Jian Hong

Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection

Odontoglossum ringspot virus (ORSV) infects numerous commercially important orchids and causes significant losses worldwide. The coat protein (CP) gene of ORSV was cloned and expressed in Escherichia coli by using the pET-32a expression vector, and the expression of recombinant protein was confirmed by Western blotting using anti-ORSV antibodies. The recombinant protein was purified using Ni-NTA agarose, and the purified protein was used as an immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Five murine MAbs against ORSV CP were obtained. Among them, two MAbs (6B4 and 1D1) also reacted with TMV CP. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) methods using the MAb (8A5) were then developed for sensitive, specific, and rapid detection of ORSV. TAS-ELISA and IC-RT-PCR could detect ORSV in the infected leaf saps with dilutions of 1:10,240 and 1:81,920 (w/v, g mL(-1)), respectively. TAS-ELISA and IC-RT-PCR detections indicated that ORSV was prevalent in orchids in the Zhejiang Province of China.

Cell-to-cell trafficking, subcellular distribution, and binding to coat protein of Broad bean wilt virus 2 VP37 protein

Broad bean wilt virus 2 (BBWV 2) is a member of the genus Fabavirus of the family Comoviridae. To date, a movement protein (MP) of BBWV 2 has not been described. Here we demonstrate that the green fluorescent protein (GFP)-VP37 fusion protein can move from initial bombarded cells to neighboring cells in Nicotiana benthamiana epidermal leaves. In addition, the GFP-VP37 fusion protein localizes as a halo around the nucleus and as punctate spots on the cell periphery in N. benthamiana epidermal leaf cells and BY-2 suspension cells. Fluorescence near the nucleus also was co-localized with the endoplasmic reticulum in BY-2 cells. Fibrous networks were found in GFP-VP37 agro-infiltrated N. benthamiana epidermal leaf cells. Deletion analyses indicated that the C-terminal region of the VP37 protein is essential for localization at the cell periphery. Using a blot overlay assay and bimolecular fluorescence complementation assay, the purified 6xHis-tagged VP37 protein was shown to bind specifically to the small coat protein of BBWV 2. The above results indicate that VP37 is a movement protein.

The VP37 protein of Broad bean wilt virus 2 induces tubule-like structures in both plant and insect cells.

VP37 protein of Broad bean wilt virus 2 (BBWV-2) is a multifunctional protein that binds single-strand nucleic acids, interacts with viral coat protein (CP) and potentiates the virus cell-to-cell movement in its host plant. In this study, tubule-like structures filled with virus-like particles were observed by Electron Microscopy in plasmodesmata in walls of Chenopodium quinoa leaf cells infected with BBWV-2. Immunogold labeling using VP37 protein specific antibody demonstrates that the VP37 is a component of the tubular structures. When VP37 was fused with the green fluorescent protein (VP37-GFP) and expressed in BY-2 protoplasts or in insect Tn cells, green fluorescent tubules of various lengths were produced, protruding from the surface of the expressing cells. These findings suggest that the movement of BBWV-2 between cells is mediated by the tubular structures that contain the VP37 protein, and the VP37 protein itself is capable of inducing these tubule-like structures in cells. Our results also suggest that the plant and insect cell factors involved in the tubule formation have conserved features.

Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

Further characterization of Maize chlorotic mottle virus and its synergistic interaction with Sugarcane mosaic virus in maize

Maize chlorotic mottle virus (MCMV) was first reported in maize in China in 2009. In this study we further analyzed the epidemiology of MCMV and corn lethal necrosis disease (CLND) in China. We determined that CLND observed in China was caused by co-infection of MCMV and sugarcane mosaic virus (SCMV). Phylogenetic analysis using four full-length MCMV cDNA sequences obtained in this study and the available MCMV sequences retrieved from GenBank indicated that Chinese MCMV isolates were derived from the same source. To screen for maize germplasm resistance against MCMV infection, we constructed an infectious clone of MCMV isolate YN2 (pMCMV) and developed an Agrobacterium-mediated injection procedure to allow high throughput inoculations of maize with the MCMV infectious clone. Electron microscopy showed that chloroplast photosynthesis in leaves was significantly impeded by the co-infection of MCMV and SCMV. Mitochondria in the MCMV and SCMV co-infected cells were more severely damaged than in MCMV-infected cells. The results of this study provide further insight into the epidemiology of MCMV in China and shed new light on physiological and cytopathological changes related to CLND in maize.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Abstract Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and be-longs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV-CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10 −6 to 10 −7 as determined by indirect enzyme-linked immunosorbent assay (ELISA). Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1:2 560 and 1:10 240 (w/v, g mL −1 ), respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not detected in any sample collected from Zhejiang or Jiangxi Province, China.

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

Citrus tristeza virus (CTV) is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein (CP) gene of CTV was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a and purified through Ni+-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines (14B10, 14H11, 20D5, and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10-7 in indirect enzyme-linked immunosorbent assay (ELISA). Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich (TAS)-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1:2560 and 1:10, 240 (w/v, g/mL), respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.

Turnip Yellow Mosaic Virus P69 Interacts with and Suppresses GLK Transcription Factors to Cause Pale-Green Symptoms in Arabidopsis

Plant viral infections often induce genetic, hormonal, and metabolic perturbations, leading to disease symptoms such as leaf yellowing, leaf distortion, stunting, wilting, necrosis, and other abnormalities (Wang et al., 2012). Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus that infects Arabidopsis thaliana and plant species in the Brassica genus and causes bright yellow mosaic symptoms (Morch et al., 1988; Skotnicki et al., 1992). The TYMV genome contains three open reading frames that encode the putative RNA-dependent RNA polymerase, the coat protein, and the viral virulence protein P69, respectively (Morch et al., 1988; Skotnicki et al., 1992).

Analysis of fig tree virus type and distribution in China

Abstract The common fig (Ficus carica L.) was one of the earliest horticultural crops to be domesticated. A number of different viruses can infect fig trees including Fig mosaic virus (FMV) that has been detected in several commercial fig trees in Xinjiang, China. However, the distribution of FMV and other fig-infecting viruses in China remains unknown. In the present study, a sample from an ancient fig tree growing in Xinjiang was investigated by electron microscopy (EM) followed by PCR/RT-PCR, and FMV, Fig badnavirus 1 (FBV-1) and Fig leaf mottle-associated virus 1 (FLMaV-1) were detected. Fig leaf samples (252) from commercial orchards across China were subjected to PCR/RT-PCR, and FMV, FBV-1 and Fig fleck-associated virus (FFkaV) were relatively abundant (44.4, 48.4 and 44%, respectively), while FLMaV-1 and Fig mild mottle-associated virus (FMMaV) were much scarcer (5.6 and 0.4%, respectively), and FLMaV-2, Fig cryptic virus (FCV), and Fig latent virus (FLV) were not detected. The presence of disease-causing viruses in fig trees presents a significant challenge for fig producers in China. This study may help to promote actions aimed at controlling fig viruses, especially FMV.