Viral Protein Sequences Research Articles

Assessment of Punicalagin activity Against Influenza a Virus Proteins via In-silico Approaches

Background: To investigate the anti-influenza mechanism of punicalagin and its inhibitory effects on influenza virus proteins, we conducted molecular docking studies targeting 11 viral proteins. Additionally, molecular dynamics simulations were performed for the protein with the lowest free energy and the minimum concentration required for binding in a simulated environment. Methods: The molecular structure and data of punicalagin were obtained from the PubChem database and converted into a PDB file. FASTA sequences of viral proteins were retrieved from UniProt, and their PDB structures were predicted using the I-TASSER server. Molecular docking was performed using AutoDock 4.2 software, while molecular dynamics simulations were conducted with Gromacs 2022 software. Results: The PB1-F2 protein exhibited the best inhibitory performance, with a binding energy of -5.76 kcal/mol and the lowest inhibition constant (Ki). Docking of punicalagin to the PB1-F2 protein led to a significant decrease in the average total energy (TE), radius of gyration (Rg), and root mean square deviation (RMSD), as well as alterations in the secondary structure of the protein (P < 0.001). The most prominent secondary structural change was a reduction in coil structures and an increase in turn structures. Conclusions: Punicalagin displayed a strong binding affinity for the PB1-F2 protein compared to other influenza viral proteins. Considering the role of PB1-F2 in exacerbating inflammation caused by influenza, punicalagin may mitigate inflammation in patients by modulating the activity of this protein.

Changes in the genome of tick-borne encephalitis virus during cultivation

The tick-borne encephalitis virus (TBEV) of the Siberian genotype was previously isolated from the brain of a deceased person. TBEV variants obtained at passages 3 and 8 on SPEV cells were inoculated into the brains of white mice for subsequent passages. Full-genome sequences of all virus variants were analyzed by high-throughput sequencing. The analysis showed the presence of 41 point nucleotide substitutions, which were mainly localized in the genes of non-structural proteins NS3 and NS5 of TBEV. In the deduced virus protein sequences, 12 amino acid substitutions were identified. After three passages through mouse brains, reverse nucleotide and amino acid substitutions were detected. Most of them were mapped in the NS5 protein gene, where 5 new nucleotide substitutions also appeared. At the same time, there was an increase in the length of the 3′-untranslated region (3′-UTR) of the viral genome by 306 nucleotides. The Y3 and Y2 3’-UTR elements were found to contain imperfect L and R repeats, which probably associated with inhibition of the activity of cellular XRN1 RNase and thus involved in the formation of sfRNA1 and sfRNA2. For all TBEV variants, high levels of infectious virus were detected both in cell culture and in the brain of white mice. The revealed changes in the genome that occur during successive passages of TBEV are most likely due to the significant genetic variability of the virus, which ensures its efficient reproduction in different hosts and active circulation in nature.

Transmission patterns of malignant catarrhal fever in sheep and cattle in Karnataka, India.

Malignant catarrhal fever (MCF) presents a sporadic yet significant threat to livestock and wildlife. A comprehensive investigation in Karnataka, India into the prevalence and transmission patterns of sheep-associated MCF (SA-MCF) was conducted. A total of 507 sheep peripheral blood leukocyte samples from 13 districts along with 27 cows and 10 buffalo samples from various regions in Karnataka were tested for SA-MCF infection i.e. Ovine gammaherpesvirus 2 (OvHV-2) using heminested PCR. Furthermore, serum samples collected from 73 cows and 15 buffalo suspected of MCF were tested using a commercially available ELISA kit. Additionally, histopathological examinations of affected tissues and phylogenetic analysis of viral tegument protein sequences were conducted. Our findings indicated a 20.11%, 33.33% and 20% positivity for OvHV-2 in sheep, cows and buffalo respectively by PCR. Statistical analysis revealed a significant association between the age of sheep and the detection of OvHV-2. Seven cows and one buffalo serum samples tested positive for ELISA. Clinical findings in bovids were consistent with typical MCF signs, and histopathological results revealed multi-organ involvement characterised by necrotising vasculitis and lymphoid hyperplasia. The nucleotide pairwise identity matrix revealed 99.5% identity between the sequences obtained in the study with sequences from other states. The phylogenetic analysis of partial tegument protein sequences from bovid and sheep samples suggested a close genetic relationship between the local OvHV-2 strains and those from various global regions. Crucially, this study underscores the widespread presence of SA-MCF in Karnataka, with significant implications for both livestock management and wildlife conservation.

A novel multi-epitope peptide vaccine targeting immunogenic antigens of Ebola and monkeypox viruses with potential of immune responses provocation in silico.

The emergence or reemergence of monkeypox (Mpox) and Ebola virus (EBOV) agents causing zoonotic diseases remains a huge threat to human health. Our study aimed at designing a multi-epitope vaccine (MEV) candidate to target both the Mpox and EBOV agents using immunoinformatics tools. Viral protein sequences were retrieved, and potential nonallergenic, nontoxic, and antigenic epitopes were obtained. Next, cytotoxic and helper T-cell (CTL and HTL, respectively) and B-cell (BCL) epitopes were predicted, and those potential epitopes were fused utilizing proper linkers. The in silico cloning and expression processes were implemented using Escherichia coli K12. The immune responses were prognosticated using the C-ImmSim server. The MEV construct (29.53kDa) included four BCL, two CTL, and four HTL epitopes and adjuvant. The MEV traits were pertinent in terms of antigenicity, non-allergenicity, nontoxicity, physicochemical characters, and stability. The MEV candidate was also highly expressed in E. coli K12. The strong affinity of MEV-TLR3 was confirmed using molecular docking and molecular dynamics simulation analyses. Immune simulation analyses unraveled durable activation and responses of cellular and humoral arms alongside innate immune responses. The designed MEV candidate demonstrated appropriate traits and was promising in the prediction of immune responses against both Mpox and EBOV agents. Further experimental assessments of the MEV are required to verify its efficacy.

Conserved sequence features in intracellular domains of viral spike proteins

Viral spike proteins mutate frequently, but conserved features within these proteins often have functional importance and can inform development of anti-viral therapies which circumvent the effects of viral sequence mutations. Through analysis of large numbers of viral spike protein sequences from several viral families, we found highly (>99%) conserved patterns within their intracellular domains. The patterns generally consist of one or more basic amino acids (arginine or lysine) adjacent to a cysteine, many of which are known to undergo acylation. These patterns were not enriched in cellular proteins in general. Molecular dynamics simulations show direct electrostatic and hydrophobic interactions between these conserved residues in hemagglutinin (HA) from influenza A and B and the phosphoinositide PIP2. Super-resolution microscopy shows nanoscale colocalization of PIP2 and several of the same viral proteins. We propose the hypothesis that these conserved viral spike protein features can interact with phosphoinositides such as PIP2.

Prevalence of Human Parvovirus B19 and Associated Risk Factors in Women with Bad Obstetric History in Babylon Province, Iraq

Abstract Background: Parvovirus infection is an important cause of fetal loss during all three trimesters of pregnancy. Objectives: This study was conducted to investigate the prevalence, susceptibility rate, and risk factors of B19-V in women aged 15 to 45 in the Al-Hamza region of Babil, Iraq, considering the negative effects of the infection. Materials and Methods: Between January 2022 and March 2023, blood samples were taken from 116 women, including those with and those without a history of bad obstetric history (BOH). Enzyme-linked immunosorbent assay (ELISA) was used to test sera taken from the study participants for B19 IgM and immunoglobulin G (IgG) antibodies. Those samples tested positive for IgG B19 were further subjected to polymerase chain reaction amplification and sequencing, targeting partial gene sequences of the nonstructural viral protein 1 (VP1) gene. Results: For human parvovirus IgG and IgM, the seropositivity was 44% and 16.5%, respectively. The following variables were associated in this study with greater seroprevalence rates of IgG and IgM, respectively: age 36–45 years (72.7%, 36.4%); abortion (80%, 32.5%); education (82.2%, 33.3%); unemployment (61%, 22.2%); and overcrowding (73.3%, 31.1%). In this case–control study, positive amplicons (700 bp) were detected from 27 samples out of 40 women of childbearing age who tested positive for IgG. Sequence analysis of the partial sequence of the VP1 gene in all positive amplicons was 100% identical with each other and with human parvovirus B19 (NC: 00088.3). According to the current study, about 66% of women are serologically negative and at risk of contracting parvovirus B19. Conclusion: Our findings conclude that pregnant women in Iraq, particularly those with BOH, must undergo frequent serological screening for B19-V during all three trimesters and be given the necessary advice to avoid B19-V infection to improve their pregnancy outcomes.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment

HIV infection compromises both the peripheral and central immune systems due to its pathogenic and neuropathogenic features. The mechanisms driving HIV-1 pathogenesis and neuropathogenesis involve a series of events, including metabolic dysregulation. Furthermore, HIV-subtype-specific variations, particularly alterations in the amino acid sequences of key viral proteins, are known to influence the severity of clinical outcomes in people living with HIV. However, the impact of amino acid sequence variations in specific viral proteins, such as Viral protein R (Vpr), on metabolites within the Tryptophan (Trp)-kynurenine (Kyn) pathway in people living with HIV remains unclear. Our research aimed to explore the relationship between variations in the Vpr amino acid sequence (specifically at positions 22, 41, 45, and 55, as these have been previously linked to neurocognitive function) and peripheral Trp-Kyn metabolites. Additionally, we sought to clarify the systems biology of Vpr sequence variation by examining the link between Trp-Kyn metabolism and peripheral inflammation, as a neuropathogenic mechanism. In this preliminary study, we analyzed a unique cohort of thirty-two (n = 32) South African cART naïve people living with HIV. We employed Sanger sequencing to ascertain blood-derived Vpr amino acid sequence variations and a targeted LC-MS/MS metabolomics platform to assess Trp-Kyn metabolites, such as Trp, Kyn, kynurenic acid (KA), and quinolinic acid (QUIN). Particle-enhanced turbidimetric assay and Enzyme-linked immunosorbent assays were used to measure immune markers, hsCRP, IL-6, suPAR, NGAL and sCD163. After applying Bonferroni corrections (p =.05/3) and adjusting for covariates (age and sex), only the Vpr G41 and A55 groups was nearing significance for higher levels of QUIN compared to the Vpr S41 and T55 groups, respectively (all p =.023). Multiple regression results revealed that Vpr amino acid variations at position 41 (adj R2 = 0.049, β = 0.505; p =.023), and 55 (adj R2 = 0.126, β = 0.444; p =.023) displayed significant associations with QUIN after adjusting for age and sex. Lastly, the higher QUIN levels observed in the Vpr G41 group were found to be correlated with suPAR (r =.588, p =.005). These results collectively underscore the importance of specific Vpr amino acid substitutions in influencing QUIN and inflammation (specifically suPAR levels), potentially contributing to our understanding of their roles in the pathogenesis and neuropathogenesis of HIV-1.

Isolation, Molecular, and Histopathological Patterns of a Novel Variant of Infectious Bursal Disease Virus in Chicken Flocks in Egypt.

After an extended period of detecting classical virulent, attenuated, and very virulent IBDV, a novel variant (nVarIBDV) was confirmed in Egypt in this study in 18, IBD vaccinated, chicken flocks aged 19-49 days. Partial sequence of viral protein 2 (VP2) [219 aa, 147-366, resembling 657 bp] of two obtained isolates (nos. 3 and 4) revealed nVarIBDV (genotype A2d) and OR682618 and OR682619 GenBank accession numbers were obtained. Phylogenetic analysis revealed that both nVarIBDV isolates were closely related to nVarIBDV strains (A2d) circulating in China, exhibiting 100% identity to SD-2020 and 99.5-98.1% similarity to ZD-2018-1, QZ, GX and SG19 strains, respectively. Similarity to USA variant strains, belonging to genotypes A2b (9109), A2c (GLS) and A2a (variant E), respectively, was 95.5-92.6%. Also, the VP2 hypervariable region in those two, A2d, isolates revealed greater similarities to Faragher 52/70 (Vaxxitek®) at 90.4% and to an Indian strain (Ventri-Plus®) and V217 (Xtreme®) at 89.7% and 86-88.9% in other vaccines. Histopathological examination of both the bursa of Fabricius and spleen collected from diseased chickens in flock no. 18 revealed severe atrophy. In conclusion, further studies are required to investigate the epidemiological situation of this novel genotype across the country, and to assess various vaccine protections against nVarIBDV. Additionally, vaccination of breeders with inactivated IBD vaccines including this nVarIBDV is essential to obtain specific maternal antibodies in their broilers.

Structure-guided discovery of anti-CRISPR and anti-phage defense proteins

Bacteria use a variety of defense systems to protect themselves from phage infection. In turn, phages have evolved diverse counter-defense measures to overcome host defenses. Here, we use protein structural similarity and gene co-occurrence analyses to screen >66 million viral protein sequences and >330,000 metagenome-assembled genomes for the identification of anti-phage and counter-defense systems. We predict structures for ~300,000 proteins and perform large-scale, pairwise comparison to known anti-CRISPR (Acr) and anti-phage proteins to identify structural homologs that otherwise may not be uncovered using primary sequence search. This way, we identify a Bacteroidota phage Acr protein that inhibits Cas12a, and an Akkermansia muciniphila anti-phage defense protein, termed BxaP. Gene bxaP is found in loci encoding Bacteriophage Exclusion (BREX) and restriction-modification defense systems, but confers immunity independently. Our work highlights the advantage of combining protein structural features and gene co-localization information in studying host-phage interactions.

PandoGen: Generating complete instances of future SARS-CoV-2 sequences using Deep Learning.

One of the challenges in a viral pandemic is the emergence of novel variants with different phenotypical characteristics. An ability to forecast future viral individuals at the sequence level enables advance preparation by characterizing the sequences and closing vulnerabilities in current preventative and therapeutic methods. In this article, we explore, in the context of a viral pandemic, the problem of generating complete instances of undiscovered viral protein sequences, which have a high likelihood of being discovered in the future using protein language models. Current approaches to training these models fit model parameters to a known sequence set, which does not suit pandemic forecasting as future sequences differ from known sequences in some respects. To address this, we develop a novel method, called PandoGen, to train protein language models towards the pandemic protein forecasting task. PandoGen combines techniques such as synthetic data generation, conditional sequence generation, and reward-based learning, enabling the model to forecast future sequences, with a high propensity to spread. Applying our method to modeling the SARS-CoV-2 Spike protein sequence, we find empirically that our model forecasts twice as many novel sequences with five times the case counts compared to a model that is 30× larger. Our method forecasts unseen lineages months in advance, whereas models 4× and 30× larger forecast almost no new lineages. When trained on data available up to a month before the onset of important Variants of Concern, our method consistently forecasts sequences belonging to those variants within tight sequence budgets.

In Silico design of a multi-epitope vaccine for Human Parechovirus: Integrating immunoinformatics and computational techniques.

Human parechovirus (HPeV) is widely recognized as a severe viral infection affecting infants and neonates. Belonging to the Picornaviridae family, HPeV is categorized into 19 distinct genotypes. Among them, HPeV-1 is the most prevalent genotype, primarily associated with respiratory and digestive symptoms. Considering HPeV's role as a leading cause of life-threatening viral infections in infants and the lack of effective antiviral therapies, our focus centered on developing two multi-epitope vaccines, namely HPeV-Vax-1 and HPeV-Vax-2, using advanced immunoinformatic techniques. Multi-epitope vaccines have the advantage of protecting against various virus strains and may be preferable to live attenuated vaccines. Using the NCBI database, three viral protein sequences (VP0, VP1, and VP3) from six HPeV strains were collected to construct consensus protein sequences. Then the antigenicity, toxicity, allergenicity, and stability were analyzed after discovering T-cell and linear B-cell epitopes from the protein sequences. The fundamental structures of the vaccines were produced by fusing the selected epitopes with appropriate linkers and adjuvants. Comprehensive physicochemical, antigenic, allergic assays, and disulfide engineering demonstrated the effectiveness of the vaccines. Further refinement of secondary and tertiary models for both vaccines revealed promising interactions with toll-like receptor 4 (TLR4) in molecular docking, further confirmed by molecular dynamics simulation. In silico immunological modeling was employed to assess the vaccine's capacity to stimulate an immune reaction. In silico immunological simulations were employed to evaluate the vaccines' ability to trigger an immune response. Codon optimization and in silico cloning analyses showed that Escherichia coli (E. coli) was most likely the host for the candidate vaccines. Our findings suggest that these multi-epitope vaccines could be the potential HPeV vaccines and are recommended for further wet-lab investigation.

Charting Peptide Shared Sequences Between 'Diabetes-Viruses' and Human Pancreatic Proteins, Their Structural and Autoimmune Implications.

Diabetes mellitus (DM) is a metabolic syndrome characterized by hyperglycaemia, polydipsia, polyuria, and weight loss, among others. The pathophysiology for the disorders is complex and results in pancreatic abnormal function. Viruses have also been implicated in the metabolic syndrome. This study charted peptides to investigate and predict the autoimmune potential of shared sequences between 8 viral species proteins (which we termed 'diabetes-viruses') and the human pancreatic proteins. The structure and immunological relevance of shared sequences between viruses reported in DM onset and human pancreatic proteins were analysed. At nonapeptide mapping between human pancreatic protein and 'diabetic-viruses', reveal 1064 shared sequences distributed among 454 humans and 4288 viral protein sequences. The viral results showed herpesviruses, enterovirus (EV), human endogenous retrovirus, influenza A viruses, rotavirus, and rubivirus sequences are hosted by the human pancreatic protein. The most common shared nonapeptide was AAAAAAAAA, present in 30 human nonredundant sequences. Among the viral species, the shared sequence NSLEVLFQG occurred in 18 nonredundant EVs protein, while occurring merely in 1 human protein, whereas LGLDIEIAT occurred in 8 influenza A viruses overlapping to 1 human protein and KDELSEARE occurred in 2 rotaviruses. The prediction of the location of the shared sequences in the protein structures, showed most of the shared sequences are exposed and located either on the surface or cleft relative to the entire protein structure. Besides, the peptides in the viral protein shareome were predicted computationally for binding to MHC molecules. Here analyses showed that the entire 1064 shared sequences predicted 203 to be either HLA-A or B supertype-restricted epitopes. Fifty-one of the putative epitopes matched reported HLA ligands/T-cell epitopes majorly coming from EV B supertype representative allele restrictions. These data, shared sequences, and epitope charts provide important insight into the role of viruses on the onset of DM and its implications.

Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods.

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for the correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similarities, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainty in distinguishing empty and filled capsids. To address these challenges, we combined two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, a selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids. Liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were then used for the capsid denaturing measurements. This multimethod combination was applied to three AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.

Involvement of cutavirus in a subset of patients with cutaneous T-cell lymphoma with an unfavorable outcome

European studies suggest an association between cutavirus (CuV) and cutaneous T-cell lymphoma (CTCL); however, the worldwide prevalence of CuV in patients with CTCL and its prognostic impact remain unknown.We investigated the prevalence and viral loads of CuV DNA using biopsy specimens from the lesional skins of 141 Japanese patients with cutaneous malignancies, including 55 patients with various types of CTCL.CuV DNA was detected significantly more frequently in biopsies from patients with mycosis fungoides (MF) (38% [13/34]; the most common subtype of CTCL) than in those from patients with other cutaneous malignancies (6% [6/107]; P<0.001). The viral-load range in patients with CuV DNA-positive MF was 23-3922 copies/103 cells and 8-65 copies/μg of DNA. A phylogenetic analysis using the partial sequences of the CuV viral capsid protein 1 (VP1)/VP2 genes revealed that the CuV sequences identified here were clustered in a Japanese-specific clade distinct from that comprising CuV sequences from European patients with MF. Kaplan-Meier curves and a log-rank test showed that CuV positivity was associated with a shorter disease-specific survival in patients with MF (P = 0.031), whereas no significant difference in overall survival was observed (P = 0.275). No significant correlation was observed between CuV DNA load and survival in patients with CuV-positive MF.Our results suggest that CuV is associated with MF in a subset of Japanese patients. Large-scale prospective studies are warranted to clarify the role of CuV status, especially regarding the viral genotype, on adverse outcomes in patients with CuV-positive MF.

Misclassified: identification of zoonotic transition biomarker candidates for influenza A viruses using deep neural network.

Introduction: Zoonotic transition of Influenza A viruses is the cause of epidemics with high rates of morbidity and mortality. Predicting which viral strains are likely to transition from their genetic sequence could help in the prevention and response against these zoonotic strains. We hypothesized that features predictive of viral hosts could be leveraged to identify biomarkers of zoonotic viral transition. Methods: We trained deep learning models to predict viral hosts based on the virus mRNA or protein sequences. Our multi-host dataset contained 848,630 unique nucleotide sequences obtained from the NCBI Influenza Virus and Influenza Research Databases. Each sequence, representing one gene from one viral strain, was classified into one of the three host categories: Avian, Human, and Swine. Trained models were analyzed using various neural network interpretation methods to identify interesting candidates for zoonotic transition biomarkers. Results: Using mRNA sequences as input led to higher prediction accuracies than amino acids, suggesting that the codon sequence contains information relevant to viral hosts that is lost during protein translation. UMAP visualization of the latent space of our classifiers showed that viral sequences clustered according to their host of origin. Interestingly, sequences from pandemic zoonotic viral strains localized at the margins between hosts, while zoonotic sequences incapable of Human-to-Human transmission localized with non-zoonotic viruses from the same host. In addition, host prediction for pandemic zoonotic sequences had low prediction accuracy, which was not the case for the other zoonotic strains. This supports our hypothesis that ambiguously predicted viral sequences bear features associated with cross-species infectivity. Finally, we compared misclassified sequences to well-classified ones to extract interesting candidates for zoonotic transition biomarkers. While features varied significantly between pairs of species and viral genes, several codons were conserved in Swine-to-Human and Avian-to-Human misclassified sequences, and in particular in the NA, HA, and NP genes, suggesting their importance for zoonosis in Humans. Discussion: Analysis of viral sequences using neural network interpretation approaches revealed important genetic differences between zoonotic viruses with pandemic potential, compared to non-zoonotic viral strains or zoonotic viruses incapable of Human-to-Human transmission.

A collection ofTrichoderma isolates from natural environments inSardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma. Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.

No Evidence for Myocarditis or Other Organ Affection by Induction of an Immune Response against Critical SARS-CoV-2 Protein Epitopes in a Mouse Model Susceptible for Autoimmunity.

After Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed into a global pandemic, not only the infection itself but also several immune-mediated side effects led to additional consequences. Immune reactions such as epitope spreading and cross-reactivity may also play a role in the development of long-COVID, although the exact pathomechanisms have not yet been elucidated. Infection with SARS-CoV-2 can not only cause direct damage to the lungs but can also lead to secondary indirect organ damage (e.g., myocardial involvement), which is often associated with high mortality. To investigate whether an immune reaction against the viral peptides can lead to organ affection, a mouse strain known to be susceptible to the development of autoimmune diseases, such as experimental autoimmune myocarditis (EAM), was used. First, the mice were immunized with single or pooled peptide sequences of the virus's spike (SP), membrane (MP), nucleocapsid (NP), and envelope protein (EP), then the heart and other organs such as the liver, kidney, lung, intestine, and muscle were examined for signs of inflammation or other damage. Our results showed no significant inflammation or signs of pathology in any of these organs as a result of the immunization with these different viral protein sequences. In summary, immunization with different SARS-CoV-2 spike-, membrane-, nucleocapsid-, and envelope-protein peptides does not significantly affect the heart or other organ systems adversely, even when using a highly susceptible mouse strain for experimental autoimmune diseases. This suggests that inducing an immune reaction against these peptides of the SARS-CoV-2 virus alone is not sufficient to cause inflammation and/or dysfunction of the myocardium or other studied organs.

Intragenomic rearrangements involving 5′-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses

BackgroundVariation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses that was first documented in an infectious isolate of the betacoronavirus SARS-CoV-2, obtained from 3 patients in Hong Kong that had a 5′-untranslated region segment at the end of the ORF6 gene that in its new location translated into an ORF6 protein with a predicted modified carboxyl terminus. While comparing the amino acid sequences of translated ORF8 genes in the GenBank database, we found a subsegment of the same 5′-UTR-derived amino acid sequence modifying the distal end of ORF8 of an isolate from the United States and decided to carry out a systematic search.MethodsUsing the nucleotide and in the case of SARS-CoV-2 also the translated amino acid sequence in three reading frames of the genomic termini of coronaviruses as query sequences, we searched for 5′-UTR sequences in regions other than the 5′-UTR in SARS-CoV-2 and reference strains of alpha-, beta-, gamma-, and delta-coronaviruses.ResultsWe here report numerous genomic insertions of 5′-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, but not delta- or gammacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions would change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain inserted 5′-UTR sequences.ConclusionThe intragenomic rearrangements involving 5′-untranslated region sequences described here, which in several cases affect highly conserved genes with a low propensity for recombination, may underlie the generation of variants homotypic with those of concern or interest and with potentially differing pathogenic profiles. Intragenomic rearrangements thus add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

Carp edema virus in Ukraine – The evidence for the furthest east presence of CEV genogroup I in Europe

Carp edema virus (CEV) is a poxvirus infecting gills of common carp (Cyprinus carpio) and causes a deadly disease called koi sleepy disease (KSD). Currently thanks to higher vigilance and screening for its presence the virus was confirmed to be widespread on the European continent and affects both ornamental koi, farmed and feral common carp. Phylogenetic research of CEV is however in its infancy. Until recently due to the lack of available sequence information, the phylogenetic analysis was limited to a p4a gene. Based on this gene two main clades were established. The genogroup I clade is of European origin and can be found in farmed or feral common carp in Europe. However, the eastern border of the geographic range of CEV from genogroup I is not yet established, therefore we decided to obtain samples from clinically affected carp from Ukraine. Furthermore, we used sequences of the DNA binding viral core protein 8 (VP8) and of the uracil DNA glycosylase (UDGS) to confirm the existence of the genogroups I and II. The results indicate that CEV is highly prevalent in Ukraine. Phylogenetic analysis using the p4a gene showed that common carps in Ukraine are infected with CEV genogroup I. The two additional genes used for the phylogenetic analysis supported the phylogenetic findings. This confirms that common carps in continental Europe are hosts for CEV from the genogroup I and the geographic range for this virus is reaching to the most eastern parts of Ukraine.