Controlling the surface charge of simple viruses

The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant viruses, and between viruses and surfaces, properties that are essential for understanding their infectivity and to their use as bionanomaterials, are largely controlled by their surface charge, which depends on pH and ionic strength. They may also depend on the charge of their contents, i.e., of their genes or–in the instance of virus-like particles–encapsidated cargo such as nucleic acid molecules, nanoparticles or drugs. In the case of enveloped viruses, the surface charge of the capsid is equally important for controlling its interaction with the lipid bilayer that it acquires and loses upon leaving and entering host cells. We have previously investigated the charge on the unenveloped plant virus Cowpea Chlorotic Mottle Virus (CCMV) by measurements of its electrophoretic mobility. Here we examine the electrophoretic properties of a structurally and genetically closely related bromovirus, Brome Mosaic Virus (BMV), of its capsid protein, and of its empty viral shells, as functions of pH and ionic strength, and compare them with those of CCMV. From measurements of both solution and gel electrophoretic mobilities (EMs) we find that the isoelectric point (pI) of BMV (5.2) is significantly higher than that of CCMV (3.7), that virion EMs are essentially the same as those of the corresponding empty capsids, and that the same is true for the pIs of the virions and of their cleaved protein subunits. We discuss these results in terms of current theories of charged colloidal particles and relate them to biological processes and the role of surface charge in the design of new classes of drug and gene delivery systems.

Genome-wide identification of endogenous viral sequences in alfalfa (Medicago sativa L.)

Endogenous viral elements (EVEs) have been for the most part described in animals and to a less extent in plants. The endogenization was proposed to contribute toward evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. During the last two decades, several full-length and fragmented EVEs of pararetroviral and non-retroviral nature have been identified in different plant genomes, both monocots and eudicots. Prior to this work, no EVEs have been reported in alfalfa (Medicago sativa L.), the most cultivated forage legume in the world. In this study, taking advantage of the most recent developments in the field of alfalfa research, we have assessed alfalfa genome on the presence of viral-related sequences. Our analysis revealed segmented EVEs resembling two dsDNA reverse-transcribing virus species: Soybean chlorotic mottle virus (family Caulimoviridae, genus Soymovirus) and Figwort mosaic virus (family Caulimoviridae, genus Caulimovirus). The EVEs appear to be stable constituents of the host genome and in that capacity could potentially acquire functional roles in alfalfa’s development and response to environmental stresses.

Maize Seed Contamination and Seed Transmission of Maize Chlorotic Mottle Virus in Kenya

Maize chlorotic mottle virus (MCMV) causes maize lethal necrosis disease in combination with a cereal infecting potyvirus, leading to high yield losses. There is limited information on seed infection or contamination rate by MCMV and its comparison to transmission rate to maize seedlings. This study was conducted to determine the extent of seed contamination in seed lots from MCMV-infected maize fields in Kenya and the transmission of MCMV from seeds to seedlings. To determine the contamination levels, whole seeds were ground, and the extract tested for the presence of MCMV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Seedling grow-outs were tested for seed transmission of MCMV using DAS-ELISA and real-time reverse transcription polymerase chain reaction (real-time RT-PCR) methods. The seed contamination rates of the four seed lots tested ranged from 4.9 to 15.9%. MCMV transmission frequency for 37,617 seedlings, tested in 820 pools of varying seed amounts by DAS-ELISA, was 0.17%, while a transmission frequency of 0.025% was obtained from 8,322 seedlings tested in 242 pools by real-time RT-PCR. Seeds from plants mechanically inoculated with MCMV had an overall seed transmission rate of 0.04% in 7,846 seedlings tested in 197 pools. The study showed that even with substantial contamination of maize seed with MCMV, the transmission of the virus from the seed to seedlings was low. Nevertheless, even low rates of transmission can be significant under field conditions where insect vectors can further spread the disease from infected seedlings, unless diseased plants are detected in time and properly managed.

Maize catalases localized in peroxisomes support the replication of maize chlorotic mottle virus

Co-infection of maize chlorotic mottle virus (MCMV) with a virus in the Potyviridae family, such as sugarcane mosaic virus, usually leads to maize lethal necrosis (MLN). Over the past decade, MCMV/MLN has emerged in many countries/regions of the world and resulted in serious yield loss in maize production. Although partial functions of some MCMV-encoded proteins have been identified, the host factors related to MCMV replication are poorly understood. Here, we show that maize peroxisomes can form aggregated bodies in MCMV-infected leaf cells. The dsRNA binding-dependent fluorescence complementation assay indicated that the aggregated peroxisomes in maize served as the major replication site of MCMV. In addition, our results revealed that all the three maize catalases were present mostly in peroxisomes in the presence or absence of MCMV. Furthermore, we determined that inhibition of catalase activity or induction of reactive oxygen species (ROS) in maize protoplasts significantly reduced the accumulation of MCMV RNA. In summary, this research reveals the replication site of MCMV and an important role of maize catalases in supporting virus replication. Our results are conducive to understanding the pathogenesis of MCMV and identifying targets for resistance breeding or gene regulation strategies.

Antiviral therapy in shrimp through plant virus VLP containing VP28 dsRNA against WSSV

The white spot syndrome virus (WSSV), currently affecting cultured shrimp, causes substantial economic losses to the worldwide shrimp industry. An antiviral therapy using double-stranded RNA interference (dsRNAi) by intramuscular injection (IM) has proven the most effective shrimp protection against WSSV. However, IM treatment is still not viable for shrimp farms. The challenge is to develop an efficient oral delivery system that manages to avoid the degradation of antiviral RNA molecules. The present work demonstrates that VLPs (virus-like particles) allow efficient delivery of dsRNAi as antiviral therapy in shrimp. In particular, VLPs derived from a virus that infects plants, such as cowpea chlorotic mottle virus (CCMV), in which the capsid protein (CP) encapsidates the dsRNA of 563 bp, are shown to silence the WSSV glycoprotein VP28 (dsRNAvp28). In experimental challenges in vivo, the VLPs- dsRNAvp28 protect shrimp against WSSV up to 40% by oral administration and 100% by IM. The novel research demonstrates that plant VLPs, which avoid zoonosis, can be applied to pathogen control in shrimp and also other organisms, widening the application window in nanomedicine.

Introgression of Maize Lethal Necrosis Resistance Quantitative Trait Loci Into Susceptible Maize Populations and Validation of the Resistance Under Field Conditions in Naivasha, Kenya

Maize lethal necrosis (MLN), resulting from co-infection by maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) can cause up to 100% yield losses in maize in Africa under serious disease conditions. Maize improvement through conventional backcross (BC) takes many generations but can significantly be shortened when molecular tools are utilized in the breeding process. We used a donor parent (KS23-6) to transfer quantitative trait loci (QTL) for resistance to MLN into nine adapted but MLN susceptible lines. Nurseries were established in Kiboko, Kenya during 2015–2017 seasons and BC3F2 progeny were developed using marker assisted backcrossing (MABC) approach. Six single nucleotide polymorphism (SNP) markers linked to QTL for resistance to MLN were used to genotype 2,400 BC3F2 lines using Kompetitive Allele Specific PCR (KASP) platform. We detected that two of the six QTL had major effects for resistance to MLN under artificial inoculation field conditions in 56 candidate BC3F2 lines. To confirm whether these two QTL are reproducible under different field conditions, the 56 BC3F2 lines including their parents were evaluated in replicated trials for two seasons under artificial MLN inoculations in Naivasha, Kenya in 2018. Strong association of genotype with phenotype was detected. Consequently, 19 superior BC3F2 lines with favorable alleles and showing improved levels of resistance to MLN under artificial field inoculation were identified. These elite lines represent superior genetic resources for improvement of maize hybrids for resistance to MLN. However, 20 BC3F2 lines were fixed for both KASP markers but were susceptible to MLN under field conditions, which could suggest weak linkage between the KASP markers and target genes. The validated two major QTL can be utilized to speed up the breeding process but additional loci need to be identified between the KASP markers and the resistance genes to strengthen the linkage.