Nearly Complete Genome Sequences of Type 2 Sabin-Like Polioviruses from Northern Nigerian Poliovirus Surveillance, 2016 to 2018.

We sequenced 109 type 2 Sabin-like poliovirus isolates that had been collected from acute flaccid paralysis patients or healthy children in Nigeria. Understanding the genetic makeup of these viruses may contribute to polio eradication efforts.

Genetic Characterization of Novel Oral Polio Vaccine Type 2 Viruses During Initial Use Phase Under Emergency Use Listing - Worldwide, March-October 2021.

The emergence and international spread of neurovirulent circulating vaccine-derived polioviruses (cVDPVs) across multiple countries in Africa and Asia in recent years pose a major challenge to the goal of eradicating all forms of polioviruses. Approximately 90% of all cVDPV outbreaks are caused by the type 2 strain of the Sabin vaccine, an oral live, attenuated vaccine; cVDPV outbreaks typically occur in areas of persistently low immunization coverage (1). A novel type 2 oral poliovirus vaccine (nOPV2), produced by genetic modification of the type 2 Sabin vaccine virus genome (2), was developed and evaluated through phase I and phase II clinical trials during 2017-2019. nOPV2 was demonstrated to be safe and well-tolerated, have noninferior immunogenicity, and have superior genetic stability compared with Sabin monovalent type 2 (as measured by preservation of the primary attenuation site [domain V in the 5' noncoding region] and significantly lower neurovirulence of fecally shed vaccine virus in transgenic mice) (3-5). These findings indicate that nOPV2 could be an important tool in reducing the risk for generating vaccine-derived polioviruses (VDPVs) and the risk for vaccine-associated paralytic poliomyelitis cases. Based on the favorable preclinical and clinical data, and the public health emergency of international concern generated by ongoing endemic wild poliovirus transmission and cVDPV type 2 outbreaks, the World Health Organization authorized nOPV2 for use under the Emergency Use Listing (EUL) pathway in November 2020, allowing for its first use for outbreak response in March 2021 (6). As required by the EUL process, among other EUL obligations, an extensive plan was developed and deployed for obtaining and monitoring nOPV2 isolates detected during acute flaccid paralysis (AFP) surveillance, environmental surveillance, adverse events after immunization surveillance, and targeted surveillance for adverse events of special interest (i.e., prespecified events that have the potential to be causally associated with the vaccine product), during outbreak response, as well as through planned field studies. Under this monitoring framework, data generated from whole-genome sequencing of nOPV2 isolates, alongside other virologic data for isolates from AFP and environmental surveillance systems, are reviewed by the genetic characterization subgroup of an nOPV working group of the Global Polio Eradication Initiative. Global nOPV2 genomic surveillance during March-October 2021 confirmed genetic stability of the primary attenuating site. Sequence data generated through this unprecedented global effort confirm the genetic stability of nOPV2 relative to Sabin 2 and suggest that nOPV2 will be an important tool in the eradication of poliomyelitis. nOPV2 surveillance should continue for the duration of the EUL.

VPipe: an Automated Bioinformatics Platform for Assembly and Management of Viral Next-Generation Sequencing Data.

Next-generation sequencing (NGS) is a powerful tool for detecting and investigating viral pathogens; however, analysis and management of the enormous amounts of data generated from these technologies remains a challenge. Here, we present VPipe (the Viral NGS Analysis Pipeline and Data Management System), an automated bioinformatics pipeline optimized for whole-genome assembly of viral sequences and identification of diverse species. VPipe automates the data quality control, assembly, and contig identification steps typically performed when analyzing NGS data. Users access the pipeline through a secure web-based portal, which provides an easy-to-use interface with advanced search capabilities for reviewing results. In addition, VPipe provides a centralized system for storing and analyzing NGS data, eliminating common bottlenecks in bioinformatics analyses for public health laboratories with limited on-site computational infrastructure. The performance of VPipe was validated through the analysis of publicly available NGS data sets for viral pathogens, generating high-quality assemblies for 12 data sets. VPipe also generated assemblies with greater contiguity than similar pipelines for 41 human respiratory syncytial virus isolates and 23 SARS-CoV-2 specimens. IMPORTANCE Computational infrastructure and bioinformatics analysis are bottlenecks in the application of NGS to viral pathogens. As of September 2021, VPipe has been used by the U.S. Centers for Disease Control and Prevention (CDC) and 12 state public health laboratories to characterize >17,500 and 1,500 clinical specimens and isolates, respectively. VPipe automates genome assembly for a wide range of viruses, including high-consequence pathogens such as SARS-CoV-2. Such automated functionality expedites public health responses to viral outbreaks and pathogen surveillance.

Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes.

Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with Ct values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with Ct values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness.

Whole-Genome Sequences of Enteroviruses D94 and D111 Isolated from Stool Specimens in Angola.

We report the whole-genome sequences of new enterovirus D94 and D111 strains, isolated from cultures from stool specimens collected from acute flaccid paralysis (AFP) cases for poliovirus surveillance in Angola during 2010.

The effect of variant interference on de novo assembly for viral deep sequencing.

BACKGROUND: Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approaches have surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. RESULTS: Our results from > 15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This "variant interference" (VI) is highly consistent and reproducible by ten commonly-used de novo assemblers, and occurs over a range of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the "rescue" of full viral genomes from fragmented contigs. CONCLUSIONS: These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses.

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 510 chip runs produced more reads at a lower cost per sample than the highest output Ion Torrent PGM 318 chip run, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 and Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 and Illumina MiSeq V2) there is less of a difference in the cost per sample between the two sequencing platforms ($5.47-$10.25 more per sample for an Ion Torrent 530 chip run when multiplexing 24 samples). These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

Outbreak of diarrhoea in piglets caused by novel rotavirus genotype G4P[49] in north-western district of Bangladesh, February 2014.

Group A rotavirus (RVA) associated diarrhoea in piglets represents one of the major causes of morbidity and mortality in pig farms worldwide. A diarrhoea outbreak occurred among nomadic piglets in north-western district of Bangladesh in February 2014. Outbreak investigation was performed to identify the cause, epidemiologic and clinical features of the outbreak. Rectal swabs and clinical information were collected from diarrhoeic piglets (n = 36). Rectal swabs were tested for RVA RNA by real-time reverse transcription polymerase chain reaction (rRT-PCR) using NSP3-specific primers. The G (VP7) and P (VP4) genes were typed by conventional RT-PCR and sanger sequencing and full genome sequences were determined using next-generation sequencing. We found the attack rate was 61% (50/82) among piglets in the nomadic pig herd, and the case fatality rate was 20% (10/50) among piglets with diarrhoea. All study piglets cases had watery diarrhoea, lack of appetite or reluctance to move. A novel RVA strain with a new P[49] genotype combined with G4 was identified among all piglets with diarrhoea. The genome constellation of the novel RVA strains was determined to be G4-P[49]-I1-R1-C1-M1-A8-N1-T7-E1-H1. Genetic analysis shows that the novel G4P[49] strain is similar to Indian and Chinese porcine or porcine-like G4 human strains and is genetically distant from Bangladeshi human G4 strains. Identification of this novel RVA strain warrants further exploration for disease severity and zoonotic potential.

Haiti Poliovirus Environmental Surveillance.

Poliovirus (PV) environmental surveillance was established in Haiti in three sites each in Port-au-Prince and Gonaïves, where sewage and fecal-influenced environmental open water channel samples were collected monthly from March 2016 to February 2017. The primary objective was to monitor for the emergence of vaccine-derived polioviruses (VDPVs) and the importation and transmission of wild polioviruses (WPVs). A secondary objective was to compare two environmental sample processing methods, the gold standard two-phase separation method and a filter method (bag-mediated filtration system [BMFS]). In addition, non-polio enteroviruses (NPEVs) were characterized by next-generation sequencing using Illumina MiSeq to provide insight on surrogates for PVs. No WPVs or VDPVs were detected at any site with either concentration method. Sabin (vaccine) strain PV type 2 and Sabin strain PV type 1 were found in Port-au-Prince, in March and April samples, respectively. Non-polio enteroviruses were isolated in 75-100% and 0-58% of samples, by either processing method during the reporting period in Port-au-Prince and Gonaïves, respectively. Further analysis of 24 paired Port-au-Prince samples confirmed the detection of a human NPEV and echovirus types E-3, E-6, E-7, E-11, E-19, E-20, and E-29. The comparison of the BMFS filtration method to the two-phase separation method found no significant difference in sensitivity between the two methods (mid-P-value = 0.55). The experience of one calendar year of sampling has informed the appropriateness of the initially chosen sampling sites, importance of an adequate PV surrogate, and robustness of two processing methods.

Nearly Complete Genome Sequence of an Echovirus 30 Strain from a Cluster of Aseptic Meningitis Cases in California, September 2017.

We report the nearly complete genome sequence of a human enterovirus, a strain of echovirus 30, obtained from a cerebrospinal fluid specimen from a teenaged patient with aseptic meningitis in September 2017.

Complete Genome Sequences of Human Astrovirus Prototype Strains (Types 1 to 8).

We report the complete genome sequences of the eight human astrovirus Oxford prototype strains. These sequences share 94.9% to 99.9% nucleotide identity with open reading frame 2 (ORF2) genes of astrovirus genomes previously deposited in GenBank and include the first complete genome of human astrovirus type 7.

Strengthening laboratory surveillance of viral pathogens: Experiences and lessons learned building next-generation sequencing capacity in Ghana.

OBJECTIVE: To demonstrate the feasibility of applying next-generation sequencing (NGS) in medium-resource reference laboratories in Africa to enhance global disease surveillance. METHODS: A training program was developed to support implementation of NGS at Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana. The program was divided into two training stages, first at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, followed by on-site training at NMIMR for a larger cohort of scientists. RESULTS: Self-assessment scores for topics covered during the NGS training program were higher post-training relative to pre-training. During the NGS Training II session at NMIMR, six enterovirus isolates from acute flaccid paralysis cases in Ghana were successfully sequenced by trainees, including two echovirus 6, two echovirus 11 and one echovirus 13. Another genome was an uncommon type (EV-B84), which has not been reported in Africa since its initial discovery from a Côte d'Ivoire specimen in 2003. CONCLUSIONS: The success at NMIMR provides an example of how to approach transferring of NGS methods to international laboratories. There is great opportunity for collaboration between institutes that have genomics expertise to ensure effectiveness and long-term success of global NGS capacity building programs.

Genetic diversity of human sapovirus across the Americas.

BACKGROUND: Sapoviruses are responsible for sporadic and epidemic acute gastroenteritis worldwide. Sapovirus typing protocols have a success rate as low as 43% and relatively few complete sapovirus genome sequences are available to improve current typing protocols. OBJECTIVE/STUDY DESIGN: To increase the number of complete sapovirus genomes to better understand the molecular epidemiology of human sapovirus and to improve the success rate of current sapovirus typing methods, we used deep metagenomics shotgun sequencing to obtain the complete genomes of 68 sapovirus samples from four different countries across the Americas (Guatemala, Nicaragua, Peru and the US). RESULTS: VP1 genotyping showed that all sapovirus sequences could be grouped in the four established genogroups (GI (n = 13), GII (n = 30), GIV (n = 23), GV (n = 2)) that infect humans. They include the near-complete genome of a GI.6 virus and a recently reported novel GII.8 virus. Sequences of the complete RNA-dependent RNA polymerase gene could be grouped into three major genetic clusters or polymerase (P) types (GI.P, GII.P and GV.P) with all GIV viruses harboring a GII polymerase. One (GII.P-GII.4) of the new 68 sequences was a recombinant virus with the hotspot between the NS7 and VP1 regions. CONCLUSIONS: Analyses of this expanded database of near-complete sapovirus sequences showed several mismatches in the genotyping primers, suggesting opportunities to revisit and update current sapovirus typing methods.

Near-Complete Genome Sequences of Several New Norovirus Genogroup II Genotypes.

We report here the near-complete genome sequences of 13 norovirus strains detected in stool samples from patients with acute gastroenteritis from Bangladesh, Ecuador, Guatemala, Peru, Nicaragua, and the United States that are classified into one existing (genotype II.22 [GII.22]), 3 novel (GII.23, GII.24 and GII.25), and 3 tentative novel (GII.NA1, GII.NA2, and GII.NA3) genotypes.

Complete Genome Sequences of Mumps and Measles Virus Isolates from Three States in the United States.

We report here the full coding sequence of nine paramyxovirus genomes, including two full-length mumps virus genomes (genotypes G and H) and seven measles virus genomes (genotypes B3 and D4, D8, and D9), from respiratory samples of patients from California, Virginia, and Alabama obtained between 2010 and 2014.

Genetic and Epidemiologic Trends of Norovirus Outbreaks in the United States from 2013 to 2016 Demonstrated Emergence of Novel GII.4 Recombinant Viruses.

Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

U50: A New Metric for Measuring Assembly Output Based on Non-Overlapping, Target-Specific Contigs.

Advances in next-generation sequencing technologies enable routine genome sequencing, generating millions of short reads. A crucial step for full genome analysis is the de novo assembly, and currently, performance of different assembly methods is measured by a metric called N50. However, the N50 value can produce skewed, inaccurate results when complex data are analyzed, especially for viral and microbial datasets. To provide a better assessment of assembly output, we developed a new metric called U50. The U50 identifies unique, target-specific contigs by using a reference genome as baseline, aiming at circumventing some limitations that are inherent to the N50 metric. Specifically, the U50 program removes overlapping sequence of multiple contigs by utilizing a mask array, so the performance of the assembly is only measured by unique contigs. We compared simulated and real datasets by using U50 and N50, and our results demonstrated that U50 has the following advantages over N50: (1) reducing erroneously large N50 values due to a poor assembly, (2) eliminating overinflated N50 values caused by large measurements from overlapping contigs, (3) eliminating diminished N50 values caused by an abundance of small contigs, and (4) allowing comparisons across different platforms or samples based on the new percentage-based metric UG50%. The use of the U50 metric allows for a more accurate measure of assembly performance by analyzing only the unique, non-overlapping contigs. In addition, most viral and microbial sequencing have high background noise (i.e., host and other non-targets), which contributes to having a skewed, misrepresented N50 value-this is corrected by U50. Also, the UG50% can be used to compare assembly results from different samples or studies, the cross-comparisons of which cannot be performed with N50.

High-Throughput Next-Generation Sequencing of Polioviruses.

The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.