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Unveiling Alternaria Alternata Botybirnavirus 1 (AaBbV1): A Novel Mycovirus Characterization
Last Updated: July 29, 2025
Author Bio: This research summary is based on the doctoral thesis of Wajeeha Shamsi, a researcher from the Atta-ur-Rahman School of Applied Biosciences at the National University of Sciences & Technology (NUST), Islamabad. Her work focuses on the identification and characterization of novel mycoviruses from environmental sources.
Introduction
In the vast and often unseen microbial world, a constant battle rages. Fungi, often viewed as infectious agents themselves, are also hosts to their own unique viruses, known as mycoviruses. The study of these fungal viruses is a rapidly expanding field, offering potential insights into fungal biology and even new avenues for biocontrol. A pivotal 2020 doctoral thesis by Wajeeha Shamsi details the discovery of a completely new species of mycovirus. This post delves into the core findings of that research, providing a detailed molecular and biological characterization of the novel Alternaria alternata botybirnavirus 1 (AaBbV1).
Identifying a New Viral Signature in A. alternata
The journey to characterize Alternaria alternata botybirnavirus 1 began with a comprehensive screening of environmental fungal isolates. Isolate 4a, collected from a diseased tomato, presented a unique profile. It manifested a grey colony growth typical of the fungus A. alternata, a fact later confirmed by ITS amplification.
Initial analysis revealed the presence of a double-stranded RNA (dsRNA) genome of approximately 6kb. The dsRNA nature was confirmed through rigorous testing, including its resistance to DNase I and S1 nuclease, which digest DNA and single-stranded nucleic acids, respectively. This discovery laid the groundwork for a deeper mycovirus characterization.
Sequencing the AaBbV1 Genome: An NGS Approach
To determine the precise genetic makeup of the virus, a Next-Generation Sequencing (NGS) approach was employed. This powerful technique revealed the presence of two distinct virus-like contigs derived from different dsRNA molecules.
The larger fragment was designated dsRNA1 and the smaller one as dsRNA2. Database searches immediately revealed that these sequences were representative of the two dsRNAs characteristic of the genus Botybirnavirus. This finding was visually supported by the thick band on an agarose gel, which indicated two dsRNAs of nearly equal size. The research team tentatively named the novel agent Alternaria alternata botybirnavirus 1 (AaBbV1).
Complete Genomic Sequence of Alternaria alternata botybirnavirus 1
Following the initial NGS results, the complete sequence of both dsRNA segments was determined by combining NGS data with RLM-RACE contigs. This comprehensive sequence data allowed for a detailed analysis of the virus’s genetic properties.
Properties of dsRNA-1
The dsRNA-1 segment of Alternaria alternata botybirnavirus 1 is 6,127 base pairs (bp) in length. Key features identified using the NCBI ORF finder program include:
- A Single Large Open Reading Frame (ORF-1): This ORF spans from nucleotide position 396 to 6032 and encodes a protein of 1878 amino acids, with a molecular mass of approximately 211 kDa.
- Proline-Rich Region: A notable proline-rich region is present at amino acid positions 212 to 252. Such regions in other viruses are known to be involved in protein-protein interactions.
- RdRp Superfamily Domain: An RNA-dependent RNA polymerase (RdRp) domain was identified at nucleotide positions 4140 to 5105. This enzyme is essential for the replication of RNA viruses.
- Untranslated Regions (UTRs): The 5′ UTR is 395 bp long, while the 3′ UTR is 95 bp long.
Properties of dsRNA-2
The dsRNA-2 segment is slightly smaller, at 5,860 bp in length. Its analysis revealed:
- A Single Large ORF-2: This ORF is located from nucleotide position 409 to 5763 and encodes a large protein of 1784 amino acids, with a molecular mass of approximately 197 kDa.
- No Functional Motifs: Unlike ORF-1, no particular functional motifs were found in the ORF-2-encoded protein through initial database searches.
- Untranslated Regions (UTRs): The 5′ UTR of dsRNA-2 is 408 bp, and the 3′ UTR is 97 bp.
The polypeptide encoded by the AaBbV1 ORF-2 shows 26–54% identity with hypothetical proteins from other botybirnaviruses, reinforcing its classification within this genus.
Phylogenetic Relationship: Where Does AaBbV1 Fit?
Phylogenetic analysis, which examines the evolutionary relationships between organisms, was conducted on the deduced protein sequence from ORF-1. This analysis compared the Alternaria alternata botybirnavirus 1 protein to those of other known viruses.
The results showed a 61% sequence similarity with the corresponding proteins of Botrytis porri botybirnavirus 1 (BpRV1) and Sclerotinia sclerotiorum botybirnavirus 3 (SsBRV3), both established members of the genus Botybirnavirus. It showed only moderate (31–40%) or low (24–29%) similarity to other botybirnaviruses or members of different viral families like Totiviridae and Chrysoviridae. This confirms that Alternaria alternata botybirnavirus 1 is a distinct new species firmly nested within the Botybirnavirus genus.
Physical and Protein Characteristics of the AaBbV1 Virion
To understand the physical structure of the virus, particles were purified using cesium chloride gradient centrifugation. The analysis revealed that the Alternaria alternata botybirnavirus 1 virions are:
- Isometric and Non-enveloped: The particles have a roughly spherical shape.
- Approximately 40 nm in diameter: This size is consistent with other related mycoviruses.
Further analysis of the viral structural proteins using SDS-PAGE identified at least five distinct protein bands with molecular weights of 110 kDa (p110), 80 kDa (p80), 73 kDa (p73), 70 kDa (p70), and 66 kDa (p66). Interestingly, the ratios of these proteins varied between different purification preparations, suggesting some degradation of structural proteins can occur during the purification process. Peptide analysis confirmed that these smaller proteins were derived from the large polyproteins encoded by ORF-1 and ORF-2.
Conclusion
The research by Wajeeha Shamsi provides the first comprehensive report of Alternaria alternata botybirnavirus 1, a novel, well-characterized fungal virus from South Asia. Its bi-segmented dsRNA genome, large ORFs, and phylogenetic relationship firmly place it as a new species in the genus Botybirnavirus. This detailed molecular and biological characterization not only expands our knowledge of mycovirus diversity but also provides a foundational resource for future studies into the complex interactions between viruses and their fungal hosts.
Source & Citations
- Thesis Title: Molecular and Biological Characterization of Mycovirus Identified from Environmental Fungal Isolates
- Researcher: Wajeeha Shamsi
- Guide (Supervisor): Dr. Muhammad Faraz Bhatti
- University: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences & Technology (NUST), Islamabad, Pakistan
- Year of Compilation: 2020
- Excerpt Page Numbers: 76, 78, 82, 85, 92, 95, 98, 100
Disclaimer: Some sentences have been lightly edited for SEO and readability. For the full, original research, please refer to the complete thesis PDF linked in the section above.
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