The emerged novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has created a global health crisis that warrants an accurate and detailed characterization of the rapidly evolving viral genome for understanding its epidemiology, pathogenesis, and containment. Here, we explored 61,485 sequences of the nucleocapsid (N) protein, a potent diagnostic and prophylactic target, for identifying the mutations to review their roles in real‐time polymerase chain reaction based diagnosis and observe consequent impacts. Compared to the Wuhan reference strain, a total of 1034 unique nucleotide mutations were identified in the mutant strains (49.15%, n = 30,221) globally. Of these mutations, 367 occupy primer binding sites including the 3′‐end mismatch to the primer‐pair of 11 well‐characterized primer sets. Noteworthily, CDC (USA) recommended the N2 primer set contained a lower mismatch than the other primer sets. Moreover, 684 amino acid (aa) substitutions were located across 317 (75.66% of total aa) unique positions including 82, 21, and 83 of those in the RNA binding N‐terminal domain (NTD), SR‐rich region, and C‐terminal dimerization domain, respectively. Moreover, 11 in‐frame deletions, mostly (n = 10) within the highly flexible linker region, were revealed, and the rest was within the NTD region. Furthermore, we predicted the possible consequence of high‐frequency mutations (≥20) and deletions on the tertiary structure of the N protein. Remarkably, we observed that a high frequency (67.94% of mutated sequences) co‐occuring mutations (R203K and G204R) destabilized and decreased overall structural flexibility. The N protein of SARS‐CoV‐2 comprises an average of 1.2 mutations per strain compared to 4.4 and 0.4 in Middle East respiratory syndrome‐related coronavirus and SARS‐CoV, respectively. Despite being proposed as the alternative target to spike protein for vaccine and therapeutics, the ongoing evolution of the N protein may challenge these endeavors, thus needing further immunoinformatics analyses. Therefore, continuous monitoring is required for tracing the ongoing evolution of the SARS‐CoV‐2 N protein in prophylactic and diagnostic interventions.

Infecting millions of people, the SARS‐CoV‐2 is evolving at an unprecedented rate, demanding advanced and specified analytic pipeline to capture the mutational spectra. In order to explore mutations and deletions in the spike (S) protein — the most‐discussed protein of SARS‐CoV‐2 — we comprehensively analyzed 35,750 complete S protein‐coding sequences through a custom Python‐based pipeline. This GISAID‐collected dataset of until 24 June 2020 covered six continents and five major climate zones. We identified 27,801 (77.77% sequences) mutated strains compared to reference Wuhan‐Hu‐1 wherein 84.40% of these strains mutated by only a single amino acid (aa). An outlier strain (EPI_ISL_463893) from Bosnia and Herzegovina possessed six aa substitutions. We also identified 11 residues with high aa mutation frequency, and each contains four types of aa variations. The infamous D614G variant has spread worldwide with ever‐rising dominance and across regions with different climatic conditions alongside L5F and D936Y mutants, which have been documented throughout all regions and climate zones, respectively. We also found 988 unique aa substitutions spanned across 660 residues, which differed significantly among different continents (p = .003) and climatic zones (p = .021) as inferred with the Kruskal–Wallis test. Besides, 17 in‐frame deletions at four sites adjacent to receptor‐binding‐domain were determined that may have a possible impact on attenuation. This study provides a fast and accurate pipeline for identifying mutations and deletions from the large dataset for coding and also non‐coding sequences as evidenced by the representative analysis on existing S protein data. By using separate multi‐sequence alignment, removing ambiguous sequences and in‐frame stop codons, and utilizing pairwise alignment, this method can derive both synonymous and non‐synonymous mutations (strain_ID reference aa:mutation position:strain aa). We suggest that the pipeline will aid in the evolutionary surveillance of any SARS‐CoV‐2 encoded proteins and will prove to be crucial in tracking the ever‐increasing variation of many other divergent RNA viruses in the future. The code is available at https://github.com/SShaminur/Mutation‐Analysis.

Background and Aim: Fowl cholera (FC) caused by Pasteurella multocida is a highly contagious bacterial disease of global importance for poultry production. The severity and incidence of FC caused by P. multocida may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment, and the bacterial strain. This study aimed to investigate the genetic diversity of multidrug-resistant P. multocida strains isolated from FC outbreaks in laying hens from commercial farms of Bangladesh. Materials and Methods: We collected 57 samples of suspected FC, including 36 live and 21 dead laying hens. P. multocida isolates were characterized by biochemical and molecular-biological methods. Results: Twenty-two strains of P. multocida were isolated from these samples through phenotypic and genotypic characterization. The strains were grouped into two distinct random amplification of polymorphic DNA (RAPD) biotypes harboring a range of pathogenic genes; exbB, ompH, ptfA, nanB, sodC, and hgbA. In this study, 90.90% and 81.82% P. multocida strains were multidrug-resistant and biofilm formers, respectively. Whole-genome sequencing of the two representative RAPD phylotypes confirmed as P. multocida type B: L2:ST122, harboring a number of virulence factors-associated genes (VFGs), and antimicrobial resistance (AMR) genes (ARGs). In addition, pan-genome analysis revealed 90 unique genes in the genomes of P. multocida predicted to be associated with versatile metabolic functions, pathogenicity, virulence, and AMR. Conclusion: This is first-ever report on the association of P. multocida genotype B: L2:ST122 and related VFGs and ARGs in the pathogenesis of FC in laying hens. This study also provides a genetic context for future researches on the evolutionary diversity of P. multocida strains and their host adaptation.

Foot‐and‐mouth disease virus (FMDV) serotype A exhibits a higher degree of genetic and antigenic diversity resulting in frequent vaccine failure due to serological mismatch between the vaccine and heterologous strains. Currently, knowledge on the molecular basis of antigenic relationships among the FMDVs is limited; nevertheless, intratype antigenic variation due to mutation(s) is widely considered as the main hurdle to appropriate FMD vaccine development. Here, we studied genetic and antigenic variations of four FMDV serotype A isolates, BAN/GA/Sa‐197/2013 (BAN‐197), BAN/CH/Sa‐304/2016 (BAN‐304), BAN/DH/Sa‐307/2016 (BAN‐307) and BAN/DH/Sa‐310/2017 (BAN‐310) circulating in Bangladesh during 2013–2017. Initially, antigenic relationships (r₁‐values) of the field isolates were evaluated by the two‐dimensional microneutralization test (2D‐MNT) using the hyperimmune antisera raised in cattle against the vaccine strain, BAN‐304. Interesingly, the results showed protective serological cross‐reactivity (r₁‐values > 0.4) between the vaccine strain and the field isolates, BAN‐307 and BAN‐310, except BAN‐197 that substantially mismatched (r₁ = 0.129 ± 0.043) with the BAN‐304. Although VP1‐based phylogeny grouped all the isolates within the same sublineage C (a subgroup of VP3^Δ59 variant) under the lineage A/ASIA/G‐VII, strikingly, computational analyses of the viral capsid proteins demonstrated significant deviation at the VP1 G‐H loop of BAN‐197 from the vaccine strain, while VP(2–4) of both isolates were structurally conserved. To bridge the gap of how the distortion of the G‐H loop and consequent antigenic hetergeneity occurred in BAN‐197, we performed in silico combinatorial substitutions of the VP1 mutant amino acids (aa) of BAN‐197 with the respective residues in BAN‐304. Remarkably, our analyses revealed that two substitutions of distantly located aa at B‐C (T48I:threonine → isoleucine) and G‐H (A143V:alanine → valine) loops, in combination, distorted the VP1 G‐H loop. Overall, this work contributes to understanding the molecular basis of antigenic relationships operating in serotype A FMDVs and the selection of suitable vaccine strain(s) for effective prophylaxis of FMD based on VP1‐based analyses.

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