Sequence-independent Amplification Research Articles

Metagenomic sequencing detects human respiratory and enteric viruses in air samples collected from congregate settings

Innovative methods for evaluating virus risk and spread, independent of test-seeking behavior, are needed to improve routine public health surveillance, outbreak response, and pandemic preparedness. Throughout the COVID-19 pandemic, environmental surveillance strategies, including wastewater and air sampling, have been used alongside widespread individual-based SARS-CoV-2 testing programs to provide population-level data. These environmental surveillance strategies have predominantly relied on pathogen-specific detection methods to monitor viruses through space and time. However, this provides a limited picture of the virome present in an environmental sample, leaving us blind to most circulating viruses. In this study, we explore whether pathogen-agnostic deep sequencing can expand the utility of air sampling to detect many human viruses. We show that sequence-independent single-primer amplification sequencing of nucleic acids from air samples can detect common and unexpected human respiratory and enteric viruses, including influenza virus type A and C, respiratory syncytial virus, human coronaviruses, rhinovirus, SARS-CoV-2, rotavirus, mamastrovirus, and astrovirus.

Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa.

White spot syndrome virus is a highly contagious pathogen affecting shrimp farming worldwide. The host range of this virus is primarily limited to crustaceans, such as shrimps, crabs, prawns, crayfish, and lobsters; however, several species of non-crustaceans, including aquatic insects, piscivorous birds, and molluscs may serve as the vectors for ecological dissemination. The present study was aimed at studying the faecal virome of domestic chickens (Gallus gallus domesticus) in Makhanda, Eastern Cape, South Africa. The cloacal swab specimens (n = 35) were collected from domestic chickens in December 2022. The cloacal swab specimens were pooled-each pool containing five cloacal swabs-for metagenomic analysis using a sequence-independent single-primer amplification protocol, followed by Nanopore MinION sequencing. While the metagenomic sequencing generated several contigs aligning with reference genomes of animal viruses, one striking observation was the presence of a White spot syndrome virus genome in one pool of cloacal swab specimens. The generated White spot syndrome virus genome was 273,795 bp in size with 88.5% genome coverage and shared 99.94% nucleotide sequence identity with a reference genome reported in China during 2018 (GenBank accession: NC_003225.3). The Neighbour-Joining tree grouped South African White spot syndrome virus genome with other White spot syndrome virus genomes reported from South East Asia. To our knowledge, this is the first report of a White spot syndrome virus genome generated from domestic chickens. The significance of White spot syndrome virus infection in domestic chickens is yet to be determined.

Exploring the Enteric Virome of Cats with Acute Gastroenteritis.

Viruses are a major cause of acute gastroenteritis (AGE) in cats, chiefly in younger animals. Enteric specimens collected from 29 cats with acute enteritis and 33 non-diarrhoeic cats were screened in PCRs and reverse transcription (RT) PCR for a large panel of enteric viruses, including also orphan viruses of recent identification. At least one viral species, including feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), feline chaphamaparvovirus, calicivirus (vesivirus and novovirus), feline kobuvirus, feline sakobuvirus A and Lyon IARC polyomaviruses, was detected in 66.1% of the samples.. Co-infections were mainly accounted for by FPV and FCoV and were detected in 24.2% of the samples. The virome composition was further assessed in eight diarrhoeic samples, through the construction of sequencing libraries using a sequence-independent single-primer amplification (SISPA) protocol. The libraries were sequenced on Oxford Nanopore Technologies sequencing platform. A total of 41 contigs (>100 nt) were detected from seven viral families infecting mammals, included Parvoviridae, Caliciviridae, Picornaviridae, Polyomaviridae, Anelloviridae, Papillomaviridae and Paramyxoviridae, revealing a broad variety in the composition of the feline enteric virome.

Whole-Genome Sequencing of Six Neglected Arboviruses Circulating in Africa Using Sequence-Independent Single Primer Amplification (SISPA) and MinION Nanopore Technologies.

On the African continent, a large number of arthropod-borne viruses (arboviruses) with zoonotic potential have been described, and yet little is known of most of these pathogens, including their actual distribution or genetic diversity. In this study, we evaluated as a proof-of-concept the effectiveness of the nonspecific sequencing technique sequence-independent single primer amplification (SISPA) on third-generation sequencing techniques (MinION sequencing, Oxford Nanopore Technologies, Oxford, UK) by comparing the sequencing results from six different samples of arboviruses known to be circulating in Africa (Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Dugbe virus (DUGV), Nairobi sheep disease virus (NSDV), Middleburg virus (MIDV) and Wesselsbron virus (WSLV)). All sequenced samples were derived either from previous field studies or animal infection trials. Using this approach, we were able to generate complete genomes for all six viruses without the need for virus-specific whole-genome PCRs. Higher Cq values in diagnostic RT-qPCRs and the origin of the samples (from cell culture or animal origin) along with their quality were found to be factors affecting the success of the sequencing run. The results of this study may stimulate the use of metagenomic sequencing approaches, contributing to a better understanding of the genetic diversity of neglected arboviruses.

Detection and Characterization of a Novel Picornavirus in European Badger (Meles meles).

The recent development of unbiased metagenomic next-generation sequencing has provided a richer view of the wild animal virome making it necessary to expand the knowledge about virus diversity in wildlife, as well as to monitor their potential transmission to domestic animals or humans. In the present study, by screening collections of enteric specimens from wild animals, a novel picornavirus was identified in the intestinal content of a badger (Meles meles). By enrichment with a sequence-independent single-primer amplification (SISPA) approach and deep sequencing with Oxford Nanopore Technologies (ONT) platform, the genome sequence of a novel picornavirus strain, Badger/3A-2019/ITA, was reconstructed. On comparison based on the polyprotein sequences, the virus was distantly related (58.7% and 59.7% sequence identity at the nucleotide and amino acid level, respectively) to the feline picornavirus strain FFUP1, identified in 2012 in Portugal and classified into genus Sakobovirus within the species Sakobuvirus A. Upon phylogenetic, pairwise homology, and distance analyses performed on the P1, 2Chel, 3Cpro, and 3Dpol proteins and the complete genomic sequence, the badger picornavirus may be considered a member of a new sakobuvirus species, which we propose as Sakobuvirus B.

The Virome of Bronchoalveolar Lavage Fluid from Patients with Fever of Unknown Origin

Background: Viral metagenomics, a high-throughput sequencing combined with virus sequence-independent amplification by random PCR, allows for unbiased detection of virtually any viruses present in samples. Materials & methods: In order to investigate the virome of bronchoalveolar lavage fluid from patients with fever of unknown origin, 58 samples collected from diseased patients were characterized and compared. Results: Some representatives of Anelloviridae were identified, we found the torque teno virus (TTV) accounts for the majority of virus communities and were more prevalent in the specimens of febrile patients. Phylogenetic analysis suggested that these anellovirus isolates were close to the previous TTV available in GenBank®. Conclusion: All these data indicate that the human anellovirus species TTV may associated with fever of unknown origin.

Molecular Identification and Characterization of a Genotype 3 Hepatitis E Virus (HEV) Strain Detected in a Wolf Faecal Sample, Italy.

Simple SummaryData on hepatitis E virus (HEV) strains circulating in carnivores are still limited. However, serological evidence suggests that their exposure to HEV infection is more common than expected. In addition, close relatives of Orthohepevirus C detected in rats and in human patients with persistent hepatitis have been detected in ferrets, minks, and foxes, providing a rationale for investigating the presence of HEV or HEV-like viruses in wild carnivores. Herewith, we report the identification and the nearly full-genome (7212 nucleotides) characterization of an HEV Gt3 strain detected in a faecal specimen collected from a wolf found dead in Northwestern Italy. This finding poses questions concerning the potential role of wolves as HEV mechanical, passive carriers, favouring the introduction and spread of HEV in the environment.Hepatitis E virus (HEV) infection is a major health problem worldwide. In developed countries, zoonotic transmission of HEV genotypes (Gt) 3 and 4 is caused by the ingestion of raw or undercooked meat of infected pigs and wild boars, the main reservoirs of HEV. However, additional animals may harbour HEV or HEV-related strains, including carnivores. In this study, we investigated the molecular epidemiology of orthohepeviruses in wild canids by screening a total of 136 archival faecal samples, collected from wolves (42) and red foxes (94) in Northwestern Italy. Orthohepevirus RNA was identified in a faecal specimen, collected from a wolf carcass in the province of La Spezia (Liguria Region, Italy). The nearly full-length (7212 nucleotides) genome of the strain HEV/81236/Wolf/2019/ITA (GenBank accession no. MZ463196) was determined by combining a sequence-independent single-primer amplification (SISPA) approach with the Oxford Nanopore Technologies sequencing platform. Upon phylogenetic analysis, the HEV detected in wolf was segregated into clade HEV-3.1, displaying the highest nucleotide (nt) identity (89.0–93.3%) to Gt3 strains belonging to subtype c. Interestingly, the wolf faecal sample also contained porcine astrovirus sequences, endorsing the hypothesis of a dietary origin of the HEV strain due to preying habits.

Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples.

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L−1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L−1, ranging from 5.49 ± 0.02 log10 GC L−1 (27th November 2019) to 6.68 ± 0.02 log10 GC L−1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from a Fish Farm in Scotland.

The aquatic virus, infectious pancreatic necrosis virus (IPNV), is known to infect various farmed fish, in particular salmonids, and is responsible for large economic losses in the aquaculture industry. Common practices to detect the virus include qPCR tests based on specific primers and serum neutralization tests for virus serotyping. Following the potential presence of IPNV viruses in a fish farm in Scotland containing vaccinated and IPNV-resistant fish, the common serotyping of the IPNV isolates was not made possible. This led us to determine the complete genome of the new IPNV isolates in order to investigate the cause of the serotyping discrepancy. Next-generation sequencing using the Illumina technology along with the sequence-independent single primer amplification (SISPA) approach was conducted to fully characterize the new Scottish isolates. With this approach, the full genome of two isolates, V1810–4 and V1810–6, was determined and analyzed. The potential origin of the virus isolates was investigated by phylogenetic analyses along with tridimensional and secondary protein structure analyses. These revealed the emergence of a new variant from one of the main virus serotypes, probably caused by the presence of selective pressure exerted by the vaccinated IPNV-resistant farmed fish.

Genetic characterisation of novel G29P[14] and G10P[11] rotavirus strains from African buffalo

We report the first description of rotavirus A strains in African buffalo (Syncerus caffer). Following RNA extraction from stool samples, cDNA was prepared, followed either by sequence-independent amplification and 454 pyrosequencing or direct sequencing on an Illumina MiSeq platform. RVA/Buffalo-wt/ZAF/4426/2002/G29P[14] exhibited a novel G29P[14] combination and an artiodactyl backbone: I2-R2-C2-M2-A11-N2-T6-E2-H3. RVA/Buffalo-wt/ZAF/1442/2007/G10P[11] also exhibited an artiodactyl backbone: I2-R2-C2-M2-A13-N2-T6-E2-H3. Characterisation of these genome constellations indicate that the two buffalo strains are moderately diverse from each other and related to South African bovine RVA strains. The detection of RVA in buffalo contribute to our understanding of the host range of rotavirus in animals.

Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic. Accurate detection of SARS‐CoV‐2 using molecular assays is critical for patient management and the control of the COVID‐19 pandemic. However, there is an increasing number of SARS‐CoV‐2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence‐independent single‐primer amplification and nanopore whole‐genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5′ end of the SARS‐CoV‐2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID‐19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real‐time RT‐PCR assay. The primers and probes are highly specific for SARS‐CoV‐2. Validation with 101 clinical specimens showed that our nsp1 RT‐PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%‐97.2%), which was similar to those of N and E gene RT‐PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%‐100%). The addition of nsp1 for multitarget detection of SARS‐CoV‐2 can avoid false‐negative results due to mutations at the primers/probes binding sites of currently available RT‐PCR assays.

A renewable DNA biosensor for sensitive detection of DNA methyltransferase activity based on cascade signal amplification

DNA methyltransferase catalyzes the methylation process of DNA. As a potential biomarker, methyltransferase activity assay has attracted more and more attention. Herein, we present a new biosensor based on cascade invasive reactions for DNA methyltransferase activity analysis. The sensing probes on the biosensor were first methylated by the Dam MTase and then cleaved by the DpnI endonuclease. The cleaved probe acted as upstream probe for the invasion reaction, triggering a highly sensitive signal amplification reaction that produced a strong fluorescent signal. After detection, the cleaved probes were extended by DNA polymerase, and regenerated to be an intact sensor. Meanwhile, as the invasion reaction was a sequence-independent signal amplification method, our proposed biosensor could be a universal strategy for other enzyme activity analysis, simply by replacing the recognition sequence.

Molecular identification and phylogeny of cucumber mosaic virus and zucchini yellow mosaic virus co-infecting Luffa cylindrica L. in Shanxi, China

To identify the viruses that induced Luffa cylindrica L. mosaic disease, the viral genomic double-strand RNA (dsRNA) was extracted, followed by sequence-independent amplification (SIA) and sequencing. Results showed that Luffa cylindrica L. was co-infected by cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV). To further characterize the Shanxi isolates of cucumber mosaic virus (CMV-SXSG) and zucchini yellow mosaic virus (ZYMV-SXSG), the MP gene sequence of CMV-SXSG and the complete genomic sequence of ZYMV-SXSG were cloned and analyzed. The genomic RNA of ZYMV-SXSG is 9474 nucleotides in length and contains one putative ORF which encodes a polyprotein of 3080 amino acids. Sequence alignments showed that CMV-SXSG shared MP nucleotide sequence identities with other CMV isolates of 78.15% to 99.4%. The nucleotide sequence similarity for the CDS region between ZYMV-SXSG and representative ZYMV strains ranged from 81.71% to 98.40%. Phylogenetic analysis suggested that CMV-SXSG and CMV-QZ shared the same origin, and that ZYMV-SXSG showed the highest sequence identity to isolate KR-PA and formed an independent branch, indicating that they have the most closely evolutionary relationship. Recombination analysis revealed that ZYMV-SXSG was a putative recombinant, and that the potential major and minor parents were the ZZ (China isolate) and KR-PA (Korean isolate), respectively. These results will provide valuable hints for more accurate analysis of CMV or ZYMV genetic diversity, and also provide an effective theoretical basis for designing better virus control strategies.

A Novel and Divergent Gyrovirus with Unusual Genomic Features Detected in Wild Passerine Birds from a Remote Rainforest in French Guiana.

Sequence-independent amplification techniques have become important tools for virus discovery, metagenomics, and exploration of viral diversity at the global scale, especially in remote areas. Here, we describe the detection and genetic characterization of a novel gyrovirus, named GyV11, present in cloacal, oral, and blood samples from neotropical wild birds in French Guiana. The molecular epidemiology revealed the presence of GyV11 only in passerine birds from three different species at a low prevalence (0.73%). This is the first characterization and prevalence study of a gyrovirus carried out in resident wild bird populations in a remote region, and provides evidence of the fecal–oral route transmission and local circulation of the virus. The molecular phylogeny of gyroviruses reveals the existence of two distinct gyrovirus lineages in which GyV11 is phylogenetically distinct from previously reported gyroviruses. Furthermore, GyV11 is placed basal in the gyrovirus phylogeny, likely owing to its ancestral origin and marked divergence. This study also provides important insights into the ecology, epidemiology, and genomic features of gyroviruses in a remote neotropical rainforest. The pathogenesis of this virus in avian species or whether GyV11 can infect humans and/or chickens needs to be further investigated.

Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics.

The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample.IMPORTANCE The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

Genetic characterization of a new astrovirus in goslings suffering from gout.

Since early 2016, the Chinese goose industry has experienced severe outbreaks of gout; however, the etiological factor of the disease is still unclear. Here, we investigated the possible involvement of viral infection in the disease. Using sequence-independent PCR amplification, astrovirus sequences were generated from a gout case. Full-length genomic sequencing and sequence analysis of three goose astrovirus (GoAstV) strains revealed that they belong to a new avastrovirus most closely related to viruses classified within species Avastrovirus 3. The GoAstV was detected in 16/16 gout cases collected from two provinces, supporting a pathogenic role for the new avastrovirus.

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.

Investigating the viral ecology of global bee communities with high-throughput metagenomics

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 4 continents and Oceania. We developed a novel pipeline to rapidly and inexpensively screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences for (+)ssRNA, (−)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 27 represented by >0.1% of the reads in a given sample, and 7 contained an RdRp or replicase sequence which could be used for robust phylogenetic analysis. This study provides a sequence-independent pipeline for viral metagenomics analysis, and greatly expands our understanding of the diversity of viruses found in bee communities.