Natural Evolution of Porcine Epidemic Diarrhea Viruses Isolated from Maternally Immunized Piglets

Simple SummaryDuring the long-term co-evolution of the virus and the host, even closely related vaccines may emerge with incomplete protective immunity due to the mutations or deletions of amino acids at specific antigenic sites. The mutation of PEDV was accelerated by the recombination of different strains and the mutation of the strains adapting to the environment. These mutations either cause immune escape from conventional vaccines or affect the virulence of the virus. Therefore, researching and developing new vaccines with cross-protection through continuous monitoring, isolation and sequencing are important to determine whether their genetic characteristics are changed and to evaluate the protective efficacy of current vaccines. The porcine epidemic diarrhea virus (PEDV) can cause severe piglet diarrhea or death in some herds. Genetic recombination and mutation facilitate the continuous evolution of the virus (PEDV), posing a great challenge for the prevention and control of porcine epidemic diarrhea (PED). Disease materials of piglets with PEDV vaccination failure in some areas of Shanxi, Henan and Hebei provinces of China were collected and examined to understand the prevalence and evolutionary characteristics of PEDV in these areas. Forty-seven suspicious disease materials from different litters on different farms were tested by multiplex PCR and screened by hematoxylin-eosin staining and immunohistochemistry. PEDV showed a positivity rate of 42.6%, infecting the small and large intestine and mesenteric lymph node tissues. The isolated strains infected Vero, PK-15 and Marc-145 multihost cells and exhibited low viral titers in all three cell types, as indicated by their growth kinetic curves. Possible putative recombination events in the isolates were identified by RDP4.0 software. Sequencing and phylogenetic analysis showed that compared with the classical vaccine strain, PEDV SX6 contains new insertion and mutations in the S region and belongs to genotype GIIa. Meanwhile, ORF3 has the complete amino acid sequence with aa80 mutated wild strains, compared to vaccine strains CV777, AJ1102, AJ1102-R and LW/L. These results will contribute to the development of new PEDV vaccines based on prevalent wild strains for the prevention and control of PED in China.

Detection of Mutations in SARS-CoV-2 Genomic Clades using COVIDSeq-NGS among International Travelers

Introduction/Objective Numerous SARS-CoV-2 variants/lineages have been identified based on genome sequencing. As of June 15, 2022 almost 11,399,573 whole genome sequences have been deposited in the GISAID-database. Severity and spread of COVID19 is based on their efficiency of infection and to multiply in host. That largely depend upon the structural mutation in spike, ORF and N proteins etc. That happens due to translation of genomic mutations during polypeptide synthesis. Also, the mutations are region/country specific. Specific mutation and combination of mutation causes the emergence of new strains. However, the strains can migrate from one region to other through travelers. The main objective of the current study is profiling of mutations in the genome of SARSCoV2 using Next- Generation-Sequencing (NGS) in international travelers and phylogenetic analysis of the sequences to find out different clades of SARSCoV2. Methods/Case Report A total of 557 SARSCoV2 genomes were sequenced on S4-sequencing flow-cell on NovaSeq 6000. For NGS of SARS-CoV-2 genome, Illumina, COVIDSeq kits and the protocols will be used strictly as recommended by the manufacturer. After NGS the analysis was done followed by FASTA sequences retrieval, mutations recording and phylogeny. Results (if a Case Study enter NA) This study reports 11 clades (19A, B, 20A, B, C, D, 20E;EU1, 20G, 20H;Beta V2, 20I: Alpha V1, 21D;and Eta) for the first time in international travelers. To best of our knowledge, this is the first report of the COVIDSeq approach for detection of mutation in SARSCoV2 genomic clades. The study revealed some dominants mutations was (Orf1a: P2018Q, K1053R, E176V, Orf1b: A520V, T2165A, S: D1127G, D614G, L18F etc. in other genes). Conclusion Profiling of common mutations among travelers could fill some gaps about the existence of SARS-CoV-2 variants information. However, further studies are needed to consolidate these findings before to be utilized for development of a potential therapeutic strategy.

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network's technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

Intra-Host Co-Existing Strains of SARS-CoV-2 Reference Genome Uncovered by Exhaustive Computational Search.

The COVID-19 pandemic caused by SARS-CoV-2 has had a severe impact on people worldwide. The reference genome of the virus has been widely used as a template for designing mRNA vaccines to combat the disease. In this study, we present a computational method aimed at identifying co-existing intra-host strains of the virus from RNA-sequencing data of short reads that were used to assemble the original reference genome. Our method consisted of five key steps: extraction of relevant reads, error correction for the reads, identification of within-host diversity, phylogenetic study, and protein binding affinity analysis. Our study revealed that multiple strains of SARS-CoV-2 can coexist in both the viral sample used to produce the reference sequence and a wastewater sample from California. Additionally, our workflow demonstrated its capability to identify within-host diversity in foot-and-mouth disease virus (FMDV). Through our research, we were able to shed light on the binding affinity and phylogenetic relationships of these strains with the published SARS-CoV-2 reference genome, SARS-CoV, variants of concern (VOC) of SARS-CoV-2, and some closely related coronaviruses. These insights have important implications for future research efforts aimed at identifying within-host diversity, understanding the evolution and spread of these viruses, as well as the development of effective treatments and vaccines against them.

Molecular Detection of Canine Parvovirus-2c (Cpv-2c) in Diseased Coyote Pups (Canis Latrans) at Northeastern Mexico

Intro: Canine parvovirus type 2 (PVC-2), Protoparvovirus genus of the Parvoviridae family, is a worldwide distribution virus that affects the Canidae family. In free-living coyotes (Canis latrans), the presence of the PCV-2a, PCV-2b and PCV-2c subtypes of PVC-2 has been reported, but there are no reports of their presence as a cause of clinical damage. The objective of this study is to report the presence of PVC-2c in an outbreak of mild gastroenteritis in three coyote pups detected in northeastern Mexico Methods: During the fall of 2019, in the suburban area of Monterrey, N.L., 3 affected coyote pups were detected with a mild gastroenteric condition consisting of mild diarrhea with loose stools, vomiting, dehydration, loss of appetite, pale mucous membranes, and low weight. Stool samples were tested for Canine Parvovirus (CPV-2), Canine Coronavirus (CCV) or Giardia antigens with a commercial kit. All samples were positive for CPV-2 and these were subsequently analyzed by PCR and sequencing of the CPV-2 VP2 gene. Using bioinformatics, the VP2 gene sequence data obtained were used to establish phylogenetic relationships with homologous sequences reported in coyotes and CPV-2 vaccines. Finding(s): The genetic sequence of VP2 obtained showed a high homology (98.1 to 100%) with CPV-2c. The sequences obtained from the pups showed 100% homology to each other. The phylogenetic tree showed that the sequences reported in coyotes are grouped in different clades and that the sequence of the VP2 gene of CPV-2c from coyote pups is grouped in a different monophyletic group. Conclusion(s): Information suggests that wild coyotes may not only act as asymptomatic reservoir hosts but may also be clinically affected by PVC-2c. It is necessary to carry out studies to know the effects of the genetic subtypes of PVC-2 in the population of coyotes and other wild canids of northeastern Mexico.Copyright © 2023

PARNAS: Objectively Selecting the Most Representative Taxa on a Phylogeny.

The use of next-generation sequencing technology has enabled phylogenetic studies with hundreds of thousands of taxa. Such large-scale phylogenies have become a critical component in genomic epidemiology in pathogens such as SARS-CoV-2 and influenza A virus. However, detailed phenotypic characterization of pathogens or generating a computationally tractable dataset for detailed phylogenetic analyses requires objective subsampling of taxa. To address this need, we propose parnas, an objective and flexible algorithm to sample and select taxa that best represent observed diversity by solving a generalized k-medoids problem on a phylogenetic tree. parnas solves this problem efficiently and exactly by novel optimizations and adapting algorithms from operations research. For more nuanced selections, taxa can be weighted with metadata or genetic sequence parameters, and the pool of potential representatives can be user-constrained. Motivated by influenza A virus genomic surveillance and vaccine design, parnas can be applied to identify representative taxa that optimally cover the diversity in a phylogeny within a specified distance radius. We demonstrated that parnas is more efficient and flexible than existing approaches. To demonstrate its utility, we applied parnas to (i) quantify SARS-CoV-2 genetic diversity over time, (ii) select representative influenza A virus in swine genes derived from over 5 years of genomic surveillance data, and (iii) identify gaps in H3N2 human influenza A virus vaccine coverage. We suggest that our method, through the objective selection of representatives in a phylogeny, provides criteria for quantifying genetic diversity that has application in the the rational design of multivalent vaccines and genomic epidemiology. PARNAS is available at https://github.com/flu-crew/parnas.

Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.

Isolation and Characterization of Porcine Sapelovirus from the PDCoV-Positive Sample and Its Molecular Epidemiology in Henan Province, China

Porcine sapelovirus (PSV) is an emerging swine enteric virus that can cause various disorders including acute diarrhea, respiratory distress, reproductive failure, and polioencephalomyelitis in pigs. In this study, we isolated a PSV strain HNHB-01 from a clinical porcine deltacoronavirus- (PDCoV-) positive intestinal content of a diarrheic piglet. PSV was first identified using the small RNA deep sequencing and assembly, and further identified by the electron microscopic observation and the immunofluorescence assay. Subsequently, this virus was serially passaged in swine testis (ST) cells, and the complete genomics of PSV HNHB-01 passage 5 (P5), P30, P60, and P100 were sequenced and analyzed. 9 nucleotide mutations and 7 amino acid changes occurred in the PSV HNHB-01 P100 strain when compared with the PSV HNHB-01 P5. Pathogenicity investigation showed that orally inoculation of PSV HNHB-01 P30 could cause obvious clinical symptoms and had broad tissue tropism in 5-day-old piglets. Epidemiological investigation revealed that PSV infections and the coinfections of diarrhea coronaviruses were highly prevalent in swine herds. The complete genomes of 8 representative PSV epidemic strains were sequenced and analyzed. Phylogenetic analysis revealed that the PSV epidemic strains were closely related to other PSV reference strains that located in the Chinese clade. Furthermore, recombination analysis revealed that the recombination events were occurred in downstream of the 2C region in our sequenced PSV HNNY-02/CHN/2018 strain. Our results provided theoretical basis for future research studies of the pathogenic mechanism, evolutionary characteristics, and the development of vaccines against PSV.

SARS-CoV-2 VARIANTS' TEMPORAL AND VL DISTRIBUTIONS IN IMMUNOCOMPROMISED PATIENTS

Background: The COVID-19 pandemic has been striking for three years and, despite the regular arise of new variants, populations are now widely immune and protected from severe symptoms. However, immunocompromised patients still have worse clinical outcomes, higher mortality and rarely develop effective immunity through vaccination or infection. Here, we studied the temporal distribution of infections, viral loads (VL) as well as the viral genetic diversity among an immunocompromised patient cohort, between January 2021 and September 2022. Method(s): Overall, 478 immunocompromised patients (solid organ transplant, HIV positive, cancer, autoimmune disease) and 234 controls (healthcare workers) from Pitie-Salpetriere and Bichat Claude-Bernard University hospitals (Paris, FRANCE) were diagnosed with SARS-CoV-2 infection by RT-qPCR. Whole genome sequencing was performed according to ARTIC protocol on Oxford Nanopore platform. All 712 full viral genomes were used to determine lineages and mapped to Wuhan-Hu-1 reference to produce a maximum likelihood phylogenetic tree (IQTree, 1000 bootstraps). Differences in temporal distributions of infections and VL were assessed using nonparametric statistical tests. Result(s): According to phylogenetic analysis, genomes from SARS-CoV- 2 infecting immunocompromised patients and those infecting healthy individuals are distributed in a similar way. No significant genetic differences can be observed between viral genomes from patients and controls within the different lineages. Temporal distribution of COVID-19 infections were also similar between immunocompromised patients and controls, with the exception of BA.2 variant for which controls were infected earlier (p< 0.001). VL were significantly lower in immunocompromised patients infected with Omicron variants (p=0.04). No differences in VL were observed for Alpha and Delta variants. Conclusion(s): At diagnosis, no intrinsic genetic divergence was observed in virus infecting immunocompromised patients compared to those circulating in the general population. Similarities in temporal distribution of infections between controls and patients suggest that these different groups become infected concomitantly. VL appeared to be lower for Omicron variants in immunocompromised patients. An earlier VL peak of Omicron and a testing of immunocompromised patients hospitalized once severe symptoms have appeared could indicate a delayed testing in these patients, once the replicative phase over. (Figure Presented).

Epidemiological and Molecular Evolution of the Hiv-1 Crf94: Birth of Crf132

Background: In 2018 we reported the emergence of the new HIV-1 recombinant CRF94-02BF2 involved in a large transmission cluster of 49 French MSM mostly infected in 2016-2017. This CRF94 raised concerns of enhanced virulence. Prevention actions were undertaken in the area and population affected. This study reported the molecular and epidemiological evolution of this CRF94 until June 2022. Method(s): In 2021-2022, French sequence databases were screened for patients infected with HIV-1 subtype CRF94 or similar strain. HIV subtyping was confirmed by phylogenetic analysis of genes encoding both protease and reverse transcriptase (1070bps), and integrase (696bps) using IQ-Tree. Five whole genomes, related but distinct from CRF94, were obtained with the DeepChek assay Whole Genome kits. Recombination breakpoints were estimated using RDP4 and SimPlot. Mann-Whitney and LogRank tests were used for statistical analyses to compare patients' characteristics. Result(s): In June 2022, 49 new HIV-1 sequences were collected: 14 clustered with the 49 previous CRF94, 32 formed a new cluster next to but distinct from CRF94, and 3 strains could not be classified. Analysis of 5 whole genomes from the new cluster revealed a new recombinant, the CRF132-94B, mainly consisting of CRF94 which recombined with subtype B in the POL and accessory genes. Vif gene changed from the F2 to the B subtype. Both CRF94 and 132 clusters involved >95% of MSM, mostly infected < 1 year before diagnosis. However, there were differences: 97% were diagnosed in 2013-2019 for CRF94 vs 90% in 2020-2022 for CRF132. At time of diagnosis, 33% of patients infected with CRF94 knew the Prep vs 95% for CRF132. In the cluster CRF94, patients were older (34 vs 30 years, p=0.02), had higher viral loads (5.42 vs 4.42 log10 copies/Ml;p< 0.001), a lower CD4 cell counts (358 vs 508 /mm3, p=0.002). On treatment, the patients with the CRF94 reached viremia < 50 copies/Ml significantly later than those infected with CRF132 (p=0.0002). The prevention activities targeting the CRF94 cluster could explained the few patients infected with this strain after 2018. The CRF132 is mainly located in another Paris region area, but no specific transmission place has been identified. Conclusion(s): After 2019, the CRF94 spread seems greatly slowed down but the very close CRF132-94B has given birth to a new highly active cluster in 2020- 2022, despite the COVID social-distancing and a strong knowledge of the Prep. CRF132 appears to be less virulent perhaps due to the Vif gene change. Identified breakpoints positions of the new HIV-1 CRF132-94B. GenBank accession numbers of the five references : ON901787 to ON901791.

Circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021-22)

Enteroviruses, beyond poliovirus, are important pathogens. Several non-polio enteroviruses (NPEVs) are causing epidemics all around the world. Limited data is available on the prevalence and diversity of these viruses from India. Objective(s): Detection and characterization of NPEVs in respiratory samples during the COVID-19 pandemic period. Material(s) and Method(s): COVID-19 negative samples from acute respiratory infections (ARI) [n = 105] and severe-acute respiratory infections (SARI) [n = 148] during the period 2021-22 were screened for NPEVs. Detection was carried out using the one step RT-PCR method targeting the 5'UTR region followed by molecular analysis. Results and Conclusion(s): Total positivity of NPEVs was noted in 35.23% and 31.08% of the ARI and SARI cases, respectively. Comparison within the two groups studied, showed significant difference in the age-wise distribution for cases>18 years of age. Year round seasonality for ARI cases while autumn seasonality for SARI cases was observed. Sequencing of representative samples of ARI cases showed prevalence of Rhinovirus A (RVA), Rhinovirus B (RVB), Rhinovirus C (RVC) and Echovirus, while predominance of RVC followed by RVA was observed for the SARI cases. Phylogenetic analysis of all the strains showed clustering of RVC strains in different clusters. Divergence was also noted in RVA and RVB strains studied. Circulation of a rare Echovirus-29 strain was noted in the ARI cases. The study highlighted significant divergence in the Rhinovirus strains studied. It warrants the need for surveillance of NPEVs, whole-genome sequencing of the circulating strains for better understanding of biodiversity among the NPEVs and the potential health burden.

Pathogenesis, Symptomatology, and Transmission of SARS-CoV-2 through Analysis of Viral Genomics and Structure

The novel coronavirus SARS-CoV-2, which emerged in late 2019, has since spread around the world and infected hundreds of millions of people with coronavirus disease 2019 (COVID-19). While this viral species was unknown prior to January 2020, its similarity to other coronaviruses that infect humans has allowed for rapid insight into the mechanisms that it uses to infect human hosts, as well as the ways in which the human immune system can respond. Here, we contextualize SARS-CoV-2 among other coronaviruses and identify what is known and what can be inferred about its behavior once inside a human host. Because the genomic content of coronaviruses, which specifies the virus's structure, is highly conserved, early genomic analysis provided a significant head start in predicting viral pathogenesis and in understanding potential differences among variants. The pathogenesis of the virus offers insights into symptomatology, transmission, and individual susceptibility. Additionally, prior research into interactions between the human immune system and coronaviruses has identified how these viruses can evade the immune system's protective mechanisms. We also explore systems-level research into the regulatory and proteomic effects of SARS-CoV-2 infection and the immune response. Understanding the structure and behavior of the virus serves to contextualize the many facets of the COVID-19 pandemic and can influence efforts to control the virus and treat the disease. IMPORTANCE COVID-19 involves a number of organ systems and can present with a wide range of symptoms. From how the virus infects cells to how it spreads between people, the available research suggests that these patterns are very similar to those seen in the closely related viruses SARS-CoV-1 and possibly Middle East respiratory syndrome-related CoV (MERS-CoV). Understanding the pathogenesis of the SARS-CoV-2 virus also contextualizes how the different biological systems affected by COVID-19 connect. Exploring the structure, phylogeny, and pathogenesis of the virus therefore helps to guide interpretation of the broader impacts of the virus on the human body and on human populations. For this reason, an in-depth exploration of viral mechanisms is critical to a robust understanding of SARS-CoV-2 and, potentially, future emergent human CoVs (HCoVs).Copyright © 2021 Rando et al.

VARIABLE REDUCTIONS IN SARS-CoV-2 VARIANT IMPORTATIONS FOLLOWING TRAVEL RESTRICTIONS

Background: Retrospectively quantifying effectiveness of interventions such as travel restrictions to counter viral introduction and transmission is critical to inform public health policy. Phylogenetic analyses of SARS-CoV-2 variants were undertaken to quantify the effects Canadian COVID-19 travel restrictions had on variant importation and transmission dynamics. Method(s): Global and Canadian GISAID sequences available up to March 2022 were subsampled proportionally to variant-specific case counts and ten phylogenies were inferred for each variant. Trees, dates, and geographies were inferred using maximum likelihood. Result(s): In response to Alpha, Canada implemented a UK flight ban from December 20, 2020-January 6, 2021, resulting in a 1.5-fold reduction in UK sublineage importation rate, with subsequent rebound (Fig. 1). Enhanced screening measures were implemented on December 24, 2020 for South African arrivals to counter Beta. Although there was a 6.3-fold reduction of Beta sublineages per week from South Africa following enhanced screening, there is low confidence in rare events. For Gamma, enhanced screening for arrivals from Brazil was implemented March 31-April 13, 2021. Proportion of Gamma sublineages from Brazil was reduced 1.6-fold within 2 weeks of the intervention, but the weekly importation rate was not significantly changed from start to end of intervention. In response to Delta, Canada issued a suspension of flights from India from April 22-September 23, 202, coinciding with a 2.4-fold reduction in sublineage importation and 3.8-fold reduction in proportion of sublineages from India. Increased importations from the USA and Europe progressively negated the ban's effectiveness. Against Omicron, Canada banned entry of all foreign nationals who had travelled through southern Africa and implemented enhanced screening for Canadians from November 26- December 18, 2021. Subsequently, the BA.1 sublineage importation rate from South Africa was maintained at a low level amid rising cases, while importations from other sources increased, reducing the proportion of sublineages from South Africa and diluting the measure's effectiveness. Conclusion(s): Flight bans and enhanced screening against SARS-CoV-2 variants were most effective when implemented rapidly and for lengthier time;however, effectiveness declined as variants became globally widespread. Ongoing genomic surveillance programs incorporating phylodynamic analyses can inform travel restriction and non-pharmaceutical intervention policy. (Figure Presented).

GENOMIC SURVEILLANCE OF SARS-CoV-2 REVEALS SEVERITY OF DELTA VARIANT IN CAMEROON

Background: At the global level, the dynamics of the COVID-19 pandemic have been driven by several epidemiological waves, determined by the emergence of new SARS-CoV-2 variants from the original viral lineage from Wuhan, China. While the SARS-CoV-2 dynamic has been described globally, there is a lack of data from Sub-Saharan African. Method(s): A laboratory-based survey was conducted in Cameroon, from March 1, 2020 to March 30, 2022, through an assessment of the evolutionary patterns of SARS-CoV-2 lineages across the four COVID-19 waves in the country. Data on full-length sequencing from all four sequencing laboratories were consecutively entered into the GISAID platform. These data were downloaded, and the molecular phylogeny of the SARS-CoV-2 sequences was performed using Nexstrain. The Mann-Whitney U test was used to calculate the correlation between the duration of each outbreak and the number of confirmed cases and between hospitalised cases and CFR, with a p value < 0.05 considered statistically significant. Result(s): A total of 3,881 samples were successfully processed, of which 38.9% (n=1,509) using PCR mutation assay, 41.5% (n=1,612) using targeted sequencing, and 19.6% (n=760) using whole-genome sequencing. The mean age of the study population was 36 years (min-max: 2-86), and 45% were within the age range 26-45. Regarding gender distribution, 50.9% were male, and 49.1% were female. Phylogenetic analysis of the 760 whole-genome sequences generated from March 2020 to March 2022 revealed that the greater proportion of SARS-CoV-2 circulating in Cameroon belonged to the viral sub-lineages of the original strain from Wuhan (74%), 15% Delta variant, 6% Omicron variant, 3% Alpha variant and 2% Beta variant.The pandemic was driven by SARS-CoV-2 lineages of origin in Wave 1 (16 weeks, 2.3% CFR), the Alpha and Beta variants in Wave 2 (21 weeks, 1.6% CFR), Delta variants in Wave 3 (11 weeks, 2.0% CFR), and Omicron variants in Wave 4 (8 weeks, 0.73% CFR), with a declining trend over time (p=0.01208). Conclusion(s): In a nutshell, the SARS-CoV-2 epidemic in Cameroon appears to have been driven by the SARS-CoV-2 lineage of origin in Wave 1, the cointroduction of the Alpha and Beta variants in Wave 2, the Delta variant in Wave 3, and the Omicron variant in Wave 4, with an overall declining trend in the wave duration, confirmed cases and hospitalisations over time.The SARS-CoV-2 lineage of origin and the Delta variant appeared to be the drivers of COVID-19 severity in Cameroon.

FROM PROMISE TO REALIZATION: SEQUENCING AND SURVEILLANCE FROM HIV-1 TO SARS-CoV-2

The advent of cheaper viral sequencing and opportunity to offer customized treatment through identification of resistance mutations in patients with HIV-1, also offered the first large-scale opportunity to use sequencing to generate insights into a global infectious disease pandemic. Using HIV-1 sequences, scientists were able to track mutations globally and within countries and use them to gain groundbreaking understanding of virus transmission and the evolution of resistance. Though invaluable in contributing to our knowledge of virus dynamics, much of what was feasible with HIV-1 was difficult to extend to other viruses due to the challenges and expense of full-genome sequences and the difficulty of obtaining samples from acute infections. More recent advances have made next-generation sequencing (NGS) possible and affordable, and growing realization of the insights sequencing can contribute has increased interest in generating sequences for an increasing variety of viruses. Against this backdrop of an advent of a new age of genomics in viral research, the SARSCoV- 2 pandemic has thrown sequencing and phylogenetics into the limelight, allowing the collection and sequencing of more samples than could even be conceived prior to 2020. It's a opportune time to consider not only where we've come from, but how the promise of 14 million sequences has been realized, and what the future holds for sequence-enabled pathogen research.

Emergence of a genotype I lineage 1 variant avian infectious bronchitis virus in Assam, India

Background: Infectious bronchitis (IB) is an acute and highly contagious viral disease of poultry affecting chicken of all ages. The causative agent IB virus (IBV) is a Gammacoronavirus within the family Coronaviridae. Viral genetic mutations and recombination events particularly in the spike protein (S1) of IBV constantly give rise to emerging IBV variants. Vaccination is considered as the most reliable approach for IBV control, but current vaccines have been found to be ineffective due to constant emergence of new variant viruses. Objective(s): The objective of our study was to detect IBV genotypes prevalent in Assam, India. Material(s) and Method(s): Oro-pharyngeal swabs and tissue samples from unvaccinated broiler chickens showing respiratory symptoms were tested using RT-PCR targeting the N gene of IBV. The virus was isolated from infected swab/tissue samples in 9 days old specific pathogen free embryonated chicken eggs through allantoic cavity route. Phylogenetic studies were done based on the S1 gene of IBV. Results and Conclusion(s): Clinically, the birds showed gasping and tracheal rales. Necropsy revealed distended ureters. Virus was isolated and identified by curling and dwarfing of the dead embryos and further confirmed by RT-PCR. Positive PCR amplicons were sequenced and phylogenetic analysis clustered the IBV isolate from Assam with genotype I lineage 1 IBV prototype sequence belonging to Beaudette and Mass 41 strains but the isolate exhibited a relatively high degree of sequence divergence with reference strains. Our findings suggest that the IBV isolate might have emerged from recombination with the local circulating virus or vaccine strains. This will have important implications for IB prevention strategies.

West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022.

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15 WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018 WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations.

Tracing the Origin of Genotype II African Swine Fever Virus in China by Genomic Epidemiology Analysis

The pandemic spread of African swine fever (ASF) has caused serious effects on the global pig industry. Virus genome sequencing and genomic epidemiology analysis play an important role in tracking the outbreaks of the disease and tracing the transmission of the virus. Here we obtained the full-length genome sequence of African swine fever virus (ASFV) in the first outbreak of ASF in China on August 3rd, 2018 and compared it with other published genotype II ASFV genomes including 9 genomes collected in China from September 2018 to October 2020. Phylogenetic analysis on genomic sequences revealed that genotype II ASFV has evolved into different genetic clusters with temporal and spatial correlation since being introduced into Europe and then Asia. There was a strong support for the monophyletic grouping of all the ASFV genome sequences from China and other Asian countries, which shared a common ancestor with those from the Central or Eastern Europe. An evolutionary rate of 1.312 × 10−5 nucleotide substitutions per site per year was estimated for genotype II ASFV genomes. Eight single nucleotide variations which located in MGF110-1L, MGF110-7L, MGF360-10L, MGF505-5R, MGF505-9R, K145R, NP419L, and I267L were identified as anchor mutations that defined genetic clusters of genotype II ASFV in Europe and Asia. This study expanded our knowledge of the molecular epidemiology of ASFV and provided valuable information for effective control of the disease.

Characterization and Evaluation of the Pathogenicity of a Natural Gene-Deleted Transmissible Gastroenteritis Virus in China

Porcine transmissible gastroenteritis virus is the major pathogen that causes fatal diarrhea in newborn piglets. In this study, a TGEV strain was isolated from the small intestine of diarrhea piglets in Sichuan Province, China, and designated SC2021. The complete genomic sequence of TGEV SC2021 was 28561 bp, revealing a new natural deletion TGEV strain. Based on phylogenetic analyses, TGEV SC2021 belonged to the Miller cluster and was closely related to CN strains. The newborn piglets orally challenged with TGEV SC2021 showed typical watery diarrhea. In addition, macro and micropathological changes in the lungs and intestines were observed. In conclusion, we isolated a new natural deletion virus strain and confirmed that the virus strain has high pathogenicity in newborn piglets. Moreover, macroscopic and microscopic lesions were observed in the lungs and intestines of all TGEV SC2021-infected piglets. In summary, we isolated a new natural deletion TGEV strain and demonstrated that the natural deletion strain showed high pathogenicity in newborn piglets. These data enrich the diversity of TGEV strains and help us to understand the genetic evolution and molecular pathogenesis of TGEV.

A Porcine Epidemic Diarrhea Virus Isolated from a Sow Farm Vaccinated with CV777 Strain in Yinchuan, China: Characterization, Antigenicity, and Pathogenicity

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus globally, causing serious economic losses to the global pig industry since 2010. Here, a PEDV CH/Yinchuan/2021 strain was isolated in a CV777-vaccinated sow farm which experienced a large-scale PEDV invasion in Yinchuan, China, in 2021. Our results demonstrated that the CH/Yinchuan/2021 isolate could efficiently propagate in Vero cells, and its proliferation ability was weaker than that of CV777 at 10 passages (P10). Phylogenetic analysis of the S gene revealed that CH/Yinchuan/2021 was clustered into subgroup GIIa, forming an independent branch with 2020-2021 isolates in China. Moreover, GII was obviously allocated into four clades, showing regional and temporal differences in PEDV global isolates. Notably, CH/Yinchuan/2021 was analyzed as a recombinant originated from an American isolate and a Chinese isolate, with a big recombinant region spanning ORF1a and S1. Importantly, we found that CH/Yinchuan/2021 harbored multiple mutations relative to CV777 in neutralizing epitopes (S10, S1A, COE, and SS6). Homology modelling showed that these amino acid differences in S protein occur on the surface of its structure, especially the insertion and deletion of multiple consecutive residues at the S10 epitope. In addition, cross-neutralization analysis confirmed that the differences in the S protein of CH/Yinchuan/2021 changed its antigenicity compared with the CV777 strain, resulting in a different neutralization profile. Animal pathogenicity test showed that CH/Yinchuan/2021 caused PEDV-typified symptoms and 100% mortality in 3-day-old piglets. These data will provide valuable information to understand the epidemiology, molecular characteristics, evolution, and antigenicity of PEDV circulating in China.