RESUMEN
Solenopsis invicta virus 4 (SINV-4), a new polycipivirus, was characterized in the host in which it was discovered, Solenopsis invicta. SINV-4 was detected in the worker and larval stages of S. invicta, but not in pupae, male or female alates, or queens. The SINV-4 titer was highest in worker ants, with a mean of 1.14 × 107 ± 5.84 ×107 SINV-4 genome equivalents/ng RNA. Electron microscopic examination of negatively stained samples from particles purified from SINV-4-infected fire ant workers revealed isometric particles with a mean diameter of 47.3 ± 1.4 nm. The mean inter-colony SINV-4 infection rate among S. invicta worker ants was 45.8 ± 38.6 in Alachua County, Florida. In S. invicta collected in Argentina, SINV-4 was detected in 22% of 54 colonies surveyed from across the Formosa region. There did not appear to be any seasonality associated with the SINV-4 infection rate among S. invicta nests. SINV-4 was successfully transmitted to uninfected S. invicta colonies by feeding. Among three colonies of S. invicta inoculated with SINV-4, two retained the infection for up to 72 days. The replicative genome strand of SINV-4 was detected in 18% (n = 11) of SINV-4-infected S. invicta colonies. Among 33 ant species examined, the plus genome strand of SINV-4 was detected in undetermined species of Dorymyrmex and Pheidole, Cyphomyrmex rimosus, Monomorium pharaonis, Pheidole obscurithorax, Solenopsis geminata, Solenopsis richteri, Solenopsis xyloni, and Solenopsis invicta. However, the replicative (minus) genome strand was only detected in S. invicta. SINV-4 infection did not impact brood production or queen fecundity in S. invicta. The mean brood rating (63.3% ± 8.8) after 31 days for SINV-4-infected colonies was not statistically different from that of uninfected colonies (48.3 ± 25.5). At the end of the 31-day test period, mean egg production was not significantly different between SINV-4-infected S. invicta colonies (287.7 ± 45.2 eggs laid/24 hours) and uninfected control colonies (193.0 ± 43.6 eggs laid/24 hours).
Asunto(s)
Hormigas , Virus ARN , Animales , Femenino , Masculino , Virus ARN/genética , Larva , Argentina , FloridaRESUMEN
The red imported fire ant (Solenopsis invicta) escaped its natural enemies when it was introduced into North America in the 1930s from South America. US efforts have focused on discovery of natural enemies, like viruses, to provide sustainable control of the ant. Nine new virus genomes were sequenced from the invasive fire ant Solenopsis invicta using metagenomic RNA sequencing. The virus genomes were verified by Sanger sequencing and random amplification of cDNA ends reactions. In addition to the nine new virus genomes, the previously described Solenopsis viruses were also detected, including Solenopsis invicta virus 1 (SINV-1), SINV-2, SINV-3, SINV-4, SINV-5, and Solenopsis invicta densovirus. The virus sequences came from S. invicta workers, larvae, pupae, and dead workers taken from midden piles collected from across the ant's native range in Formosa, Argentina. One of the new virus genomes (Solenopsis invicta virus 6) was also detected in populations of North American S. invicta. Phylogenetic analysis of the RNA dependent RNA polymerase, the entire nonstructural polyprotein, and genome characteristics were used to tentatively taxonomically place these new virus genome sequences; these include four new species of Dicistroviridae, one Polycipiviridae, one Iflaviridae, one Totiviridae, and two genome sequences that were too taxonomically divergent to be placed with certainty. The S. invicta virome is the best characterized from any ant species and includes 13 positive-sense, single-stranded RNA viruses (Solenopsis invicta virus 1 to Solenopsis invicta virus 13), one double-stranded RNA virus (Solenopsis midden virus), and one double-stranded DNA virus (Solenopsis invicta densovirus). These new additions to the S. invicta virome offer potentially new classical biological control agents for S. invicta.
Asunto(s)
Hormigas/virología , Dicistroviridae/genética , Metagenómica , Virus ARN/genética , Animales , Argentina , Dicistroviridae/aislamiento & purificación , Genoma Viral/genética , Virus ARN/aislamiento & purificación , ARN Viral/genética , Análisis de Secuencia de ARNRESUMEN
Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.
Asunto(s)
Hormigas/virología , Virus ARN/patogenicidad , Animales , Argentina , Genes Virales , Metagenómica , Sistemas de Lectura Abierta , Filogenia , Virus ARN/clasificación , Virus ARN/genéticaRESUMEN
The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan in an attempt to infer demographic information about the recent S. invicta introduction into Taiwan by phylogenetic analysis. Nucleotide sequences were calculated to exhibit an overall identity of >90% between geographically-separated samples. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. Examination of the amino acid alignment for the RdRp revealed sequence identity >98% with only nine amino acid changes observed. Seven of these changes occurred in less than 4.3% of samples, while 2 (at positions 1266 and 1285) were featured prominently. Changes at positions 1266 and 1285 accounted for 36.2% and 34.0% of the samples, respectively. Two distinct groups were observed based on the amino acid residue at position 1266, Threonine or Serine. In cases where this amino acid was a Threonine, 90% of these sequences possessed a corresponding Valine at position 1285; only 10% of the Threonine(1266)-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine(1266) group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine(1266)/Valine(1285) and Serine(1266)/Isoleucine(1285) combinations are predominant phenotypes.
Asunto(s)
Hormigas/virología , Demografía , Dicistroviridae/genética , Dicistroviridae/patogenicidad , Genoma Viral/genética , Mutación/genética , Secuencia de Aminoácidos , Animales , Argentina , Datos de Secuencia Molecular , Filogenia , Poliproteínas/análisis , Taiwán , Estados UnidosRESUMEN
Solenopsis invicta virus 3 (SINV-3) is a recently described positive-strand RNA virus that infects the red imported fire ant, S. invicta. The genome of an Argentinean isolate of Solenopsis invicta virus 3 (SINV-3(ArgSF )) obtained from the Santa Fe region of Argentina was sequenced in entirety. Assembly of nine overlapping fragments yielded a consensus genome sequence 10,386 nucleotides long, excluding the poly(A) tail present on the 3' end (Genbank accession number GU017972). With the exception of the poly(A) tail, the genome length of SINV-3(ArgSF ) was identical to the North American isolate (SINV-3(USDM )). The SINV-3(ArgSF ) genome possessed three major open reading frames (ORFs) (comprised of >or=100 codons) in the sense orientation; SINV-3(USDM ) possessed only two. ORFs 1 and 2 had identical start and stop genome positions for both isolates. Blastp analysis of the translated ORF 1 of SINV-3(ArgSF ) recognized conserved domains for helicase, protease, and RNA-dependent RNA polymerase. These domains and their corresponding positions were identical to those reported for SINV-3(USDM ). ORF 2a, unique to the SINV-3(ArgSF ) genome, was also found in frame 2 and had a canonical start codon located at nucleotide position 8,351 and a stop codon ending at position 8,827. Blastp analysis of the translated amino acid sequence of ORF 2a revealed no significant similarity in the Genbank database. The two SINV-3 isolates exhibited 96.2% nucleotide sequence identity across the entire genome. The amino acid sequences of ORFs 1 and 2 exhibited higher identities (99.0 and 98.2%, respectively) than the corresponding nucleotide regions within the genome. These data indicated that the nucleotide differences between the SINV-3 isolates were largely synonymous. This observation was corroborated by codon substitution rate analysis. Thus, the majority of the SINV-3 codon changes were silent in the two polyproteins, indicating purifying selection pressure on the viral genome.
Asunto(s)
Hormigas/virología , Genoma Viral , Virus ARN/genética , Animales , Argentina , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mutación Puntual , Virus ARN/aislamiento & purificación , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
Multiplex reverse transcription and polymerase chain reaction (PCR) methods were developed to detect Solenopsis invicta viruses -1, -2, and -3 simultaneously in their host, the red imported fire ant, S. invicta. cDNA synthesis was conducted in a single reaction containing an oligonucleotide primer specific for each virus. Multiplex PCR was subsequently conducted with oligonucleotide primer pairs specific for each virus. The method was specific and sensitive, capable of detecting as few as 500 copies of the viral genomes consistently. Specificity was verified by PCR and amplicon sequencing. The method was evaluated against field-collected samples of ant workers from colonies in Argentina (n=135 ant colonies) and the United States (n=172 ant colonies). The prevalence of each virus in fire ant colonies varied considerably from site to site. A number of colonies exhibited multiple virus infections. However, the multiple SINV infection rate was lower than for single infections. Comparison of viral infection prevalence between S. invicta colonies in Argentina and the U.S. showed no statistical differences, regardless of infection category. This method is anticipated to facilitate epidemiological and related studies concerning the S. invicta viruses in fire ants.