Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

Is the Intergenic Region of Aedes aegypti Totivirus a Recombination Hotspot?

The genus totivirus in the family Totiviridae contains double-stranded RNA viruses. Their genome has two open reading frames (ORFs) that encode capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). The toti-like viruses recently identified in Anopheles sp. and Aedes aegypti mosquitoes (AaTV) share the same genome organization as other totiviruses. The AaTVs that have been described in distinct geographical regions are monophyletic. In this study, we show that AaTV sequences can be grouped into at least three phylogenetic clades (named A, B, and C). Clades A and B are composed of AaTV sequences from mosquitoes collected in the Caribbean region (Guadeloupe), and clade C contains sequences from the USA. These clades may represent AaTV lineages that are locally adapted to their host populations. We also identified three recombinant AaTV strains circulating in mosquitoes in Guadeloupe. Although these strains have different chimeric patterns, the position of the recombination breakpoint was identical in all strains. Interestingly, this breakpoint is located in a hairpin-like structure in the intergenic region of the AaTV genome. This RNA structure may stall RNA polymerase processivity and consequently induce template switching. In vitro studies should be conducted to further investigate the biological significance of AaTV's intergenic region as a recombination hotspot.

2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology.

2A is an oligopeptide sequence that mediates a ribosome "skipping" effect and can mediate a co-translation cleavage of polyproteins. These sequences are widely distributed from insect to mammalian viruses and could act by accelerating adaptive capacity. These sequences have been used in many heterologous co-expression systems because they are versatile tools for cleaving proteins of biotechnological interest. In this work, we review and update the occurrence of 2A/2A-like sequences in different groups of viruses by screening the sequences available in the National Center for Biotechnology Information database. Interestingly, we reported the occurrence of 2A-like for the first time in 69 sequences. Among these, 62 corresponded to positive single-stranded RNA species, six to double stranded RNA viruses, and one to a negative-sense single-stranded RNA virus. The importance of these sequences for viral evolution and their potential in biotechnological applications are also discussed.

A multiplex RT-PCR method to detect papaya meleira virus complex in adult pre-flowering plants.

Papaya sticky disease (PSD), which can destroy orchards, was first attributed to papaya meleira virus (PMeV). However, the discovery of papaya meleira virus 2 (PMeV2) associated with PSD plants impose the need to detect this viral complex. We developed a multiplex RT-PCR (mPCR) technique capable of detecting two viruses in a single assay from pre-flowering plant samples, which is a useful tool for early diagnosis of PSD. We also determined the limit of detection (LOD) using asymmetric plasmid dilutions of both PMeV and PMeV2, which revealed that a higher titer of one virus prevents detection of the other. Thus, this technique is an alternative method for detecting PMeV and PMeV2 in a single reaction.

Novel infectious myonecrosis virus (IMNV) genotypes associated with disease outbreaks on Penaeus vannamei shrimp farms in Indonesia.

Infectious myonecrosis virus (IMNV) is one of the most pathogenic viruses that affect Penaeus vannamei shrimp. In 2018, IMNV was reported in grow-out ponds of P. vannamei in Situbondo, Indonesia. Diseased animals displayed clinical signs of infectious myonecrosis (IMN) characterized by white discoloration of skeletal muscle. Histopathology of affected shrimp revealed lesions that are pathognomonic of IMNV infection. The major capsid protein (MCP) gene was amplified and sequenced from representative samples showing IMN pathology. Multiple alignment of predicted amino acid sequences of the MCP gene with known IMNV genotypes in the GenBank database revealed three unique genotypes, SB-A, SB-B and SB-C,in Situbondo samples. The number of amino acid changes in SB-A, SB-B and SB-C compared to known IMNV genotypes ranged from 7-710, including the isolate SB-B, which contains deletion of 622 aa. A phylogenetic analysis using homologous sequences from Brazil and Indonesia showed that these three isolates represent new IMNV genotypes.

Evolutionary origin of 2A-like sequences in Totiviridae genomes.

In recent years there has been a significant increase in the number of new species potentially belonging to the Totiviridae family. Most of these new viruses have not yet been covered by the Committee on Taxonomy of Viruses (ICTV) official classification. In this study, a phylogenetic analysis including new sequences of Totiviridae candidates revealed a clade including Giardiavirus and a great diversity of new totiviruses, which infect arthropods, protozoa and mollusc. This expanded Giardiavirus clade comprises two monophyletic groups, one of them including Giardia lamblia virus (GLV) grouped with viruses that infect arthropods and vertebrates (GLV-like group), and the other includes the previously proposed Artivirus group (IMNV-like group). A screening of the members of the GLV-like group in search of genomic elements already described in IMNV-like group revealed the existence of sites with a high propensity to become 2 A-like oligopeptides, mainly in a specific subgroup of arthropod viruses, suggesting that these viruses preserved ancestral characteristics. The existence of these "pseudo 2 A-sites" associated to phylogenetic reconstruction indicates that these sequences appear at a decisive stage for viral evolution. If they are changed to functional 2 A-like sequences, an irreversible route to increase the genome complexity will be initiated.

Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures.

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5 µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease.

Papaya sticky disease, or "meleira", is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.

New insights about ORF1 coding regions support the proposition of a new genus comprising arthropod viruses in the family Totiviridae.

Analyzing the positions of 2A-like polypeptide cleavage sites in all available genomes of arthropod totiviruses we propose the limits of all ORF1 coding sequences and observed that two proteins previously predicted in infectious myonecrosis virus genome are unique in the arthropod totiviruses group. A putative protein cleavage site upstream the major capsid protein was also identified only in these genomes. In addition, protein models generated using ab initio and threading approaches revealed conserved structures possibly related to formation of viral protrusions and RNA packaging, clarifying the mechanisms involved in the extracellular transmission. These data appoints that the group formed by arthropod totiviruses are sufficient distinctive to be clustered in new genus belonging to the Totiviridae family, in agreement with previous phylogenetic analysis.

RNAi-based inhibition of infectious myonecrosis virus replication in Pacific white shrimp Litopenaeus vannamei.

Disease in Pacific white shrimp Litopenaeus vannamei caused by the infectious myonecrosis virus (IMNV) causes significant socioeconomic impacts in infection-prone shrimp aquaculture regions. The use of synthetic dsRNA to activate an RNA interference (RNAi) response is being explored as a means of disease prophylaxis in farmed shrimp. Here, survival was tracked in L. vannamei injected with long synthetic dsRNAs targeted to IMNV open reading frame (ORF) 1a, ORF1b, and ORF2 genome regions prior to injection challenge with IMNV, and real-time RT-PCR was used to track the progress of IMNV infection and mRNA expression levels of the host genes sid1, dicer2, and argonaute2. Injection of dsRNAs targeting the ORF1a and ORF1b genes but not the ORF2 gene strongly inhibited IMNV replication over a 3 wk period following IMNV challenge, and resulted in 90 and 83% shrimp survival, respectively. Host gene mRNA expression data indicated that the Sid1 protein, which forms a transmembrane channel involved in cellular import/export of dsRNA, increased in abundance most significantly in shrimp groups that were most highly protected by virus-specific dsRNA injection. Subclinical IMNV infections present in the experimental L. vannamei used increased markedly in the 2 d between injection of any of the 4 virus-specific or non-specific dsRNAs tested and IMNV challenge. While handling and injection stress are implicated in increasing IMNV replication levels, the underlying molecular factors that may have been involved remain to be elucidated.

Extended genome sequences of penaeid shrimp infectious myonecrosis virus strains from Brazil and Indonesia.

New sequencing studies of the nonsegmented dsRNA genome of penaeid shrimp infectious myonecrosis virus (IMNV), a tentatively assigned member of the family Totiviridae, identified previously unread sequences at both genome termini in three previously analyzed IMNV strains, one from Brazil (the prototype strain of IMNV) and two from Indonesia. The new sequence determinations add >600 nt to the 5' end of the genomic plus strand of each strain, increasing the length of the 5' nontranslated region to at least 469-472 nt and the length of the upstream open reading frame (ORF1) translation product by at least 48 aa. These new findings are similar to recent ones for two other IMNV strains (GenBank KF836757.1 and KJ556923.1) and thereby corroborate important amendments to the full-length IMNV genome sequence.

Analysis of new isolates reveals new genome organization and a hypervariable region in infectious myonecrosis virus (IMNV).

Infectious myonecrosis virus (IMNV) has been the cause of many losses in shrimp farming since 2002, when the first myonecrosis outbreak was reported at Brazilian's northeast coast. Two additional genomes of Brazilian IMNV isolates collected in 2009 and 2013 were sequenced and analyzed in the present study. The sequencing revealed extra 643 bp and 22 bp, at 5' and 3' ends of IMNV genome respectively, confirming that its actual size is at least 8226 bp long. Considering these additional sequences in genome extremities, ORF1 can starts at nt 470, encoding a 1708 aa polyprotein. Computational predictions reveal two stem loops and two pseudoknots in the 5' end and a putative stem loop and a slippery motif located at 3' end, indicating that these regions can be involved in the start and termination of translation. Through a careful phylogenetic analysis, a higher genetic variability among Brazilian isolates could be observed, comparing with Indonesian IMNV isolates. It was also observed that the most variable region of IMNV genome is located in the first half of ORF1, coinciding with a region which probably encodes the capsid protrusions. The results presented here are a starting point to elucidate the viral's translational regulation and the mechanisms involved in virulence.

Genetic diversification of penaeid shrimp infectious myonecrosis virus between Indonesia and Brazil.

Infectious myonecrosis virus (IMNV) is a pathogen of penaeid shrimp, most notably the whiteleg shrimp Litopenaeus vannamei. First discovered in L. vannamei from Brazilian aquaculture farms in 2003, IMNV was additionally confirmed in L. vannamei from Indonesian farms in 2006 and has since been found in numerous provinces there. Only two complete sequences of IMNV strains have been reported to date, one strain from the Brazilian state of Piauí collected in 2003 and another from the Indonesian province of East Java collected in 2006. In this study, we determined the complete sequences of two additional Indonesian strains, one from Lampung province collected in 2011 and another from East Java province collected in 2012. We also determined partial sequences for six other strains to enhance phylogenetic comparisons, which have heretofore been limited by the small number of reported sequences, including only one for an Indonesian strain. The new results demonstrate clear genetic diversification of IMNV between Indonesia and Brazil, as well as within Indonesia. Analyses of conserved sequence motifs suggest a revised RNA pseudoknot prediction for ribosomal frameshifting.

Species typing of Cuban Trichomonas vaginalis virus by RT-PCR, and association of TVV-2 with high parasite adhesion levels and high pathogenicity in patients.

The viral infection of the parasite with T. vaginalis virus (TVV) may have important implications for trichomonal virulence. In this study we identified the TVV species isolated from Cuban T. vaginalis, using specie specific Reverse Transcriptase-PCR. Of the 37 clinical isolates studied, 21 were infected with TVV, 6 contained TVV-1, 12, TVV- 2 and 3 were co-infected with TVV-1 and -2. The strains infected with TVV showing highest adhesion level in comparison to not infected strains, with high statistical significance. The strains infected only with TVV-2 showing highest adhesion level in comparison to strains infected with TVV-1, with high statistical significance. The parasites classified as mild symptomatic are infected only with TVV-1, however the severe only with TVV-2. According to our results, it seems that only two TVV species are infecting the Cuban isolates. Further studies using higher number of strains should be conducted in order to corroborate these results.

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family.

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.