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MPLA derivatives are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine.

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.

BackgroundThe 2009 pandemic H1N1 influenza A virus (pdm09) continue to evolve, and few studies have systemically analyzed the evolution, replication, and transmission of pmd09 viruses in China.MethodsTo better understand the evolution and pathogenicity of pdm09 viruses, we systematically analyzed viruses that were confirmed in 2009–2020 in China and characterized their replication and transmission ability. We extensively analyzed the evolution characteristics of pdm/09 in China over the past decades. The replication ability of 6B.1 and 6B.2 lineages on Madin-Darby canine kidney (MDCK) and human lung adenocarcinoma epithelial (A549) cells and their pathogenicity and transmission in guinea pigs were also compared.ResultsIn total, 3,038 pdm09 viruses belonged to clade 6B.1 (62% of all pdm09 viruses) and clade 6B.2 (4%). Clade 6B.1 pdm09 viruses are the predominant clade, with proportions of 54.1%, 78.9%, 57.2%, 58.6%, 61.7%, 76.3%, and 66.6% in the North, Northeast, East, Central, South, Southwest, and Northeast regions in China, respectively. The isolation proportion of clade 6B.1 pdm/09 viruses was 57.1%, 74.3%, 96.1%, 98.2%, 86.7%, and 78.5% in 2015–2020, respectively. A clear differentiation time point appeared in 2015 before which the evolution trend of pdm09 viruses in China was similar to that in North America but then showed a different trend after that point. To characterize pdm09 viruses in China after 2015, we further analyzed 33 pdm09 viruses isolated in Guangdong in 2016–2017, among which A/ Guangdong/33/2016 and A/Guangdong/184/2016 (184/2016) belonged to clade 6B.2, and the other 31 strains belonged to clade 6B.1. A/Guangdong/887/2017 (887/2017) and A/Guangdong/752/2017 (752/2017) (clade 6B.1), 184/2016 (clade 6B.2) and A/California/04/2009 (CA04) replicated efficiently in MDCK cells and A549 cells, as well as the turbinates of guinea pigs. 184/2016 and CA04 could transmit among guinea pigs through physical contact.ConclusionOur findings provide novel insights into the evolution, pathogenicity, and transmission of pdm09 virus. The results show that enhancing surveillance of pdm09 viruses and timely evaluation of their virulence are essential.

sThe COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319–541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319–583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.

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.

In recent years, the chikungunya virus (CHIKV) has continued to spread from local epidemics to nonnative habitats until eventually reaching pandemic status. Nonendemic areas such as China have also emerged as potential epidemic areas of CHIKV. Serological detection of CHIKV is the key to diagnosing and controlling the prevalence of this virus. In this study, we review the progress of the serological detection of the envelope (E) protein in CHIKV, and we provide a novel research assay and ideas for the serological detection of CHIKV. The luciferase immunosorbent assay (LISA) does not require species-specific labeled secondary antibodies for detection, making it universally suitable for tracking samples from various animals or carriers. At present, most research on CHIKV antigen detection technology tends to combine two or more proteins to avoid the decrease in detection ability caused by antigen mutation. Our results indicate that two or more kinds of CHIKV E antigens combined with LISA detection can improve the detection rate of anti-CHIKV immunoglobulin G (IgG) antibodies in CHIKV-infected patient sera and detect antibodies in the early stage of infection accurately and sensitively. After 235 days of infection, the anti-CHIKV IgG antibodies could still be detected in CHIKV-infected patients. All serum samples were tested with a detection rate of 100% after combining various recombinant CHIKV E antigens. Our proposed CHIKV-specific LISA could be a useful tool for serum diagnosis of CHIKV infection and serum epidemic research in areas where CHIKV is endemic, which would help to manage potential epidemics in the future.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is the etiological agent of chikungunya fever. To investigate the prevalence and genetic characteristics of CHIKV in China, we performed a molecular epidemiological study during the 2014–2019 period. Phylogenetic analysis of CHIKV in the 2014-2019 shows that it is currently clustered geographically, which means that CHIKV’s local adaptability is gradually strengthening. Focusing on CHIKV adaptive mutation E1-A226V is not obvious in our study. The CHIKV E1 amino acid non-synonymous mutation results revealed the common mutation site M343I. Most mutation sites belong to the American epidemic strains, and the mutation rate is 2.8%. In 31 sequences, 19 non-synonymous mutations were found in CHIKV E1 (total mutation rate 20/499 = 4%), and 37 non-synonymous mutations in CHIKV E2 (total mutation rate 30/425 = 7%). It is worth noting that the mutation of CHIKV E2 has changed more than that of CHIKV E1 between 2014 and 2019. CHIKV E2 screened out common mutation sites, and the results showed that there are five common mutation sites, namely S119G, L182M, G206D, S300N, and A345T. Eighty-three percent of the CHIKV E2 receptor binding domain mutations in the American strains, namely T3I and N6H, may cause immune escape.We constructed a new luciferase immunosorbent assay using CHIKV E2 antigen and mutant E2 antigen to test the serum of the CHIKV infected patients, and the detected samples and dilution ratios were different. The T3I, N6H, S119G, L182M, G206D, S300N, and A345T mutations in CHIKV E2 could explain these differences between patients.IMPORTANCECHIKV originated in Africa more than 500 years ago, with a common lineage dividing into two distinct branches called West Africa (West African, WA) and East/Central/South Africa (East/Central/South African, ECSA). Moreover, the E1-A226V mutation enhanced the replication and transmission capacity of CHIKV in Aedes albopictus. However, it should be noted that E1-A226V does not explain the CHIKV epidemic that has occurred in the Americas in recent years. We analyzed CHIKV samples in the 2014-2019, and the results showed that the mutation of CHIKV E1 protein occurred was not mainly concentrated in E1-A226V, but concentrated in M343I. This also means that CHIKV constantly mutates in the natural environment and is formed under natural conditions. Analysis of the common mutation sites of CHIKV E2 showed that there were seven common mutation sites S119G/L182M/ G206D /S300N/A345T, and the new mutation of CHIKV E2 may affect serological antibody detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been redetected after discharge in some coronavirus disease 2019 (COVID-19) patients. The reason for the recurrent positivity of the test and the potential public health concern due to this occurrence are still unknown. Here, we analyzed the viral data and clinical manifestations of 289 domestic Chinese COVID-19 patients and found that 21 individuals (7.3%) were readmitted for hospitalization after detection of SARS-CoV-2 after discharge. First, we experimentally confirmed that the virus was involved in the initial infection and was not a secondary infection. In positive retests, the virus was usually found in anal samples (15 of 21, 71.4%). Through analysis of the intracellular viral subgenomic messenger RNA (sgmRNA), we verified that positive retest patients had active viral replication in their gastrointestinal tracts (3 of 16 patients, 18.7%) but not in their respiratory tracts. Then, we found that viral persistence was not associated with high viral titers, delayed viral clearance, old age, or more severe clinical symptoms during the first hospitalization. In contrast, viral rebound was associated with significantly lower levels of and slower generation of viral receptor-binding domain (RBD)-specific IgA and IgG antibodies. Our study demonstrated that the positive retest patients failed to create a robust protective humoral immune response, which might result in SARS-CoV-2 persistence in the gastrointestinal tract and possibly in active viral shedding. Further exploration of the mechanism underlying the rebound in SARS-CoV-2 in this population will be crucial for preventing virus spread and developing effective vaccines.

ObjectivesTo investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:−.MethodsA total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014–19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms.ResultsWe identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles.ConclusionsOur findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Background Southern China is at risk for arborvirus disease transmission, including Zika virus and dengue. Patients often present to clinical care with non-specific acute febrile illnesses (AFI). To better describe the etiology of AFI, we implemented a two-year AFI surveillance project at five sentinel hospitals in Yunnan and Guangdong Provinces. Methods Between June 2017 and August 2019, we enrolled patients between 2 and 65 years of age presenting at one sentinel hospital in Mengla County, Yunnan, and four in Jiangmen City, Guangdong, with symptoms of AFI (acute onset of fever ≥ 37.5°C within the past 7 days) without respiratory symptoms or diarrhea. Demographic, epidemiologic, and clinical information was obtained and entered into a web-based AFI surveillance database. A custom TaqMan Array card (TAC) was used to test patients’ whole blood specimens for 27 different pathogens using real-time polymerase chain reaction assays. Results During the two-year project period, 836 patients were enrolled; 443 patients from Mengla County and 393 patients from Jiangmen City. The median age was 33 years [range: 2–65], and most were hospitalized [641, 77%]. Of 796 patients with valid TAC results, 341 (43%) were positive for at least one of the 10 unique pathogens detected. This included 205 (26%) patients positive for dengue virus, 60 (8%) for Orientia tsutsugamushi, and 42 (5%) for Coxiella burnetii. Ten patients (1%) in Jiangmen City tested positive for malaria, 8 of whom reported recent travel outside of China. TAC results were negative for 455 (57%) patients. None of the patients had a positive TAC detection for Zika virus. Conclusions The project detected variability in the etiology of AFI in Southern China and highlighted the importance of differential diagnosis. Dengue, O. tsutsugamushi, and C. burnetii were the most frequently identified pathogens among enrolled AFI patients. As a non-notifiable disease, the frequent detection of C. burnetii is noteworthy and warrants additional investigation. The project provided a framework for routine surveillance for persons presenting with AFI.

Background In this study, a comprehensive evaluation of management for pathogenic microbiology laboratories is performed based on a combination of Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) and Rank Sum Ratio (RSR) methods; in addition, the basis for improving laboratory management is provided. Methods Using the laboratory evaluation tool developed by World Health Organization and a combination of TOPSIS and RSR methods, a system of evaluation indicators for the management of Chinese pathogenic microbiology laboratories is established for comprehensively evaluating the pathogenic microbiology laboratories of seven provincial Centers for Disease Control and Prevention. Results The evaluation system includes 12 primary indicators and 37 secondary indicators. In terms of laboratory management, the seven laboratories were ranked as D, G, E, C, F, B, and A in descending order. None of these laboratories were evaluated as “good” or “poor.” One of the laboratories was marked as “relatively poor” (A), two as “medium” (B and F), and four as “relatively good” (C, E, G, and D). Conclusion In this study, a method for evaluating laboratory management using the TOPSIS and RSR methods is proposed, and a basis for comprehensively evaluating laboratory management for pathogenic microbiology laboratories is provided to reflect management practices.

In vaccinees who were infected with SARS-CoV in 2003, we observed greater antibody responses against spike and nucleoprotein of both SARS-CoV-2 and SARS-CoV after a single dosage of inactivated SARS-CoV-2 vaccine. After receiving the second vaccination, antibodies against RBD of SARS-CoV-2 Wuhan, Beta, Delta, and recently emerged Omicron are significantly higher in SARS-CoV experienced vaccinees than in SARS-CoV naïve vaccinees. Neutralizing activities measured by authentic viruses and pseudoviruses of SARS-CoV, SARS-CoV-2 Wuhan, Beta, and Delta are greater in SARS-CoV experienced vaccinees. In contrast, only weak neutralizing activities against SARS-CoV-2 and variants were detected in SARS-CoV naïve vaccinees. By 6 months after the second vaccination, neutralizing activities were maintained at a relatively higher level in SARS-CoV experienced vaccinees but were undetectable in SARS-CoV naïve vaccinees. These findings suggested a great possibility of developing a universal vaccine by heterologous vaccination using spike antigens from different SARS-related coronaviruses.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 μg or 50 μg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

The appearance and magnitude of the immune response and the related factors correlated with SARS-CoV-2 vaccination need to be defined. Here, we enrolled a prospective cohort of 52 participants who received two doses of inactivated vaccines (BBIBP-CorV). Their serial plasma samples (n = 260) over 2 months were collected at five timepoints. We measured antibody responses (NAb, S-IgG and S-IgM) and routine blood parameter. NAb seroconversion occurred in 90.7% of vaccinated individuals and four typical NAb kinetic curves were observed. All of the participants who seroconverted after the first dose were females and had relatively high prevaccine estradiol levels. Moreover, those without seroconversion tended to have lower lymphocyte counts and higher serum SAA levels than those who experienced seroconversion. The NAb titers in young vaccine recipients had a significantly higher peak than those in elderly recipients. S-IgG and S-IgM dynamics were accompanied by similar trends in NAb. Here, we gained insight into the dynamic changes in NAbs and preliminarily explored the prevaccine blood parameters related to the kinetic subclasses, providing a reference for vaccination strategies.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a new coronavirus causing Coronavirus Disease 2019 (COVID-19), is a major topic of global human health concern. The Delta and Omicron variants have caused alarming responses worldwide due to their high transmission rates and a number of mutations. During a one-year follow-up (from June 2020 to June 2021), we included 114 patients with SARS-CoV-2 infection to study the long-term dynamics and the correlative factors of neutralizing antibodies (NAbs) in convalescent patients. The blood samples were collected at two detection time points (at 6 and 12 months after discharge). We evaluated the NAbs response of discharged patients by performing a micro-neutralization assay using a SARS-CoV-2 wild type. In addition, a total of 62 serum samples from discharged COVID-19 patients with Alpha, Beta, Delta, and Omicron variants of infection were enrolled to perform cross-neutralization tests using the original SARS-CoV-2 strain and VOCs variants (including Alpha, Beta, Gamma, Delta, and Omicron variants) and to assess the ability of NAbs against the SARS-CoV-2 variants. NAbs seroconversion occurred in 91.46% of patients (n = 82) in the first timepoint and in 89.29% of patients (n = 84) in the second detection point, and three kinds of NAbs kinetics curves were perceived. The NAbs levels in young patients had higher values than those in elder patients. The kinetics of disease duration was accompanied by an opposite trend in NAbs levels. Despite a declining NAbs response, NAbs activity was still detectable in a substantial proportion of recovered patients one year after discharge. Compared to the wild strain, the Omicron strain could lead to a 23.44-, 3.42-, 8.03-, and 2.57-fold reduction in neutralization capacity in “SAlpha”, “SBeta”, “SDelta”, and “SOmicron”, respectively, and the NAbs levels against the Omicron strain were significantly lower than those of the Beta and Delta variants. Remarkably, the NAbs activity of convalescent serum with Omicron strain infection was most obviously detectable against six SARS-CoV-2 strains in our study. The role of the vaccination history in NAbs levels further confirmed the previous study that reported vaccine-induced NAbs as the convincing protection mechanism against SARS-CoV-2. In conclusion, our findings highlighted the dynamics of the long-term immune responses after the disappearance of symptoms and revealed that NAbs levels varied among all types of convalescent patients with COVID-19 and that NAbs remained detectable for one year, which is reassuring in terms of protection against reinfection. Moreover, a moderate correlation between the duration of disease and Nabs titers was observed, whereas age was negatively correlated with Nabs titers. On the other hand, compared with other VOCs, the Omicron variant was able to escape the defenses of the immune system more significantly, and the convalescent serum infected with the Omicron variant played a critical part in protection against different SARS-CoV-2 variants. Recovery serum from individuals vaccinated with inactivated vaccine preceding infection with the Omicron strain had a high efficacy against the original strain and the VOCs variants, whereas the convalescent serum of persons vaccinated by inactivated vaccine prior to infection with the Delta variant was only potent against the wild-type strain.

Most COVID-19 convalescents can build effective anti-SARS-CoV-2 humoral immunity, but it remains unclear how long it can maintain and how efficiently it can prevent the reinfection of the emerging SARS-CoV-2 variants. Here, we tested the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest known epicenter. SARS-CoV-2 immunoglobulins G (IgG) were well maintained in most patients and potently neutralizes the infection of the original strain and the B.1.1.7 variant. However, varying degrees of immune escape was observed on the other tested variants in a patient-specific manner, with individuals showing remarkably broad neutralization potency. The immune escape can be largely attributed to several critical spike mutations. These results suggest that SARS-CoV-2 can elicit long-lasting immunity but escaped by the emerging variants., graphical Abstract

SummaryThe COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.

SARS-CoV-2 vaccination has been launched worldwide to build effective population-level immunity to curb the spread of this virus. The effectiveness and duration of protective immunity is a critical factor for public health. Here, we report the kinetics of the SARS-CoV-2 specific immune response in 204 individuals up to 1-year after recovery from COVID-19. RBD-IgG and full-length spike-IgG concentrations and serum neutralizing capacity decreases during the first 6-months, but is maintained stably up to 1-year after hospital discharge. Even individuals who had generated high IgG levels during early convalescent stages had IgG levels that had decreased to a similar level one year later. Notably, the RBD-IgG level positively correlates with serum neutralizing capacity, suggesting the representative role of RBD-IgG in predicting serum protection. Moreover, viral-specific cellular immune protection, including spike and nucleoprotein specific, persisted between 6 months and 12 months. Altogether, our study supports the persistence of viral-specific protective immunity over 1 year., The quality of immune response to SARS-CoV-2 is thought to wane over time, but it is unclear how long it can persist. Here the authors show persistent immune responses in a large number of patients over the course of a 1-year follow-up from the time of recovery from COVID-19.

Background: A lot of recent researches have focused on the duration of the nature immunity elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. An improved understanding of the immunity offered by the antibodies developed against SARS-CoV-2 in recovered patients is critical for the development of diagnostic tests and vaccines. Methods: We enrolled 114 donors, which providing blood samples after discharge for half a year and one year. Neutralizing antibodies (NAbs) were tested using a micro-neutralization assay. Results: In two tests, 82 of 114 recovered patients completed the first test half a year after discharge and NAbs remained detectable in the vast majority of patients (75/82, 91.46%). In the comparison of the two intervals, 50% (27/54) of individuals had increased NAbs titers. when 31.48% (17/54) of patients remained unchanged. Conclusion: Our results suggest that immune ability is acquired in most individuals infected with SARS-CoV-2 and is sustained in a majority of patients for up to a year after recovery.

Most COVID-19 patients can build effective humoral immunity against SARS-CoV-2 after recovery(1, 2). However, it remains unknown how long the protection can maintain and how efficiently it can protect people from the reinfection of the emerging SARS-CoV-2 variants. Here we evaluated the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest epicenter of SARS-CoV-2. We demonstrated that the SARS-CoV-2 immunoglobulin G (IgG) maintains at a high level and potently neutralizes the infection of the original strain (WT) and the B.1.1.7 variant in most patients. However, they showed varying degrees of efficacy reduction against the other variants of concern (P.1, B.1.525, and especially B.1.351) in a patient-specific manner. Mutations in RBD including K417N, E484K, and E484Q/L452R (B.1.617) remarkably impair the neutralizing activity of the convalescents’ sera. Encouragingly, we found that a small fraction of patients’ sera showed broad neutralization potency to multiple variants and mutants, suggesting the existence of broadly neutralizing antibodies recognizing the epitopes beyond the mutation sites. Our results suggest that the SARS-CoV-2 vaccination effectiveness relies more on the timely re-administration of the epitope-updated vaccine than the durability of the neutralizing antibodies.

The spread of influenza A/H3N2 variants possessing the hemagglutinin 121 K mutation and the unexpectedly high incidence of influenza in the 2017–2018 northern hemisphere influenza season have raised serious concerns about the next pandemic. We summarized the national surveillance data of seasonal influenza in China and identified marked differences in influenza epidemics between northern and southern China, particularly the predominating subtype and the presence of an additional summer peak in southern China. Notably, a minor spring peak of influenza caused by a different virus subtype was also observed. We also revealed that the 3C.2a lineage was dominant from the summer of 2015 to the end of the 2015–2016 peak season in China, after which the 3C.2a2 lineage predominated despite the importation and co-circulation of the 121 K variants of 3C.2a1 and 3C.2a3 lineages at the global level. Finally, an analysis based on genetic distances revealed a delay in A/H3N2 vaccine strain update. Overall, our results highlight the complicated circulation pattern of seasonal influenza in China and the necessity for a timely vaccine strain update worldwide.

A safe, efficacious and deployable vaccine is urgently needed to control COVID-19 pandemic. We report here the preclinical development of a COVID-19 vaccine candidate, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and NHPs, and also elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 μg or 50 μg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea and bronchi, with milder lung lesions. No evidence of disease enhancement is observed in both models. ZF2001 is being evaluated in the ongoing international multi-center Phase 3 trials (NCT04646590) and has been approved for emergency use in Uzbekistan.

The ongoing of coronavirus disease 2019 (COVID-19) pandemic caused by novel SARS-CoV-2 coronavirus, resulting in economic losses and seriously threating the human health in worldwide, highlighting the urgent need of a stabilized, easily produced and effective preventive vaccine. The SARS-COV-2 spike protein receptor binding region (RBD) plays an important role in the process of viral binding receptor angiotensin-converting enzyme 2 (ACE2) and membrane fusion, making it an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticles vaccine candidates, RBD-Ferritin (24-mer), RBD-mi3 (60-mer) and RBD-I53-50 (120-mer), with the application of covalent bond linking by SpyTag-SpyCatcher system. It was demonstrated that the neutralizing capability of sera from mice immunized with three RBD-conjugated nanoparticles adjuvanted with AddaVax or Sigma Systerm Adjuvant (SAS) after each immunization was ~8-to 120-fold greater than monomeric RBD group in SARS-CoV-2 pseudovirus and authentic virus neutralization assay. Most importantly, sera from RBD-conjugated NPs groups more efficiently blocked the binding of RBD to ACE2 or neutralizing antibody in vitro, a further proof of promising immunization effect. Besides, high physical stability and flexibility in assembly consolidated the benefit for rapid scale-up production of vaccine. These results supported that our designed SARS-CoV-2 RBD-conjugated nanoparticle was competitive vaccine candidate and the carrier nanoparticles could be adopted as universal platform for future vaccine development.

Noroviruses (NoVs) are the leading cause of acute gastroenteritis outbreaks. The specific geographical distribution and expanding diversity of NoVs has posed a challenge to NoV surveillance and interventions. Here, we describe the long-term dynamic correlation between NoV distribution in sewage and in the local population by using high-throughput sequencing and operational taxonomic unit (OTU) analysis. The NoV viral loads in sewage were closely associated with the number of NoV outbreaks in the population. Compared with the viral distributions in outbreaks, the dominance of the newly emerged variants, such as GII.P17-GII.17 and GII.P16-GII.2, could be detected two months ahead in sewage. In addition, the dynamics of pre-epidemic variants, which were rarely detected from clinics, could be captured through sewage surveillance, thus improving our understanding of the viral origin and evolution. Our data highlight that the high-throughput environmental screening should become a critical part of the response to infectious diseases.

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain (RBD, residues 319-541) and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice. The antibodies also effectively suppressed SARS-CoV-2 RBD binding to soluble ACE2, indicating that RBD-mFc may be further developed as a safe and effective SARS-CoV-2 vaccine.

In this study, we designed a set of SARS‐CoV‐2 enrichment probes to increase the capacity for sequence‐based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS‐CoV‐2 enrichment probe set contains 502 120nt ssDNA biotin‐labeled probes designed based on all available SARS‐CoV‐2 viral sequences and it can be used to enrich for SARS‐CoV‐2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from a cell culture, three nasopharyngeal swab samples (cycle threshold [Ct] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID‐19 (positive control) and four throat swab samples from patients without COVID‐19 (negative controls), respectively. Moreover, based on these high‐quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication, and its phylogenetic relationship with other selected high‐quality samples from The Genome Variation Map (GVM). Therefore, this universal SARS‐CoV‐2 enrichment probe system can capture and enrich SARS‐CoV‐2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles. This article is protected by copyright. All rights reserved.

This manuscript is based on the method we developed urgently to deal with the research requirement in the conflict between achieving a complete genome sequence for the evolutionary history of SARS-CoV-2 study and the low viral RNA concentration. Here, in this manuscript, we developed a set of SARS-CoV-2 enrichment probes to increase the sensitivity of sequence-based virus detection and characterization via obtaining the comprehensive genome sequence. Following the CDC health and safety guidelines, we test the concept using the culturing supernatant contain SARS-CoV-2 particles, and its full-length sequence was used for further analysis. The fraction of SARS-CoV-2 endogenous DNA was 93.47% with Cluster Factor about 1.1, which demonstrate that the numbers of mapped reads to SARS-CoV-2 reference sequence significantly increased, compared to metagenomic sequencing technology, following SARS-CoV-2 probe enrichment. Moreover, based on the high-quality sequence, we discussed the heterozygosity and viral expression during replication of coronavirus, and its phylogenetic relationship with other selected high-quality samples from The Genome Variation Map (GVM) (on 2020/03/22). We believe this manuscript is valuable for all the researchers who are interested in using clinical warp samples to obtain the high coverage of SARS-CoV-2 genome sequence with a relatively low concentration of viral particles. This would allow the clinician to correlate the diagnostic data with molecular monitoring in viral evolutional, the most importantly, to track the functional mutation of SARS-CoV-2.

Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

Dengue viruses (DENVs) affect tropical and subtropical regions, including the Guangdong province of China. Dengue cases are reported almost every year in Shantou city in the Guangdong province. To understand the molecular characteristics of DENVs isolated in Shantou, we performed a molecular epidemiological study based on the envelope protein (E) gene of the DENVs isolated from the cases. Total 174 serum samples were collected from 174 dengue-suspected patients during 2015-2017. A total of 33.9% (59/174) were diagnosed with dengue. Serotypes of DENVs were identified in 27 samples; 37% (10/27), 55.6% (15/27), 3.7% (1/27), 3.7% (1/27) were serotype 1 DENV (DENV-1), serotype 2 DENV (DENV-2), serotype 3 DENV (DENV-3), and serotype 4 DENV (DENV4), respectively. Genotypes Ⅰ and Ⅳ were detected in DENV-1, while only the Cosmopolitan genotype was detected in DENV-2. The replacement of the predominant serotype (genotype), which takes place every year, implied that the dengue endemic in Shantou might be caused by an imported infection rather than by local populations. Our results suggested that DENVs in Shantou are closely related to the strains circulating in Southeast Asian countries, which were possibly transmitted to Shantou through some relay point cities.

BackgroundOn December 31, 2019, an outbreak of COVID-19 in humans was reported in Wuhan, and then spread fast to other provinces, China. We analyzed data from field investigations and genetic sequencing to describe the evidence and characteristics of human-to-human transmission in Guangdong Province.MethodsA confirmed COVID-19 case was defined if a suspected case was verified with positive of SARS-CoV-2 in throat swabs, nasal swabs, bronchoalveolar lavage fluid (BALF), or endotracheal aspirates by real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) or genetic sequencing. Field investigations were conducted for each confirmed case. Clinical and demographic data of confirmed cases were collected from medical records. Exposure and travel history were obtained by interview.ResultsA total of 1,151 confirmed cases were identified as of February 10, 2020 in Guangdong Province, China. Of them, 697 (60.1%) cases were from 234 cluster infections. Two hundred and fourteen (18.6%) were secondary cases, in which 144 cases were from family cluster infections. With the epidemic continuing, although familial cluster events were dominated, community cluster events increased with a nosocomial event. The whole genomes within the same family cluster infections were identical, and presented a few unique single nucleotide variants (SNVs) compared with SARS-CoV-2 identified on December 2019 in Wuhan.ConclusionsWe observed evident human-to-human transmissions of SARS-CoV-2 in Guangdong, China. Although most of them were from family cluster infections, community and nosocomial infections were increasing. Our findings indicate that human-to-human transmission risks are transferring from family to community in Guangdong Province.

Global spread of Zika virus in 2015 and 2016 highlighted the importance of surveillance to rapidly detect, report, and respond to emerging infections. We describe the lessons learned from the development and deployment of a web-based surveillance reporting system for Zika virus and other acute febrile illnesses (AFI) in Guangdong and Yunnan provinces, China. In less than 2 months, we customized the China Epidemiologic Dynamic Data Collection (EDDC) platform to collect, manage, and visualize data in close to real time. According to provincial and sentinel hospital staff requirements, the customized platform provided specific user authorization management, online/offline data collection, data quality control, and secure data transmission and protection and visualization tools. AFI case data and laboratory test results were entered through a web-based interface by hospital and provincial-level staff and saved on a China CDC server in Beijing. The dashboard visualized aggregate data by hospital, age, onset date, and laboratory test results. From June 2017 to December 2018, data from 768 patients with AFI were entered into the web-based surveillance system and visualized by hospital and provincial-level decision makers. Input from provincial and hospital staff was essential for developing the AFI case-reporting and feedback tools appropriate for specific settings and decision-making requirements. Web-based systems (eg, EDDC, data collection, and visualization tools that can be easily adapted to meet local surveillance needs) can help shorten time for system deployment, thereby strengthening global health security to rapidly detect and respond to emerging infections and outbreaks.

Background: A novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing pneumonia-associated respiratory disease pandemic across the world. Until now, the viral prevalence patterns, radiological characters, and antibody responses at initial stage of SARS-CoV-2 infection have been reported. The prolonged viral RNA shedding and coexistence with viral specific IgG antibody in discharged patients have also been reported recently. To date, however, correlations among viral specific RNA shedding, radiological pattern shift, and serum neutralizing antibody response covering the whole acute- and convalescent- phase in patients infected with SARS-CoV-2 are unknown. Focusing on a family cluster, present study is aim to give a bird view on the correlation of neutralizing antibody responses with viral clearance and lung disease prognosis from acute to convalescent phases. Methods: We conducted a surveillance study on a familial cluster of COVID-19 patients from Jan 17 to Apr 27 (99 days), 2020, in Guangzhou, Guangdong province, China. Epidemiological study was performed to clarify the virus transmission inside a family. NPAs, stool, and serum samples were collected and conducted RT-PCR to test the presence of SARS-CoV-2. Serum was used to perform neutralizing antibody tests to determine dynamic levels of neutralizing antibody. Radiological images as well as high-resolution CT scans for each patients were continually recorded to monitor outcomes of lung recovery. Findings: In this family cluster, three members (P2, P3, P4) in Guangzhou were infected by SARS-CoV-2 due to a close contact with P1 (index), who came back Guangzhou from Wuhan on Jan, 17, 2020, while another two members (H1 and H2) were unsusceptible and negative to SARS-CoV-2 test. The patients (P1, P2, P3) presented varying degrees of clinical symptoms on admission including recurrent fever, cough, and dyspnea, while P4 was asymptomatic. CT images showed ground-glass opacities in lungs of P1-3. The median length from illness onset to discharge for four-confirmed cases was 21.3 (4.7) days (range from 15 to 26). However, all patients returned positive for SARS-CoV-2 in follow-up investigation. The mean time for RNA shedding was 36.5 (3.2) days in P1, P2, and P4. The neutralizing antibody of P1, P2, and P4 was detectable during illness day 16 to 22. Then, the level was apparently ascent to a relative high titer and remained stable during 34-40, 31-41, and 35 days of illness onset for P1, P2, and P4, respectively, while declined gradually with time. The last positive time for SARS-CoV-2 test for P1, P2, and P4 was 40, 35, and 34 days of illness onset, respectively. Follow-up chest CT showed good prognosis in lung disease of P1 and P2, and CT scan was normal for P4. P3 was an exceptional case in this family cluster, who experienced the longest RNA shedding time (up to 68 days) as well as lung disease. In particular, radiological worsen was observed at day 34 of illness onset, high-resolution CT image revealed a glass-ground opacity in middle segment of right lung since day 45 to 99 of illness onset. The neutralizing antibody level in P3 was low during the entire study and significantly lower than that in other patients. Interpretation: The efficiency of SARS-CoV-2 virus clearance and lung disease prognosis were closely related to the levels of neutralizing antibody in COVID-19 patients. Viral clearance was observed along with high level of neutralizing antibody in P1, P2, and P4. However, the level of neutralizing antibody in P3 was not high enough to clear the virus completely, leading a prolong RNA shedding and lung disease. We proposed that high level of neutralizing antibody was essential for viral clearance and lung recovery, and virus was thoroughly eliminated during the period when neutralizing antibody reached high level in serum. Funding Statement: This work was supported by grants from the Guangzhou Science and Technology Program (201904010012), Guangdong Provincial Science and Technology Program (2018B020207006), Guangdong Provincial Novel Coronavirus Scientific and Technological Project (2020111107001). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Institutional Review Board of the Guangdong Second Provincial General Hospital. All patients involved in this study signed the clinical research informed consent.

Background: COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world Remarkable achievements have been made in d

The SARS-CoV-2 Delta (B.1.617.2) strain is a variant of concern (VOC) that has become the dominant strain worldwide in 2021. Its transmission capacity is approximately twice that of the original strain, with a shorter incubation period and higher viral load during infection. Importantly, the breakthrough infections of the Delta variant have continued to emerge in the first-generation vaccine recipients. There is thus an urgent need to develop a novel vaccine with SARS-CoV-2 variants as the major target. Here, receptor binding domain (RBD)-conjugated nanoparticle vaccines targeting the Delta variant, as well as the early and Beta/Gamma strains, are developed. Under both a single-dose and a prime-boost strategy, these RBD-conjugated nanoparticle vaccines induce the abundant neutralizing antibodies (NAbs) and significantly protect hACE2 mice from infection by the authentic SARS-CoV-2 Delta strain, as well as the early and Beta strains. Furthermore, the elicitation of the robust production of broader cross-protective NAbs against almost all the notable SARS-CoV-2 variants including the Omicron variant in rhesus macaques by the third re-boost with trivalent vaccines is found. These results suggest that RBD-based monovalent or multivalent nanoparticle vaccines provide a promising second-generation vaccine strategy for SARS-CoV-2 variants.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-COV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of $10^{-17}$ M (approximately 6 copies/$\mu$L). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of $10^{-16}$ M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis., Comment: 39 pages, 6 figures

Inoculation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is ongoing worldwide. However, the emergence of SARS-CoV-2 variants could cause immune evasion. We developed a bivalent nanoparticle vaccine that displays the receptor binding domains (RBDs) of the D614G and B.1.351 strains. With a prime-boost or a single-dose strategy, this vaccine elicits a robust neutralizing antibody and full protection against infection with the authentic D614G or B.1.351 strain in human angiotensin-converting enzyme 2 transgene mice. Interestingly, 8 months after inoculation with the D614G-specific vaccine, a new boost with this bivalent vaccine potently elicits cross-neutralizing antibodies for SARS-CoV-2 variants in rhesus macaques. We suggest that the D614G/B.1.351 bivalent vaccine could be used as an initial single dose or a sequential enforcement dose to prevent infection with SARS-CoV-2 and its variants., Graphical abstract, Yuan et al. construct a bivalent vaccine based on the RBD sequence of two SARS-CoV-2 variants, D614G and B.1.351. Vaccination with a single dose or a prime-boost regimen is protective against SARS-CoV-2 challenge in mice. The bivalent vaccine elicits nAbs against SARS-CoV-2 variants in rhesus macaques with a third-dose regimen

H6 subtype avian influenza virus (AIV) was prevalent in poultry and could sporadically infect humans. Here, a total of 196 novel H6 AIVs isolated from poultry in eight provinces of China from 2014 to 2016 were phylogenetically characterized. Our analysis revealed that they could be divided into two clades in the Asian H6 HA lineage, A/wild duck/Shantou/2853/2003(H6N2) (ST2853-like) (85.7%) and A/duck/Shantou/339/2000(H6N2) (ST339-like) (14.3%), in which ST2853-like strains predominate. These novel strains belonged to the H6N6 (n = 165, 84.2%), H6N2 (n = 30, 15.3%), and H6N3 (n = 1, 0.51%) subtypes, which could be classified into 36 genotypes including 12 novel genotypes described in this study. In particular, several strains possessed the V190 and S228 mutations in HA (H3 numbering), which is critical for human receptor binding and identical to the human-derived strain A/Taiwan/2/2013(H6N1). Furthermore, 10.3% of the H6N6 isolates possessed the N6-∆11b (59-69) deletion. In summary, we describe phylogenetic and molecular characterizations of H6 AIVs in southern China and highlight the constant prevalence of H6 AIVs in poultry as well as adaptation to mammalian hosts.

Introduction Dengue has become a more serious human health concern in China, with increased incidence and expanded outbreak regions. The knowledge of the cross-sectional and longitudinal epidemiological characteristics and the evolutionary dynamics of dengue in high-risk areas of China is limited. Methods Records of dengue cases from 2013 to 2016 were obtained from the China Notifiable Disease Surveillance System. Full envelope gene sequences of dengue viruses detected from the high-risk areas of China were collected. Maximum Likelihood tree and haplotype network analyses were conducted to explore the phylogenetic relationship of viruses from high-risk areas of China. Results A total of 56,520 cases was reported in China from 2013 to 2016. During this time, Yunnan, Guangdong and Fujian provinces were the high-risk areas. Imported cases occurred almost year-round, and were mainly introduced from Southeast Asia. The first indigenous case usually occurred in June to August, and the last one occurred before December in Yunnan and Fujian provinces but in December in Guangdong Province. Seven genotypes of DENV 1–3 were detected in the high-risk areas, with DENV 1-I the main genotype and DENV 2-Cosmopolitan the secondary one. The Maximum Likelihood trees show that almost all the indigenous viruses separated into different clusters. DENV 1-I viruses were found to be clustered in Guangdong Province, but not in Fujian and Yunnan, from 2013 to 2015. The ancestors of the Guangdong viruses in the cluster in 2013 and 2014 were most closely related to strains from Thailand or Singapore, and the Guangdong virus in 2015 was most closely related to the Guangdong virus of 2014. Based on closest phylogenetic relationships, viruses from Myanmar possibly initiated further indigenous cases in Yunnan, those from Indonesia in Fujian, while viruses from Thailand, Malaysia, Singapore and Indonesia were predominant in Guangdong Province. Conclusions Dengue is still an imported disease in China, although some genotypes continued to circulate in successive years. Viral phylogenies based on the envelope gene suggested periodic introductions of dengue strains into China, primarily from Southeast Asia, with occasional sustained, multi-year transmission in some regions of China., Author summary Dengue is the most prevalent and rapidly spreading mosquito-borne viral disease globally. Because of the multiple introductions, dengue outbreaks occurred in epidemic seasons in Southern China, supported by suitable weather conditions. Surveillance data from 2013 to 2016 in China showed that Guangdong, Yunnan and Fujian provinces were the high-risk areas, with dengue outbreaks occurring almost every year. However, knowledge has been lacking of the epidemiological characteristics and the evolution pattern of dengue virus in these high-risk areas. This study shows a variety of epidemiological characteristics and sources of imported cases among the high-risk areas in China, with likely origins primarily from countries in Southeast Asia. Seven genotypes of the DENV 1–3 variety co-circulated with DENV1-I, the main genotype, and DENV 2-Cosmopolitan, the secondary. Genetic relationships among viral strains suggest that the indigenous viruses in the high-risk areas arose from imported viruses and sometimes persisted between years into the next epidemic season, especially in Guangdong Province. Population movement has played a vital role in dengue epidemics in China. This information may be useful in dengue control, especially during epidemic seasons and in the development of an early warning system within the region, in collaboration with bordering countries.

During 2016 in Guangzhou, China, we detected infectious avian influenza viruses (AIVs) in 39.8% of samples from chicken carcasses slaughtered at live poultry markets but none from carcasses supplied to supermarkets by facilities bypassing live poultry markets. Promoting supply chains with high biosecurity may reduce the risk for zoonotic AIV transmission.