Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C.

In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India.

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

The Coat Protein of Citrus Yellow Vein Clearing Virus Interacts with Viral Movement Proteins and Serves as an RNA Silencing Suppressor.

Citrus yellow vein clearing virus is a newly accepted member of the genus Mandarivirus in the family Alphaflexiviridae. The triple gene block proteins (TGBp1, TGBp2 and TGBp3) encoded by plant viruses in this family function on facilitating virus movement. However, the protein function of citrus yellow vein clearing virus (CYVCV) have never been explored. Here, we showed in both yeast two-hybrid (Y2H) and bimolecular fluorescence (BiFC) assays that the coat protein (CP), TGBp1 and TGBp2 of CYVCV are self-interacting. Its CP also interacts with all three TGB proteins, and TGBp1 and TGBp2 interact with each other but not with TGBp3. Furthermore, the viral CP colocalizes with TGBp1 and TGBp3 at the plasmodesmata (PD) of epidermal cells of Nicotiana benthamiana leaves, and TGBp1 can translocate TGBp2 from granular-like structures embedded within ER networks to the PD. The results suggest that these proteins could coexist at the PD of epidermal cells of N. benthamiana. Using Agrobacterium infiltration-mediated RNA silencing assays, we show that CYVCV CP is a strong RNA silencing suppressor (RSS) triggered by positive-sense green fluorescent protein (GFP) RNA. The presented results provide insights for further revealing the mechanism of the viral movement and suppression of RNA silencing.

p15 encoded by Garlic virus X is a pathogenicity factor and RNA silencing suppressor.

Garlic virus X (GarVX) encodes a 15 kDa cysteine-rich protein (CRP). To investigate the function(s) of p15, its subcellular localization, role as a symptom determinant and capacity to act as a viral suppressor of RNA silencing (VSR) were analysed. Results showed that GFP-tagged p15 was distributed in the cytoplasm, nucleus and nucleolus. Expression of p15 from PVX caused additional systemic foliar malformation and led to increased accumulation of PVX, showing that p15 is a virulence factor for reconstructed PVX-p15. Moreover, using a transient agro-infiltration patch assay and a Turnip crinkle virus (TCV) movement complementation assay, it was demonstrated that p15 possesses weak RNA silencing suppressor activity. Removal of an amino acid motif resembling a nuclear localization signal (NLS) prevented p15 from accumulating in the nucleus but did not abolish its silencing suppression activity. This study provides the first insights into the multiple functions of the GarVX p15 protein.

Characterization of the complete genome of euonymus yellow vein associated virus, a distinct member of the genus Potexvirus, family Alphaflexiviridae, isolated from Euonymus bungeanus Maxim in Liaoning, Northern China.

In August 2016, a yellow vein disease was observed on leaves of Euonymus bungeanus Maxim (Euonymus, Celastraceae) in Liaoning, China. Virions measuring 750 × 13 nm were observed in a sample from the diseased plant. A potexvirus was detected in the sample by small-RNA deep sequencing analysis and recovered by traditional cloning. The genome of this potexvirus consists of 7,279 nucleotides, excluding the poly(A) tail at the 3' end, and contains five open reading frames (ORFs). Based on the nucleotide and amino acid sequences of the coat protein gene, the virus shared the highest sequence similarity with white clover mosaic virus (WCMV, X16636) (40.1%) and clover yellow mosaic virus (ClYMV, D00485) (37.1%). Phylogenetic analysis showed that the virus clustered with potexviruses and is most closely related to strawberry mild yellow edge virus. These results indicate that this virus is a distinct member of the genus Potexvirus, for which the name euonymus yellow vein associated virus (EuYVAV) is proposed. To our knowledge, this is the first report of a potexvirus on E. bungeanus.

Comparative detection of a large population of grapevine viruses by TaqMan® RT-qPCR and ELISA.

Grapevine (Vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. Reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. Biological indexing and ELISA are currently the standard methods for screening propagation material, and PCR-methods are becoming increasingly popular. Due to the diversity of virus isolates, it is essential to verify that the tests allow the detection of the largest possible virus populations. We developed three quadruplex TaqMan® RT-qPCR assays for detecting nine different viruses that cause considerable damage in many vineyards world-wide. Each assay is designed to detect three viruses and the grapevine Actin as an internal control. A large population of grapevines from diverse cultivars and geographic location was tested for the presence of nine viruses: Arabis mosaic virus (ArMV), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses (GLRaV-1, -2, -3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), and Grapevine virus B (GVB). In general, identical results were obtained with multiplex TaqMan® RT-qPCR and ELISA although, in some cases, viruses could be detected by only one of the two techniques.

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection.

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.

Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widespread virus infecting Vitis spp. Although it has established a compatible viral interaction in Vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse Vitis species. The changes induced by GRSPaV in V. vinifera cv 'Bosco', an Italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order to provide comprehensive information about the global effects of GRSPaV. In two years, this virus caused a moderate decrease in physiological efficiency, yield performance, and sugar content in berries associated with several transcriptomic alterations. Transcript profiles were analysed by a microarray technique in petiole, leaf, and berry samples collected at véraison and by real-time RT-PCR in a time course carried out at five grapevine developmental stages. Global gene expression analyses showed that transcriptomic changes were highly variable among the different organs and the different phenological phases. GRSPaV triggers some unique responses in the grapevine at véraison, never reported before for other plant-virus interactions. These responses include an increase in transcripts involved in photosynthesis and CO(2) fixation, a moderate reduction in the photosynthesis rate and some defence mechanisms, and an overlap with responses to water and salinity stresses. It is hypothesized that the long co-existence of grapevine and GRSPaV has resulted in the evolution of a form of mutual adaptation between the virus and its host. This study contributes to elucidating alternative mechanisms used by infected plants to contend with viruses.

Expressing a whitefly GroEL protein in Nicotiana benthamiana plants confers tolerance to tomato yellow leaf curl virus and cucumber mosaic virus, but not to grapevine virus A or tobacco mosaic virus.

Transgenesis offers many ways to obtain virus-resistant plants. However, in most cases resistance is against a single virus or viral strain. We have taken a novel approach based on the ability of a whitefly endosymbiotic GroEL to bind viruses belonging to several genera, in vivo and in vitro. We have expressed the GroEL gene in Nicotiana benthamiana plants, postulating that upon virus inoculation, GroEL will bind to virions, thereby interfering with pathogenesis. The transgenic plants were inoculated with the begomovirus tomato yellow leaf curl virus (TYLCV) and the cucumovirus cucumber mosaic virus (CMV), both of which interacted with GroEL in vitro, and with the trichovirus grapevine virus A (GVA) and the tobamovirus tobacco mosaic virus (TMV), which did not. While the transgenic plants inoculated with TYLCV and CMV presented a high level of tolerance, those inoculated with GVA and TMV were susceptible. The amounts of virus in tolerant transgenic plants was lower by three orders of magnitude than those in non-transgenic plants; in comparison, the amounts of virus in susceptible transgenic plants were similar to those in non-transgenic plants. Leaf extracts of the tolerant plants contained GroEL-virus complexes. Hence, tolerance was correlated with trapping of viruses in planta. This study demonstrated that multiple resistances to viruses belonging to several different taxonomic genera could be achieved. Moreover, it might be hypothesized that plants expressing GroEL will be tolerant to those viruses that bind to GroEL in vitro, such as members of the genera Begomovirus, Cucumovirus, Ilarvirus, Luteovirus, and Tospovirus.

Analysis of the complete genome of peach chlorotic mottle virus: identification of non-AUG start codons, in vitro coat protein expression, and elucidation of serological cross-reactions.

The entire genome of peach chlorotic mottle virus (PCMV), originally identified as Prunus persica cv. Agua virus (4N6), was sequenced and analysed. PCMV cross-reacts with antisera to diverse viruses, such as plum pox virus (PPV), genus Potyvirus, family Potyviridae; and apple stem pitting virus (ASPV), genus Foveavirus, family Flexiviridae. The PCMV genome consists of 9005 nucleotides (nts), excluding a poly(A) tail at the 3' end of the genome. Five open reading frames (ORFs) were identified with four untranslated regions (UTR) including a 5', a 3', and two intergenic UTRs. The genome organisation of PCMV is similar to that of ASPV and the two genomes share a nucleotide (nt) sequence identity of 58%. PCMV ORF1 encodes the replication-associated protein complex (Mr 241,503), ORF2-ORF4 code for the triple gene block proteins (TGBp; Mr 24,802, 12,370, and 7320, respectively), and ORF5 encodes the coat protein (CP) (Mr 42,505). Two non-AUG start codons participate in the initiation of translation: 35AUC and 7676AUA initiate translation of ORF1 and ORF5. In vitro expression with subsequent Western blot analysis confirmed ORF5 as the CP-encoding gene and confirmed that the codon AUA is able to initiate translation of the CP. Expression of a truncated CP fragment (Mr 39, 689) was demonstrated, and both proteins are expressed in vivo, since both were observed in Western blot analysis of PCMV-infected peach and Nicotiana occidentalis. The expressed proteins cross-reacted with an antiserum against ASPV. The amino acid sequences of the CPs of PCMV and ASPV CP share only 37% identity, but there are 11 shared peptides 4-8 aa residues long. These may constitute linear epitopes responsible for ASPV antiserum cross reactions. No significant common linear epitopes were associated with PPV. Extensive phylogenetic analysis indicates that PCMV is closely related to ASPV and is a new and distinct member of the genus Foveavirus.