Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

Visualized RNA detection of SARS-CoV-2 in a closed tube by coupling RT-PCR with nested invasive reaction.

A simple, cost-effective and reliable diagnosis of pathogen nucleic acids assay is much required for controlling a pandemic of a virus disease, such as COVID-19. Our previously developed visualized detection of pathogen DNA in a single closed tube is very suitable for POCT. However, virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance. To enable this visualized assay to detect virus RNA directly, various types of reverse transcriptase were investigated, and finally we found that HiScript II reverse transcriptase could keep active and be well adapted to the one-pot visualized assay in optimized conditions. Reverse transcription, template amplification and amplicon identification by PCR coupled with invasive reaction, as well as visualization by self-assembling of AuNP probes could be automatically and sequentially performed in a closed tube under different temperature conditions, achieving "sample (RNA)-in-result (red color)-out" only by a simple PCR engine plus the naked eye. The visualized RT-PCR is sensitive to unambiguous detection of 5 copies of the N and ORFlab genes of SARS-CoV-2 RNA comparing favourably with qPCR methods (commercialized kit), is specific to genotype 3 variants (Alpha, Beta and Omicron) of SARS-CoV-2, and is very accurate for picking up 0.01% Omicron variant from a large amount of sequence-similar backgrounds. The method is employed in detecting 50 clinical samples, and 10 of them were detected as SARS-CoV-2 positive samples, identical to those by conventional RT-PCR, indicating that the method is cost-effective and labor-saving for pathogen RNA screening in resource-limited regions.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the serum cytokine and chemokine levels in asymptomatic, mild, moderate, severe, and convalescent SARS-CoV-2-infected cases. Proinflammatory cytokine and chemokine production induced by SARS-CoV-2 were observed not only in symptomatic patients but also in asymptomatic cases, and returned to normal after recovery. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Moreover, a set of cytokine and chemokine profiles were significantly higher in SARS-CoV-2-infected male than female patients. The serum levels of MCP-1, G-CSF, and VEGF were weakly and positively correlated with viral titers. We suggest that combinatorial analysis of serum cytokines and chemokines with clinical classification may contribute to evaluation of the severity of COVID-19 and optimize the therapeutic strategies.

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae.

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.

Outbreak of Vaccinia Virus Infection from Occupational Exposure, China, 2017.

Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV.

Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay.

BACKGROUND: As an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device). RESULTS: This assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples. CONCLUSIONS: Thus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Co-circulation of multiple genotypes of influenza A (H7N9) viruses in eastern China, 2016-2017.

Five epidemic waves of human infection with influenza A (H7N9) virus have emerged in China since spring 2013. We previously described the epidemiological characterization of the fifth wave in Jiangsu province. In this study, 41 H7N9 viruses from patients and live-poultry markets were isolated and sequenced to further elucidate the genetic features of viruses of the fifth wave in Jiangsu province. Phylogenetic analysis revealed substantial genetic diversity in the internal genes, and 18 genotypes were identified from the 41 H7N9 virus strains. Furthermore, our data revealed that 41 isolates from Jiangsu contained the G186V and Q226L/I mutations in their haemagglutinin (HA) protein, which may increase the ability of these viruses to bind the human receptor. Four basic amino acid insertions were not observed in the HA cleavage sites of 167 H7N9 viruses from Jiangsu, which revealed that highly pathogenic avian influenza (HPAI) H7N9 viruses did not spread to Jiangsu province in the fifth wave. These findings revealed that multiple genotypes of H7N9 viruses co-circulated in the fifth wave in Jiangsu province, which indicated that the viruses have undergone ongoing evolution with genetic mutation and reassortment. Our study highlights the need to constantly monitor the evolution of H7N9 viruses and reinforce systematic influenza surveillance of humans, birds, and pigs in China.

Identification and genetic characterization of a novel circular single-stranded DNA virus in a human upper respiratory tract sample.

Metagenomic analysis through high-throughput sequencing is a tool for detecting both known and novel viruses. Using this technique, a novel circular single-stranded DNA (ssDNA) virus genome was discovered in respiratory secretions from a febrile traveler. The virus, named human respiratory-associated PSCV-5-like virus (HRAPLV), has a genome comprising 3,018 bases, with two major putative ORFs inversely encoding capsid (Cap) and replicase (Rep) protein and separated by two intergenic regions. One stem-loop structure was predicted in the larger intergenic region (LIR). The predicted amino acid sequences of the Cap and Rep proteins of HRAPLV showed highest identity to those of porcine stool-associated circular virus 5 isolate CP3 (PoSCV 5) (53.0% and 48.9%, respectively). The host tropism of the virus is unknown, and further study is warranted to determine whether this novel virus is associated with human disease.

Pyrosequencing with di-base addition for single nucleotide polymorphism genotyping.

We develop color code-based pyrosequencing with di-base addition for analysis of single nucleotide polymorphisms (SNPs). When a di-base is added into the polymerization, one or several two-color code(s) containing the type and the number of incorporated nucleotides will be produced. The code information obtained in a single run is useful to genotype SNPs as each allelic variant will give a specific pattern compared to the two other variants. Special care has to be taken while designing the di-base dispensation order. Here, we present a detailed protocol for establishing sequence-specific di-base addition to avoid nonsynchronous extension at the SNP sites. By using this technology, as few as 50 copies of DNA templates were accurately sequenced. Higher signals were produced and thus a relatively lower sample amount was required. Furthermore, the read length of per flow was increased, making simultaneous identification of multiple SNPs in a single sequencing run possible. Validation of the method was performed by using templates with two SNPs covering 37 bp and with three SNPs covering 58 bp as well as 82 bp. These SNPs were successfully genotyped by using only a sequencing primer in a single PCR/sequencing run. Our results demonstrated that this technology could be potentially developed into a powerful methodology to accurately determine SNPs so as to diagnose clinical settings.