Developing both rapid- and long-acting antiviral drugs for single-dose administration can improve medication adherence and protect people at risk of infection. To provide proof of this concept, here, we designed multimerized form of viral receptor-binding domains (RBDs) to immediately occupy viral receptors to block infection and subsequently induce virus-specific protective immunity. We engineered SARS-CoV-2 RBD, enhancing its affinity to ACE2 and immunogenicity through multimerization and Fc modification. A single administration of 4RBD-Fc not only effectively blocked ACE2-dependent SARS-CoV-2 infections but also elicited robust virus-specific mucosal and systemic immunity in the absence of adjuvants, providing superior early and long-lasting protection compared to adjuvanted vaccines in mice. These findings demonstrate the feasibility and efficacy of engineered viral RBD as immediate-acting and long-lasting single-dose antiviral drugs through rapid receptor blocking and ensuing adaptive immunity induction.

BACKGROUND: Noroviruses are a predominant cause of acute gastroenteritis (AGE) outbreaks globally, many outbreaks are associated with waterborne transmission. However, waterborne AGE outbreaks caused by the GII.9[P7] strain are relatively rare. METHODS: In April 2024, an AGE outbreak occurred among high school students on an educational excursion in Guangdong, China. Feces or anal swabs from clinical cases and asymptomatic canteen staffs, water samples from septic tank and tap water, along with food samples were collected for pathogen detection by real-time RT-PCR, and positive samples were subsequently characterized through gene sequencing analysis. RESULTS: From 12 April to 14 April 2024, a total of 84 individuals met the case definitions. The cases occurred continuously throughout the excursion without a distinct epidemic peak and the number of cases decreased significantly after the students left on April 13. Norovirus GII was detected in 12 symptomatic cases (12/24) and an asymptomatic food handler (co-infected with rotavirus A,1/7) and all water samples (7/7). The norovirus strain was identified as GII.9[P7] based on phylogenetic analysis, with 100% nucleotide sequence identity among the clinical cases and water samples, implying that the causative agent of the outbreak originates from contaminated drinking water. CONCLUSIONS: This study identified GII.9[P7] norovirus as the causative agent of this outbreak. This was the first reported waterborne outbreak of GII.9[P7] norovirus in China. Our study highlights the necessity of an integrated environmental and clinical case surveillance system for prevention and control of norovirus-associated gastroenteritis outbreak.

The Pearl River Delta (PRD) region in southern China is a densely populated area and a hotspot for Vibrio parahaemolyticus infections. However, systematic research on this pathogen, particularly comparing clinical and environmental strains, remains limited. This study analyzed the molecular epidemiology and antimicrobial resistance of 200 V. parahaemolyticus isolates from 12 cities in the PRD region from 2022 to 2023. The results indicated that the most prevalent serotypes were O3:K6 (39.5 %) and O10:K4 (27.5 %), predominantly found in clinical isolates. Most clinical isolates exhibited the characteristics of toxRS/new+, tdh + , and trh- , along with the sequence type 3 (ST3), while environmental isolates did not possess these genetic markers. Antimicrobial susceptibility testing showed that although clinically recommended antibiotics remain effective, some isolates have exhibited resistance, with environmental isolates displaying higher rates of antimicrobial resistance than clinical isolates. Moreover, a total of 26 antibiotic resistance genes (ARGs) associated with 10 antibiotic categories were identified, showing variations in distribution patterns among isolates from different sources. Phylogenetic analysis indicated that clinical isolates formed a distinct lineage, contrasting with the greater diversity observed in environmental isolates. Whole-genome analysis further revealed significant differences in pathogenicity- related genes between the two groups, with genes associated with biofilm formation and antimicrobial resistance being more commonly found in environmental isolates. These findings underscore the genetic variability and distinct patterns of antimicrobial resistance between clinical and environmental V. parahaemolyticus strains, highlighting the need for ongoing surveillance and targeted interventions to effectively address foodborne illnesses.

ABSTRACT Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009–2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance. IMPORTANCE Salmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Background: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. Objective: We sought to design ASOs that can block SARSCoV-2 by downregulating ACE2 in human airway. Methods: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. Results: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, , ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. Conclusions: ACE2-targeting ASOs can effectively block SARSCoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high- risk populations. (J Allergy Clin Immunol 2024;154:1044-59.)

Objective: To understand the serotype distribution, drug resistance and molecular characterization of invasive non-typhoid Salmonella (iNTS) in Guangdong Province from 2018 to 2022 and provide scientific evidence for the prevention and treatment of blood flow infection caused by Salmonella. Methods: Serological identification, antimicrobial susceptibility testing, multilocus sequence typing (MLST), and whole genome sequencing were performed on Salmonella isolated from blood and stool samples in Guangdong from 2018 to 2022. Simultaneously, annotated the sequencing results for drug resistance genes and virulence factors by a microbial gene annotation system. Results: The 136 iNTS strains were divided into 25 serotypes, and Salmonella enteritidis accounted for 38.24% (52/136). The OR of other iNTS serotypes were calculated with Salmonella typhimurium as the control. The OR values of Oreninburg, Rysson, and Pomona serotypes were the highest, which were 423.50, 352.92, and 211.75, respectively. The drug resistance rate of iNTS was 0.74%-66.91%, which was lower than that of non-iNTS (3.90%-77.21%). The main iNTS of drug resistance were ampicillin and tetracycline, with resistance rates of 66.91% (91/136) and 50.00% (68/136), respectively, while the resistance rates to ciprofloxacin (5.88%,8/136), ceftazidime (5.88%,8/136), gentamicin (5.13%,7/136) and cefoxitin (0.74%, 1/136) were relatively low. iNTS carried a variety of drug-resistance genes and virulence factors, but no standard virulence factor distribution has been found. MLST cluster analysis showed that iNTS was divided into 26 sequence types, and ST11 accounted for 38.24% (52/136). Conclusions: The iNTS strains in Guangdong were dominated by Salmonella enteritidis, of which three serotypes, Oreninburg, Rison, and Pomona, may be associated with a higher risk of invasive infection during 2018 to 2022. iNTS was sensitive to clinical first-line therapeutic drugs (cephalosporins and fluoroquinolones), with highly diverse sequences and clear phylogenetic branches. ST11 was the local dominant clone group.

Objective:This study aimed to understand the epidemic status and phylogenetic relationships of rotavirus group A (RVA) in the Pearl River Delta region of Guangdong Province, China.Methods:This study included individuals aged 28 days-85 years. A total of 706 stool samples from patients with acute gastroenteritis collected between January 2019 and January 2020 were analyzed for 17 causative pathogens, including RVA, using a Gastrointestinal Pathogen Panel, followed by genotyping, virus isolation, and complete sequencing to assess the genetic diversity of RVA.Results:The overall RVA infection rate was 14.59% (103/706), with an irregular epidemiological pattern. The proportion of co-infection with RVA and other pathogens was 39.81% (41/103). Acute gastroenteritis is highly prevalent in young children aged 0-1 year, and RVA is the key pathogen circulating in patients 6-10 months of age with diarrhea. G9P[8] (58.25%, 60/103) was found to be the predominant genotype in the RVA strains, and the 41 RVA-positive strains that were successfully sequenced belonged to three different RVA genotypes in the phylogenetic analysis. Recombination analysis showed that gene reassortment events, selection pressure, codon usage bias, gene polymorphism, and post-translational modifications (PTMs) occurred in the G9P[8] and G3P[8] strains.Conclusion:This study provides molecular evidence of RVA prevalence in the Pearl River Delta region of China, further enriching the existing information on its genetics and evolutionary characteristics and suggesting the emergence of genetic diversity. Strengthening the surveillance of genotypic changes and gene reassortment in RVA strains is essential for further research and a better understanding of strain variations for further vaccine development.

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies—YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

This study aimed to compare the sensitivity of two-phase separation and the filtration method using a mixed cellulose ester (MCE) membrane to detect enteroviruses in sewage samples. From December 2015 to July 2016, four domestic sewage samples (1 L/sample) were collected monthly from the Guangzhou Liede Sewage Treatment Plant, and each sewage sample was divided into two aliquots (500 mL). The sewage sample was concentrated using the two-phase separation and the filtration method using an MCE membrane, and the treated solutions were inoculated into cells for enterovirus isolation. Polymerase chain reaction amplification, VP1 sequencing, and enterovirus molecular typing were performed on the positive isolates. The detection rates of poliovirus (PV) and non-polio enterovirus (NPEV) obtained using the filtration method using an MCE membrane were higher than those using the two-phase separation method. McNemar's test showed that the detection rates of PV, NPEV, type 1 Sabin-like (SL1), type 2 Sabin-like (SL2), and type 3 Sabin-like (SL3) strain were not statistically significant (P > 0.05). In Guangdong Province, China, the detection rates for PV and NPEV were 53.13% and 62.50% (20/32), respectively. Twenty-seven PVs were isolated, three highly variable strains of the type 1 vaccine, with seven nucleotide substitutions in the VP1 region, compared with the type 1 Sabin strains. Eighty-seven strains of NPEV were isolated and nine serotypes were identified, among which coxsackievirus B3 (CVB3), echovirus 6(E6), and echovirus 11(E11) were the dominant strains. The filtration method using an MCE membrane is more sensitive than two-phase separation and can be used as a robust, sensitive, and cost-effective method to isolate enteroviruses from sewage.

Estuarine ecosystems undergo pronounced and intricate changes due to the mixing of freshwater and saltwater. Additionally, urbanization and population growth in estuarine regions result in shifts in the planktonic bacterial community and the accumulation of antibiotic resistance genes (ARGs). The dynamic changes in bacterial communities, environmental factors, and carriage of ARGs from freshwater to seawater, as well as the complex interrelationships among these factors, have yet to be fully elucidated. Here, we conducted a comprehensive study based on metagenomic sequencing and full-length 16S rRNA sequencing, covering the entire Pearl River Estuary (PRE) in Guangdong, China. The abundance and distribution of the bacterial community, ARGs, mobile genetic elements (MGEs), and bacterial virulence factors (VFs) were analyzed on a site-by-site basis through sampling along the salinity gradient in PRE, from upstream to downstream. The structure of the planktonic bacterial community undergoes continuous changes in response to variations in estuarine salinity, with the phyla Proteobacteria and Cyanobacteria being dominant bacterial throughout the entire region. The diversity and abundance of ARGs and MGEs gradually decreased with the direction of water flow. A large number of ARGs were carried by potentially pathogenic bacteria, especially in Alpha-proteobacteria and Beta-proteobacteria. Multi-drug resistance genes have the highest abundance and subtypes in PRE. In addition, ARGs are more linked to some MGEs than to specific bacterial taxa and disseminate mainly by HGT and not by vertical transfer in the bacterial communities. Various environmental factors, such as salinity and nutrient concentrations, have a significantly impact on the community structure and distribution of bacteria. In conclusion, our results represent a valuable resource for further investigating the intricate interplay between environmental factors and anthropogenic disturbances on bacterial community dynamics. Moreover, they contribute to a better understanding of the relative impact of these factors on the dissemination of ARGs.

Dear editor, We read with great interest the recent study by Cailard et al.1Caillard S. Laugel E. Benotmane I. Kremer S.F. Molecular evolution of the SARS-CoV-2 omicron BA.2 variant in kidney transplant recipients with prolonged viral shedding.J Infect. 2023; https://doi.org/10.1016/j.jinf.2023.02.001Abstract Full Text Full Text PDF Scopus (1) Google Scholar who reported the evolution of SARS-CoV-2 Omicron BA.2 variants in kidney transplant recipients (KTRs). They found that some KTRs could shed SARS-CoV-2 for a prolonged time and the viruses in these KTRs accumulated rare mutations which were associated with immune escape and symptom deterioration. Here, we describe a potential mechanism for the emergence of SARS-CoV-2 variants of concern (VOCs) that can rapidly spread globally, change clinical presentations, or decrease effectiveness of vaccines. Some of these VOCs, such as Alpha, Delta and Omicron, rapidly outcompeted previous variants with increased transmissibility.2Domingo P. de Benito N. Alpha variant SARS-CoV-2 infection: how it all starts.EBioMedicine. 2021; 74103703https://doi.org/10.1016/j.ebiom.2021.103703Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 3Callaway E. Delta coronavirus variant: scientists brace for impact.Nature. 2021; 595: 17-18https://doi.org/10.1038/d41586-021-01696-3Crossref PubMed Scopus (183) Google Scholar, 4Karim S.S.A. Karim Q.A. Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic.Lancet. 2021; 398: 2126-2128https://doi.org/10.1016/S0140-6736(21)02758-6Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar There was one epidemic spike of SARS-CoV-2 infection in Guangzhou city in China between May 21 and June 19 in 2021 (Fig. S1). Whole genome sequence analysis showed that these viruses were highly homogenous, with the majority of them (66%) identical to each other, suggesting that they were derived from a single source. Since these sequences formed a distinct cluster within the large Delta variant group, they were named Delta-x (Figs. S2-S3). Delta-x acquired 12 new signature mutations not commonly found in other variants (Fig. 1A). The mutations were randomly scattered across the whole viral genome (Fig. 1B). We analyzed all available 1,698,654 good quality sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database by May 21, 2021. All 12 mutations were at the high variable sites (Fig. 1B). These Delta-x signature mutations were among the top 12% of mutations that occurred more than 1,000 times except one at position 25,562 (Fig. 1B). At each site, there are three possible mutations. Interestingly, the frequencies of all 12 mutations were the highest among them (Fig. 1C). This suggests that Delta-x signature mutations are a unique combination of 12 highly mutable nucleotides. Examining the frequencies of all possible combinations of mutations showed that the vast majority (91.40%) of the sequences did not contain any of the Delta-x unique mutations. Among the 145,929 sequences carrying any Delta-x mutations, the sequences with one mutation were predominant (92.15%), while the sequences with 2, 5, or 6 mutations were more frequent than others (Fig. 1D). No sequences with more than nine signature mutations were detected. All predominant haplotypes for the sequences with the same number of mutations accounted for at least 35% of the population (Fig. 1E). The predominant haplotypes for the combinations of higher numbers of mutations (5−9) were nearly exclusive (93%−100%). These results suggest that the predominant haplotypes present at high frequency or exclusively are more viable than others. Interestingly, each predominant haplotype generally differed from that with one fewer mutation by gaining an additional mutation (Fig. 1E). The predominant haplotypes with the most detectable mutations (8 or 9) were rare, but each was the only haplotype detected in that population. The sequences containing eight or nine Delta-x mutations were found in different countries in four continents (Fig. S4), suggesting that the ancestors of Delta-x had been widely presented in different human populations. When compared to Delta and Alpha in cell culture, the Delta-x replicated at a delayed rate (Fig. 1F, S5). To more accurately compare their fitness, both Delta and Delta-x were cultured together. The Delta virus quickly outcompeted (88.2%) the Delta-x virus, demonstrating that Delta-x was less fit than Delta (Fig. 1G). When tested with the sera from the donors infected with original and Delta-x strains, all three viruses were similarly neutralized (Fig. 1H), indicating that both Delta-x and Delta have a similar neutralization profile. To investigate if signature mutations in other VOCs were also generated through the similar mechanism, we obtained good quality SARS-CoV-2 whole genome sequences (8,846,680) from GISAID by March 5, 2022. As seen with Delta-x, the majority of VOC signature mutations in all five VOCs (Alpha, Beta, Delta, Gamma, and Omicron) were also those with the highest mutation rates at their positions (Fig. 2). Thus, they were all generated through acquiring a set of the highest mutable nucleotides at the majority of signature mutation sites. For the earlier VOCs, combinations of signature mutations in the same viral genome were either not found (Beta and Alpha) or very rare (Delta and Gamma). However, many sequences with 9 or fewer Omicron signature mutations were found among 473,719 sequences. Examination of the predominant haplotypes for sequences with 2–9 mutations showed that those with higher numbers of mutations were also generated by gaining additional mutations, as observed for Delta-x (Fig. S6). Among 12 Delta-x mutations, 5 were synonymous mutations and only 1 (V1176F) was found in the end of the S gene (Table S1). Therefore, all those mutations should not be driven by neutralizing antibodies. This was in good agreement with the observation that no significant differences were observed between Delta and Delta-x. However, four mutations (Y1920H, V1176F, Q57R and L116F) in NSP3, S, ORF3a, and ORF7a, respectively, were found in the known CD8 restricted T cell epitopes.5Grifoni A. Sidney J. Vita R. Peters B. Crotty S. Weiskopf D. et al.SARS-CoV-2 human T cell epitopes: adaptive immune response against COVID-19.Cell Host Microbe. 2021; 29: 1076-1092https://doi.org/10.1016/j.chom.2021.05.010Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Thus, escaping from T cell immune responses may play a critical role in the generation of the Delta-x variant. The majority of Delta-x signature mutations (11 of 12) were transition mutations (A:G or T:C), which are favored by the ExoN defective viruses.6Smith E.C. Blanc H. Surdel M.C. Vignuzzi M. Denison M.R. Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.PLOS Pathog. 2013; 9e1003565https://doi.org/10.1371/journal.ppat.1003565Crossref Scopus (290) Google Scholar, 7Tian D. Sun Y. Xu H. Ye Q. The emergence and epidemic characteristics of the highly mutated SARS-CoV-2 Omicron variant.J Med Virol. 2022; 94: 2376-2383https://doi.org/10.1002/jmv.27643Crossref PubMed Scopus (171) Google Scholar Similar results were also observed for other VOCs. Therefore, accumulation of these mutable nucleotides is likely an intrinsic property of SARS-CoV-2 when the function of the proofreading enzyme ExoN is affected. Our study also confirmed that the high transmissibility of VOCs is not necessarily associated with their replication capacity and pathogenicity.8Karim F. Moosa M. Gosnell B. Cele S. Giandhari J. Pillay S. et al.Persistent SARS-CoV-2 infection and intra-host evolution in association with advanced HIV infection.medRxiv. 2021; https://doi.org/10.1101/2021.06.03.21258228Crossref Scopus (0) Google Scholar, 9Shuai H. Chan J.F. Hu B. Chai Y. Yuen T.T. Yin F. et al.Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron.Nature. 2022; 603: 693-699https://doi.org/10.1038/s41586-022-04442-5Crossref PubMed Scopus (255) Google Scholar, 10Chen J. Pathogenicity and transmissibility of 2019-nCoV-A quick overview and comparison with other emerging viruses.Microbes Infect. 2020; 22: 69-71https://doi.org/10.1016/j.micinf.2020.01.004Crossref PubMed Scopus (505) Google Scholar Understanding of the mechanisms of generation of new VOCs can have important implications. Since the accumulation of highly mutable nucleotides in the SARS-CoV-2 genome may be the intrinsic property of its replication-associated enzymes, the new combinations of these mutable nucleotides are more likely to produce viable variants with higher transmissibility. This may explain why new VOCs constantly emerge and rapidly replace the previous variants.2Domingo P. de Benito N. Alpha variant SARS-CoV-2 infection: how it all starts.EBioMedicine. 2021; 74103703https://doi.org/10.1016/j.ebiom.2021.103703Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 3Callaway E. Delta coronavirus variant: scientists brace for impact.Nature. 2021; 595: 17-18https://doi.org/10.1038/d41586-021-01696-3Crossref PubMed Scopus (183) Google Scholar, 4Karim S.S.A. Karim Q.A. Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic.Lancet. 2021; 398: 2126-2128https://doi.org/10.1016/S0140-6736(21)02758-6Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar Highly mutable nucleotides, especially those at known biological function domains or epitopes targeted by immune responses, should be closely monitored. Furthermore, modeling of such highly mutable nucleotides may be used to predict new VOCs in the future. This work was supported by the National Key Research and Development Program of China (Grant Nos. 2021YFC2301500 and 2021YFC0863300), the Project of Medicine Discipline of Guangzhou (2021-2023-11).

Polio cases can be missed by acute flaccid paralysis (AFP) case surveillance alone, emphasizing the importance of environmental surveillance (ES). In this study, to investigate the serotype distribution and epidemiological trends of poliovirus (PV), we characterized PV isolated from domestic sewage in Guangzhou City, Guangdong Province, China from 2009 to 2021. A total of 624 sewage samples were collected from the Liede Sewage Treatment Plant, and the positive rates of PV and non-polio enteroviruses were 66.67% (416/624) and 78.37% (489/624), respectively. After sewage sample treatment, each sewage sample was inoculated in six replicate tubes of three cell lines, and 3370 viruses were isolated during the 13-year surveillance period. Among these, 1086 isolates were identified as PV, including type 1 PV (21.36%), type 2 PV (29.19%), and type 3 PV (49.48%). Based on VP1 sequences, 1057 strains were identified as Sabin-like, 21 strains were high-mutant vaccines, and eight strains were vaccine-derived poliovirus (VDPV). The numbers and serotypes of PV isolates in sewage were influenced by the vaccine switch strategy. After type 2 OPV was removed from the trivalent oral PV (OPV) vaccine and a bivalent OPV (bOPV) was adopted in May 2016, the last type 2 PV strain was isolated from sewage, with no detection thereafter. Type 3 PV isolates increased significantly and became the dominant serotype. Before and after the second vaccine switch in January 2020, that is, from the first dose of IPV and second-fourth doses of bOPV to the first two doses of IPV and third-fourth doses of bOPV, there was also a statistical difference in PV positivity rates in sewage samples. Seven type 2 VDPVs and one type 3 VDPV were identified in sewage samples in 2009-2021, and phylogenetic analysis indicated that all VDPVs isolated from ES in Guangdong are newly discovered VDPVs, different from VDPV previously discovered in China, and were classified as ambiguous VDPV. It is noteworthy that no VDPV cases were reported in AFP case surveillance in the same period. In conclusion, continued PV ES in Guangzhou since April 2008 has been a useful supplement to AFP case surveillance, providing an important basis for evaluating the effectiveness of vaccine immunization strategies. ES improves early detection, prevention, and control; accordingly, this strategy can curb the circulation of VDPVs and provide a strong laboratory basis for maintaining a polio-free status.

Objective:To analyze the evolutionary characteristics and variation of etiological agent in an acute hemorrhagic conjunctivitis (AHC) outbreak in a city of Guangdong province in May, so as to provide scientific basis for formulating a new round of measures for prevention and control of AHC epidemic.Methods:In this study, 20 conjunctival swabs were collected from AHC patients, and enterovirus, human enterovirus 70 (HEV70) and coxsackievirus A 24 variant (CVA24v) nucleic acids were detected by real-time fluorescence quantitative PCR. In addition, the VP1 and 3Cpro regions of the CVA24v positive samples were sequenced to analyze their evolutionary relationship with the CVA24v strains circulating in China and abroad.Results:All the 20 eye swab samples were EV-positive, and CVA24v-positive, with a positive rate of 100.00%, and all were HEV70-negative.The genomes of CVA24v in VP1 and 3Cpro regions of CVA24v in 5 and 7 samples were successfully sequenced. Based on molecular characterization analysis of VP1 and 3Cpro regions, it was found that the CVA24v isolated in this outbreak had the greatest nucleotide similarity with the CVA24v strains isolated in Thailand in 2014 and French Reunion Islands in 2015. The phylogenetic analysis of 3Cpro and VP1 regions showed that the CVA24v isolated in this outbreak is clustered together with the CVA24v that was prevalent in Thailand in 2014 and the French Reunion Islands in 2015, and have high affinity. Compared with CVA24v isolated in Guangdong in 2010, Thailand in 2014, and French Reunion Islands in 2015, CVA24v isolated in this outbreak was replaced at 4 amino acid sites in 3Cpro region and 1 amino acid site in VP1 region.Conclusions:The cause of this outbreak is enterovirus CVA24v, which has the highest similarity to CVA24v isolated in Thailand in 2014 and in the French Reunion Islands in 2015. There were new amino acid mutations in both 3Cpro and VP1 regions.

This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed.

The banning of colistin as a feed additive for food-producing animals in mainland China in 2017 caused the decline in the prevalence of Escherichia coli-mobilized colistin resistance (mcr-1) in China. Salmonella Typhimurium and its monophasic 1,4,[5],12:i:- variants are also the main species associated with the spread of mcr-1; however, the evidence of the prevalence and transmission of mcr-1 among Salmonella is lacking. Herein, the 5,354 Salmonella isolates recovered from fecal samples of diarrheal patients in Guangdong, Southern China, from 2009 to 2019 were screened for colistin resistance and mcr-1, and mcr-1-positive isolates were characterized based on whole-genome sequencing (WGS) data. Relatively high prevalence rates of colistin resistance and mcr-1 (4.05%/4.50%) were identified, and more importantly, the prevalence trends of colistin-resistant and mcr-1-positive Salmonella isolates had a similar dynamic profile, i.e., both were first detected in 2012 and rapidly increased during 2013 to 2016, followed by a sharp decrease since 2017. WGS and phylogenetic analysis indicate that, whether before or after the ban, the persistence and cross-hospital transmission of mcr-1 are primarily determined by IncHI2 plasmids with similar backbones and sequence type 34 (ST34) Salmonella in specific clades that are associated with a high prevalence of IncHI2 plasmids and clinically important antimicrobial resistance genes, including blaCTX-M-14-fosA3-oqxAB-floR genotypes. Our work reveals the difference in the prevalence rate of mcr-1 in clinical Salmonella before and after the Chinese colistin ban, whereas mcr-1 transmission was closely linked to multidrug-resistant IncHI2 plasmid and ST34 Salmonella across diverse hospitals over 10 years. Continued surveillance is required to explore the factors related to a sharp decrease in mcr-1 after the recent ban and determine whether the ban has affected the carriage of mcr-1 in Salmonella circulating in the health care system. IMPORTANCE Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae. However, the emergence of the mobilized colistin resistance (mcr-1) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting. In this study, we investigated the existence of mcr-1 in clinical Salmonella from a 10-year continuous surveillance and genomic study. Overall, the colistin resistance rate and mcr-1 carriage of Salmonella in tertiary hospitals in Guangdong (2009 to 2019) were relatively high and, importantly, rapidly increased from 2013 to 2016 and significantly decreased after the Chinese colistin withdrawal. However, before or after the ban, the MDR IncHI2 plasmid with a similar backbone and ST34 Salmonella were the main vectors involved in the spread of mcr-1. Interestingly, these Chinese mcr-1-carrying Salmonella obtain phylogenetically and phylogeographically distinct patterns compared with those from other continents and are frequently associated with clinically important ARGs including the extended-spectrum β-lactamases. Our data confirmed that the national stewardship intervention seems to be successful in blocking antibiotic resistance determinants and that continued surveillance of colistin resistance in clinical settings, farm animals, and related products is necessary.

Toll-like receptor 4 (TLR4) is a reliable target for the development of vaccine adjuvants. To identify novel TLR4 ligands with improved immunological properties for use as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine, herein, natural E. coli monophosphoryl lipid A (MPLA) and nine of its derivatives were designed and synthesized. Immunological evaluation showed that compounds 1, 3, 5 and 7 exhibited comparative or better adjuvant activity than clinically used Al adjuvants, and are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine. Preliminary structure-activity relationship analysis of the MPLA derivatives showed that the replacement of the functional groups at the C-1, C-4' or C-6' position of E. coli MPLA has an effect on its biological activity. In addition, we found that the combination of MPLA and Al was feasible for immunotherapy and could further enhance immune responses, providing a new direction toward the immunological enhancement of RBD-hFc based SARS-CoV-2 vaccines.

ObjectiveTo study the epidemic and genetic characteristics of coxsackievirus A8 (CVA8) in Guangdong province. MethodsFrom all specimens sampled in hand, foot, and mouth disease (HFMD) cases reported during 2010 – 2020 in Guangdong province, we collected those of other enterovirus (EV) – non-EV71/CVA16/CVA6. After amplified VP1 gene based-genotype identification, CVA8 strains were isolated from the specimens using rhabdomyosarcoma (RDa) cells. The entire VP1 region of the isolated CVA8 strains were amplified and sequenced and then phylogenetically analyzed using DNASTAR6.0 and MEGA6.06 software packages. ResultsOf totally 2650 other EV specimens collected during the period, 2.6% (69) were positive for CVA8 and the annual ratio of CVA8 positive specimens ranged from 0.5% to 12.0%, with the highest ratio in 2020. The CVA8 positive specimens were detected in 13 cities of the province and the highest clustering of positive specimens (28) was detected in Shantou city during July – November 2020. CVA8 strains were isolated from 73.9% (51) of the positive specimens. The phylogenetic analysis results indicated that CVA8 could be divided into five genotypes: A, B, C, D and E. The 44 CVA8 strains isolated in this study shared 77.8% – 100.0% homologies in nucleotide and 92.9% – 100.0% in amino acid, including genotype D (5 strains) and genotype E (39 strains), and the nucleotide homology of 28 CVA8 strains isolated in Shantou city during July – November 2020 was 99.3% – 100.0%. ConclusionCVA8 was prevalent sporadically in Guangdong province during 2010 – 2020 generally but highly prevalence among local HFMD cases in Shantou city during July – November 2020. CVA8 strains of genotype D and E were prevalent and strains of genotype E are currently prevalent in Guangdong province.

Salmonella is widely known as one of the most common foodborne pathogens, and antibiotics remain effective in clinical therapies against its infections. To guide better clinical antibiotic treatment, we analyzed the antimicrobial resistance (AMR) profiles of a nation-wide collection of 36,822 Salmonella isolates derived from sporadic diarrhea cases in China from 2014 to 2021. A panel of 15 antibiotics, including 10 critically important and 5 highly important antimicrobial agents for human medicine based on the WHO CIA List, was selected for AMR surveillance. Salmonella enterica serovar Typhimurium, Enteritidis, I 4,[5],12:i:-, London, and Stanley were turns to be the top five serotypes from human diarrhea cases in China. Antimicrobial susceptibility testing revealed that the majority of the isolates (87.2%) were resistant to at least one antimicrobial agent, and 66.5% were multidrug resistant (MDR). Salmonella isolates were found to be highly resistant to ampicillin (73.4%) while sensitive to imipenem (98.73%). Over the eight years, the isolates were demonstrated generally an increase in resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and azithromycin, but displayed varied resistance profiles in terms of serotypes. Stanley (0.66–27.18%) and Agona (0.78–45.30%) had lower resistance rates compared to Typhimurium (1.11–85.6%), Enteritidis (1.55–91.29%), and I 4,[5],12:i:- (1.02–94.28%). In conclusion, our results provide systematic data on the resistance characteristics of Salmonella isolates from human diarrhea cases in China. Furthermore, this data identifies priorities for the clinical treatments of antibiotics.

Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae . However, the emergence of the mobilized colistin resistance ( mcr-1 ) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting.

Abstract Currently circulating SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading acute-infection-induced germline antibodies isolated earlier in the pandemic. We identified that memory B cells from Delta variant breakthrough-infection patients expressed antibodies with more extensive somatic hypermutations (SHMs) allowing isolation of a number of broadly neutralizing antibodies with activities against heterologous variants of concerns (VOCs) including Omicron variant. Structural studies identified that SHM introduced altered amino acids and highly unusual HCDR2 insertions respectively in two representative broadly neutralizing antibodies - YB9-258 and YB13-292. Previously, insertion/deletion were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. Identified SHMs involved heavily in epitope recognition, they broadened neutralization breadth by rendering antibodies resistant to VOC mutations highly detrimental to previously isolated antibodies targeting similar epitopes. These data provide molecular mechanisms for enhanced immunity to heterologous SARS-CoV-2 variants after repeated antigen exposures with implications for future vaccination strategy.