Prevalence of Non-Polio Enteroviruses in the Sewage of Guangzhou City, China, from 2013 to 2021.

Continuous surveillance of enteroviruses (EVs) in urban domestic sewage can timely reflect the circulation of EVs in the environment and crowds, and play a predictive and early warning role in EV-related diseases. To better understand the long-term epidemiological trends of circulating EVs and EV-related diseases, we conducted a 9-year (2013 to 2021) surveillance study of non-polio EVs (NPEVs) in urban sewage in Guangzhou city, China. After concentrating and isolating the viruses from the sewage samples, NPEVs were detected and molecular typing was performed. Twenty-one different NPEV serotypes were identified. The most isolated EVs were echovirus 11 (E11), followed by coxsackievirus (CV) B5, E6, and CVB3. EV species B prevailed in sewage samples, but variations in the annual frequency of different serotypes were also observed in different seasons, due to spatial and temporal factors. E11 and E6 were detected continuously before 2017, and the number of isolates was relatively stable during the surveillance period. However, after their explosive growth in 2018 and 2019, their numbers suddenly decreased significantly. CVB3 and CVB5 had alternating trends; CVB5 was most frequently detected in 2013 to 2014 and 2017 to 2018, while CVB3 was most frequently detected in 2015 to 2016 and 2020 to 2021. Phylogenetic analysis showed that at least two different transmission chains of CVB3 and CVB5 were prevalent in Guangzhou City. Our results show that in the absence of a comprehensive and systematic EV-related disease surveillance system in China, environmental surveillance is a powerful and effective tool to strengthen and further investigate the invisible transmission of EVs in the population. IMPORTANCE 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. We detected 21 different non-polio enteroviruses (NPEVs) with yearly variations in prevalence and peak seasons. In addition, this study is very important for understanding the epidemiology of EVs during the COVID-19 pandemic, as the detection frequency and serotypes of EVs in sewage changed considerably around 2020. We believe that our study makes a significant contribution to the literature because our results strongly suggest that environmental surveillance is an exceptionally important tool, which can be employed to detect and monitor organisms of public health concern, which would otherwise be missed and under-reported by case-based surveillance systems alone.

A Synthetic SARS-CoV-2-Derived T-Cell and B-Cell Peptide Cocktail Elicits Full Protection against Lethal Omicron BA.1 Infection in H11-K18-hACE2 Mice.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developing the capacity for immune evasion and resistance to existing vaccines and drugs. To address this, development of vaccines against coronavirus disease 2019 (COVID-19) has focused on universality, strong T cell immunity, and rapid production. Synthetic peptide vaccines, which are inexpensive and quick to produce, show low toxicity, and can be selected from the conserved SARS-CoV-2 proteome, are promising candidates. In this study, we evaluated the effectiveness of a synthetic peptide cocktail containing three murine CD4+ T-cell epitopes from the SARS-CoV-2 nonspike proteome and one B-cell epitope from the Omicron BA.1 receptor-binding domain (RBD), along with aluminum phosphate (Al) adjuvant and 5' cytosine-phosphate-guanine 3' oligodeoxynucleotide (CpG-ODN) adjuvant in mice. The peptide cocktail induced good Th1-biased T-cell responses and effective neutralizing-antibody titers against the Omicron BA.1 variant. Additionally, H11-K18-hACE2 transgenic mice were fully protected against lethal challenge with the BA.1 strain, with a 100% survival rate and reduced pulmonary viral load and pathological lesions. Subcutaneous administration was found to be the superior route for synthetic peptide vaccine delivery. Our findings demonstrate the effectiveness of the peptide cocktail in mice, suggesting the feasibility of synthetic peptide vaccines for humans. IMPORTANCE Current vaccines based on production of neutralizing antibodies fail to prevent the infection and transmission of SARS-CoV-2 Omicron and its subvariants. Understanding the critical factors and avoiding the disadvantages of vaccine strategies are essential for developing a safe and effective COVID-19 vaccine, which would include a more effective and durable cellular response, minimal effects of viral mutations, rapid production against emerging variants, and good safety. Peptide-based vaccines are an excellent alternative because they are inexpensive, quick to produce, and very safe. In addition, human leukocyte antigen T-cell epitopes could be targeted at robust T-cell immunity and selected in the conserved region of the SARS-CoV-2 variants. Our study showed that a synthetic SARS-CoV-2-derived peptide cocktail induced full protection against lethal infection with Omicron BA.1 in H11-K18-hACE2 mice for the first time. This could have implications for the development of effective COVID-19 peptide vaccines for humans.

A Beta Strain-Based Spike Glycoprotein Vaccine Candidate Induces Broad Neutralization and Protection against SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) are circulating worldwide, making it resistant to existing vaccines and antiviral drugs. Therefore, the evaluation of variant-based expanded spectrum vaccines to optimize the immune response and provide broad protectiveness is very important. In this study, we expressed spike trimer protein (S-TM) based on the Beta variant in a GMP-grade workshop using CHO cells. Mice were immunized twice with S-TM protein combined with aluminum hydroxide (Al) and CpG Oligonucleotides (CpG) adjuvant to evaluate its safety and efficacy. BALB/c immunized with S-TM + Al + CpG induced high neutralizing antibody titers against the Wuhan-Hu-1 strain (wild-type, WT), the Beta and Delta variants, and even the Omicron variant. In addition, compared with the S-TM + Al group, the S-TM + Al + CpG group effectively induced a stronger Th1-biased cell immune response in mice. Furthermore, after the second immunization, H11-K18 hACE2 mice were well protected from challenge with the SARS-CoV-2 Beta strain, with a 100% survival rate. The virus load and pathological lesions in the lungs were significantly reduced, and no virus was detected in mouse brain tissue. Our vaccine candidate is practical and effective for current SARS-CoV-2 VOCs, which will support its further clinical development for potential sequential immune and primary immunization. IMPORTANCE Continuous emergence of adaptive mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge the use and development of existing vaccines and drugs. The value of variant-based vaccines that are capable of inducing a higher and broader protection immune response against SARS-CoV-2 variants is currently being evaluated. This article shows that a recombinant prefusion spike protein based on a Beta variant was highly immunogenic and could induced a stronger Th1-biased cell immune response in mice and was effectively protective against challenge with the SARS-CoV-2 Beta variant. Importantly, this Beta-based SARS-CoV-2 vaccine could also offer a robust humoral immune response with effectively broad neutralization ability against the wild type and different variants of concern (VOCs): the Beta, Delta, and Omicron BA.1 variants. To date, the vaccine described here has been produced in a pilot scale (200L), and the development, filling process, and toxicological safety evaluation have also been completed, which provides a timely response to the emerging SARS-CoV-2 variants and vaccine development.

A highly efficient needle-free-injection delivery system for mRNA-LNP vaccination against SARS-CoV-2.

Despite the various vaccines that have been developed to combat the coronavirus disease 2019 (COVID-19) pandemic, the persistent and unpredictable mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require innovative and unremitting solutions to cope with the resultant immune evasion and establish a sustainable immune barrier. Here we introduce a vaccine-delivery system with a combination of a needle-free injection (NFI) device and a SARS-CoV-2-Spike-specific mRNA-Lipid Nanoparticle (LNP) vaccine. The benefits are duller pain and a significant increase of immunogenicity compared to the canonical needle injection (NI). From physicochemical and bioactivity analyses, the structure of the mRNA-LNP maintains stability upon NFI, contradictory to the belief that LNPs are inclined towards destruction under the high-pressure conditions of NFI. Moreover, mRNA-LNP vaccine delivered by NFI induces significantly more binding and neutralizing antibodies against SARS-CoV-2 variants than the same vaccine delivered by NI. Heterogeneous vaccination of BA.5-LNP vaccine with NFI enhanced the generation of neutralizing antibodies against Omicron BA.5 variants in rabbits previously vaccinated with non-BA.5-specific mRNA-LNP or other COVID-19 vaccines. NFI parameters can be adjusted to deliver mRNA-LNP subcutaneously or intramuscularly. Taken together, our results suggest that NFI-based mRNA-LNP vaccination is an effective substitute for the traditional NI-based mRNA-LNP vaccination.

Highly diverse ribonucleic acid viruses in the viromes of eukaryotic host species in Yunnan province, China.

Background: The diversity in currently documented viruses and their morphological characteristics indicates the need for understanding the evolutionary characteristics of viruses. Notably, further studies are needed to obtain a comprehensive landscape of virome, the virome of host species in Yunnan province, China. Materials and methods: We implemented the metagenomic next-generation sequencing strategy to investigate the viral diversity, which involved in 465 specimens collected from bats, pangolins, monkeys, and other species. The diverse RNA viruses were analyzed, especially focusing on the genome organization, genetic divergence and phylogenetic relationships. Results: In this study, we investigated the viral composition of eight libraries from bats, pangolins, monkeys, and other species, and found several diverse RNA viruses, including the Alphacoronavirus from bat specimens. By characterizing the genome organization, genetic divergence, and phylogenetic relationships, we identified five Alphacoronavirus strains, which shared phylogenetic association with Bat-CoV-HKU8-related strains. The pestivirus-like virus related to recently identified Dongyang pangolin virus (DYPV) strains from dead pangolin specimens, suggesting that these viruses are evolving. Some genomes showed higher divergence from known species (e.g., calicivirus CS9-Cali-YN-CHN-2020), and many showed evidence of recombination events with unknown or known strains (e.g., mamastroviruses BF2-astro-YN-CHN-2020 and EV-A122 AKM5-YN-CHN-2020). The newly identified viruses showed extensive changes and could be assigned as new species, or even genus (e.g., calicivirus CS9-Cali-YN-CHN-2020 and iflavirus Ifla-YN-CHN-2020). Moreover, we identified several highly divergent RNA viruses and estimated their evolutionary characteristics among different hosts, providing data for further examination of their evolutionary dynamics. Conclusion: Overall, our study emphasizes the close association between emerging viruses and infectious diseases, and the need for more comprehensive surveys.

Preclinical immunological evaluation of an intradermal heterologous vaccine against SARS-CoV-2 variants.

The recent emergence of COVID-19 variants has necessitated the development of new vaccines that stimulate the formation of high levels of neutralizing antibodies against S antigen variants. A new strategy involves the intradermal administration of heterologous vaccines composed of one or two doses of inactivated vaccine and a booster dose with the mutated S1 protein (K-S). Such vaccines improve the immune efficacy by increasing the neutralizing antibody titers and promoting specific T cell responses against five variants of the RBD protein. A viral challenge test with the B.1.617.2 (Delta) variant confirmed that both administration schedules (i.e. "1 + 1" and "2 + 1") ensured protection against this strain. These results suggest that the aforementioned strategy is effective for protecting against new variants and enhances the anamnestic immune response in the immunized population.

Antibody Response to COVID-19 Virus - Heilongjiang Province and Gansu Province, China, 2020.

WHAT IS ALREADY KNOWN ABOUT THIS TOPIC?: Coronavirus disease 2019 (COVID-19) has become a global pandemic, while the profile of antibody response against the COVID-19 virus has not been well clarified. WHAT IS ADDED BY THIS REPORT?: In this study, 210 serum samples from 160 confirmed COVID-19 cases with different disease severities were recruited. The IgM, IgA, IgG, and neutralizing antibodies (NAb) against COVID-19 virus were determined. Our findings indicated that four antibodies could be detectable at low levels within 2 weeks of disease onset, then rapidly increasing and peaking from the 3rd to 5th Weeks. NAb decreased between 5th and 9th Weeks, and a higher IgM/IgA level was observed in the groups with mild/moderate severity within 2 weeks (p<0.05), while all 4 types of antibodies were higher in the group with severe/critical severity after 4 weeks (p<0.05). WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: Our study on the dynamics of serological antibody responses against COVID-19 virus among COVID-19 patients complements the recognition regarding the humoral immune response to COVID-19 virus infection. The findings will help in the interpretation of antibody detection results for COVID-19 patients and be beneficial for the evaluation of vaccination effects.

COVID-19 Cases from the First Local Outbreak of the SARS-CoV-2 B.1.1.7 Variant in China May Present More Serious Clinical Features: A Prospective, Comparative Cohort Study.

The SARS-CoV-2 B.1.1.7 variant has increased sharply in numbers worldwide and is reported to be more contagious than the nonvariant. Little is known regarding the detailed clinical features of B.1.1.7 variant infection. Data on 74 COVID-19 cases from two outbreaks in two districts of Beijing, China were extracted from a cloud database, including 41 cases from Shunyi District (Shunyi B.1.470 group) and 33 from Daxing (Daxing B.1.1.7 group) from December 25, 2020 to January 17, 2021. We conducted a comparison of the clinical characteristics. Seven clinical indicators of the Daxing B.1.1.7 group were significantly higher than those of the Shunyi group, including the proportion with fever over 38°C, the levels of C-reactive protein (CRP), serum amyloid A (SAA), creatine kinase (CK), d-dimer (DD), and CD4+ T lymphocytes (CD4+ T), and the proportion with ground-glass opacity (GGO) in the lung (P values of ≤0.05). After adjusting for age, B.1.1.7 variant infection was a risk factor for elevated CRP (P = 0·045), SAA (P = 0·011), CK (P = 0·034), and CD4+ T (P = 0.029) and for the presence of GGO (P = 0.005). The median threshold cycle (CT) value of reverse transcriptase quantitative PCR (RT-qPCR) tests of the N gene target in the Daxing B.1.1.7 group was significantly lower (P = 0.036) than that in the Shunyi B.1.470 group. Clinical features, including a more serious inflammatory response, pneumonia, and a possibly higher viral load, were detected in the cases infected with B.1.1.7 SARS-CoV-2. The B.1.1.7 variant may have increased pathogenicity. IMPORTANCE The SARS-CoV-2 B.1.1.7 variant, which was first identified in the United Kingdom, has increased sharply in numbers worldwide and was reported to be more contagious than the nonvariant. To our knowledge, no studies investigating the detailed clinical features of COVID-19 cases infected with the B.1.1.7 variant have been published. Local epidemics have rarely occurred in China, but occasionally, a small clustered outbreak triggered by an imported SARS-CoV-2 strain with only one chain of transmission could happen. From late 2020 to early 2021, two clustered COVID-19 outbreaks occurred in Beijing, one of which was caused by the B.1.1.7 variant. The COVID-19 patients from the two outbreaks received similar clinical tests, diagnoses, and treatments. We found that the B.1.1.7 variant infection could lead to a more serious inflammatory response, acute response process, more severe pneumonia, and probably higher viral loads. This therefore implies that the B.1.1.7 variant may have increased pathogenicity.

Long Distance Transmission of SARS-CoV-2 from Contaminated Cold Chain Products to Humans - Qingdao City, Shandong Province, China, September 2020.

What is already known about this topic? Though coronavirus disease 2019 (COVID-19) has largely been controlled in China, several outbreaks of COVID-19 have occurred from importation of cases or of suspected virus-contaminated products. Though several outbreaks have been traced to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated on the outer packaging of cold chain products, live virus has not been obtained. What is added by this report? In September 2020, two dock workers were detected as having asymptomatic SARS-CoV-2 infection using throat swabs during routine screening in Qingdao, China. Epidemiological information showed that the two dock workers were infected after contact with contaminated outer packaging, which was confirmed by genomic sequencing. Compared to the Wuhan reference strain, the sequences from the dock workers and the package materials differed by 12-14 nucleotides. Furthermore, infectious virus from the cold chain products was isolated by cell culture, and typical SARS-CoV-2 particles were observed under electron microscopy. What are the implications for public health practice? The international community should pay close attention to SARS-CoV-2 transmission mode through cold chain, build international cooperative efforts in response, share relevant data, and call on all countries to take effective prevention and control measures to prevent virus contamination in cold-chain food production, marine fishing and processing, transportation, and other operations.

COVID-19 Outbreak Caused by Contaminated Packaging of Imported Cold-Chain Products - Liaoning Province, China, July 2020.

What is known about this topic? Few major outbreaks of coronavirus disease 2019 (COVID-19) have occurred in China after major non-pharmaceutical interventions and vaccines have been deployed and implemented. However, sporadic outbreaks that had high possibility to be linked to cold chain products were reported in several cities of China.. What is added by this report? In July 2020, a COVID-19 outbreak occurred in Dalian, China. The investigations of this outbreak strongly suggested that the infection source was from COVID-19 virus-contaminated packaging of frozen seafood during inbound unloading personnel contact. What are the implications for public health practice? Virus contaminated paper surfaces could maintain infectivity for at least 17-24 days at -25 ℃. Exposure to COVID-19 virus-contaminated surfaces is a potential route for introducing the virus to a susceptible population. Countries with no domestic transmission of COVID-19 should consider introducing prevention strategies for both inbound travellers and imported goods. Several measures to prevent the introduction of the virus via cold-chain goods can be implemented.

Excretion of SARS-CoV-2 through faecal specimens.

Coronavirus disease 2019 (COVID-19) has become a pandemic with increasing numbers of cases worldwide. SARS-CoV-2, the causative virus of COVID-19, is mainly transmitted through respiratory droplets or through direct and indirect contact with an infected person. The possibility of potential faecal-oral transmission was investigated in this study. We collected 258 faecal specimens from nine provinces in China and detected the nucleic acid of SARS-CoV-2 using real-time RT-PCR. Vero cells were used to isolate the virus from SARS-CoV-2 nucleic acid positive samples, after which sequencing of Spike gene in eight samples was performed. In all, 93 of 258 (36%) stool samples were positive for SARS-CoV-2 RNA. The positive rates of critical, severe, moderate, and mild patients were 54.4%, 56.1%, 30.8%, and 33.3%, respectively. The content of nucleic acid increased within 2 weeks after the onset of the disease. From the perspective of clinical typing, the nucleic acid can be detected in the faeces of critical patients within two weeks and until four to five weeks in the faeces of severe and mild patients. SARS-CoV-2 was isolated from stool specimens of two severe patients. Four non-synonymous mutations in Spike gene were newly detected in three stool samples. A small number of patients had strong faecal detoxification ability. The live virus in faeces could be an important source of contamination, which may lead to infection and further spread in areas with poor sanitary conditions. The findings of this study have public health significance and they should be considered when formulating disease control strategies.