Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Estimation of SARS-CoV-2 fitness gains from genomic surveillance data without prior lineage classification.

The emergence of SARS-CoV-2 variants with increased fitness has had a strong impact on the epidemiology of COVID-19, with the higher effective reproduction number of the viral variants leading to new epidemic waves. Tracking such variants and their genetic signatures, using data collected through genomic surveillance, is therefore crucial for forecasting likely surges in incidence. Current methods of estimating fitness advantages of variants rely on tracking the changing proportion of a particular lineage over time, but describing successful lineages in a rapidly evolving viral population is a difficult task. We propose a method of estimating fitness gains directly from nucleotide information generated by genomic surveillance, without a priori assigning isolates to lineages from phylogenies, based solely on the abundance of single nucleotide polymorphisms (SNPs). The method is based on mapping changes in the genetic population structure over time. Changes in the abundance of SNPs associated with periods of increasing fitness allow for the unbiased discovery of new variants, thereby obviating a deliberate lineage assignment and phylogenetic inference. We conclude that the method provides a fast and reliable way to estimate fitness advantages of variants without the need for a priori assigning isolates to lineages.

Hound: a novel tool for automated mapping of genotype to phenotype in bacterial genomes assembled de novo.

Increasing evidence suggests that microbial species have a strong within species genetic heterogeneity. This can be problematic for the analysis of prokaryote genomes, which commonly relies on a reference genome to guide the assembly process. Differences between reference and sample genomes will therefore introduce errors in final assembly, jeopardizing the detection from structural variations to point mutations-critical for genomic surveillance of antibiotic resistance. Here we present Hound, a pipeline that integrates publicly available tools to assemble prokaryote genomes de novo, detect user-given genes by similarity to report mutations found in the coding sequence, promoter, as well as relative gene copy number within the assembly. Importantly, Hound can use the query sequence as a guide to merge contigs, and reconstruct genes that were fragmented by the assembler. To showcase Hound, we screened through 5032 bacterial whole-genome sequences isolated from farmed animals and human infections, using the amino acid sequence encoded by blaTEM-1, to detect and predict resistance to amoxicillin/clavulanate which is driven by over-expression of this gene. We believe this tool can facilitate the analysis of prokaryote species that currently lack a reference genome, and can be scaled either up to build automated systems for genomic surveillance or down to integrate into antibiotic susceptibility point-of-care diagnostics.

Motifs in SARS-CoV-2 evolution.

We present a novel framework enhancing the prediction of whether novel lineage poses the threat of eventually dominating the viral population. The framework is based purely on genomic sequence data, without requiring prior established biological analysis. Its building blocks are sets of coevolving sites in the alignment (motifs), identified via coevolutionary signals. The collection of such motifs forms a relational structure over the polymorphic sites. Motifs are constructed using distances quantifying the coevolutionary coupling of pairs and manifest as coevolving clusters of sites. We present an approach to genomic surveillance based on this notion of relational structure. Our system will issue an alert regarding a lineage, based on its contribution to drastic changes in the relational structure. We then conduct a comprehensive retrospective analysis of the COVID-19 pandemic based on SARS-CoV-2 genomic sequence data in GISAID from October 2020 to September 2022, across 21 lineages and 27 countries with weekly resolution. We investigate the performance of this surveillance system in terms of its accuracy, timeliness, and robustness. Lastly, we study how well each lineage is classified by such a system.

Reduced Likelihood of Hospitalization with the JN.1 or HV.1 SARS-CoV-2 Variants Compared to the EG.5 Variant.

Within a multi-state viral genomic surveillance program, we evaluated whether proportions of SARS-CoV-2 infections attributed to the JN.1 variant and to XBB-lineage variants (including HV.1 and EG.5) differed between inpatient and outpatient care settings during periods of cocirculation. Both JN.1 and HV.1 were less likely than EG.5 to account for infections among inpatients versus outpatients (aOR=0.60 [95% CI: 0.43-0.84; p=0.003] and aOR=0.35 [95% CI: 0.21-0.58; p<0.001], respectively). JN.1 and HV.1 variants may be associated with a lower risk of severe illness. The severity of COVID-19 may have attenuated as predominant circulating SARS-CoV-2 lineages shifted from EG.5 to HV.1 to JN.1.

Genomic diversity and antimicrobial susceptibility of invasive Neisseria meningitidis in South Africa, 2016-2021.

BACKGROUND: Invasive meningococcal isolates in South Africa have in previous years (<2008) been characterized by serogroup B, C, W and Y lineages over time, with penicillin intermediate resistance (peni) at 6%. We describe the population structure and genomic markers of peni among invasive meningococcal isolates in South Africa, 2016-2021. METHODS: Meningococcal isolates were collected through national, laboratory-based invasive meningococcal disease (IMD) surveillance. Phenotypic antimicrobial susceptibility testing and whole-genome sequencing were performed, and the mechanism of reduced penicillin susceptibility was assessed in silico. RESULTS: Of 585 IMD cases reported during the study period, culture and PCR-based capsular group was determined for 477/585 (82%); and 241/477 (51%) were sequenced. Predominant serogroups included NmB (210/477; 44%), NmW (116/477; 24%), NmY (96/477; 20%) and NmC (48/477; 10%). Predominant clonal complexes (CC) were CC41/44 in NmB (27/113; 24%), CC11 in NmW (46/56; 82%), CC167 in NmY (23/44; 53%), and CC865 in NmC (9/24; 38%). Peni was detected in 16% (42/262) of isolates, and was due to the presence of a penA mosaic, with the majority harboring penA7, penA9 or penA14. CONCLUSION: IMD lineages circulating in South Africa were consistent with those circulating prior to 2008, however peni was higher than previously reported, and occurred in a variety of lineages.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Reemergence of Clade IIb-Associated Mpox, Germany, July-December 2023.

In July 2023, clade IIb-associated mpox reemerged in Germany at low levels, mainly affecting men who have sex with men. We report a representative case and phylogeny of available genome sequences. Our findings underscore the need for standardized surveillance and indication-based vaccination to limit transmission and help prevent endemicity.

Phylogenomics of Dengue Virus Isolates Causing Dengue Outbreak, São Tomé and Príncipe, 2022.

We determined that the dengue outbreak in São Tomé and Príncipe during 2022 was caused by dengue virus serotype 3 genotype III. Phylogenomic analyses showed that the outbreak strain was closely related to the newly identified GIII-American-II lineage and that the virus probably was introduced from the Americas.

Novel Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2023.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.

Evolution and Spread of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Wild Birds, South Korea, 2022-2023.

During October 2022-March 2023, highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus caused outbreaks in South Korea, including 174 cases in wild birds. To understand the origin and role of wild birds in the evolution and spread of HPAI viruses, we sequenced 113 HPAI isolates from wild birds and performed phylogenetic analysis. We identified 16 different genotypes, indicating extensive genetic reassortment with viruses in wild birds. Phylodynamic analysis showed that the viruses were most likely introduced to the southern Gyeonggi-do/northern Chungcheongnam-do area through whooper swans (Cygnus cygnus) and spread southward. Cross-species transmission occurred between various wild bird species, including waterfowl and raptors, resulting in the persistence of HPAI in wild bird populations and further geographic spread as these birds migrated throughout South Korea. Enhanced genomic surveillance was an integral part of the HPAI outbreak response, aiding in timely understanding of the origin, evolution, and spread of the virus.

Early Genomic Surveillance and Phylogeographic Analysis of Getah Virus, a Reemerging Arbovirus, in Livestock in China.

Getah virus (GETV) mainly causes disease in livestock and may pose an epidemic risk due to its expanding host range and the potential of long-distance dispersal through animal trade. Here, we used metagenomic next-generation sequencing (mNGS) to identify GETV as the pathogen responsible for reemerging swine disease in China and subsequently estimated key epidemiological parameters using phylodynamic and spatially-explicit phylogeographic approaches. The GETV isolates were able to replicate in a variety of cell lines, including human cells, and showed high pathogenicity in a mouse model, suggesting the potential for more mammal hosts. We obtained 16 complete genomes and 79 E2 gene sequences from viral strains collected in China from 2016 to 2021 through large-scale surveillance among livestock, pets, and mosquitoes. Our phylogenetic analysis revealed that three major GETV lineages are responsible for the current epidemic in livestock in China. We identified three potential positively selected sites and mutations of interest in E2, which may impact the transmissibility and pathogenicity of the virus. Phylodynamic inference of the GETV demographic dynamics identified an association between livestock meat consumption and the evolution of viral genetic diversity. Finally, phylogeographic reconstruction of GETV dispersal indicated that the sampled lineages have preferentially circulated within areas associated with relatively higher mean annual temperature and pig population density. Our results highlight the importance of continuous surveillance of GETV among livestock in southern Chinese regions associated with relatively high temperatures. IMPORTANCE Although livestock is known to be the primary reservoir of Getah virus (GETV) in Asian countries, where identification is largely based on serology, the evolutionary history and spatial epidemiology of GETV in these regions remain largely unknown. Through our sequencing efforts, we provided robust support for lineage delineation of GETV and identified three major lineages that are responsible for the current epidemic in livestock in China. We further analyzed genomic and epidemiological data to reconstruct the recent demographic and dispersal history of GETV in domestic animals in China and to explore the impact of environmental factors on its genetic diversity and its diffusion. Notably, except for livestock meat consumption, other pig-related factors such as the evolution of live pig transport and pork production do not show a significant association with the evolution of viral genetic diversity, pointing out that further studies should investigate the potential contribution of other host species to the GETV outbreak. Our analysis of GETV demonstrates the need for wider animal species surveillance and provides a baseline for future studies of the molecular epidemiology and early warning of emerging arboviruses in China.

Developing a next level integrated genomic surveillance: Advances in the molecular epidemiology of HIV in Germany.

Advances in the molecular epidemiological studies of the Human Immunodeficiency Virus (HIV) at the Robert Koch Institute (RKI) by laboratory and bioinformatic automation should allow the processing of larger numbers of samples and more comprehensive and faster data analysis in order to provide a higher resolution of the current HIV infection situation in near real-time and a better understanding of the dynamic of the German HIV epidemic. The early detection of the emergence and transmission of new HIV variants is important for the adaption of diagnostics and treatment guidelines. Likewise, the molecular epidemiological detection and characterization of spatially limited HIV outbreaks or rapidly growing sub-epidemics is of great importance in order to interrupt the transmission pathways by regionally adapting prevention strategies. These aims are becoming even more important in the context of the SARS-CoV2 pandemic and the Ukrainian refugee movement, which both have effects on the German HIV epidemic that should be monitored to identify starting points for targeted public health measures in a timely manner. To this end, a next level integrated genomic surveillance of HIV is to be established.

Highly sensitive wastewater surveillance of SARS-CoV-2 variants by targeted next-generation amplicon sequencing provides early warning of incursion in Victoria, Australia.

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread.IMPORTANCEThis study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method's flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

Prevalence and diversity of imported severe acute respiratory syndrome coronavirus 2 variants in China, 2021-2022.

The causative agent of coronavirus disease 2019 (COVID-19), known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread accumulatively to 240 countries and continues to evolve. To gain a comprehensive understanding of the epidemiological characteristics of imported variants in China and their correlation with global circulating variants, genomic surveillance data from 11 139 imported COVID-19 cases submitted by Chinese provincial CDC laboratories between 2021 and 2022 were analyzed. Consensus sequences underwent rigorous quality checks, followed by amino acid mutations analysis using Nextclade. Sequences with satisfactory quality control status were classified according to the Pango nomenclature. The results showed that the dominant variants in imported cases reflected the global epidemic trend. An increase in the number of imported SARS-CoV-2 lineages monitored in China in the second half of 2022, and the circulating Omicron subvariants changed from the ancestral lineages of BA.5 and BA.2 into the lineages containing key amino acid mutations of spike protein. There was significant variation in the detection of Omicron subvariants among continents (χ2 = 321.968, p < 0.001) in the second half of 2022, with four lineages (BA.2.3.7, BA.2.2, BA.5.2.7, and XBB.1.2) identified through imported surveillance mainly prevalent respectively in Taiwan, China, Hong Kong SAR, China, Russian Federation, and Singapore. These findings revealed the alterations in circulating imported variants from 2021 to 2022 in China, reflecting the higher diversity of lineages in the second half of 2022, and revealed the predominant lineages of countries or regions that are in close contacts to China, providing new insights into the global prevalence of SARS-CoV-2.

What is the functional reach of wastewater surveillance for respiratory viruses, pathogenic viruses of concern, and bacterial antibiotic resistance genes of interest?

BACKGROUND: Despite a clear appreciation of the impact of human pathogens on community health, efforts to understand pathogen dynamics within populations often follow a narrow-targeted approach and rely on the deployment of specific molecular probes for quantitative detection or rely on clinical detection and reporting. MAIN TEXT: Genomic analysis of wastewater samples for the broad detection of viruses, bacteria, fungi, and antibiotic resistance genes of interest/concern is inherently difficult, and while deep sequencing of wastewater provides a wealth of information, a robust and cooperative foundation is needed to support healthier communities. In addition to furthering the capacity of high-throughput sequencing wastewater-based epidemiology to detect human pathogens in an unbiased and agnostic manner, it is critical that collaborative networks among public health agencies, researchers, and community stakeholders be fostered to prepare communities for future public health emergencies or for the next pandemic. A more inclusive public health infrastructure must be built for better data reporting where there is a global human health risk burden. CONCLUSIONS: As wastewater platforms continue to be developed and refined, high-throughput sequencing of human pathogens in wastewater samples will emerge as a gold standard for understanding community health.

Use of strips of rapid diagnostic tests as a source of ribonucleic acid for genomic surveillance of viruses: an example of SARS-CoV-2.

BACKGROUND: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. METHOD: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. RESULT: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. CONCLUSION: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2.

A regional genomic surveillance program is implemented to monitor the occurrence and emergence of SARS-CoV-2 variants in Yubei District, China.

BACKGROUND: In December 2022, Chongqing experienced a significant surge in coronavirus disease 2019 (COVID-19) epidemic after adjusting control measures in China. Given the widespread immunization of the population with the BA.5 variant, it is crucial to actively monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant evolution in Chongqing's Yubei district. METHODS: In this retrospective study based on whole genome sequencing, we collected oropharyngeal and nasal swab of native COVID-19 cases from Yubei district between January to May 2023, along with imported cases from January 2022 to January 2023. Through second-generation sequencing, we generated a total of 578 genomes. RESULTS: Phylogenetic analyses revealed these genomes belong to 47 SARS-CoV-2 Pango lineages. BA.5.2.48 was dominant from January to April 2023, rapidly replaced by XBB* variants from April to May 2023. Bayesian Skyline Plot reconstructions indicated a higher evolutionary rate (6.973 × 10-4 subs/site/year) for the XBB.1.5* lineage compared to others. The mean time to the most recent common ancestor (tMRCA) of BA.5.2.48* closely matched BA.2.75* (May 27, 2022). Using multinomial logistic regression, we estimated growth advantages, with XBB.1.9.1 showing the highest growth advantage (1.2, 95% HPI:1.1-1.2), followed by lineage FR.1 (1.1, 95% HPI:1.1-1.2). CONCLUSIONS: Our monitoring reveals the rapid replacement of the previously prevalent BA.5.2.48 variant by XBB and its sub-variants, underscoring the ineffectiveness of herd immunity and breakthrough BA.5 infections against XBB variants. Given the ongoing evolutionary pressure, sustaining a SARS-CoV-2 genomic surveillance program is imperative.

Investigating the cause of a 2021 winter wave of COVID-19 in a border region in eastern Germany: a mixed-methods study, August to November 2021.

It is so far unclear how the COVID-19 winter waves started and what should be done to prevent possible future waves. In this study, we deciphered the dynamic course of a winter wave in 2021 in Saxony, a state in Eastern Germany neighbouring the Czech Republic and Poland. The study was carried out through the integration of multiple virus genomic epidemiology approaches to track transmission chains, identify emerging variants and investigate dynamic changes in transmission clusters. For identified local variants of interest, functional evaluations were performed. Multiple long-lasting community transmission clusters have been identified acting as driving force for the winter wave 2021. Analysis of the dynamic courses of two representative clusters indicated a similar transmission pattern. However, the transmission cluster caused by a locally occurring new Delta variant AY.36.1 showed a distinct transmission pattern, and functional analyses revealed a replication advantage of it. This study indicated that long-lasting community transmission clusters starting since early autumn caused by imported or locally occurring variants all contributed to the development of the 2021 winter wave. The information we achieved might help future pandemic prevention.

Long-term surveillance of SARS-CoV-2 in the school community from Campo Grande, Brazil.

BACKGROUND: The COVID-19 pandemic has significantly impacted education systems worldwide, with Brazil being one of the countries with the longest school closures. Over a million children and teenagers have been affected, leading to increased hunger and nutritional deficiencies. This study aimed to implement long-term surveillance of SARS-CoV-2 infections in public and private schools in Campo Grande, Brazil, after returning to in-person classes. METHODS: The study involved testing and genomic surveillance at 23 public and private schools in Campo Grande, Mato Grosso do Sul, Brazil, from October 18, 2021 to November 21, 2022. The participants eligible for enrollment were students aged 6-17 years and staff members from school institutions. At the time of collection, participants were asked if they had symptoms in the last two weeks. Whole-genome sequencing of SARS-CoV-2 was conducted to identify circulating variants and to compare them with those detected in the municipality. The demographic data and clinical history of the participants were described, and a logistic regression model was used to understand how the RT-qPCR results could be related to different characteristics. RESULTS: The study included 999 participants, most of whom were women. A total of 85 tests were positive, with an overall positivity rate of 3.2%. The dynamics of case frequency were consistent with those observed in the municipality during the study period. The most common symptoms reported were cough, rhinorrhea, headache, and sore throat. Symptoms were significantly associated with SARS-CoV-2 infection. Eleven lineages were identified in school community samples, with a frequency of occurrence per period similar to that found in the sequences available for the municipality. The most prevalent lineages within the sampling period were BA.2 (59.3%) and BA.5 (29.6%). CONCLUSIONS: Our findings demonstrate that schools can play a crucial role in epidemiological surveillance, helping trigger rapid responses to pathogens such as SARS-CoV-2. Long-term surveillance can be used to track outbreaks and assess the role of children and adults in transmission. It can also contribute to pandemic preparedness, enabling a rapid response to emergencies, such as COVID-19.