Genome-wide study of globally distributed respiratory syncytial virus (RSV) strains implicates diversification utilizing phylodynamics and mutational analysis.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes mild cold-like symptoms and severe lower respiratory tract infections, causing hospitalizations in children, the elderly and immunocompromised individuals. Due to genetic variability, this virus causes life-threatening pneumonia and bronchiolitis in young infants. Thus, we examined 3600 whole genome sequences submitted to GISAID by 31 December 2022 to examine the genetic variability of RSV. While RSVA and RSVB coexist throughout RSV seasons, RSVA is more prevalent, fatal, and epidemic-prone in several countries, including the United States, the United Kingdom, Australia, and China. Additionally, the virus's attachment glycoprotein and fusion protein were highly mutated, with RSVA having higher Shannon entropy than RSVB. The genetic makeup of these viruses contributes significantly to their prevalence and epidemic potential. Several strain-specific SNPs co-occurred with specific haplotypes of RSVA and RSVB, followed by different haplotypes of the viruses. RSVA and RSVB have the highest linkage probability at loci T12844A/T3483C and G13959T/C2198T, respectively. The results indicate that specific haplotypes and SNPs may significantly affect their spread. Overall, this analysis presents a promising strategy for tracking the evolving epidemic situation and genetic variants of RSV, which could aid in developing effective control, prophylactic, and treatment strategies.

In Silico Functional Characterization of a Hypothetical Protein From Pasteurella Multocida Reveals a Novel S-Adenosylmethionine-Dependent Methyltransferase Activity.

Genomes may now be sequenced in a matter of weeks, leading to an influx of "hypothetical" proteins (HP) whose activities remain a mystery in GenBank. The information included inside these genes has quickly grown in prominence. Thus, we selected to look closely at the structure and function of an HP (AFF25514.1; 246 residues) from Pasteurella multocida (PM) subsp. multocida str. HN06. Possible insights into bacterial adaptation to new environments and metabolic changes might be gained by studying the functions of this protein. The PM HN06 2293 gene encodes an alkaline cytoplasmic protein with a molecular weight of 28352.60 Da, an isoelectric point (pI) of 9.18, and an overall average hydropathicity of around -0.565. One of its functional domains, tRNA (adenine (37)-N6)-methyltransferase TrmO, is a S-adenosylmethionine (SAM)-dependent methyltransferase (MTase), suggesting that it belongs to the Class VIII SAM-dependent MTase family. The tertiary structures represented by HHpred and I-TASSER models were found to be flawless. We predicted the model's active site using the Computed Atlas of Surface Topography of Proteins (CASTp) and FTSite servers, and then displayed it in 3 dimensional (3D) using PyMOL and BIOVIA Discovery Studio. Based on molecular docking (MD) results, we know that HP interacts with SAM and S-adenosylhomocysteine (SAH), 2 crucial metabolites in the tRNA methylation process, with binding affinities of 7.4 and 7.5 kcal/mol, respectively. Molecular dynamic simulations (MDS) of the docked complex, which included only modest structural adjustments, corroborated the strong binding affinity of SAM and SAH to the HP. Evidence for HP's possible role as an SAM-dependent MTase was therefore given by the findings of Multiple sequence alignment (MSA), MD, and molecular dynamic modeling. These in silico data suggest that the investigated HP might be used as a useful adjunct in the investigation of Pasteurella infections and the development of drugs to treat zoonotic pasteurellosis.

Genomic variability correlates with biofilm phenotypes in multidrug resistant clinical isolates of Pseudomonas aeruginosa.

The multifactorial nature of Pseudomonas aeruginosa biofilm development and genomic variabilities implicates its resistance to conventional antimicrobials and virulence. Therefore, genetic determinants need to be extensively studied to block the early steps of biofilm or already formed biofilms. In this study, a total of 20 multidrug resistant (MDR) clinical P. aeruginosa isolates were evaluated for their biofilm forming abilities and related genes. Of the isolates tested, all of them showed surface attachment tendencies in nutrient limiting conditions, and classified as strong (SBF = 45%), moderate (MBF = 30%) and weak (WBF = 25%) biofilm formers. Complete genome sequencing of representative strong (DMC-27b), moderate (DMC-20c) and weak biofilm former (DMC-30b) isolates was performed. Analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98-100% sequence identity to the reference PAO1 strain. Complete and partial sequence data of LecB proteins from tested isolates indicate that isolates containing PA14-like LecB sequences produced strong biofilms. All of the 7 pel operon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide sequence variation with other tested isolates, and their corresponding proteins are 99% identical with the pel operon proteins of PA7. Bioinformatics analyses identified divergent sequence and structural features that separate PA7 like pel operon proteins from reference PAO1-like pel operon. Congo red and pellicle forming assays revealed that the sequence and structure variations may have interfered with the Pel production pathway and resulted in impaired Pel production in isolate 30b that has a PA7 like pel operon. Expression analysis also showed that both pelB and lecB genes were about 5 to 6 folds upregulated after 24 h in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes of P. aeruginosa strains that affect their biofilm phenotypes.

Characterization of multidrug and heavy metal resistance of carbapenemases producing Klebsiella pneumoniae from poultry samples in Bangladesh.

BACKGROUND: Resistance to multiple drugs in Klebsiella pneumoniae (KPN) is an important issue in human and animal medicine. KPN phenotypic and genotypic aspects in poultry samples have not been comprehensively explored in Bangladesh. METHODS: This research focused on the prevalence of antibiotic resistance and the characterization of KPN in Bangladeshi poultry isolates using both phenotypic and genotypic approaches. RESULTS: A total of 32 poultry samples were randomly obtained from a commercial poultry farm in Narsingdi, Bangladesh, and 43.90% (18/41) of the isolates were confirmed to be KPN, whereas all isolates were biofilm producers. The antibiotic sensitivity test revealed the most remarkable (100%) antibiotic resistance level against Ampicillin, Doxycycline and Tetracycline while remaining susceptible to Doripenem, Meropenem, Cefoxitin and Polymyxin B. Resistance to Nalidixic acid, Nitrofurantoin, Trimethoprim, Levofloxacin, Ciprofloxacin, Cefuroxime and Chloramphenicol ranges from 18% to 70%. Minimum inhibitory concentrations for carbapenem-resistant KPN ranged from 128 to 512 mg/mL for Meropenem, Imipenem, Gentamycin and Ciprofloxacin, respectively. [Correction added on 15 June 2023, after first online publication: 512 g/mL was corrected to 512 mg/mL in the preceding sentence]. Carbapenemase-producing KPN isolates harboured a single or multiple ß-lactamase genes, blaSIM-1 , blaIMP-4 and blaOXA-48 as well as one ESBL gene (blaTEM ) and plasmid-mediated quinolone resistance gene (qnrB). Furthermore, Cr and Co outperformed Cu and Zn in antibacterial effectiveness. CONCLUSIONS: The results of this investigation showed that the high prevalence of multidrug-resistant pathogenic KPN in our chosen geographic location had displayed sensitivity to FOX/PB/Cr/Co, which might be regarded as an alternate treatment to reduce pressure of use on carbapenems.

Bacterial profile, antimicrobial resistance, and molecular detection of ESBL and quinolone resistance gene of uropathogens causing urinary tract infection in the southeastern part of Bangladesh.

Humans frequently contract urinary tract infections (UTIs), which can be brought on by uropathogens (UPs) that are multi-drug resistant. Treatment for UTIs brought on by pathogenic UPs that produce extended-spectrum lactamases (ESBLs) is more costly and potentially fatal. As a result, the objective of this study was to use culture, biochemical, and 16S rRNA sequencing to identify and characterize UPs isolated from outpatients in Noakhali, Bangladesh, who had symptoms of UTIs. ESBL gene identification and quinolone resistance gene typing were then performed on the isolates using polymerase chain reaction (PCR). Throughout the trial's 8-month duration, 152 (76%) of 200 urine samples were positive for the presence of UPs. The overall number of UPs recovered was 210, with 39 individuals having multiple UPs present in their samples. Among all of the isolates, Escherichia coli (45.24%, 95/210; 95% confidence interval (CI): 35.15-57.60%), Enterobacter spp. (24.76%, 52/210; CI: 19.15-35.77%), Klebsiella spp. (20.95%; 44/210; CI: 15.15-30.20%), and Providencia spp. (9.05%; 19/210; CI: 4.95-19.25%) were the four most prevalent bacteria found in the isolates. The UPs displayed a very high level of resistance to piperacillin 96.92% (126/130), ampicillin 90% (117/130), nalidixic acid 77.69% (101/130), cefazolin 70% (91/130), amoxicillin 50% (55/130), cefazolin 42.31% (55/130), nitrofurantoin 43.08% (56/130), and ciprofloxacin 33.08% (43/130), whereas resistance to netilmicin (3.85%), amikacin (4.62%), and imipenem (9.23%) was low. Individually, every species of E. coli and Providencia spp. showed greater ampicillin, amikacin, cefazolin, cefazolin, and nalidixic acid resistance than the others. The bivariate results indicate several antibiotic pairings, and isolates had meaningful associations. All MDR isolates were subjected to PCR, which revealed that blaCTX-M-15 genes predominated among the isolates, followed by the blaTEM class (37%). Isolates also had the qnrS, aac-6´-Ib-cr, and gyrA genes. The findings provide worrying indications of a major expansion of MDR isolates in the study locations, particularly the epidemiological balCTX-M 15, with the potential for the transmission of multi-drug-resistant UP strains in the population.

Epidemiology and molecular characterization of avian influenza virus in backyard poultry of Chattogram, Bangladesh.

Ducks, the natural reservoir of avian influenza virus (AIV), act as reassortment vessels for HPAI and low pathogenic avian influenza (LPAI) virus for domestic and wild bird species. In Bangladesh, earlier research was mainly focused on AIV in commercial poultry and live bird markets, where there is scanty literature reported on AIV in apparently healthy backyard poultry at the household level. The present cross-sectional study was carried out to reveal the genomic epidemiology of AIV of backyard poultry in coastal (Anowara) and plain land (Rangunia) areas of Bangladesh. We randomly selected a total of 292 households' poultry (having both chicken and duck) for sampling. We administered structured pre-tested questionnaires to farmers through direct interviews. We tested cloacal samples from birds for the matrix gene (M gene) followed by H5 and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). All AIV-positive samples were subjected to four-gene segment sequencing (M, PB1, HA, and NA gene). We found that the prevalence of AIV RNA at the household level was 6.2% (n = 18; N = 292), whereas duck and chicken prevalence was 3.6% and 3.2%, respectively. Prevalence varied with season, ranging from 3.1% in the summer to 8.2% in the winter. The prevalence of subtypes H5 and H9 in backyard poultry was 2.7% and 3.3%, respectively. The phylogenetic analysis of M, HA, NA, and PB1 genes revealed intra-genomic similarity, and they are closely related to previously reported AIV strains in Bangladesh and Southeast Asia. The findings indicate that H5 and H9 subtypes of AIV are circulating in the backyard poultry with or without clinical symptoms. Moreover, we revealed the circulation of 2.3.2.1a (new) clade among the chicken and duck population without occurring outbreak which might be due to vaccination. In addition to routine surveillance, molecular epidemiology of AIV will assist to gain a clear understanding of the genomic evolution of the AIV virus in the backyard poultry rearing system, thereby facilitating the implementation of effective preventive measures to control infection and prevent the potential spillover to humans.

Antibiogram and virulence profiling reveals multidrug resistant Staphylococcus aureus as the predominant aetiology of subclinical mastitis in riverine buffaloes.

BACKGROUND: Staphylococcus spp. are the major causal agents of mastitis in dairy animals worldwide leading to profound economic losses and public health threats. Recently, Staphylococcus aureus has emerged as a multidrug resistant and zoonotic pathogen. This study aimed to characterize S. aureus in subclinical mastitis (SCM) milk samples of riverine buffaloes in Bangladesh through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. METHOD: We characterized S. aureus in SCM milk samples (N = 500) of riverine buffaloes through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. RESULTS: Out of 500 milk samples tested, 188 (37.6%) were found positive for SCM. From 188 SCM samples, 291 isolates were obtained with a prevalence of S. aureus in 37.4% (109/291) isolates. Phylogenetic analysis revealed the evolutionary divergence of S. aureus isolates in bubaline SCM milk samples. The antibiogram profiling showed that about 96.0% S. aureus isolates were multidrug resistant (MDR). Notably, 29 and 16 isolates harboured methicillin-resistant (mecA) and panton-valentine leucocidin (pvl) genes, respectively, and 46 plasmid-bearing isolates were MDR. Nine Staphylococcal enterotoxins (SEs/SEls) including sea (11.9%), sec (7.4%), sed (4.6%), seg (3.7%), and seh (3.7%) were detected with 72.48% toxinotypes comprising a single gene. CONCLUSION: This study therefore suggests S. aureus as the single-most aetiology (∼37.0%) of SCM in riverine buffaloes, and emergence of MDR, enterotoxin producing, and virulent S. aureus strains could impose potential threats to animal welfare and public health.

Transmission Dynamics and Genomic Epidemiology of Emerging Variants of SARS-CoV-2 in Bangladesh.

With the progression of the global SARS-CoV-2 pandemic, the new variants have become more infectious and continue spreading at a higher rate than pre-existing ones. Thus, we conducted a study to explore the epidemiology of emerging variants of SARS-CoV-2 that circulated in Bangladesh from December 2020 to September 2021, representing the 2nd and 3rd waves. We collected new cases and deaths per million daily data with the reproduction rate. We retrieved 928 SARS-CoV-2 sequences from GISAID and performed phylogenetic tree construction and mutation analysis. Case counts were lower initially at the end of 2020, during January-February and April-May 2021, whereas the death toll reached the highest value of 1.587 per million on the first week of August and then started to decline. All the variants (α, ß, δ, η) were prevalent in the capital city, Dhaka, with dispersion to large cities, such as Sylhet and Chattogram. The B.1.1.25 lineage was prevalent during December 2020, but the B.1.617.2/δ variant was later followed by the B.1.351/ß variant. The phylogeny revealed that the various strains found in Bangladesh could be from numerous countries. The intra-cluster and inter-cluster communication began in Bangladesh soon after the virus arrived. The prominent amino acid substitution was D614G from December 2020 to July 2021 (93.5 to 100%). From February-April, one of the VOC's important mutations, N501Y substitution, was also estimated at 51.8%, 76.1%, and 65.1% for the α, ß and γ variants, respectively. The γ variant's unique mutation K417T was detected only at 1.8% in February. Another frequent mutation was P681R, a salient feature of the δ variant, detected in June (88.2%) and July (100%). Furthermore, only one γ variant was detected during the entire second and third wave, whereas no η variant was observed in this period. This rapid growth in the number of variants identified across Bangladesh shows virus adaptation and a lack of strict quarantine, prompting periodic genomic surveillance to foresee the spread of new variants, if any, and to take preventive measures as soon as possible.

Designing potential siRNA molecules for silencing the gene of the nucleocapsid protein of Nipah virus: A computational investigation.

Nipah virus (NiV), a zoonotic virus, engenders severe infections with noticeable complications and deaths in humans and animals. Since its emergence, it is frightening, this virus has been causing regular outbreaks in various countries, particularly in Bangladesh, India, and Malaysia. Unfortunately, no efficient vaccine or drug is available now to combat this baneful virus. NiV employs its nucleocapsid protein for genetic material packaging, which is crucial for viral replication inside the host cells. The small interfering RNAs (siRNAs) can play a central role in inhibiting the expression of disease-causing viral genes by hybridization and subsequent inactivation of the complementary target viral mRNAs through the RNA interference (RNAi) pathway. Therefore, potential siRNAs as molecular therapeutics against the nucleocapsid protein gene of NiV were designed in this study. First, ten prospective siRNAs were identified using the conserved nucleocapsid gene sequences among all available NiV strains collected from various countries. After that, off-target binding, GC (guanine-cytosine) content, secondary structure, binding affinity with the target, melting temperature, efficacy analysis, and binding capacity with the human argonaute protein 2 (AGO2) of these siRNAs were evaluated to predict their suitability. These designed siRNA molecules bear promise in silencing the NiV gene encoding the nucleocapsid protein and thus can alleviate the severity of this dangerous virus. Further in vivo experiments are recommended before using these designed siRNAs as alternative and effective molecular therapeutic agents against NiV.

Transmission dynamics and susceptibility patterns of SARS-CoV-2 in domestic, farmed and wild animals: Sustainable One Health surveillance for conservation and public health to prevent future epidemics and pandemics.

The exact origin of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and source of introduction into humans has not been established yet, though it might be originated from animals. Therefore, we conducted a study to understand the putative reservoirs, transmission dynamics, and susceptibility patterns of SARS-CoV-2 in animals. Rhinolophus bats are presumed to be natural progenitors of SARS-CoV-2-related viruses. Initially, pangolin was thought to be the source of spillover to humans, but they might be infected by human or other animal species. So, the virus spillover pathways to humans remain unknown. Human-to-animal transmission has been testified in pet, farmed, zoo and free-ranging wild animals. Infected animals can transmit the virus to other animals in natural settings like mink-to-mink and mink-to-cat transmission. Animal-to-human transmission is not a persistent pathway, while mink-to-human transmission continues to be illuminated. Multiple companions and captive wild animals were infected by an emerging alpha variant of concern (B.1.1.7 lineage) whereas Asiatic lions were infected by delta variant, (B.1.617.2). To date, multiple animal species - cat, ferrets, non-human primates, hamsters and bats - showed high susceptibility to SARS-CoV-2 in the experimental condition, while swine, poultry, cattle showed no susceptibility. The founding of SARS-CoV-2 in wild animal reservoirs can confront the control of the virus in humans and might carry a risk to the welfare and conservation of wildlife as well. We suggest vaccinating pets and captive animals to stop spillovers and spillback events. We recommend sustainable One Health surveillance at the animal-human-environmental interface to detect and prevent future epidemics and pandemics by Disease X.

Genomic characterization of the dominating Beta, V2 variant carrying vaccinated (Oxford-AstraZeneca) and nonvaccinated COVID-19 patient samples in Bangladesh: A metagenomics and whole-genome approach.

Bangladesh is experiencing a second wave of COVID-19 since March 2021, despite the nationwide vaccination drive with ChAdOx1 (Oxford-AstraZeneca) vaccine from early February 2021. Here, we characterized 19 nasopharyngeal swab (NPS) samples from COVID-19 suspect patients using genomic and metagenomic approaches. Screening for SARS-CoV-2 by reverse transcriptase polymerase chain reaction and metagenomic sequencing revealed 17 samples of COVID-19 positive (vaccinated = 10, nonvaccinated = 7) and 2 samples of COVID-19 negative. We did not find any significant correlation between associated factors including vaccination status, age or sex of the patients, diversity or abundance of the coinfected organisms/pathogens, and the abundance of SARS-CoV-2. Though the first wave of the pandemic was dominated by clade 20B, Beta, V2 (South African variant) dominated the second wave (January 2021 to May 2021), while the third wave (May 2021 to September 2021) was responsible for Delta variants of the epidemic in Bangladesh including both vaccinated and unvaccinated infections. Noteworthily, the receptor binding domain (RBD) region of S protein of all the isolates harbored similar substitutions including K417N, E484K, and N501Y that signify the Beta, while D614G, D215G, D80A, A67V, L18F, and A701V substitutions were commonly found in the non-RBD region of Spike proteins. ORF7b and ORF3a genes underwent a positive selection (dN/dS ratio 1.77 and 1.24, respectively), while the overall S protein of the Bangladeshi SARS-CoV-2 isolates underwent negative selection pressure (dN/dS = 0.621). Furthermore, we found different bacterial coinfections like Streptococcus agalactiae, Neisseria meningitidis, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Pseudomonas plecoglossicida, expressing a number of antibiotic resistance genes such as tetA and tetM. Overall, this approach provides valuable insights on the SARS-CoV-2 genomes and microbiome composition from both vaccinated and nonvaccinated patients in Bangladesh.

Spatial epidemiology and genetic diversity of SARS-CoV-2 and related coronaviruses in domestic and wild animals.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed susceptibility to diverse animal species. We conducted this study to understand the spatial epidemiology, genetic diversity, and statistically significant genetic similarity along with per-gene recombination events of SARS-CoV-2 and related viruses (SC2r-CoVs) in animals globally. We collected a number of different animal species infected with SARS-CoV-2 and its related viruses. Then, we retrieved genome sequences of SARS-CoV-2 and SC2r-CoVs from GISAID and NCBI GenBank for genomic and mutational analysis. Although the evolutionary origin of SARS-CoV-2 remains elusive, the diverse SC2r-CoV have been detected in multiple Rhinolophus bat species and in Malayan pangolin. To date, human-to-animal spillover events have been reported in cat, dog, tiger, lion, gorilla, leopard, ferret, puma, cougar, otter, and mink in 25 countries. Phylogeny and genetic recombination events of SC2r-CoVs showed higher similarity to the bat coronavirus RaTG13 and BANAL-103 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the N protein from bats and pangolin showed close resemblance to SARS-CoV-2. The clustering of animal and human strains from the same geographical area has proved human-to-animal transmission of the virus. The Alpha, Delta and Mu-variant of SARS-CoV-2 was detected in dog, gorilla, lion, tiger, otter, and cat in the USA, India, Czech Republic, Belgium, and France with momentous genetic similarity with human SARS-CoV-2 sequences. The mink variant mutation (spike_Y453F) was detected in both humans and domestic cats. Moreover, the dog was affected mostly by clade O (66.7%), whereas cat and American mink were affected by clade GR (31.6 and 49.7%, respectively). The α-variant was detected as 2.6% in cat, 4.8% in dog, 14.3% in tiger, 66.7% in gorilla, and 77.3% in lion. The highest mutations observed in mink where the substitution of D614G in spike (95.2%) and P323L in NSP12 (95.2%) protein. In dog, cat, gorilla, lion, and tiger, Y505H and Y453F were the common mutations followed by Y145del, Y144del, and V70I in S protein. We recommend vaccine provision for pet and zoo animals to reduce the chance of transmission in animals. Besides, continuous epidemiological and genomic surveillance of coronaviruses in animal host is crucial to find out the immediate ancestor of SARS-CoV-2 and to prevent future CoVs threats to humans.

Genome-wide identification and prediction of SARS-CoV-2 mutations show an abundance of variants: Integrated study of bioinformatics and deep neural learning.

Genomic data analysis is a fundamental system for monitoring pathogen evolution and the outbreak of infectious diseases. Based on bioinformatics and deep learning, this study was designed to identify the genomic variability of SARS-CoV-2 worldwide and predict the impending mutation rate. Analysis of 259044 SARS-CoV-2 isolates identified 3334545 mutations with an average of 14.01 mutations per isolate. Globally, single nucleotide polymorphism (SNP) is the most prevalent mutational event. The prevalence of C > T (52.67%) was noticed as a major alteration across the world followed by the G > T (14.59%) and A > G (11.13%). Strains from India showed the highest number of mutations (48) followed by Scotland, USA, Netherlands, Norway, and France having up to 36 mutations. D416G, F106F, P314L, UTR:C241T, L93L, A222V, A199A, V30L, and A220V mutations were found as the most frequent mutations. D1118H, S194L, R262H, M809L, P314L, A8D, S220G, A890D, G1433C, T1456I, R233C, F263S, L111K, A54T, A74V, L183A, A316T, V212F, L46C, V48G, Q57H, W131R, G172V, Q185H, and Y206S missense mutations were found to largely decrease the structural stability of the corresponding proteins. Conversely, D3L, L5F, and S97I were found to largely increase the structural stability of the corresponding proteins. Multi-nucleotide mutations GGG > AAC, CC > TT, TG > CA, and AT > TA have come up in our analysis which are in the top 20 mutational cohort. Future mutation rate analysis predicts a 17%, 7%, and 3% increment of C > T, A > G, and A > T, respectively in the future. Conversely, 7%, 7%, and 6% decrement is estimated for T > C, G > A, and G > T mutations, respectively. T > G\A, C > G\A, and A > T\C are not anticipated in the future. Since SARS-CoV-2 is mutating continuously, our findings will facilitate the tracking of mutations and help to map the progression of the COVID-19 intensity worldwide.

First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera.

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.

Evolutionary Dynamics and Epidemiology of Endemic and Emerging Coronaviruses in Humans, Domestic Animals, and Wildlife.

Diverse coronavirus (CoV) strains can infect both humans and animals and produce various diseases. CoVs have caused three epidemics and pandemics in the last two decades, and caused a severe impact on public health and the global economy. Therefore, it is of utmost importance to understand the emergence and evolution of endemic and emerging CoV diversity in humans and animals. For diverse bird species, the Infectious Bronchitis Virus is a significant one, whereas feline enteric and canine coronavirus, recombined to produce feline infectious peritonitis virus, infects wild cats. Bovine and canine CoVs have ancestral relationships, while porcine CoVs, especially SADS-CoV, can cross species barriers. Bats are considered as the natural host of diverse strains of alpha and beta coronaviruses. Though MERS-CoV is significant for both camels and humans, humans are nonetheless affected more severely. MERS-CoV cases have been reported mainly in the Arabic peninsula since 2012. To date, seven CoV strains have infected humans, all descended from animals. The severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) are presumed to be originated in Rhinolopoid bats that severely infect humans with spillover to multiple domestic and wild animals. Emerging alpha and delta variants of SARS-CoV-2 were detected in pets and wild animals. Still, the intermediate hosts and all susceptible animal species remain unknown. SARS-CoV-2 might not be the last CoV to cross the species barrier. Hence, we recommend developing a universal CoV vaccine for humans so that any future outbreak can be prevented effectively. Furthermore, a One Health approach coronavirus surveillance should be implemented at human-animal interfaces to detect novel coronaviruses before emerging to humans and to prevent future epidemics and pandemics.

Molecular Epidemiology of SARS-CoV-2 in Diverse Environmental Samples Globally.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swamped the global environment greatly in the current pandemic. Wastewater-based epidemiology (WBE) effectively forecasts the surge of COVID-19 cases in humans in a particular region. To understand the genomic characteristics/footprints and diversity of SARS-CoV-2 in the environment, we analyzed 807 SARS-CoV-2 sequences from 20 countries deposited in GISAID till 22 May 2021. The highest number of sequences (n = 638) were reported in Austria, followed by the Netherlands, China, and Bangladesh. Wastewater samples were highest (40.0%) to successfully yield the virus genome followed by a 24 h composite wastewater sample (32.6%) and sewage (18.5%). Phylogenetic analysis revealed that SARS-CoV-2 environmental strains are a close congener with the strains mostly circulating in the human population from the same region. Clade GRY (32.7%), G (29.2%), GR (25.3%), O (7.2%), GH (3.4%), GV (1.4%), S (0.5%), and L (0.4%) were found in environmental samples. Various lineages were identified in environmental samples; nevertheless, the highest percentages (49.4%) of the alpha variant (B.1.1.7) were detected in Austria, Liechtenstein, Slovenia, Czech Republic, Switzerland, Germany, and Italy. Other prevalent lineages were B.1 (18.2%), B.1.1 (9.2%), and B.1.160 (3.9%). Furthermore, a significant number of amino acid substitutions were found in environmental strains where the D614G was found in 83.8% of the sequences. However, the key mutations-N501Y (44.6%), S982A (44.4%), A570D (43.3%), T716I (40.4%), and P681H (40.1%) were also recorded in spike protein. The identification of the environmental belvedere of SARS-CoV-2 and its genetic signature is crucial to detect outbreaks, forecast pandemic harshness, and prepare with the appropriate tools to control any impending pandemic. We recommend genomic environmental surveillance to trace the emerging variants and diversity of SARS-CoV-2 viruses circulating in the community. Additionally, proper disposal and treatment of wastewater, sewage, and medical wastes are important to prevent environmental contamination.

Multilocus sequence typing of multidrug-resistant Salmonella strains circulating in poultry farms of Bangladesh.

Salmonella is one of the most important foodborne zoonotic pathogens, and becoming multidrug-resistant (MDR), which represents a serious public health concern worldwide. This study aimed to identify the circulating MDR strains of Salmonella through cutting edge molecular techniques including gene specific PCR, RAPD-PCR, ribosomal gene sequencing, and multilocus sequence types (MLST) in the poultry industry of Bangladesh. Two hundred Salmonella isolates were retrieved from 154 samples comprising droppings (n = 60), cloacal swabs (n = 60), feeds (n = 14), feeding water (n = 14), and handler's swab (n = 6) from 14 commercial layer farms of Bangladesh. The isolates were confirmed as Salmonella through invA gene specific PCR, and further genotyping was done by RAPD-PCR, and 16S rRNA sequencing. The isolates were distributed into 18 different genotypes according to RAPD typing. The phylogenetic analysis identified three diverging phylogroups such as S. enterica Litchfield, S. enterica Enteritidis and S. enterica Kentucky with 11, 8, and 6 strains, respectively. The in vitro antibiogram profiling the Salmonella isolates through disc diffusion method using 13 commercially available antibiotics revealed highest resistance against doxycycline (91.5%) followed by tetracycline and ampicillin (86.0%, in each), and 72.0% isolates as MDR, being resistant to ≥ 5 antibiotics. The MLST typing was carried out based on the PCR amplification of seven housekeeping genes (aroC, hisD, hemD, purE, secA, thrA, and dnaN). MLST typing also revealed three sequence types (STs) such as ST11, ST198, and ST214 in these isolates, and eBURST analysis showed ST11 as the founder genotype. The three STs were highly resistant to tetracyclines and quinolone group of antibiotics, and all of the isolates harboring S. enterica Litchfield showed the highest resistance. Circulating common MLSTs with MDR properties in different farms confirmed the possibility of a common route of intra-farm transmission. We report for the first time of the association serovar Litchfield (ST11) in avian salmonellosis with MDR properties which is an urgent public health concern in Bangladesh.

Temporal landscape of mutational frequencies in SARS-CoV-2 genomes of Bangladesh: possible implications from the ongoing outbreak in Bangladesh.

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh-originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA X, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in nonspecific proteins (NSP2-3, NSP-12(RdRp), NSP-13(Helicase)), S-Spike, ORF3a, and N-Nucleocapsid protein were common in the circulating strains with varying degrees and the most unique mutations (UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, Envelope protein (E) and accessory factors (NSP7a, ORF 6, ORF7b) suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed a significant association (p = 0.003) with sex and age of the COVID-19-positive cases. So, identification of these mutational accumulation patterns may greatly facilitate vaccine development deciphering the age and the sex-dependent differential susceptibility to COVID-19.

Assessment of basic reproduction number (R0), spatial and temporal epidemiological determinants, and genetic characterization of SARS-CoV-2 in Bangladesh.

Epidemiological and molecular characterization of SARS-CoV-2 is essential for identifying the source of the virus and for effective control of the spread of local strains. We estimated case fatality rate, cumulative recovery number, basic reproduction number (R0) and future incidence of COVID-19 in Bangladesh. We illustrated the spatial distribution of cases throughout the country. We performed phylogenetic and mutation analysis of SARS-CoV-2 sequences from Bangladesh. As of July 31, 2020, Bangladesh had a case fatality rate of 1.32%. The cases were initially clustered in Dhaka and its surrounding districts in March but spreads throughout the country over time. The R0 calculated as 1.173 in Exponential Growth method. For the projection, a 20% change in R0 with subsequent infection trend has been calculated. The genomic analysis of 292 Bangladeshi SARS-CoV-2 strains suggests diverse genomic clades L, O, S, G, GH, where predominant circulating clade was GR (83.9%; 245/292). The GR clades' phylogenetic analysis revealed distinct clusters (I to XIII) with intra-clade variations. The mutation analysis revealed 1634 mutations where 94.6% and 5.4% were non-synonymous and unique mutation, respectively. The Spike, Nucleocapsid, NSP2, and RdRP showed substantially high mutation but no mutation was recorded in NSP9 and NSP11 protein. In spike (S) protein, 355 predominant mutations were recorded, highest in D614G. Alternatively, I120F in NSP2 protein, R203K and G204R in nucleocapsid protein, and P323L in RdRp were more recurrent. The Bangladeshi genomes belonged to phylogenetic lineages A, B, B.1, B.1.1, B.1.1.23, B.1.1.25, B.1.113, and B.1.36, among which 50.0% sequences owned by the pangolin lineage B.1.1.25. The study illustrates the spatial distribution of SARS-CoV-2, and molecular epidemiology of Bangladeshi isolates. We recommend continuous monitoring of R0 and genomic surveillance to understand the transmission dynamics and detect new variants of SARS-CoV-2 for proper control of the current pandemic and evaluate the effectiveness of vaccination globally.

Genome-wide genetic marker analysis and genotyping of Escherichia fergusonii strain OTSVEF-60.

Poultry originated Escherichia fergusonii (POEF), an emerging bacterial pathogen, causes a wide range of intestinal and extra-intestinal infections in the poultry industry which incurred significant economic losses worldwide. Chromosomal co-existence of antibiotics and metal resistance genes has recently been the focal point of POEF isolates besides its pathogenic potentials. This study reports the complete genome analysis of POEF strain OTSVEF-60 from the poultry originated samples of Bangladesh. The assembled draft genome of the strain was 4.2 Mbp containing 4503 coding sequences, 120 RNA (rRNA = 34, tRNA = 79, ncRNA = 7), and three intact phage signature regions. Forty-one broad range antibiotic resistance genes (ARGs) including dfrA12, qnrS1, blaTEM-1, aadA2, tet(A), and sul-2 along with multiple efflux pump genes were detected, which translated to phenotypic resistant patterns of the pathogen to trimethoprim, fluoroquinolones, ß-lactams, aminoglycoside, tetracycline, and sulfonamides. Moreover, 22 metal resistance genes were found co-existing within the genome of the POEF strain, and numerous virulence genes (VGs) coding for cit (AB), feo (AB), fep (ABCG), csg (ABCDEFG), fliC, ompA, gadA, ecpD, etc. were also identified throughout the genome. In addition, we detected a class I integron gene cassette harboring dfrA12, ant (3″)-I, and qacEΔ-sul2 genes; 42 copies of insertion sequence (IS) elements; and two CRISPR arrays. The genomic functional analysis predicted several metabolic pathways related to motility, flagellar assembly, epithelial cell invasion, quorum sensing, biofilm formation, and biosynthesis of vitamin, co-factors, and secondary metabolites. We herein for the first time detected multiple ARGs, VGs, mobile genetic elements, and some metabolic functional genes in the complete genome of POEF strain OTSVEF-60, which might be associated with the pathogenesis, spreading of ARGs and VGs, and subsequent treatment failure against this emerging avian pathogen with currently available antimicrobials.