Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5′ regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses.

Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5′ cap structure, and 3′ polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates. 5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations. The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors.

Microplastics and antibiotic resistance genes are two new pollutants in water environments, and they have potential risks to human health and ecological safety. On the basis of the accumulation of pollutants and microorganisms in sediment, macrobenthic invertebrates are considered as potential practitioners of microplastic degradation and antibiotic resistance gene (ARG) transfer. However, whether microplastic degradation can affect ARG transfer in aquatic environments, especially in the gut of macrobenthic invertebrates, remains unclear. In this study, we demonstrated that microplastics including polyethylene terephthalate (PET), polyvinyl chloride(PVC), polyamide (PA), polystyrene (PS), polypropylene (PP), polyethylene (PE), and polyurethane (PU), and ARGs including tetA, sul1, sul2, and sul3 were widely distributed in sediment and benthic invertebrates in Nansi lake. The distribution of ARGs was related to the number and size of microplastic particles. In particular, it was found for the first time that the content of ARGs corresponding to individual particles was linearly and negatively correlated with the size of microplastics. The results of animal feeding experiments showed that microplastic degradation in the gut of Chironomidae larvae could promote the conjugative transfer of ARGs. The underlying molecular mechanism was SOS response. This study provides a new method for the analysis of the interaction effect of multiple pollutants in freshwater environments.

Background: A variety of cis-acting RNA elements with structures in the 5′- or 3′-untranslated region (UTR) of viral genomes play key roles in viral translation. Cherry virus A (CVA) is a member of the genus Capillovirus in the family Betaflexiviridae. It has a positive single-stranded RNA genome of ~ 7400 nucleotides (nt). The length of the CVA 5′-UTR is ~ 100 nt; however, the function of this long UTR has not yet been reported. Methods: Molecular and phylogenetic analyses were performed on 75 CVA sequences, which could be divided into four groups, and the RNA secondary structure was predicted in four CVA 5′-UTR types. These four CVA 5′-UTR types were then inserted upstream of the firefly luciferase reporter gene FLuc (FLuc), and in vitro translation of the corresponding transcripts was evaluated using wheat germ extract (WGE). Then, in-line structure probing was performed to reveal the conserved RNA structures in CVA-5′UTR. Results: The four CVA 5′-UTR types appeared to have a conserved RNA structure, and the FLuc construct containing these four CVA 5′-UTR types increased the translation of FLuc by 2–3 folds, suggesting weak translation enhancement activity. Mutations in CVA 5′-UTR suppressed translation, suggesting that the conserved RNA structure was important for function. Conclusion: The conserved RNA secondary structure was identified by structural evolution analysis of different CVA isolates and was found to regulate translation.

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus in the family Tombusviridae. MNSV is naturally transmitted by the soil-inhabiting fungus Olpidium radicale and by seeds and can also be transmitted by mechanical inoculation (Gonzalez-Garza, 1979). MNSV infects mainly cucurbits including melon (Cucumis melon), watermelon (Citrullus lanatus) andcucumber (Cucumis sativus) and causes necrotic spots on leaves, stems and fruits as well as curled and wilted symptoms on leaves (Gonzalez-Garza, 1979). MNSV was originally reported in Japan (Kishi, 1966) and is widely distributed in Asia, Europe, and North America (Herrera et al., 2010). In China, MNSV was first reported in melon in Jiangsu Province in 2008 (Gu et al., 2008) and then occurred in Hami melon in Xinjiang Province in 2014 (Ref. to GenBank KP406627). During October of 2014, necrotic spots and curled symptom as well as mosaic, mottled and/or crinkle symptoms occurred on watermelon and melon in greenhouses in Tai’an city in the Shandong Province, China. To identify the potential existence of MNSV in these samples, total RNAs were extracted from diseased leaves using Trizol reagent, and then reverse transcription was performed using M-MLV reverse transcriptase with primer MN-1937-R (5-GTCTCCAGTGCCTTACCATGTC-3), complementary to the middle region of RNA-dependent RNA polymerase (RdRp) gene of MNSV. The reverse transcripts were used as template to amplify by PCR the potential fragment of 911-1937 of MNSV using primers MN-1937-R and MN-911-F (5-CTCGCTGGGTTCTGTACTTCG-3), corresponding to RdRp coding region of MNSV. PCR products with the size of about 1000 bp were detected in samples of watermelon and melon. The purified fragments were cloned into pMD18-T (Takara) and sequenced using universal primers M13F and M13R. Blast analysis of obstained sequencesshowed highest similarity with MNSV (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These partial RdRp sequences were submitted to GenBank obtaining number KU522434 for the watermelon and KU522435 for the melon isolate. They shared a nucleotide identity of 99.71 %. In addition, KU522434 (watermelon) shared 95.91 % identity with the previously reported MNSV-HM sequence from China (GenBank GU480022), while its identity with other reported MNSV sequences ranged from 70.20 % to 96.49 % (Table 1). Similar results were obtained with the corresponding sequence of the melon isolate (GenBank KU522435) (Table 1). DAS-ELISA was also successfully performed to confirm the presence of MNSV from diseased leaves. MNSV samples diluted by PBS buffer at 1: (20-26) were detected by DAS-ELISA using rabbit polyclonal antibody of MNSV (from NEOGEN, 1024) as capture antibody at 1/1000 dilution and detection antibody at 1/1000 dilution respectively. This is the first report of MNSV naturally infecting watermelon in China. This result suggests that MNSV has spread during past years in different hosts and regions in China. Quick and reliable detection on MNSV should be developed to avoid potential further losses of watermelon trade in China.