A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Background Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. Results In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. Conclusions Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

Broad neutralizing nanobody against SARS-CoV-2 engineered from pre-designed synthetic library

SARS-CoV-2 infection is initiated with Spike glycoprotein binding to the receptor of human angiotensin converting enzyme 2 via its receptor binding domain. Blocking this interaction is considered as an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody, VHH60, directly produced from a humanized synthetic nanobody library. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein with a KD of 2.56 nM, inhibits infections of both live SARS-CoV-2 and pseudotyped viruses harboring wildtype, escape mutations and prevailing variants at nanomolar level. VHH60 also suppresses SARS-CoV-2 infection and propagation 50-fold better and protects mice from death two times longer than that of control group after live virus inoculation on mice. VHH60 therefore is a powerful synthetic nanobody with a promising profile for disease control against COVID19.

Incomplete antiviral treatment may induce longer durations of viral shedding during SARS-CoV-2 infection

The duration of viral shedding is determined by a balance between de novo infection and removal of infected cells. That is, if infection is completely blocked with antiviral drugs (100% inhibition), the duration of viral shedding is minimal and is determined by the length of virus production. However, some mathematical models predict that if infected individuals are treated with antiviral drugs with efficacy below 100%, viral shedding may last longer than without treatment because further de novo infections are driven by entry of the virus into partially protected, uninfected cells at a slower rate. Using a simple mathematical model, we quantified SARS-CoV-2 infection dynamics in non-human primates and characterized the kinetics of viral shedding. We counterintuitively found that treatments initiated early, such as 0.5 d after virus inoculation, with intermediate to relatively high efficacy (30–70% inhibition of virus replication) yield a prolonged duration of viral shedding (by about 6.0 d) compared with no treatment.

Variation of resistance response of orchids induced by Rhizoctonia against the infection of Odontoglossum ringspot virus (ORSV) based on percentage of disease development

Orchid is one of the largest groups of flowering plants that are in great demand by society. Besides, the various flowers with a long vase life, the price of orchids is also stable and affordable. Phalaenopsis and Dendrobium are the most popular types of orchids. To anticipate this, orchid cultivation needs to be improved. However, orchid cultivation is not always accompanied by the effort of disease prevention, particularly those caused by viruses. The virus that most infects orchids is the Odontoglossum ringspot virus (ORSV). Symptoms that appear on the leaves are chlorosis, mosaic with a pattern of lines, circles, and necrotic rings/rings. Control of viral infections can utilize organisms that are able to induce resistance, namely mycorrhizae. Mycorrhizae will penetrate into the root cortex tissue and provide nutrients for the orchid. Mycorrhizae that are quite often found are Rhizoctonia and are applied by induction to orchids. Induction was undertaken by planting orchids in Rhizoctonia inoculum until anatomically a peloton structure (solid coils) was formed on the orchid roots. The aim of this study was to study the response of orchid plants in the form of percentage incidence and intensity of the disease and the level of plant resistance to ORSV infection in Phalaenopsis amabilis and Dendrobium discolour which had been induced by Rhizoctonia. This research was conducted at the Laboratory of Botany, Biology, FMIPA, Universitas Lampung, using a factorial completely randomized design (CRD). Factor 1 was the type of orchid and factor 2 was mycorrhizal induction, virus inoculation, and a combination of both with 4 replications. The finding indicated that Phalaenopsis amabilis was more susceptible to ORSV infection than Dendrobium discolour based on a higher incidence and intensity of the disease. Mycorrhizal induction and virus inoculation (MAV) in Dendrobium discolour showed an increase of resistance response compared to Phalaenopsis amabilis.

Silver Nanoparticles as A Highly Viricidal Agent to Deter Plant-Infecting Viruses and Disrupt their Acquisition and Transmissibility by Vector Aphid

Silver nanoparticles (AgNPs) are a potentially effective tool for deterring viral plant pathogens. This study was carried out to evaluate the efficacy of AgNPs to defeat Bean yellow mosaic virus (BYMV) on faba bean plants from the host, virus and vector aphid tripartite interactions side. The antiviral capabilities were evaluated during a foliar protective and curative scheme. Furthermore, the efficiency of AgNPs on virus acquisition and transmission by its vector aphid was investigated. Results indicated that the AgNPs had greatly exhibited curative viricidal activities for inactivation BYMV when applied 48 h post-virus inoculation. The disease occurrence was entirely inhibited with AgNPs rate as low as 100 mg.l− 1, while the infectivity was completely arrested when plants were preventively exposed to 200 mg.l− 1 24 h pre-virus inoculation. AgNPs proved high bio-reactivity by binding to viral particles, suppressing their replication and accumulation within the plant tissues. Moreover, it was noticeably showed to upregulate the pathogenesis-related gene (PR-1) and promote the defense-related enzymes and protein profiles in treated plants irrespective of concentration. Exposure of aphids to AgNPs-treated plants before virus acquisition excitingly reduced the BYMV acquisition and transmission efficiency by 40.65% up to 100 % 24 h post-application and the virus acquisition was affected for 10 days by 6.89 Up to 79.64 % depending on the AgNPs rate. These results concluded that the AgNPs have a high curing viricidal activity by targeting the virus envelop, and more excitingly it can affect the virus-vector combination, suggesting that it may contribute to alleviating the natural disease occurrence and virus transmission under field conditions. Therefore, according to the available literature, this study provides the first report on the deterring activity of nanomaterials against plant virus acquisition and transmission by its vector insects.

Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

Effect of Cucumber Mosaic Virus on Proline and Hydrogen Peroxide Content in Some Pepper Hybrids Grown in Lattakia Governorate, Syria

Al-Ajouriyeh, H., I. Ismail, B. Samra and F. Sahyouni. 2021. Effect of Cucumber Mosaic Virus on Proline and Hydrogen Peroxide Content in Some Pepper Hybrids Grown in Lattakia Governorate, Syria. Arab Journal of Plant Protection, 39(1): 39-46. A study was conducted at Al-Muturki village in Lattakia Governorate during 2019/2020 growing season to investigate the effect Cucumber mosaic virus (CMV) infection on the proline and hydrogen peroxide (H2O2) content of four pepper hybrids (Taline F1, Amani F1, Harek F1 and Marvilo F1) at three periods post viral inoculation (15, 30 and 45 days). The experiment was carried out by using complete randomized block design with 8 treatments and 8 replicates per treatment. The results showed that the viral infection affected the proline and hydrogen peroxidase content 15 days after virus inoculation. The highest effect of the viral infection was at 30 days after virus inoculation, where the values of proline and hydrogen peroxidase of the infected pepper hybrids significantly increased compared to control plants. However, 45 days after virus inoculation, the effect of viral infection decreased with plant age. Keywords: Pepper, Cucumber mosaic virus, proline, hydrogen peroxidase.

Study on the Dynamic Proliferation of JEV in BHK-21 Cells

<b><i>Introduction:</i></b> Epidemic Japanese encephalitis is one of the most important zoonotic diseases that cause central nervous system damage. The vaccination has become the most effective and economical measure for its control. Hence, real-time monitoring of Japanese encephalitis virus (JEV) proliferation is crucial to optimize virus inoculation, culturing conditions, and virus harvest time. <b><i>Methods:</i></b> The proliferation dynamics of JEV in BHK-21 cells was studied by combining the established quantitative PCR method with the conventional TCID₅₀ assay in this study. <b><i>Results:</i></b> The proliferation curve determined by the 2 methods has a definite parallel relationship, but the quantitative real-time PCR method (4 h) is faster and more sensitive than the TCID₅₀ method (3–4 days). The determination results of TCID₅₀ showed that the highest viral titer was 10^5.44 TCID₅₀/0.1 mL and 10^4.86 TCID₅₀/0.1 mL in cell suspension and culture supernate, respectively, while the virus RNA copies reached the peak at 1.0 × 10^7.5 copies/µL and 1.0 × 10^5.6 copies/µL in cell suspension and culture supernate, respectively. <b><i>Conclusion:</i></b> The comprehensive analysis showed that the best time for JEV proliferation in BHK-21 cell was 60 h post infection.

Inhibition of Rehmannia mosaic virus Infection by Ganoderma sp. Extract

In fruiting bodies and mycelia of several fungi belonging to a Basidiomycetes group, among others, Ganoderma contains active polysaccharides and has potential as an antiviral substances. This study aimed to determine the effect of mycelium and fruiting body extract of Ganoderma sp. against Rehmannia mosaic virus (ReMV) infections on Chenopodium amaranticolor with variations of dilution and application times. The results showed that mycelium and fruiting body extract of Ganoderma sp. could inhibit ReMV infections. Antiviral extract which were applied 4 hours before, and at the same time with virus inoculation resulted in a higher viral inhibition rates than when applied 4 hours after virus inoculation. Fruiting body extract at 10-1 and 10-2 dilutions delayed symptom appearance caused by ReMV infection when applied 4 hours before virus inoculation. Mycelium extract at dilution of 10-2 and 10-3 applied 4 hours before virus inoculation resulted in the highest inhibition rate (100%) which was indicated as the number of local lesions on Chenopodium amaranticolor leaves. Meanwhile the fruiting body extract dilution of 10-1 resulted in highest inhibition (99.55%) when applied 4 hours before virus inoculation.