Epidemiology of norovirus infection in Nigeria: a systematic review and meta-analysis.

Human norovirus (HuNoV) is responsible for most cases of gastroenteritis worldwide, but information about the prevalence and diversity of HuNoV infections in lower-income settings is lacking. In order to provide more information about the burden and distribution of norovirus in Nigeria, we systematically reviewed original published research articles on the prevalence of HuNoV in Nigeria by accessing databases, including PubMed, Web of Science, ScienceDirect, Google Scholar, and African Journals Online (AJOL). The protocol for the review was registered on PROSPERO (registration number CRD42022308857). Thirteen relevant articles were included in the review, and 10 of them were used for meta-analysis. The pooled prevalence of HuNoV-associated gastroenteritis among children below 5 years of age in Nigeria, determined using the random-effects model, was 10.9% (95% CI, 6.7-16.7%). Among children below the age of 5 presenting with HuNoV infections, the highest prevalence was in children ≤2 years old (n = 127, 83%). The prevalence of HuNoV infections was seen to decrease with increasing age. In addition, HuNoV was detected in asymptomatic food handlers, bats, and seafoods. A total of 85 sequences of HuNoV isolates from Nigeria have been determined, and based on those sequences, the most prevalent norovirus genogroup was GII (84%). Genotypes GII.4 and GI.3 were the most frequently identified genotypes, with GII.4 constituting 46% of all of the HuNoVs identified in Nigeria. These results suggest a risk associated with cocirculation of emerging variants with known genotypes because of their recombination potential. Larger molecular epidemiological studies are still needed to fully understand the extent and pattern of circulation of HuNoVs in Nigeria.

Near-source passive sampling for monitoring viral outbreaks within a university residential setting.

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for the population-level monitoring of pathogens, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For assessment, several wastewater sampling regimes and methods of viral concentration have been investigated, mainly targeting SARS-CoV-2. However, the use of passive samplers in near-source environments for a range of viruses in wastewater is still under-investigated. To address this, near-source passive samples were taken at four locations targeting student hall of residence. These were chosen as an exemplar due to their high population density and perceived risk of disease transmission. Viruses investigated were SARS-CoV-2 and its variants of concern (VOCs), influenza viruses, and enteroviruses. Sampling was conducted either in the morning, where passive samplers were in place overnight (17 h) and during the day, with exposure of 7 h. We demonstrated the usefulness of near-source passive sampling for the detection of VOCs using quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS). Furthermore, several outbreaks of influenza A and sporadic outbreaks of enteroviruses (some associated with enterovirus D68 and coxsackieviruses) were identified among the resident student population, providing evidence of the usefulness of near-source, in-sewer sampling for monitoring the health of high population density communities.

Research needs for optimising wastewater-based epidemiology monitoring for public health protection.

Wastewater-based epidemiology (WBE) is an unobtrusive method used to observe patterns in illicit drug use, poliovirus, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The pandemic and need for surveillance measures have led to the rapid acceleration of WBE research and development globally. With the infrastructure available to monitor SARS-CoV-2 from wastewater in 58 countries globally, there is potential to expand targets and applications for public health protection, such as other viral pathogens, antimicrobial resistance (AMR), pharmaceutical consumption, or exposure to chemical pollutants. Some applications have been explored in academic research but are not used to inform public health decision-making. We reflect on the current knowledge of WBE for these applications and identify barriers and opportunities for expanding beyond SARS-CoV-2. This paper critically reviews the applications of WBE for public health and identifies the important research gaps for WBE to be a useful tool in public health. It considers possible uses for pathogenic viruses, AMR, and chemicals. It summarises the current evidence on the following: (1) the presence of markers in stool and urine; (2) environmental factors influencing persistence of markers in wastewater; (3) methods for sample collection and storage; (4) prospective methods for detection and quantification; (5) reducing uncertainties; and (6) further considerations for public health use.

Site Specific Relationships between COVID-19 Cases and SARS-CoV-2 Viral Load in Wastewater Treatment Plant Influent.

Wastewater based epidemiology (WBE) has become an important tool during the COVID-19 pandemic, however the relationship between SARS-CoV-2 RNA in wastewater treatment plant influent (WWTP) and cases in the community is not well-defined. We report here the development of a national WBE program across 28 WWTPs serving 50% of the population of Scotland, including large conurbations, as well as low-density rural and remote island communities. For each WWTP catchment area, we quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. Daily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ > 0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from 25 cases for larger plants to a single case in smaller plants. SARS-CoV-2 viral RNA load can be used to predict the number of cases detected in the WWTP catchment area, with a clear statistically significant relationship observed above site-specific case thresholds.

SARS-CoV-2 and other pathogenic microorganisms in the environment.

The title of the Virtual Special Issue (VSI) "SARS-CoV-2 and other pathogenic microorganisms in the environment", clearly indicates a main focus not only on the virus causing the current pandemic, but also on other pathogenic microorganisms and their spatial and temporal dynamics in environmental compartments. Overall, the VSI has received more than 100 submissions relating to most of the possible fields connected to the pandemic, many of them of high scientific value. A rigorous peer-reviewing process has been carried out, with a panel of experts making a great work to evaluate that important number of submissions. As a result, those manuscripts reaching the highest scientific standards were selected for publication. We think that the papers included constitute a set of high-quality contributions, which should help to improve the overall scientific perspective regarding this crucial issue. In this piece, the Editors comment some issues on the papers accepted for publication, and include additional reflections.

Fish polyomaviruses belong to two distinct evolutionary lineages.

The Polyomaviridae is a diverse family of circular double-stranded DNA viruses. Polyomaviruses have been isolated from a wide array of animal hosts. An understanding of the evolutionary and ecological dynamics of these viruses is essential to understanding the pathogenicity of polyomaviruses. Using a high throughput sequencing approach, we identified a novel polyomavirus in an emerald notothen (Trematomus bernacchii) sampled in the Ross sea (Antarctica), expanding the known number of fish-associated polyomaviruses. Our analysis suggests that polyomaviruses belong to three main evolutionary clades; the first clade is made up of all recognized terrestrial polyomaviruses. The fish-associated polyomaviruses are not monophyletic, and belong to two divergent evolutionary lineages. The fish viruses provide evidence that the evolution of the key viral large T protein involves gain and loss of distinct domains.

The Ancient Evolutionary History of Polyomaviruses.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Genome sequences of a capulavirus infecting Plantago lanceolata in the Åland archipelago of Finland.

The discovery and full-genome sequences of two isolates of a fourth capulavirus species are reported. The viruses were discovered during a viral metagenomics survey of uncultivated Plantago lanceolata plants in the Åland archipelago of south western Finland. The newly discovered viruses apparently produce no symptoms in P. lanceolata. They have a genome organization that is very similar to that of the three known capulavirus species and additionally share between 62.9 and 67.1% genome-wide sequence identity with the isolates of these species. It is therefore proposed that these viruses be assigned to a new capulavirus species named "Plantago lanceolata latent virus".

Identification of an avian polyomavirus associated with Adélie penguins (Pygoscelis adeliae).

Little is known about viruses associated with Antarctic animals, although they are probably widespread. We recovered a novel polyomavirus from Adélie penguin (Pygoscelis adeliae) faecal matter sampled in a subcolony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adélie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organization with three ORFs that encoded capsid proteins on the one strand and two non-structural protein-coding ORFs on the complementary strand. The genome of AdPyV shared ~60 % pairwise identity with all avipolyomaviruses. Maximum-likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences showed that the T-Ag of AdPyV clustered with those of avipolyomaviruses, sharing between 48 and 52 % identities. Only three viruses associated with Adélie penguins have been identified at a genomic level, avian influenza virus subtype H11N2 from the Antarctic Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and Royds on Ross Island.

Identification of novel Bromus- and Trifolium-associated circular DNA viruses.

The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromus hordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifolium resupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99 % sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74 % identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand.

Simultaneous detection and characterization of common respiratory pathogens in wastewater through genomic sequencing.

Genomic surveillance of SARS-CoV-2 has given insight into the evolution and epidemiology of the virus and its variant lineages during the COVID-19 pandemic. Expanding this approach to include a range of respiratory pathogens can better inform public health preparedness for potential outbreaks and epidemics. Here, we simultaneously sequenced 38 pathogens including influenza viruses, coronaviruses and bocaviruses, to examine the abundance and seasonality of respiratory pathogens in urban wastewater. We deployed a targeted bait capture method and short-read sequencing (Illumina Respiratory Virus Oligos Panel; RVOP) on composite wastewater samples from 8 wastewater treatment plants (WWTPs) and one associated hospital site. By combining seasonal sampling with whole genome sequencing, we were able to concurrently detect and characterise a range of common respiratory pathogens, including SARS-CoV-2, adenovirus and parainfluenza virus. We demonstrated that 38 respiratory pathogens can be detected at low abundances year-round, that hospital pathogen diversity is higher in winter vs. summer sampling events, and that significantly more viruses are detected in raw influent compared to treated effluent samples. Finally, we compared detection sensitivity of RT-qPCR vs. next generation sequencing for SARS-CoV-2, enteroviruses, influenza A/B, and respiratory syncytial viruses. We conclude that both should be used in combination; RT-qPCR allowed accurate quantification, whilst genomic sequencing detected pathogens at lower abundance. We demonstrate the valuable role of wastewater genomic surveillance and its contribution to the field of wastewater-based epidemiology, gaining rapid understanding of the seasonal presence and persistence for common respiratory pathogens. By simultaneously monitoring seasonal trends and early warning signs of many viruses circulating in communities, public health agencies can implement targeted prevention and rapid response plans.

Assessment of wastewater derived pollution using viral monitoring in two estuaries.

Human wastewater-derived pollution of the environment is an emerging health risk that increases the number of waterborne and foodborne illnesses globally. To better understand and mitigate such health risks, we investigated the prevalence of faecal indicator bacteria, Escherichia coli, and indicator virus (crAssphage) along with human and animal enteric viruses (adenoviruses, noroviruses, sapoviruses, hepatitis E virus) in shellfish and water samples collected from two shellfish harvesting areas in the UK. Human noroviruses were detected at higher detection rates in oyster and water samples compared to mussels with peaks during the autumn-winter seasons. Human enteric viruses were sporadically detected during the warmer months, suggesting potential introduction by tourists following the relaxation of COVID-19 lockdown measures. Our results suggest that viral indicators are more suitable for risk assessment and source tracking than E. coli. The detection of emerging hepatitis and sapoviruses, support the need for comprehensive viral monitoring in shellfish harvesting areas.

National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches.

Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes.

Examining the stability of viral RNA and DNA in wastewater: Effects of storage time, temperature, and freeze-thaw cycles.

Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope - E, Nucleocapsid - N and Spike protein - S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at -80 °C, -20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses - both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at -80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temperatures.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.

Prevalence and relative risk of Rotavirus Gastroenteritis in children under five years in Nigeria: a systematic review and meta-analysis.

Rotavirus is responsible for most cases of gastroenteritis and mortality in children below 5 years of age, especially in developing countries, including Nigeria. Nonetheless, there is limited data on the nationwide estimate for the prevalence of rotavirus. This systematic review and meta-analysis sought to determine the pooled prevalence of rotavirus infections and its relative risk among children below 5 years of age in Nigeria. Eligible published studies between 1982 and 2021 were accessed from 'PubMed', 'Science Direct', 'Google Scholar' and 'African Journal Online', 'Web of Science', 'Springer', 'Wiley' were systematically reviewed. The pooled prevalence, relative risk and regional subgroup analyses were calculated using the random effects model at 95% confidence interval (CI). A total of 62 selected studies, including 15 studies case-control studies, were processed in this review from a pooled population of 18,849 children. The nationwide pooled prevalence of rotavirus among children below 5 years of age in Nigeria was 23% (CI 95%; 19-27). Regional subgroup analysis showed that the Southern region had a prevalence of 27% (CI 95%; 21-32) while the Northern region had a 20% (CI 95%; 16-25%) prevalence, although the difference was not significant (P = 0.527). Rotavirus was implicated in most cases of acute gastroenteritis with a relative risk of 5.7 (95% CI: 2.9-11.2). The high prevalence and relative risk of rotavirus infections among children in Nigeria shows that rotavirus is an important cause of acute gastroenteritis in Nigeria. Thus, there is a need for further surveillance, especially at community levels together with the introduction of rotavirus vaccines into the national immunization program.

Development and validation of a duplex RT-qPCR assay for norovirus quantification in wastewater samples.

Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.

Diverse Microvirus Genomes Identified in the Stomach of a Sharp-Spined Notothen (Trematomus pennellii) from the Ross Sea (East Antarctica).

Sharp-spined notothen (Trematomus pennellii) is an icefish endemic to the southern ocean. From the stomach of an individual, we identified the genomes of 51 microviruses (family Microviridae). The major capsid proteins of most of these share the closest similarities to those identified in other marine organisms.

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater.

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 µM and 100 µM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 µM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed -0.52-1.15, 0.9-1.51, and 0.31-1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.