Geographical distribution, genetic studies and vector transmission of alfalfa enamovirus-1 infecting alfalfa crop in Argentina.

Alfalfa (Medicago sativa L.) growing areas of Argentina were surveyed between 2010 and 2018 to determine the geographical distribution and analyse the genetic diversity among alfalfa enamovirus-1 (AEV-1) isolates. The virus was detected in all 17 surveyed alfalfa-producing provinces, with a prevalence of 64%. The plant virus AEV-1 is widely distributed in the country, and its transmission vector has been unknown until now. Here we show that the black aphid Aphis craccivora can transmit AEV-1. The CP sequence identity among 16 AEV-1 isolates from Argentina was from 98 to 100% and from 98.9 to 100% at nucleotide and amino acid levels, respectively, indicating a low level of sequence variation among these isolates. The phylogenetic analysis based on the complete nucleotide sequence of the CP gene indicated that AEV-1 isolates are closely related and clustered in a monophyletic group. These results suggest that AEV-1 has spread very recently in Argentina. In the present study, we report the geographical distribution of AEV-1 in the main alfalfa-growing areas of Argentina and, for the first time, identify an insect vector and describe the CP gene diversity of an enamovirus worldwide.

Distribution and genetic variability of alfalfa dwarf virus, a cytorhabdovirus associated with alfalfa dwarf disease in Argentina.

In 2010, a novel cytorhabdovirus named alfalfa dwarf virus (ADV) was detected for the first time in lucerne crops in Argentina showing dwarfism, in mixed infections with several other viruses. ADV appears to be endemic to Argentina and has not been reported elsewhere. In this study, we have investigated the genetic variability of ADV based on the complete nucleoprotein (N) gene of 13 isolates from different lucerne-growing regions in Argentina. Phylogenetic and sequence identity analyses showed that all ADV isolates are closely related and have not diverged more than 1% in the N gene despite geographical separation. These data provide further evidence that ADV is new to science and emerged and spread very recently. A total of 43 single-nucleotide polymorphisms were identified between the ADV isolates studied. Analysis of N gene ORF sequence revealed a mutational bias, with more transitions than transversions. In all cases, the ratio of non-synonymous/synonymous nucleotide changes was < 1, indicating that ADV N gene is under predominantly purifying selection.

Genome characterization of an Argentinean isolate of alfalfa leaf curl virus.

We investigated the molecular characteristics of an Argentinean isolate of alfalfa leaf curl virus (ALCV-Arg), a virus of the genus Capulavirus in the family Geminiviridae that was isolated from alfalfa plants showing dwarfism. The genome was found to be 2,750 nucleotides in length. In pairwise comparisons, this ALCV isolate shared 83.2% to 92.6% sequence identity with European ALCV isolates. Sequence comparisons and phylogenetic analysis showed that this isolate combines features of strains A and B of ALCV. Recombination analysis showed that ALCV-Arg is a recombinant isolate that was generated by intraspecific recombination between ALCV strains A and B. The results of this study not only show that ALCV-Arg is unique because it combines features of strains A and B but also show that ALCV naturally infects this forage crop on the American continent.

The Effects of Bean Leafroll Virus on Life History Traits and Host Selection Behavior of Specialized Pea Aphid (Acyrthosiphon pisum, Hemiptera: Aphididae) Genotypes.

Intraspecific specialization by insect herbivores on different host plant species contributes to the formation of genetically distinct "host races," but the effects of plant virus infection on interactions between specialized herbivores and their host plants have barely been investigated. Using three genetically and phenotypically divergent pea aphid clones (Acyrthosiphon pisum L.) adapted to either pea (Pisum sativum L.) or alfalfa (Medicago sativa L.), we tested how infection of these hosts by an insect-borne phytovirus (Bean leafroll virus; BLRV) affects aphid performance and preference. Four important findings emerged: 1) mean aphid survival rate and intrinsic rate of population growth (Rm) were increased by 15% and 14%, respectively, for aphids feeding on plants infected with BLRV; 2) 34% of variance in survival rate was attributable to clone × host plant interactions; 3) a three-way aphid clone × host plant species × virus treatment significantly affected intrinsic rates of population growth; and 4) each clone exhibited a preference for either pea or alfalfa when choosing between noninfected host plants, but for two of the three clones tested these preferences were modestly reduced when selecting among virus-infected host plants. Our studies show that colonizing BLRV-infected hosts increased A. pisum survival and rates of population growth, confirming that the virus benefits A. pisum. BLRV transmission affected aphid discrimination of host plant species in a genotype-specific fashion, and we detected three unique "virus-association phenotypes," with potential consequences for patterns of host plant use by aphid populations and crop virus epidemiology.

Complete genome sequence of a new enamovirus from Argentina infecting alfalfa plants showing dwarfism symptoms.

Alfalfa dwarf disease, probably caused by synergistic interactions of mixed virus infections, is a major and emergent disease that threatens alfalfa production in Argentina. Deep sequencing of diseased alfalfa plant samples from the central region of Argentina resulted in the identification of a new virus genome resembling enamoviruses in sequence and genome structure. Phylogenetic analysis suggests that it is a new member of the genus Enamovirus, family Luteoviridae. The virus is tentatively named "alfalfa enamovirus 1" (AEV-1). The availability of the AEV-1 genome sequence will make it possible to assess the genetic variability of this virus and to construct an infectious clone to investigate its role in alfalfa dwarfism disease.

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants.

Vaccines produced in transgenic plants constitute a promising alternative to conventional immunogens, presenting the possibility of stimulating secretory and systemic immunity against enteric pathogens when administered orally. Protection against enteric pathogens affecting newborn animals requires, in most cases, the stimulation of lactogenic immunity. Here, the group presents the development of an experimental immunogen based on expression of an immunorelevant peptide, eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as harbouring at least one neutralizing epitope as well as being responsible for the adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently expressed in transgenic alfalfa as a translational fusion protein with the highly stable reporter enzyme beta-glucuronidase (betaGUS), which served as a carrier, stabilized the synthesized peptide and facilitated screening for the higher expression levels in plants. Correlation of expression of the eBRV4 epitope in plants with those presenting the highest betaGUS activities was confirmed by a Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in plants was effective in inducing an anti-rotavirus antibody response in adult female mice when administered either intraperitoneally or orally and, more importantly, suckling mice born from immunized female mice were protected against oral challenge with virulent rotavirus. These results demonstrate the feasibility of inducing lactogenic immunity against an enteric pathogen using an edible vaccine produced in transgenic plants.