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The COVID-19 pandemic is caused by a novel and rapidly mutating coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although several drugs are already in clinical use or under emergency authorization, there is still an urgent need to develop new drugs. Through the mining and analysis of 2776 genomes of the SARS-CoV-2 virus, we identified papain-like protease (PLpro), which is a critical enzyme required for coronavirus to generate a functional replicase complex and manipulate post-translational modifications on host proteins for evasion against host antiviral immune responses, as a conserved molecular target for the development of anti-SARS-CoV-2 therapy. We then made an infection model using the NCI-H1299 cell line stably expressing SARS-CoV-2 PLpro protein (NCI-H1299/PLpro). To investigate the effect of targeting and degrading PLpro mRNA, a compact CRISPR-Cas13 system targeting PLpro mRNA was developed and validated, which was then delivered to the aforementioned NCI-H1299/PLpro cells. The results showed that CRISPR-Cas13 mediated mRNA degradation successfully reduced the expression of viral PLpro protein. By combining the power of AAV and CRISPR-Cas13 technologies, we aim to explore the potential of attenuating viral infection by targeted degradation of important viral mRNAs via safe and efficient delivery of AAV carrying the CRISPR-Cas13 system. This study demonstrated a virus-against-virus gene therapy strategy for COVID-19 and provided evidence for the future development of therapies against SARS-CoV-2 and other RNA viral infections.

As of 2024, SARS-CoV-2 continues to propagate and drift as an endemic virus, impacting healthcare for years. The largest sequencing initiative for any species was initiated to combat the virus, tracking changes over time at a full virus base-pair resolution. The SARS-CoV-2 sequencing represents a unique opportunity to understand selective pressures and viral evolution but requires cross-disciplinary approaches from epidemiology to functional protein biology. Within this work, we integrate a two-year genotyping window with structural biology to explore the selective pressures of SARS-CoV-2 on protein insights. Although genotype and the Spike (Surface Glycoprotein) protein continue to drift, most SARS-CoV-2 proteins have had few amino acid alterations. Within Spike, the high drift rate of amino acids involved in antibody evasion also corresponds to changes within the ACE2 binding pocket that have undergone multiple changes that maintain functional binding. The genotyping suggests selective pressure for receptor specificity that could also confer changes in viral risk. Mapping of amino acid changes to the structures of the SARS-CoV-2 co-transcriptional complex (nsp7-nsp14), nsp3 (papain-like protease), and nsp5 (cysteine protease) proteins suggest they remain critical factors for drug development that will be sustainable, unlike those strategies targeting Spike.

Enteroviruses are RNA viruses that initiate infections through the gastrointestinal (GI) tract and are associated with enteric illness in individuals of all ages. Most serious infections of enteroviruses are in infants and young children where it is the common cause of aseptic meningitis and other systemic diseases, leading to a high mortality rate. Enteroviruses belong to the small non-enveloped family of the Picornaviridae family. The virus can spread mainly through fecal–oral and respiratory routes. In the Arabian Gulf countries, the incidence of enteroviral infections is only restricted to a few reports, and thus, knowledge of the epidemiology, characteristics, and pathogenesis of the virus in the gulf countries remains scarce. In this minireview, we sought to provide an overview of the characteristics of enterovirus and its pathogenesis, in addition to gathering the reports of enterovirus infection prevalence in Gulf Cooperation Council (GCC) countries. We also present a summary of the common methods used in its detection.

Marek’s disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek’s disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

As of 2024, SARS-CoV-2 continues to propagate and drift as an endemic virus, impacting healthcare for years. The largest sequencing initiative for any species was initiated to combat the virus, tracking changes over time at a full virus base-pair resolution. The SARS-CoV-2 sequencing represents a unique opportunity to understand selective pressures and viral evolution but requires cross-disciplinary approaches from epidemiology to functional protein biology. Within this work, we integrate a two-year genotyping window with structural biology to explore the selective pressures of SARS-CoV-2 on protein insights. Although genotype and the Spike (Surface Glycoprotein) protein continue to drift, most SARS-CoV-2 proteins have had few amino acid alterations. Within Spike, the high drift rate of amino acids involved in antibody evasion also corresponds to changes within the ACE2 binding pocket that have undergone multiple changes that maintain functional binding. The genotyping suggests selective pressure for receptor specificity that could also confer changes in viral risk. Mapping of amino acid changes to the structures of the SARS-CoV-2 co-transcriptional complex (nsp7-nsp14), nsp3 (papain-like protease), and nsp5 (cysteine protease) proteins suggest they remain critical factors for drug development that will be sustainable, unlike those strategies targeting Spike.

Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus “Zetapartitivirus” and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family “Mycoaspirividae”, respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp.

A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder (Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events (12,352) than the R group (11,452). The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.

JC polyomavirus (JCPyV) is the causative agent of the fatal, incurable, neurological disease, progressive multifocal leukoencephalopathy (PML). The virus is present in most of the adult population as a persistent, asymptotic infection in the kidneys. During immunosuppression, JCPyV reactivates and invades the central nervous system. A main predictor of disease outcome is determined by mutations within the hypervariable region of the viral genome. In patients with PML, JCPyV undergoes genetic rearrangements in the noncoding control region (NCCR). The outcome of these rearrangements influences transcription factor binding to the NCCR, orchestrating viral gene transcription. This study examines 989 NCCR sequences from patient isolates deposited in GenBank to determine the frequency of mutations based on patient isolation site and disease status. The transcription factor binding sites (TFBS) were also analyzed to understand how these rearrangements could influence viral transcription. It was determined that the number of TFBS was significantly higher in PML samples compared to non-PML samples. Additionally, TFBS that could promote JCPyV infection were more prevalent in samples isolated from the cerebrospinal fluid compared to other locations. Collectively, this research describes the extent of mutations in the NCCR that alter TFBS and how they correlate with disease outcome.

A hypovirulent SZ-2-3y strain isolated from diseased Paris polyphylla was identified as Botrytis cinerea. Interestingly, SZ-2-3y was coinfected with a mitovirus, two botouliviruses, and a 3074 nt fusarivirus, designated Botrytis cinerea fusarivirus 8 (BcFV8); it shares an 87.2% sequence identity with the previously identified Botrytis cinerea fusarivirus 6 (BcFV6). The full-length 2945 nt genome sequence of the mitovirus, termed Botrytis cinerea mitovirus 10 (BcMV10), shares a 54% sequence identity with Fusarium boothii mitovirus 1 (FbMV1), and clusters with fungus mitoviruses, plant mitoviruses and plant mitochondria; hence BcMV10 is a new Mitoviridae member. The full-length 2759 nt and 2812 nt genome sequences of the other two botouliviruses, named Botrytis cinerea botoulivirus 18 and 19 (BcBoV18 and 19), share a 40% amino acid sequence identity with RNA-dependent RNA polymerase protein (RdRp), and these are new members of the Botoulivirus genus of Botourmiaviridae. Horizontal transmission analysis showed that BcBoV18, BcBoV19 and BcFV8 are not related to hypovirulence, suggesting that BcMV10 may induce hypovirulence. Intriguingly, a partial BcMV10 sequence was detected in cucumber plants inoculated with SZ-2-3y mycelium or pXT1/BcMV10 agrobacterium. In conclusion, we identified a hypovirulent SZ-2-3y fungal strain from P. polyphylla, coinfected with four novel mycoviruses that could serve as potential biocontrol agents. Our findings provide evidence of cross-kingdom mycoviral sequence transmission.

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

Seasonal flu is an acute respiratory disease that exacts a massive toll on human populations, healthcare systems and economies. The disease is caused by an enveloped Influenza virus containing eight ribonucleoprotein (RNP) complexes. Each RNP incorporates multiple copies of nucleoprotein (NP), a fragment of the viral genome (vRNA), and a viral RNA-dependent RNA polymerase (POL), and is responsible for packaging the viral genome and performing critical functions including replication and transcription. A complete model of an Influenza RNP in atomic detail can elucidate the structural basis for viral genome functions, and identify potential targets for viral therapeutics. In this work we construct a model of a complete Influenza A RNP complex in atomic detail using multiple sources of structural and sequence information and a series of homology-modeling techniques, including a motif-matching fragment assembly method. Our final model provides a rationale for experimentally-observed changes to viral polymerase activity in numerous mutational assays. Further, our model reveals specific interactions between the three primary structural components of the RNP, including potential targets for blocking POL-binding to the NP-vRNA complex. The methods developed in this work open the possibility of elucidating other functionally-relevant atomic-scale interactions in additional RNP structures and other biomolecular complexes.

Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25−30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity.

The complete sequence of a novel RNA virus isolated from Tetrastichus brontispae (TbRV-1) was determined to be 12,239 nucleotides in length with five non-overlapping, linearly arranged coding sequences (CDS), potentially encoding nucleoproteins, hypothetical proteins, matrix proteins, glycoproteins, and RNA-dependent RNA polymerases. Sequence analysis indicated that the RNA-dependent RNA polymerase of TbRV-1 shares a 65% nucleotide and 67% amino acid sequence identity with Hubei dimarhabdovirus 2, suggesting that TbRV-1 is a member of the dimarhabdovirus supergroup. This corresponded to the result of the phylogenetic analysis. The affiliation of TbRV-1 with members of the family Rhabdoviridae was further validated by similar transcription termination motifs (GGAACUUUUUUU) to the Drosophila sigmavirus. The prevalence of TbRV-1 in all tissues suggested that the virus was constitutive of, and not specific to, any wasp tissue. To our knowledge, this is the first report on the complete genome sequence of a dimarhabdovirus in parasitoids.

The COVID-19 pandemic represents an unprecedented opportunity to exploit the advantages of personalized medicine for the prevention, diagnosis, treatment, surveillance and management of a new challenge in public health. COVID-19 infection is highly variable, ranging from asymptomatic infections to severe, life-threatening manifestations. Personalized medicine can play a key role in elucidating individual susceptibility to the infection as well as inter-individual variability in clinical course, prognosis and response to treatment. Integrating personalized medicine into clinical practice can also transform health care by enabling the design of preventive and therapeutic strategies tailored to individual profiles, improving the detection of outbreaks or defining transmission patterns at an increasingly local level. SARS-CoV2 genome sequencing, together with the assessment of specific patient genetic variants, will support clinical decision-makers and ultimately better ways to fight this disease. Additionally, it would facilitate a better stratification and selection of patients for clinical trials, thus increasing the likelihood of obtaining positive results. Lastly, defining a national strategy to implement in clinical practice all available tools of personalized medicine in COVID-19 could be challenging but linked to a positive transformation of the health care system. In this review, we provide an update of the achievements, promises, and challenges of personalized medicine in the fight against COVID-19 from susceptibility to natural history and response to therapy, as well as from surveillance to control measures and vaccination. We also discuss strategies to facilitate the adoption of this new paradigm for medical and public health measures during and after the pandemic in health care systems.

Soybean ( Glycine max L.) is one of the most important crop plants in the Republic of Korea. Here, we conducted a soybean virome study. We harvested a total of 172 soybean leaf samples showing disease symptoms from major soybean-growing regions in the Republic of Korea. Individual samples were examined for virus infection by RT-PCR. Moreover, we generated eight libraries representing eight provinces by pooling samples and four libraries from single samples. RNA-seq followed by bioinformatics analyses revealed 10 different RNA viruses infecting soybean. The proportion of viral reads in each transcriptome ranged from 0.2 to 31.7%. Coinfection of different viruses in soybean plants was very common. There was a single dominant virus in each province, and this geographical difference might be related to the soybean seeds that transmit viruses. In this study, 32 viral genome sequences were assembled and successfully used to analyze the phylogenetic relationships and quasispecies nature of the identified RNA viruses. Moreover, RT-PCR with newly developed primers confirmed infection of the identified viruses in each library. Taken together, our soybean virome study provides a comprehensive overview of viruses infecting soybean in eight geographical regions in the Republic of Korea and four single soybean plants in detail.