Assessing the hidden diversity underlying consensus sequences of SARS-CoV-2 using VICOS, a novel bioinformatic pipeline for identification of mixed viral populations.

INTRODUCTION: Coinfection with two SARS-CoV-2 viruses is still a very understudied phenomenon. Although next generation sequencing methods are very sensitive to detect heterogeneous viral populations in a sample, there is no standardized method for their characterization, so their clinical and epidemiological importance is unknown. MATERIAL AND METHODS: We developed VICOS (Viral COinfection Surveillance), a new bioinformatic algorithm for variant calling, filtering and statistical analysis to identify samples suspected of being mixed SARS-CoV-2 populations from a large dataset in the framework of a community genomic surveillance. VICOS was used to detect SARS-CoV-2 coinfections in a dataset of 1,097 complete genomes collected between March 2020 and August 2021 in Argentina. RESULTS: We detected 23 cases (2%) of SARS-CoV-2 coinfections. Detailed study of VICOS's results together with additional phylogenetic analysis revealed 3 cases of coinfections by two viruses of the same lineage, 2 cases by viruses of different genetic lineages, 13 were compatible with both coinfection and intra-host evolution, and 5 cases were likely a product of laboratory contamination. DISCUSSION: Intra-sample viral diversity provides important information to understand the transmission dynamics of SARS-CoV-2. Advanced bioinformatics tools, such as VICOS, are a necessary resource to help unveil the hidden diversity of SARS-CoV-2.

First Complete Genome Sequence of Potato leafroll virus from Argentina.

In this study, we determined for the first time the complete genomic sequence of an Argentinian isolate of Potato leafroll virus (PLRV), the type species of the genus Polerovirus The isolate sequenced came from a Solanum tuberosum plant that had been naturally infected with the virus. Isolate PLRV-AR had a nucleotide sequence identity between 94.4 and 97.3% with several known PLRV isolates worldwide.

Identification of a New Cotton Disease Caused by an Atypical Cotton Leafroll Dwarf Virus in Argentina.

An outbreak of a new disease occurred in cotton (Gossypium hirsutum) fields in northwest Argentina starting in the 2009-10 growing season and is still spreading steadily. The characteristic symptoms of the disease included slight leaf rolling and a bushy phenotype in the upper part of the plant. In this study, we determined the complete nucleotide sequences of two independent virus genomes isolated from cotton blue disease (CBD)-resistant and -susceptible cotton varieties. This virus genome comprised 5,866 nucleotides with an organization similar to that of the genus Polerovirus and was closely related to cotton leafroll dwarf virus, with protein identity ranging from 88 to 98%. The virus was subsequently transmitted to a CBD-resistant cotton variety using Aphis gossypii and symptoms were successfully reproduced. To study the persistence of the virus, we analyzed symptomatic plants from CBD-resistant varieties from different cotton-growing fields between 2013 and 2015 and showed the presence of the same virus strain. In addition, a constructed full-length infectious cDNA clone from the virus caused disease symptoms in systemic leaves of CBD-resistant cotton plants. Altogether, the new leafroll disease in CBD-resistant cotton plants is caused by an atypical cotton leafroll dwarf virus.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection.

Population genetics structure of glyphosate-resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance.

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.

Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse genetic approach.

Glyphosate resistance in perennial Sorghum halepense (Johnsongrass), endowed by reduced glyphosate translocation and leaf uptake.

BACKGROUND: In a large cropping area of northern Argentina, Sorghum halepense (Johnsongrass) has evolved towards glyphosate resistance. This study aimed to determine the molecular and biochemical basis conferring glyphosate resistance in this species. Experiments were conducted to assess target EPSPS gene sequences and (14)C-glyphosate leaf absorption and translocation to meristematic tissues. RESULTS: Individuals of all resistant (R) accessions exhibited significantly less glyphosate translocation to root (11% versus 29%) and stem (9% versus 26%) meristems when compared with susceptible (S) plants. A notably higher proportion of the applied glyphosate remained in the treated leaves of R plants (63%) than in the treated leaves of S plants (27%). In addition, individuals of S. halepense accession R(2) consistently showed lower glyphosate absorption rates in both adaxial (10-20%) and abaxial (20-25%) leaf surfaces compared with S plants. No glyphosate resistance endowing mutations in the EPSPS gene at Pro-101-106 residues were found in any of the evaluated R accessions. CONCLUSION: The results of the present investigation indicate that reduced glyphosate translocation to meristems is the primary mechanism endowing glyphosate resistance in S. halepense from cropping fields in Argentina. To a lesser extent, reduced glyphosate leaf uptake has also been shown to be involved in glyphosate-resistant S. halepense.

The complete genome sequence of a virus associated with cotton blue disease, cotton leafroll dwarf virus, confirms that it is a new member of the genus Polerovirus.

Cotton blue disease is the most important virus disease of cotton in the southern part of America. The complete nucleotide sequence of the ssRNA genome of the cotton blue disease-associated virus was determined for the first time. It comprised 5,866 nucleotides, and the deduced genomic organization resembled that of members of the genus Polerovirus. Sequence homology comparison and phylogenetic analysis confirm that this virus (previous proposed name cotton leafroll dwarf virus) is a member of a new species within the genus Polerovirus.

Functional and biochemical properties of Mal de Río Cuarto virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts.

Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.

Mal de Río Cuarto virus (MRCV) genomic segment S3 codes for the major core capsid protein.

Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. Compared with other plant-infecting genus of the family, the function and localization of MRCV and other Fijivirus proteins are poorly understood. Through analysis of viral particle purifications, we positively identified five structural proteins of approximately 170, 140, 130, 66, and 62 kDa. The protein encoded by MRCV S3 genomic segment was expressed as a fusion protein in Escherichia coli, purified and used for rabbit immunization. The resulting antiserum reacted with the 140 kDa structural protein and with incomplete virus particles in immunoelectron microscopy assays, suggesting that MRCV S3 codes for the major core capsid protein.

Virus infection elevates transcriptional activity of miR164a promoter in plants.

BACKGROUND: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations. RESULTS: Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction. CONCLUSION: This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.

Sequence and phylogenetic analysis of genome segments S1, S2, S3 and S6 of Mal de Río Cuarto virus, a newly accepted Fijivirus species.

Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of four MRCV genome segments was determined. MRCV S1, S2, S3 and S6 were predicted to encode proteins of 168.4, 134.4, 141.7 and 90 kDa, respectively. MRCV S1 encodes a basic protein that contains conserved RNA-dependent RNA polymerase motifs, and is homologous to Rice black streaked dwarf virus (RBSDV), Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV) polymerases as well as to corresponding proteins of members of other genera of the Reoviridae. MRCV S2 codes for a protein with intermediate homology to the ones coded by RBSDV S4 and FDV S3 'B' spike, which is presumably the B-spike protein. MRCV S3 most probably encodes the major core protein and is highly homologous to corresponding proteins of RBSDV S2 and FDV S3. MRCV S6-encoded protein has low homology to the proteins of unknown function coded by RBSDV S6 and FDV S6. The identity levels between all analyzed MRCV coded proteins and their RBSDV counterparts varied between 84.5 and 44.8%. The analysis of the reported sequences allowed a phylogenetic comparison of MRCV with other reovirus and supported its taxonomic status within the genus.