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.

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.

The EVVA Cohort Study: Anal and Cervical Type-Specific Human Papillomavirus Prevalence, Persistence, and Cytologic Findings in Women Living With HIV.

Background: The risk of anal cancer due to high-risk human papillomavirus (HR-HPV) is higher in women living with human immunodeficiency virus (HIV) than in the general population. We present findings of cervical and anal HPV and cytologic tests at baseline in the EVVA cohort study and HPV persistence data 6 months after baseline. Methods: Semiannual visits included questionnaires, chart reviews, cervical/anal cytologic and cervical/anal HPV testing for 2 years. Genotyping for 36 HPV genotypes was performed using the Roche Linear Array HPV genotyping test. Results: A total of 151 women living with HIV were recruited. At baseline, 75% had anal HPV, 51% had anal HR-HPV, 50% had cervical HPV, and 29% had cervical HR-HPV. Anal HPV-16 and HPV-51 were more frequent in women born in Canada (31% and 29%, respectively, compared with ≤16% for other women). Most anal HR-HPV types detected at 6 months (57%-93%) were persistent from baseline. Findings of anal cytologic tests were abnormal for 37% of women. Conclusions: Anal HPV is highly prevalent in women living with HIV, and type distribution varies by place of birth. High-resolution anoscopy was indicated in more than one third of results. As anal cancer is potentially preventable, these important findings need to be considered when selecting the best approach for anal cancer screening programs.

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.

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.

Managing emotions in the age of political polarization: A randomized controlled trial comparing mindfulness to cognitive reappraisal.

Emotional appraisals of political stimuli (e.g., videos) have been shown to drive shared neural encoding, which correspond to shared, yet divisive, interpretations of such stimuli. However, mindfulness practice may entrain a form of emotion regulation that de-automatizes social biases, possibly through alteration of such neural mechanisms. The present study combined a naturalistic neuroimaging paradigm and a randomized controlled trial to examine the effects of short-term mindfulness training (MT) (n = 35) vs structurally equivalent Cognitive Reappraisal training (CT) (n = 37) on politically-situated emotions while evaluating the mechanistic role of prefrontal cortical neural synchrony. Participants underwent functional near-infrared spectroscopy (fNIRS) recording while viewing inflammatory partisan news clips and continuously rating their momentary discrete emotions. MT participants were more likely to respond with extreme levels of anger (odds ratio = 0.12, p < .001) and disgust (odds ratio = 0.08, p < .001) relative to CT participants. Neural synchrony-based analyses suggested that participants with extreme emotion reactions exhibited greater prefrontal cortical neural synchrony, but that this pattern was less prominent in participants receiving MT relative to CT (CT > MT; channel 1 ISC = .040, p = .030).

Complete genome sequences of six S19 poliovirus reference strains.

We report the complete genome sequences of six S19 poliovirus reference strains for all three poliovirus serotypes, including three Sabin vaccine-derived and three wild-type-derived strains. The S19 strains are extensively attenuated and genetically stable when compared to the reference poliovirus strains, while maintaining the same antigenicity and immunogenicity.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

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.

Genome Sequences of 16 Enterovirus Isolates from Environmental Sewage in Guatemala, 2019 to 2021.

Enteroviruses can cause human infectious disease. We report 16 near-complete genome sequences of enteroviruses that were isolated through environmental surveillance of wastewater in Guatemala.

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.

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.

Identification, structure, and characterization of an exopolysaccharide produced by Histophilus somni during biofilm formation.

BACKGROUND: Histophilus somni, a gram-negative coccobacillus, is an obligate inhabitant of bovine and ovine mucosal surfaces, and an opportunistic pathogen responsible for respiratory disease and other systemic infections in cattle and sheep. Capsules are important virulence factors for many pathogenic bacteria, but a capsule has not been identified on H. somni. However, H. somni does form a biofilm in vitro and in vivo, and the biofilm matrix of most bacteria consists of a polysaccharide. RESULTS: Following incubation of H. somni under growth-restricting stress conditions, such as during anaerobiosis, stationary phase, or in hypertonic salt, a polysaccharide could be isolated from washed cells or culture supernatant. The polysaccharide was present in large amounts in broth culture sediment after H. somni was grown under low oxygen tension for 4-5 days (conditions favorable to biofilm formation), but not from planktonic cells during log phase growth. Immuno-transmission electron microscopy showed that the polysaccharide was not closely associated with the cell surface, and was of heterogeneous high molecular size by gel electrophoresis, indicating it was an exopolysaccharide (EPS). The EPS was a branched mannose polymer containing some galactose, as determined by structural analysis. The mannose-specific Moringa M lectin and antibodies to the EPS bound to the biofilm matrix, demonstrating that the EPS was a component of the biofilm. The addition of N-acetylneuraminic acid to the growth medium resulted in sialylation of the EPS, and increased biofilm formation. Real-time quantitative reverse transcription-polymerase chain reaction analyses indicated that genes previously identified in a putative polysaccharide locus were upregulated when the bacteria were grown under conditions favorable to a biofilm, compared to planktonic cells. CONCLUSIONS: H. somni is capable of producing a branching, mannose-galactose EPS polymer under growth conditions favorable to the biofilm phase of growth, and the EPS is a component of the biofilm matrix. The EPS can be sialylated in strains with sialyltransferase activity, resulting in enhanced density of the biofilm, and suggesting that EPS and biofilm formation may be important to persistence in the bovine host. The EPS may be critical to virulence if the biofilm state is required for H. somni to persist in systemic sites.

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.

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.

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.

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.

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.