stripe Virus.

Rice stripe virus (RSV) is one of the most important viral pathogens of rice in East Asia. The origin and dispersal of RSV remain poorly understood, but an emerging hypothesis suggests that: (i) RSV originates from Yunnan, a southwest province of China; and (ii) some places of eastern China have acted as a center for the international dissemination of RSV. This hypothesis, however, has never been tested rigorously. Using a data set comprising more than 200 time-stamped coat protein gene sequences of RSV from Japan, China and South Korea, we reconstructed the phylogeographic history of RSV with Bayesian phylogeographic inference. Unexpectedly, the results did not support the abovementioned hypothesis. Instead, they suggested that RSV originates from Japan and Japan has been the major center for the dissemination of RSV in the past decades. Based on these data and the temporal dynamics of RSV reported recently by another group, we proposed a new hypothesis to explain the origin and dispersal of RSV. This new hypothesis may be valuable for further studies aiming to clarify the epidemiology of RSV. It may also be useful in designing management strategies against this devastating virus.

The Spatial Diffusion of Cherry Leaf Roll Virus Revealed by a Bayesian Phylodynamic Analysis.

Cherry leaf roll virus (CLRV) is an important plant pathogen that causes severe and detrimental effects on cherry and other fruit plants. Despite recent progress in plant pathology, molecular biology, and population genetics of CLRV, the spatiotemporal spread of this virus remains poorly studied. In this study, we employed a Bayesian phylodynamics framework to investigate the spatial diffusion patterns of CLRV by analyzing the coat protein gene sequences of 81 viral isolates collected from five different countries. Consistent with the trade of cherry, our Bayesian phylodynamic analyses pointed to viral origins in New Zealand and identified multiple migration pathways between Germany and other countries, suggesting that Germany has played an important role in CLRV transmission. The results of our study will be useful in developing sustainable management strategies to control this pathogen.

The cap-snatching frequency of a plant bunyavirus from nonsense mRNAs is low but is increased by silencing of UPF1 or SMG7.

Bunyaviruses cleave host cellular mRNAs to acquire cap structures for their own mRNAs in a process called cap-snatching. How bunyaviruses interact with cellular mRNA surveillance pathways such as nonsense-mediated decay (NMD) during cap-snatching remains poorly understood, especially in plants. Rice stripe virus (RSV) is a plant bunyavirus threatening rice production in East Asia. Here, with a newly developed system allowing us to present defined mRNAs to RSV in Nicotiana benthamiana, we found that the frequency of RSV to target nonsense mRNAs (nsRNAs) during cap-snatching was much lower than its frequency to target normal mRNAs. The frequency of RSV to target nsRNAs was increased by virus-induced gene silencing of UPF1 or SMG7, each encoding a protein component involved in early steps of NMD (in an rdr6 RNAi background). Coincidently, RSV accumulation was increased in the UPF1- or SMG7-silenced plants. These data indicated that the frequency of RSV to target nsRNAs during cap-snatching is restricted by NMD. By restricting the frequency of RSV to target nsRNAs, NMD may impose a constraint to the overall cap-snatching efficiency of RSV. Besides a deeper understanding for the cap-snatching of RSV, these findings point to a novel role of NMD in plant-bunyavirus interactions.

The phylogeographic history of tomato mosaic virus in Eurasia.

Tomato mosaic virus (ToMV) is a tobamovirus affecting solanaceous crops worldwide. The process of its emergence, however, is poorly understood. Here, Bayesian phylogenetic framework was employed to reconstruct the phylogeography of ToMV in Eurasia. The results showed that the ToMV in Europe, Middle East and East Asia has been evolving at a rate of 4.05 × 10-4 substitutions/site/year (95% credibility interval 2.43 × 10-4 - 5.62 × 10-4). Their most recent common ancestor (MRCA), most probably first appeared in Europe, was dated to around 1757 Common Era. The first introduction of ToMV into Middle East occurred in 1920s, with Europe as the source, while the first introduction of ToMV into East Asia occurred shortly afterwards, with Middle East as the source. From about 1950 onwards, inter-regional migrations of ToMV between Europe, Middle East and East Asia have been common. Overall, these data provide a glimpse into the phylogeographic history of ToMV in Eurasia.

Four distinct isolates of Helminthosporium victoriae virus 190S identified from Bipolaris maydis.

Helminthosporium victoriae virus 190S (HvV190S) is the type species of the genus Victorivirus under the family Totiviridae. To date, HvV190S has never been found in places outside of the USA and has Helminthosporium victoriae as its only know natural host fungus in the field. Here, we report the identification of 4 double-stranded RNA (dsRNA) viruses from Bipolaris maydis in Hubei province of China. Interestingly, the genomes of the 4 viruses show 81.2 %-85.5 % nucleotide sequence identities to HvV190S. Their capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) share 95.5-97.9 % and 94.6-96.6 % amino acid sequence identities to corresponding proteins of HvV190S. Therefore, the 4 viruses, which show 81.8-87.3 % pairwise genome sequence identities, should be considered as distinct isolates of HvV190S. Our finding suggests that HvV190S is widely distributed in the world and may infect fungal species other than H. victoriae.

C4, the Pathogenic Determinant of Tomato Leaf Curl Guangdong Virus, May Suppress Post-transcriptional Gene Silencing by Interacting With BAM1 Protein.

Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1.

A convenient in vivo cap donor delivery system to investigate the cap snatching of plant bunyaviruses.

Like their animal-infecting counterparts, plant bunyaviruses use capped RNA leaders cleaved from host cellular mRNAs to prime viral genome transcription in a process called cap-snatching, but in vivo systems to investigate the details of this process are lacking for them. Here, we report that Rice stripe tenuivirus (RSV) and Tomato spotted wilt tospovirus (TSWV) cleave capped RNA leaders from mRNAs transiently expressed by agroinfiltration, which makes it possible to artificially deliver defined cap donors to the two plant bunyaviruses with unprecedented convenience. With this system, some ideas regarding how plant bunyaviruses select and use capped RNA leaders can be tested easily. We were also able to obtain clear evidence that the capped RNA leaders selected by TSWV are generally longer than those by RSV. TSWV frequently uses the prime-and-realign mechanism in transcription primed by capped RNA leaders shorter than a certain length, like that has been demonstrated recently for RSV.

The complete genome sequence of a novel cytorhabdovirus identified in strawberry (Fragaria ananassa Duch.).

A cytorhabdovirus, tentatively named "strawberry-associated virus 1" (SaV1), was identified in strawberry (Fragaria ananassa Duch.), and its complete genome sequence was determined. Its negative-sense single-stranded RNA genome is composed of 14,159 nucleotides and contains eight open reading frames (ORFs) in the canonical order 3'-N-P-P3-M-G-P6-P7-L-5. The ORFs are separated by conserved intergenic sequences, and the genome coding region is flanked by 3' and 5' untranslated regions of 179 and 856 nt, respectively. SaV1 N and L genes shares 32-57% and 38-64% amino acid sequence identity with those of nine reported cytorhabdoviruses, respectively. Phylogenetic analysis showed that SaV1 clustered with high confidence with representative cytorhabdoviruses and is most closely related to tomato yellow mottle-associated virus. There are two additional small genes of unknown function between the G and L genes. We propose that SaV1 should be considered a member of a novel species in the genus Cytorhabdovirus, family Rhabdoviridae.

EasyCodeML: A visual tool for analysis of selection using CodeML.

The genomic signatures of positive selection and evolutionary constraints can be detected by analyses of nucleotide sequences. One of the most widely used programs for this purpose is CodeML, part of the PAML package. Although a number of bioinformatics tools have been developed to facilitate the use of CodeML, these have various limitations. Here, we present a wrapper tool named EasyCodeML that provides a user-friendly graphical interface for using CodeML. EasyCodeML has a custom running mode in which parameters can be adjusted to meet different requirements. It also offers a preset running mode in which an evolutionary analysis pipeline and publication-quality tables can be exported by a single click. EasyCodeML allows visualized, interactive tree labelling, which greatly simplifies the use of the branch, branch-site, and clade models of selection. The program allows comparison of major codon-based models for analyses of selection. EasyCodeML is a stand-alone package that is supported in Windows, Mac, and Linux operating systems, and is freely available at https://github.com/BioEasy/EasyCodeML.

Bayesian phylodynamic analysis reveals the dispersal patterns of tobacco mosaic virus in China.

Tobacco mosaic virus (TMV) is widespread in China and causes considerable economic losses to tobacco production. The molecular epidemiology of this virus is, however, poorly understood. In this study, we sequenced the genomes of 51 TMV isolates from five tobacco-producing regions in China and investigated the dispersal patterns of this virus. Our phylogenetic analysis showed that TMV might have been introduced to China in the early 1900s, probably first to southwest China. However, TMV then moved to the north of the country, where it expanded. The north became the main seeding region for the subsequent movements of the virus within China. The north-to-south movement of TMV coincides with a shift of major tobacco-producing areas from north to south in this century, suggesting a link between human activities and the dispersal of TMV in China.

Europe was a hub for the global spread of potato virus S in the 19th century.

Potato virus S (PVS) is a major plant pathogen that causes considerable losses in global potato production. Knowledge of the evolutionary history and spatio-temporal dynamics of PVS is vital for developing sustainable management schemes. In this study, we investigated the phylodynamics of the virus by analysing 103 nucleotide sequences of the coat protein gene, sampled between 1985 and 2014. Our Bayesian phylogenetic analyses showed that PVS has been evolving at a rate of 3.32 × 10-4 substitutions/site/year (95% credibility interval 1.33 × 10-4-5.58 × 10-4). We dated the crown group to the year 1325 CE (95% credibility interval 762-1743 CE). Our phylogeographic analyses pointed to viral origins in South America and identified multiple migration pathways between Europe and other regions, suggesting that Europe has been a major hub for PVS transmission. The results of our study have potential implications for developing effective strategies for the control of this pathogen.

Rapid evolutionary dynamics of pepper mild mottle virus.

Pepper mild mottle virus (PMMoV) is one of the most destructive pathogens of pepper crops and has major impacts on global crop yields. Some aspects of the molecular biology of PMMoV have been studied intensively, but estimates of its evolutionary rate have shown considerable variation. We investigated the phylodynamics of PMMoV by analysing 171 nucleotide sequences of the coat protein gene, sampled between 1980 and 2016. Our Bayesian phylogenetic analyses, using the structured coalescent, dated the crown group to 1949 (95% credibility interval 1935-1962). We reveal that PMMoV has been evolving at a rate of 9.363 × 10-4 substitutions/site/year (95% credibility interval 7.362 × 10-4-1.138 × 10-3). This is similar to evolutionary rates estimated for animal RNA viruses, indicating that PMMoV has been undergoing rapid evolutionary dynamics.

Genetic diversity of the families Aeshnidae, Gomphidae and Libellulidae through COI gene from South China.

Adult dragonflies (Anisoptera) were collected from different localities of South China covering eight provinces. Representative sequences were sixty-one, including 16 species, 11 genera and three families (Aeshnidae, Gomphidae and Libellulidae), under cytochrome oxidase subunit I (COI) gene. After alignment of sequences by BioEdit v6, genetic interaction and divergence were computed by MEGA 7 whereas all the indices of genetic diversity were calculated by DnaSP v5 software. Phylogenetic trees were constructed through Neighbor-Joining method under Jukes-Cantor model, and all species of respective families were assembled with each other into individual groups. Maximum divergence was observed by Trithemis genus (18.69%), followed by Orthetrum genus (18.16%), whereas a minimum value of divergence was noted for Pantala genus (0.31%). On the other hand, maximum genetic diversity was recorded for Orthetrum genus up to 142 mutations, followed by Trithemis genus (126 mutations), while the minimum value (two mutations) was observed for Pantala genus. Genetic diversity for overall and Libellulidae family sequences was much higher, up to 404 mutations and 344 mutations, respectively. Current results suggest a high diversity of odonates in the South China region and results are valuable in gaining a total obligation of the diversity of Asian odonates and conservation measures of this insect group.

Complete genome sequences of three novel cycloviruses identified in a dragonfly (Odonata: Anisoptera) from China.

Three cycloviruses (genus Cyclovirus, family Circoviridae) were recovered from a dragonfly (Odonata: Anisoptera) captured in Fuzhou, China. The three cycloviruses, named dragonfly associated cyclovirus 9, 10 and 11 (DfCyV-9, -10, -11), respectively, show 56.1-79.6% genome-wide identity to known cycloviruses and 61.6-65.1% among themselves. Thus, according to the current species demarcation criteria, they represent three novel cycloviruses. Notably, DfCyV-10 has a predicted replication-associated protein (Rep) that is most similar to that of bat associated cyclovirus 2 (BatACyV-2), a cyclovirus discovered in China, with 79.4% amino acid sequence identity, but a putative capsid protein (Cp) most similar to that of BatACyV-10, a cyclovirus discovered in Brazil, with 71.7% amino acid sequence identity. These data are useful for understanding the diversity and evolution of cycloviruses, especially those found in insects.

Genetic diversity and molecular evolution of Ornithogalum mosaic virus based on the coat protein gene sequence.

Ornithogalum mosaic virus (OrMV) has a wide host range and affects the production of a variety of ornamentals. In this study, the coat protein (CP) gene of OrMVwas used to investigate the molecular mechanisms underlying the evolution of this virus. The 36 OrMV isolates fell into two groups which have significant subpopulation differentiation with an FST value of 0.470. One isolate was identified as a recombinant and the other 35 recombination-free isolates could be divided into two major clades under different evolutionary constraints with dN/dS values of 0.055 and 0.028, respectively, indicating a role of purifying selection in the differentiation of OrMV. In addition, the results from analysis of molecular variance (AMOVA) indicated that the effect of host species on the genetic divergence of OrMV is greater than that of geography. Furthermore, OrMV isolates from the genera Ornithogalum, Lachenalia and Diuri tended to group together, indicating that OrMV diversification was maintained, in part, by host-driven adaptation.

A nation-wide genetic survey revealed a complex population structure of Bemisia tabaci in Pakistan.

The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex distributed worldwide. In Pakistan, B. tabaci poses a serious threat to agriculture production. To understand its diversity in Pakistan, a large-scale sampling was conducted from various locations of all four provinces of the country and Mitochondrial cytochrome oxidase I (mtCOI) gene sequencing was used to determine the whiteflies genetically. The study revealed the presence of five different cryptic species in Pakistan namely Asia II-1, Asia II-5, Asia II-7, Asia II-8 and MEAM-1, respectively. Among them, Asia II-1, which was previously reported from a few areas in the country, had been found now to be prevalent all over the country covering 88.7% of all the sequenced samples. Based on the mtCOI sequences and genetic distance analyses, the diversity of Asia II-1 was much greater than all other cryptic species, which exist only in small patches.

Genetic diversity of begomoviruses in Pakistan captured through a vector based survey.

Begomoviruses (Geminiviridea), transmitted by whiteflies, constitute one of the most dangerous groups of plant viruses posing a severe threat to economically important crops in tropical and sub-tropical areas. In this study, whiteflies were collected from various locations all over Pakistan. The begomoviruses carried by these whiteflies were detected by PCR with the degenerative primers pair AV94/Dep3. Analysis of the 177 sequences obtained in our study, revealed 14 distinct begomovirus species, including five which were not previously reported in this country. Putative novel strains of Corchorus yellow vein virus (CoYVV) and Chilli leaf curl virus (ChiLCV) showing less than 90% identity with the previously available taxa were also identified. The greatest number of begomoviruses per single site was detected in Sindh province, where up to five different begomovirus species were identified from the same cropping field. Moreover, Cotton leaf curl Multan virus - Rajasthan (CLCuMuV-Ra) was found prevalent in all the cotton growing areas. The data reported here may be useful in the development of control measures against begomoviruses.

Genetic interaction and diversity of the families Libellulidae and Gomphidae through COI gene from China and Pakistan.

A total of 300 dragonflies (Odonata) were collected from six different localities of China and Pakistan. Sixty seven representative samples were selected to sequence their mitochondrial cytochrome oxidase subunit I (COI). An examination of the resultant sequences identified 21 different dragonfly species, belonging to 15 distinct genera, two families, Libellulidae and Gomphidae. Sequence alignment was executed using Clustal-W in BioEdit v6. The phylogenetic tree was constructed through Neighbor-joining method by using Jukes-Cantor model, and genetic divergence was calculated via Kimura 2-parameter using MEGA7, while Genetic diversity was calculated by DnaSP v5. The maximum genetic divergence was observed for Crocothemis servilia, at 20.49%, followed by Libellulidae sp. with 22.30% while minimum divergence (0.82%) was observed for Melligomphus ardens. Likewise, a significant genetic diversity was observed for all species. However, Crocothemis servilia species presented maximum value (176 mutations) followed by Libellulidae spp. (150 mutations), whereas minimum value (3 mutations) was observed by Orthetrum testaceum. Interestingly, the diversity of C. servilia, all of which are collected from a single location of China, is much higher than those from Pakistan, which were collected from 5 different places with a spatial distance exceeding 500 Kms. Our results are useful in gaining a full appreciation of the global diversity of dragonflies and the development of conservation measures of this insect.