Metagenomic fingerprinting of waterborne enteric viruses and their inhibition using phyto-nanomaterials

Abstract

Waterborne enteric viruses are major causes of diseases including gastroenteritis, hepatitis, encephalitis, meningitis, conjunctivitis, and respiratory disorders. Transmission occurs primarily through the faecal-oral channel via contaminated water or food, constituting significant public health risks. These viruses have been reported in South Africa following exposure to contaminated surface waters, dams, and wastewater. Nevertheless, limited data exist on their prevalence, diversity and abundance, especially in wastewater treatment plants (WWTPs) in eThekwini Municipality, Durban, KwaZulu-Natal, South Africa, necessitating metagenomic surveillance. In addition, the development of resistance to conventional drugs and high treatment costs necessitates the development of novel approaches to identify inexpensive and efficient antiviral medications. Interestingly, Spondias mombin and Macaranga barteri extracts have exhibited antiviral activity against several enteric viruses while Dicerocaryum eriocarpum with reported antibacterial activity remain unexplored as a reservoir of antiviral drug candidates. Accordingly, for the first time, this study profile the prevalence and diversity of viral families, including human and plant pathogenic viruses at the selected WWTPs and receiving rivers in eThekwini, Durban, Kwazulu-Natal, South Africa, using metagenomics next-generation sequencing (mNGS) approach and evaluates metabolites from S. mombin, M. barteri and D. eriocarpum against severe acute respiratory syndromecoronavirus-2 (SARS-CoV-2) and rotavirus A (RVA) using in silico techniques. The mNGS viral surveillance investigation identified viral families with a wide range of hosts including vertebrates (19.46%), plant (6.89%), insect (1.90%), fungal (0.13%), unclassified (7.25%) and phages (64.37%). Altogether, 30 viral families encompassing 26 different human pathogenic viral species were identified including enteroviruses, hepatovirus A, rotavirus A, norovirus, and astrovirus A. Conversely, plant viruses accounted for eight families dominated by the Virgaviridae. Most phytopathogenic reads belonged to the Tobamovirus genus, with additional representation from the family Bromoviridae detected across all sampling sites except RIVER4. These findings highlight the significance of wastewater surveillance in tracking known infections and unveiling possible emerging viral threats. RVA, one of the commonest waterborne enteric viruses detected in this study and SARS-CoV-2, although not detected in this study, probably due to low circulation within the communities at the time of sampling, were selected due to their clinical relevance and therapeutic limitations for in silico drug discovery from selected antiviral plants (S. mombin, M. barteri and D. eriocarpum). Molecular docking identified top-ranked compounds with higher negative docking scores against SARS-CoV-2 and RVA targets from the 77 pooled metabolites identified from the three selected medicinal plants against each of the investigated targets [main protease and RNAdependent RNA polymerase (RdRp)] of SARS-CoV-2 and RVA [capsid protein (VP7A, VP7C, VP7D), RdRp (VP1), spike protein (VP5*, VP8*)]. A minimum of three top-ranked compounds were subsequently identified owing to their physicochemical, pharmacokinetic and toxicological profiles suitable for drug development. The profiled top ranked compounds against the selected druggable targets of SARS-CoV-2 and RVA were subsequently subjected to 120-ns molecular dynamics (MD) simulations using the in-house HEAL1361 program with the Amber 18 package of the Centre for High Performance Computing (CHPC), South Africa. At least three of the top-ranked compounds exhibited higher negative binding free energy across the evaluated targets, suggesting structural and thermodynamic compatibility of the ligands at the binding domain of the targets and their suitability for drug development thereby validating the antiviral potential of the selected medicinal plants. However, against SCMP, only quercetin 3-O-rhamnoside had a higher negative binding free energy, suggesting the need for optimization of other metabolites. Notably, chrysoeriol7-glucuronide and spiraeoside demonstrated broad-spectrum affinity for RdRp of SARS-CoV-2 and RVA. Furthermore, (2S)- 6-(gamma,gamma-dimethylallyl)-3’,4’-dimethoxy-6’’,6’’- dimethylpyran[2’’,3’’:7,8]flavanone (2SG) and sericetin had better broad-spectrum affinities for both RVA VP5* and VP8*, while 2SG, apigenin-4 ′-glucoside and gnetin L exhibited better broad-spectrum affinities for RVA VP7A, VP7C and VP7D, suggesting their potential as multitarget antiviral candidates. However, some compounds induce higher thermodynamic entropy within the proteins relative to the standard. For instance, against VP5*, 2SG (-20.98 kcal/mol, 1.88 Å), sericetin (-20.46 kcal/mol, 1.93 Å) and sacranoside (-16.96 kcal/mol, 1.50 Å), though having higher negative binding free energies, exhibited higher RMSD values compared to the standard (1.30 Å) and unbound VP5* (1.45 Å). A partly similar trend was illustrated against VP8*, where sericetin (-18.31 kcal/mol, 1.27 Å) and 2SG (-20.98 kcal/mol, 1.18 Å) exhibited higher negative binding free energies yet had higher RMSD and RMSF values respectively, compared to the standard (1.19 Å, 1.01 Å). This observation highlights the need for optimizing these leads to improve their thermodynamic stability while boosting their affinity for the target proteins. Thus, due to their critical role in viral entry and infection, silver complexation optimization was limited to VP5* and VP8* of RVA to block the first and most vulnerable stage of infection. Accordingly, the silver complexation approach was employed to model possible silver nanoparticle conjugates for each lead compound, treating the compound as the reducing and capping agent. Thereafter, the effect of the complexation was evaluated whether complexation of the lead compound with silver enhances their binding affinity in silico. Improved binding affinity enhances drug activity through silver complexation which help mitigate drug resistance, broaden activity spectrum and enhance potency in vitro and in vivo. Thus, the study adopted silver complexation approach by conjugating silver with identified lead compounds against VP5* and VP8* via in silico modelling to achieve improved activity, reduced toxicity and maintain favorable pharmacokinetic profiles. To comprehend the effect of the complexation, quantum chemical calculations (density functional theory) were used to compare the molecular features of the silver-complexed leads against the lead compounds alongside assessment of drug-likeness and toxicity profiles. Thereafter, 12 and 19 silvercomplexed leads were docked against VP5* and VP8* respectively, followed by comparative interaction analysis between lead and silver-complexed compounds. Notably, the silvercomplexed leads exhibited enhanced molecular properties, suggesting better reactivity than their corresponding lead compounds without significantly altering their pharmacokinetic properties and toxicity profiles; hence, they remain suitable for drug development. Remarkably, higher negative docking scores were observed in all silver-complexed leads docked against VP5* compared to their corresponding lead compounds, except in the sacranoside A series. Conversely, lower negative docking scores were reported in the apigenin-4'-glucoside series, gnetin L A-E, and sericetin B relative to their lead compounds, while the remaining silvercomplexed leads exhibited improved binding affinity. Comparative interaction analysis identified 2SGC (-6.59 kcal/mol) and 2SGA (-6.77 kcal/mol) against VP5* and 2SGC (-8.06 kcal/mol) and SERA (-7.26 kcal/mol) against VP8* as promising candidates. Further computational investigation, including MD simulation, is recommended to elucidate the thermodynamics profile of the resulting protein and silver-complexed leads. Summarily, the observed enhanced binding capability of the silver-complexed leads could be attributed to their higher reactivity and increased interactions with essential amino acids at the binding pocket of the targets investigated. To validate the therapeutic potential of the profiled silver-complexed leads in combating viral infections, complementary in vitro and in vivo studies are strongly recommended.