Signaling and Resistosome Formation in Plant Innate Immunity to Viruses: Is There a Common Mechanism of Antiviral Resistance Conserved across Kingdoms?

Virus-specific proteins, including coat proteins, movement proteins, replication proteins, and suppressors of RNA interference are capable of triggering the hypersensitive response (HR), which is a type of cell death in plants. The main cell death signaling pathway involves direct interaction of HR-inducing proteins with nucleotide-binding leucine-rich repeats (NLR) proteins encoded by plant resistance genes. Singleton NLR proteins act as both sensor and helper. In other cases, NLR proteins form an activation network leading to their oligomerization and formation of membrane-associated resistosomes, similar to metazoan inflammasomes and apoptosomes. In resistosomes, coiled-coil domains of NLR proteins form Ca2+ channels, while toll-like/interleukin-1 receptor-type (TIR) domains form oligomers that display NAD+ glycohydrolase (NADase) activity. This review is intended to highlight the current knowledge on plant innate antiviral defense signaling pathways in an attempt to define common features of antiviral resistance across the kingdoms of life.

New Cherry-Adapted Plum Pox Virus Phylogroups Discovered in Russia.

Plum pox virus (PPV) is the most pathogenic virus of stone fruit crops worldwide. Unusual PPV isolates were discovered on sour cherry (Prunus cerasus L.) and steppe cherry (P. fruticosa Pall.) in the Republic of Tatarstan and the Middle Ural region, Russia. They induced typical sharka symptoms and tested positive for PPV by ELISA and RT-PCR, but were not detected by PCR using known strain-specific primers. Their complete genomes were determined using high-throughput sequencing. Phylogenetic analysis allocated new isolates to four clearly distinguished lineages (SC, TAT, Y, Tat-26) within a cluster of PPV cherry-adapted strains. The phylogroups SC and TAT had 84.5 to 86.9% average nucleotide identity to each other and strain CR, with which they comprised a common subcluster. Isolates from the Middle Ural region (group Y) were closer to strain C, sharing 96.9% identity. The fourth lineage is represented by the isolate Tat-26, which was a recombinant of strain CR and C isolates as major and minor parents, respectively. These results show that the genetic diversity of PPV is higher than thought and may contribute to a better understanding of the origin and evolution of cherry-adapted strains of the virus. P. fruticosa was reported as a new natural PPV host for the first time.

Environmentally friendly method of RNA isolation.

The diversity of organisms, tissues and cells is so great that, to date, no universal method for RNA extraction from these biological materials exist. The RNA isolation technique with a mix of guanidine thiocyanate, phenol, and chloroform is most widely used. Extraction and purification of RNA methods using selling guanidinium-phenol (TRIzol)-based and silica-based column kits have limitations on toxicity, or RNA isolation, particularly for plants, and scaling. The agents' toxicity is particularly relevant when employing for mass analysis in practice while gaining RNA preparations during the pandemics, epizootics, and epiphytotic. In modern diagnostics of infections at the molecular level, powerful RT-PCR technology is used, which amplifies the detection of RNA pathogens by hundreds of millions of times. We proposed obtaining RNA samples from viruses, bacteria, and plants for the reverse transcription reactions with a subsequent amplification of cDNAs by the polymerase chain reaction using potent and nontoxic chaotropic agent ammonium trichloroacetate. The method works in the analytical and preparative range and can be useful in the case of extraordinary circumstances during mass infections. Potentially this method can be adapted for obtaining RNA samples ready for the RT-isothermal PCR in the field.

Atypical structure of the nuclear membrane, distribution of nuclear pores and lamin B1 in spermatozoa of patients with complete and partial globozoospermia.

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

Biodiversity of Phototrophs and Culturable Fungi in Gobustan Caves.

Unique natural objects, such as the caves of the Gobustan National Historical and Artistic Preserve, are also of great cultural and historical value due to rock art and sites of ancient people. A favorable microclimate makes these habitats convenient for colonization by microbiota, including phototrophs. In arid regions with intense seasonal fluctuations of microclimatic parameters, the conditions for survival are the least favorable; therefore, it becomes especially important to determine the composition of communities that are the most adapted to specific conditions. This work aimed to identify the biodiversity of communities of caves and grottoes of the Gobustan Reserve. The studies were carried out in July 2019. Samples were analyzed for cyanobacteria and algae by microscopy and cultivation methods, microfungi were isolated by soil dilution, and the fouling glass method was also used. In total, 29 taxa of cyanobacteria and algae, 18 taxa of fungi, and 3 species of mosses were identified. The studied habitats were dominated by the algae Chlorella vulgaris, Aphanocapsa sp., and Stichococcus bacillaris; the subdominants were Jaaginema subtilissimum, Leptolyngbya tenuis, Chlorococcum minutum, and Humidophila contenta. Microfungi had the highest occurrence of Aspergillus niger, Aureobasidium pullulans, Alternaria alternata, and Talaromyces ruber. It was noted that cyanobacteria dominated in morphologically differentiated biofilms and green algae on the rocks. The greatest number of microfungi was found in the aphotic zone and bryophyte tufts. The dominance of green algae is atypical for most caves of other regions and may be associated with intense lighting of habitats. The absence of protonema is a consequence of the aridity and low moisture content of the substrates.

Polyclonal antibodies against potato spindle tuber viroid RNA.

Ribonucleic acid (RNA) can act as a hapten in the direct immunization of animals. For antigen synthesis, 65 mg of viroid RNA were obtained by in vitro transcription of the recombinant DNA. We received a reasonable immune response in mice and rabbits with synthesized conjugate viroid RNA-lysozyme. Analyses of polyclonal mouse and rabbit antisera as well as estimates of antibody specificity were performed by dot-Enzyme Linked Immunosorbent Assay (ELISA), sandwich ELISA, and northern immunoblotting. Antiserum obtained showed strong cross-reactions with cellular RNA. The viroid polyclonal antibody cross-reactions with cellular RNAs were depleted via titration antibodies by the plant cellular or commercial yeast RNA. We successfully used antibodies against the viroid RNA-lysozyme antigen to detect the wild-type potato viroid and diagnose potato viroid infection. We presume that intrinsic cross-reactions of RNA antibodies are potentially dangerous after nucleic acid vaccination. Research into the specificity of antibodies against viral RNAs is underway.

Plum Pox Virus Strain C Isolates Can Reduce Sour Cherry Productivity.

The impact of plum pox virus (PPV) on sour cherry (Prunus cerasus L.) productivity has been studied by comparing the yield of PPV-infected and PPV-free fruit-bearing trees. A total of 152 16- to 17-year-old trees of nine cultivars and hybrids were surveyed in the production orchards (cultivar collection and hybrid testing plots) in the Republic of Tatarstan, Russia. Sixty trees tested positive for PPV using ELISA and RT-PCR. Among them, 58 PPV isolates belonged to the strain C and the other 2 isolates to the strain CV. For the cultivars Sevastyanovskaya, Shakirovskaya, hybrids 88-2 and 80-8, the average (2012 to 2019) productivity of infected trees was 38% to 45% lower than for PPV-free trees of the same cultivar or hybrid. No ilarviruses (prunus necrotic ringspot virus, prune dwarf virus, apple mosaic virus, American plum line pattern virus) were detected in PPV-infected trees, suggesting that reduced cherry productivity was attributed to the PPV infection. Thus, it was shown for the first time that PPV can reduce the productivity of at least some sour cherry cultivars and hybrids, and strain C isolates are responsible for crop losses.

COVID-19: Multiorgan Dissemination of SARS-CoV-2 Is Driven by Pulmonary Factors.

Multi-organ failure is one of the common causes of fatal outcome in COVID-19 patients. However, the pathogenetic association of the SARS-CoV-2 viral load (VL) level with fatal dysfunctions of the lungs, liver, kidneys, heart, spleen and brain, as well as with the risk of death in COVID-19 patients remains poorly understood. SARS-CoV-2 VL in the lungs, heart, liver, kidneys, brain, spleen and lymph nodes have been measured by RT qPCR using the following formula: NSARS-CoV-2/NABL1 × 100. Dissemination of SARS-CoV-2 in 30.5% of cases was mono-organ, and in 63.9% of cases, it was multi-organ. The average SARS-CoV-2 VL in the exudative phase of diffuse alveolar damage (DAD) was 60 times higher than in the proliferative phase. The SARS-CoV-2 VL in the lungs ranged from 0 to 250,281 copies. The "pulmonary factors" of SARS-CoV-2 multi-organ dissemination are the high level of SARS-CoV-2 VL (≥4909) and the exudative phase of DAD. The frequency of SARS-CoV-2 dissemination to lymph nodes was 86.9%, heart-56.5%, spleen-52.2%, liver-47.8%, kidney-26%, and brain-13%. We found no link between the SARS-CoV-2 VL level in the liver, kidneys, and heart and the serum level of CPK, LDH, ALP, ALT, AST and Cr of COVID-19 patients. Isolated detection of SARS-CoV-2 RNA in the myocardium of COVID-19 patients who died from heart failure is possible. The pathogenesis of COVID-19-associated multi-organ failure requires further research in a larger cohort of patients.

Viral Load and Patterns of SARS-CoV-2 Dissemination to the Lungs, Mediastinal Lymph Nodes, and Spleen of Patients with COVID-19 Associated Lymphopenia.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18-1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810-250281), lymph nodes-832 copies (range 96-11586), and spleen-71.5 copies (range 0-2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

Sequence Analysis of Plum pox virus Strain C Isolates from Russia Revealed Prevalence of the D96E Mutation in the Universal Epitope and Interstrain Recombination Events.

The understanding of genetic diversity, geographic distribution, and antigenic properties of Plum pox virus (PPV) is a prerequisite to improve control of sharka, the most detrimental viral disease of stone fruit crops worldwide. Forty new PPV strain C isolates were detected in sour cherry (Prunus cerasus) from three geographically distant (700⁻1100 km) regions of European Russia. Analysis of their 3'-terminal genomic sequences showed that nineteen isolates (47.5%) bear the D96E mutation in the universal epitope of the coat protein. Almost all of them cannot be detected by the monoclonal antibody 5B in triple antibody sandwich enzyme-linked immunosorbent assayand Western blot analysis that may potentially compromise serological PPV detection in cherries. Full-length genomes of seven PPV-C isolates were determined employing next-generation sequencing. Using the Recombination Detection Program (RDP4), the recombination event covering the region from (Cter)P1 to the middle of the HcPro gene was predicted in all the available PPV-C complete genomes. The isolates Tat-4, belonging to the strain CV, and RU-17sc (PPV-CR) were inferred as major and minor parents, respectively, suggesting possible pathways of evolution of the cherry-adapted strains. Downy cherry (P. tomentosa) was identified as the natural PPV-C host for the first time.

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV­based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod­shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

Immunogenicity and protective efficacy of candidate universal influenza A nanovaccines produced in plants by Tobacco mosaic virus-based vectors.

A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.