Key Aspects of Coronavirus Avian Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is an enveloped and positive-sense single-stranded RNA virus. IBV was the first coronavirus to be discovered and predominantly causes respiratory disease in commercial poultry worldwide. This review summarizes several important aspects of IBV, including epidemiology, genetic diversity, antigenic diversity, and multiple system disease caused by IBV as well as vaccination and antiviral strategies. Understanding these areas will provide insight into the mechanism of pathogenicity and immunoprotection of IBV and may improve prevention and control strategies for the disease.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

Features of liver function in pediatric patients infected with Delta variant versus Omicron variant of severe acute respiratory syndrome Coronavirus 2

Objective: To summarize and analyze the features of liver function in pediatric patients infected with Delta variant versus Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: In this study, an analysis was performed for the liver function test results of the locally transmitted or imported pediatric patients with SARS-CoV-2 infection during isolation who were admitted to Guangzhou Eighth People's Hospital, Guangzhou Medical University, since May 21, 2021, and the clinical data and the constituent ratio of liver injury were compared between the pediatric patients infected with Delta variant and those infected with Omicron variant. The independent samples t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. Results: A total of 85 pediatric patients infected with SARS-CoV-2 were enrolled, among whom there were 32 (37.6%) pediatric patients infected with Delta variant and 53 (62.4%) pediatric patients infected with Omicron variant, and there were no significant differences between the two groups in age, sex, body height, body weight, and comorbidities (all P > 0.05). There were no significant differences between the two groups in elating aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, total bilirubin, albumin, and cholinesterase (all P > 0.05), and the pediatric patients infected with Omicron variant had a significantly higher level of total bile acid (TBA) than those infected with Delta variant (Z=-2.336, P=0.020). However, the median values of TBA were within the normal range and the ratios of abnormal TBA were no significant difference between the two groups (P > 0.05). Among the 85 pediatric patients, 10 (11.8%) had a mild increase in liver function parameters, among whom 7 had an increase in TBA, 1 had an increase in ALT, 1 had increases in ALT and AST, and 1 had an increase in ALP. The analysis of liver injury in the pediatric patients infected with Delta variant or Omicron variant showed that there was no significant difference in the constituent ratio of liver injury caused by the two variants (6.3% vs 15.1%, X2=0.691, P=0.406). Conclusion: Mild liver injury is observed in pediatric patients infected with Delta and Omicron variants of SARS-CoV-2, but further studies are needed to evaluate the long-term influence of such infection on liver function.

Coronavirus Endoribonuclease Ensures Efficient Viral Replication and Prevents Protein Kinase R Activation.

Coronavirus (CoV) nsp15 is an endoribonuclease conserved throughout the CoV family. The enzymatic activity and crystal structure of infectious bronchitis virus (IBV) nsp15 are undefined, and the protein's role in replication remains unclear. We verified the uridylate-specific endoribonuclease (EndoU) activity of IBV and found that the EndoU active sites were located in the C-terminus of nsp15 and included His223, His238, Lys278 and Tyr334. We further constructed an infectious clone of the IBV-rSD strain (rSD-wild-type [WT]) and EndoU-deficient IBVs by changing the codon for the EndoU catalytic residues to alanine. Both the rSD-WT and EndoU-deficient viruses propagated efficiently in embryonated chicken eggs. Conversely, EndoU-deficient viral propagation was severely impaired in chicken embryonic kidney cells, which was reflected in the lower viral mRNA accumulation and protein synthesis. After infecting chickens with the parental rSD-WT strain and EndoU-deficient viruses, the EndoU-deficient-virus-infected chickens presented reduced mortality, tissue injury and viral shedding.IMPORTANCE Coronaviruses can emerge from animal reservoirs into naive host species to cause pandemic respiratory and gastrointestinal diseases with significant mortality in humans and domestic animals. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal and reproductive systems, causing millions of dollars in lost revenue worldwide annually. Mutating the viral endoribonuclease resulted in an attenuated virus and prevented protein kinase R activation. Therefore, EndoU activity is a virulence factor in IBV infections, thus providing an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.

Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Configurational patterns for COVID-19 related social media rumor refutation effectiveness enhancement based on machine learning and fsQCA.

Infodemics are intertwined with the COVID-19 pandemic, affecting people's perception and social order. To curb the spread of COVID-19 related false rumors, fuzzy-set qualitative comparative analysis (fsQCA) is used to find configurational pathways to enhance rumor refutation effectiveness. In this paper, a total of 1,903 COVID-19 related false rumor refutation microblogs on Sina Weibo are collected by a web crawler from January 1, 2022 to April 20, 2022, and 10 main conditions affecting rumor refutation effectiveness index (REI) are identified based on "three rules of epidemics". To reduce data redundancy, five ensemble machine learning models are established and tuned, among which Light Gradient Boosting Machine (LGBM) regression model has the best performance. Then five core conditions are extracted by feature importance ranking of LGBM. Based on fsQCA with the five core conditions, REI enhancement can be achieved through three different pathway elements configurations solutions: "Highly influential microblogger * high followers' stickiness microblogger", "high followers' stickiness microblogger * highly active microblogger * concise information description" and "high followers' stickiness microblogger * the sentiment tendency of the topic * concise information description". Finally, decision-making suggestions for false rumor refutation platforms and new ideas for improving false rumor refutation effectiveness are proposed. The innovation of this paper reflects in exploring the REI enhancement strategy from the perspective of configuration for the first time.

Vitamin A in children's pneumonia for a COVID-19 perspective: A systematic review and meta-analysis of 15 trials.

BACKGROUND: To systematically review and meta-analyze the efficacy of vitamin A as an adjuvant therapy for pneumonia in children. METHODS: We searched in PubMed, the Cochrane Library, Chinese National Knowledge Infrastructure, WanFang Database and Chongqing VIP information network from libraries building to March 2022, screening randomized controlled trials (RCT) about vitamin A combined with conventional therapy for pneumonia in children. Two researchers used the Cochrane risk of bias tool to assess the quality of included studies dependently. Data analysis was conducted in the RevMan 5.3. RESULTS: 15 trials involving 3496 patients (treated group: 1898; control group: 1598) were analyzed in this study. The Meta-analysis showed that vitamin A combined with conventional therapy improved clinical efficacy (P < .05), shortened the duration of fever and cough, negative time of chest X-ray, and the hospitalization, lung rale disappearance, choking milk disappearance, shortness of breath disappearance and perilabial cyanosis disappearance (P < .05). However, vitamin A combined with conventional therapy did not reduce the mortality of pneumonia in children (P > .05). CONCLUSION: Vitamin A contributes to relieve the clinical symptoms and signs, and also shorten the hospitalization.

Association between fear of COVID-19 and hoarding behavior during the outbreak of the COVID-19 pandemic: The mediating role of mental health status

Hoarding behavior can effectively improve people's ability to resist risks, so as to reduce the negative effects of risks. However, excessive hoarding behavior will seriously reduce people's quality of life. The COVID-19 pandemic can cause excessive hoarding in a large number of people in a short period of time, and also cause a series of economic problems such as social material shortage. It is unclear how hoarding levels are linked to fear and negative emotions caused by COVID-19 among people of different educational backgrounds and social status. The purpose of this study was to explore the relationship between fear of COVID-19 and hoarding behavior in different populations in school and social contexts, as well as the mediating role of negative emotions and the moderating role of subjective/objective social status and education level in this process. An online cross-sectional survey was conducted in various provinces in China in January 2022. Demographic information, the MacArthur Scale of Subjective Social Status, the Fear of COVID-19 scale, the Depression Anxiety Stress-21, and the Saving Inventory-Revised were used to evaluate the severity of individual hoarding symptoms, the frequency of hoarding, the degree of fear, and the negative emotions (depression, anxiety, stress) caused by COVID-19. Research data showed that fear of COVID-19 was significantly correlated with hoarding behavior (p < 0.05). Fear of COVID-19 was significantly lower in the student sample than in the nonstudent sample (p < 0.05). Negative emotions played a mediating role in the relationship between fear of COVID-19 and hoarding behavior (p < 0.05). Educational and economic levels moderated this process, but social status did not. Compared with the student sample, educational background and income had less of a moderating effect on the depression, anxiety, and stress caused by fear of COVID-19 in the nonstudent sample. However, these factors had a more regulative effect on the clutter and excessive acquisition behavior caused by depression, anxiety, and stress, although not on difficulty discarding. These findings suggest that reduce negative emotions in the population, improve cognitive levels, and provide financial support from governments may be effective ways to reduce hoarding symptoms.

LncRNA-Mediated Adipogenesis in Different Adipocytes.

Long-chain noncoding RNAs (lncRNAs) are RNAs that do not code for proteins, widely present in eukaryotes. They regulate gene expression at multiple levels through different mechanisms at epigenetic, transcription, translation, and the maturation of mRNA transcripts or regulation of the chromatin structure, and compete with microRNAs for binding to endogenous RNA. Adipose tissue is a large and endocrine-rich functional tissue in mammals. Excessive accumulation of white adipose tissue in mammals can cause metabolic diseases. However, unlike white fat, brown and beige fats release energy as heat. In recent years, many lncRNAs associated with adipogenesis have been reported. The molecular mechanisms of how lncRNAs regulate adipogenesis are continually investigated. In this review, we discuss the classification of lncRNAs according to their transcriptional location. lncRNAs that participate in the adipogenesis of white or brown fats are also discussed. The function of lncRNAs as decoy molecules and RNA double-stranded complexes, among other functions, is also discussed.

A smartphone-based platform for ratiometric visualization of SARS-CoV-2 via an oligonucleotide probe.

COVID-19 necessitates the development of reliable and convenient diagnostic tools. In this work, a facile 3D-printed smartphone platform was constructed that achieved reliable visual detection of SARS-CoV-2 by eliminating the effect of ambient light and fixing the camera position relative to the sample. The oligonucleotide probe is modified with orange-red-emitting TAMRA working as an internal standard and green-emitting FAM serving as a sensitive sensing agent. Under 365-nm UV excitation, the emission wavelengths of TAMRA and FAM are 580 nm and 518 nm, respectively. When the probes interact with the targets, the green fluorescence gradually restores while the orange-red fluorescence remains stable. Thus, a striking color transition from orange-red to green could be observed by the naked eye. The detection limit of SARS-CoV-2 nucleic acid is 0.23 nM, and the entire process of color change could be completed in 25 min. Furthermore, the RGB value analysis of the sample solution was conducted using a smartphone for reliable and reproducible discrimination of SARS-CoV-2. The proposed smartphone platform might establish a general method for visual detection of SARS-CoV-2 nucleic acid as well as other virus-related diseases.

Attenuated Viral Replication of Avian Infectious Bronchitis Virus with a Novel 82-Nucleotide Deletion in the 5a Gene Indicates a Critical Role for 5a in Virus-Host Interactions.

We previously found that a deletion in γ-coronavirus Infectious bronchitis virus (IBV) accessory gene 5a is critical for decreased viral pathogenicity in chickens. Here, we systematically analyzed IBV virus infection: invasion, genome replication, subgenomic mRNA (sgmRNA) synthesis, protein synthesis, and virion release. The ability of the mutant IBV strain rYN-Δ5a to invade susceptible cells was not significantly different from that of parental rYN. However, compared with rYN, the level of sgmRNA synthesis and genome replication after cell entry by rYN-Δ5a was significantly lower in the early stage, resulting in a significantly lower level of nucleoprotein (N) synthesis and a consequent significantly lower number of offspring viruses released into the supernatant. The detected 5a protein was diffusely distributed in the cytoplasm and perinuclear area. We identified 16 differentially expressed host proteins, 8 of which were found to be host nuclear and cytoplasmic transport-related proteins. Coimmunoprecipitation revealed an interaction between hemagglutinin (HA)-tagged TNPO1, TNPO3, XPO1, XPOT, RanBP1, and EIF2B4 proteins and Flag-tagged 5a protein, and laser confocal microscopy confirmed 5a protein colocalization with these proteins, indicating that 5a protein can cause changes in the host protein localization. These host proteins promote the nuclear localization of N proteins, so we believe that 5a protein can hijack host nucleoplasmic transport-related proteins to help N enter the nucleus. This may involve regulating the cell cycle to promote the optimal intracellular conditions for virus assembly or by participating in the regulation of nucleolar function as a strategy to optimize virus replication. IMPORTANCE Coronaviruses (CoVs) have a huge impact on humans and animals. It is important for the prevention and control of the viruses to assess the molecular mechanisms related to virulence attenuation. Here, we systematically analyzed a single cycle of virus infection by γ-CoV IBV lacking accessory protein 5a. We observed that a 5a deletion in the IBV genome affected virus replication and sgmRNA synthesis early in the virus life cycle, leading to decreases in protein synthesis, offspring virus assembly, and virion release in chicken embryonic kidney cells. IBV 5a protein was found to interact with multiple host nuclear and cytoplasmic transport- and translation-related proteins, which can also interact with IBV N and relocate it into the cell nucleus. These findings provide a comprehensive view regarding the importance of IBV accessory protein 5a and an important theoretical basis for studying the interaction between coronavirus and host cell proteins.

Role of Stress Granules in Suppressing Viral Replication by the Infectious Bronchitis Virus Endoribonuclease.

Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.

VisualCommunity: a platform for archiving and studying communities.

VisualCommunity is a platform designed to support community or neighborhood scale research. The platform integrates mobile, AI, visualization techniques, along with tools to help domain researchers, practitioners, and students collecting and working with spatialized video and geo-narratives. These data, which provide granular spatialized imagery and associated context gained through expert commentary have previously provided value in understanding various community-scale challenges. This paper further enhances this work AI-based image processing and speech transcription tools available in VisualCommunity, allowing for the easy exploration of the acquired semantic and visual information about the area under investigation. In this paper we describe the specific advances through use case examples including COVID-19 related scenarios.

Pathological effect of different avian infectious bronchitis virus strains on the bursa of Fabricius of chickens.

Infectious bronchitis is an acute and highly contagious disease caused by avian infectious bronchitis virus (IBV). As well as the typical clinical respiratory signs, such as dyspnoea and tracheal rales, QX genotype strains can also cause damage to the urinary system and reproductive system. Our previous studies found that chickens infected with QX-type IBV also displayed damage to the bursa of Fabricius. To investigate the effects of different genotypes of IBV on the bursa of Fabricius, we challenged one-week-old SPF chickens with Mass, QX and TW genotype IBV strains and compared the clinical signs, gross lesions, histopathological damage, viral loads, and expression levels of inflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-α,ß, γ and TNF-α). The results showed that all three strains caused tissue damage, while significant temporal variations in the viral loads of the different infected groups were detected. IBV infection seriously interfered with the natural immune response mediated by inflammatory cytokines (IFN-α, IFN-ß, IL-6 and IFN-γ) in chickens. Our results suggested that IBV has potential immunological implications for chickens that may lead to poor production efficiency. RESEARCH HIGHLIGHTSAvian coronavirus IBV is an important pathogen of chickens.IBV has potential immunological implications in chickens.The bursal viral load of different IBV strains varies significantly.

Development of a Nanoparticle Multiepitope DNA Vaccine against Virulent Infectious Bronchitis Virus Challenge.

To develop a safe and effective nanoparticle (NP) multiepitope DNA vaccine for controlling infectious bronchitis virus (IBV) infection, we inserted the multiepitope gene expression box SBNT into a eukaryotic expression vector pcDNA3.1(+) to construct a recombinant plasmid pcDNA/SBNT. The NP multiepitope DNA vaccine pcDNA/SBNT-NPs were prepared using chitosan to encapsulate the recombinant plasmid pcDNA/SBNT, with a high encapsulation efficiency of 94.90 ± 1.35%. These spherical pcDNA/SBNT-NPs were 140.9 ± 73.2 nm in diameter, with a mean ζ potential of +16.8 ± 4.3 mV. Our results showed that the chitosan NPs not only protected the plasmid DNA from DNase degradation but also mediated gene transfection in a slow-release manner. Immunization with pcDNA/SBNT-NPs induced a significant IBV-specific immune response and partially protected chickens against homologous IBV challenge. Therefore, the chitosan NPs could be a useful gene delivery system, and NP multiepitope DNA vaccines may be a potential alternative for use in the development of a novel, safe, and effective IBV vaccine.

[Applications of chromatography in giant complex drug-organism system].

Chromatography is an important branch of analytical chemistry that focuses on the separation and analysis of complex structures. Following more than 100 years of development and improvement, chromatography theory and technology have gradually become sophisticated. It has become a coalition of science, technology, and art. Recently, chromatography has been successfully used in combination with mass spectrometry, nuclear magnetic resonance spectroscopy, and atomic emission spectroscopy. Chromatography and the combination with other techniques has significantly improved the analysis of complex systems, such as the environment, food, petrochemicals, biological specimens, and medicine. As one of the oldest healing systems, Traditional Chinese Medicine (TCM) has served to maintain the health of people in China and worldwide for thousands of years. Therefore, it has become a core representative of traditional Chinese culture. In the past two years, TCM has been widely used to treat COVID-19, especially in patients with mild symptoms. Recently, Chinese government emphasized the inheritance and innovation of TCM and stepped up efforts to promote its modernization. TCM includes herbal medicine, acupuncture, moxibustion, massage, food therapy, and physical exercise, such as Tai Chi. In most cases, the patients are administered a mixture of TCM formulas containing more than two herbal medicines, resulting in a highly complicated compound mixture. There is no doubt that long-term clinical practices have demonstrated the safety and therapeutic effect of TCM. However, the compound mixture must be simplified to identify the active compounds. This is mainly because of the existence of carcinogenic compounds, pesticides, and heavy metal residues introduced through plantation and production processes. Moreover, enzymes within the human system generate further new compounds in response to the entry of the TCM containing thousands of components. Consequently, the complex TCM and organism systems interact with each other, constituting a giant complex drug-organism system. The analysis of this giant complex system is acknowledged as a key aspect in the modernization process of TCM. In the last 20 years, many studies have been conducted to screen and identify effective compounds in TCM. These effective compounds can be either the original compounds or new metabolic components generated in vivo. All these efforts are aimed at simplifying the components of TCM and elucidating the therapeutic mechanism. It is well known that chromatography can provide technical support for complex systems owing to its unique advantage of outstanding separation and analysis capabilities. Therefore, chromatography and its combination with other technologies have become mainstream technologies for promoting the compilation of molecular structure, information, digitalization, and modernization of TCM. This paper reviews the research and application of chromatography and combination technologies in a giant complex TCM formula-organism system. Furthermore, the authors briefly introduce and summarize the understanding, research ideas, and activities of the authors' team on the modernization of TCM. "Liang Guanxi" and "He strategy" are proposed as novel strategies for studying the giant complex drug-organism system. A distinguished technology integrated with mathematical model of causal relation, combined receptor chromatography, identification of chemical molecular structure and evaluating of pharmacological activities was established. It was successfully employed to determine the core effector-response substances of "Liang Guanxi" herb pairs in a giant complex drug-organism system. Subsequently, utilizing the proposed technology of Combination of Traditional Chinese Medicine Molecular Chemistry, the author's team designed and developed four series of innovative drugs. Inspired by the hundred years of chromatography history and thousands of years of TCM culture, the future development of chromatographic technology is expected. Furthermore, the mechanisms of TCM in medical healthcare, prevention, and treatment of diseases are likely be explained through chromatography, leading to a new strategy to realize the molecularization and digitalization of TCM, which is beneficial to the development of original new drugs.

Two newly isolated GVI lineage infectious bronchitis viruses in China show unique molecular and pathogenicity characteristics.

During 2016 to 2020, GVI-1 type infectious bronchitis virus (IBV) strains were sporadically reported across China, indicating a new epidemic trend of the virus. Here we investigated the molecular characteristics and pathogenicity of two newly isolated GVI-1 type IBV virus strains (CK/CH/TJ1904 and CK/CH/NP2011) from infected chicken farms in China. Genetic evolution analysis of the S1 gene showed the highest homology with the GVI-1 representative strain, TC07-2. Phylogenetic analysis and recombination analysis of the virus genomes indicated that newly isolated strains in China may be independently derived from recombination events that occurred between GI-19 and GI-22 strains and early GVI-1 viruses. Interestingly, unlike the deduced parental GI-19 or GI-22 strains, CK/CH/TJ1904 and CK/CH/NP2011 showed affinity for the trachea rather than the kidney and were less pathogenic. This difference may be because of recombination events that occurred during the long co-existence of the GVI-1 viruses with prevalent GI-19 and GI-22 strains. Considering the new trend, it is very important to permanently monitor circulating strains and to develop new vaccines to counteract emerging new-type IBVs.

Identification of Novel T-Cell Epitopes on Infectious Bronchitis Virus N Protein and Development of a Multi-epitope Vaccine.

Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining, and carboxyfluorescein succinimidyl ester (CFSE) lymphocyte proliferation assays; among them, three peptides (N211-230, N271-290, and N381-400) were cytotoxic T lymphocyte (CTL) epitopes, and one peptide (N261-280) was a dual-specific T-cell epitope, which can be recognized by both CD8+ and CD4+ T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus-expressing IBV multi-epitope genes represent a safe and effective vaccine candidate for controlling infectious bronchitis. IMPORTANCE T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So, T-cell-mediated immune responses play an important role in the control of viral infection, and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation.

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

The furin-S2' site in avian coronavirus plays a key role in central nervous system damage progression.

The furin cleavage site plays an important role in virus pathogenicity. The spike protein of SARS-CoV-2 harbors a furin cleavage site insertion in contrast to SARS-CoV, which may be related to its stronger communicability. An avian coronavirus with an extra furin cleavage site upstream of the fusion peptide (S2' site) infected monocyte cells and neuron cells leading to viremia or encephalitis, respectively. Immunohistochemistry and real-time quantitative polymerase chain reaction were used to follow disease progression and demonstrated differences between the parent avian coronavirus and mutated avian coronavirus with a furin-S2' site. Magnetic resonance imaging and biological dye to evaluate the blood-brain barrier permeability showed that avian coronavirus with a furin-S2' site had increased permeability compared with parent avian coronavirus. Immunohistochemistry of brains after intracerebral injection of avian coronavirus and immunofluorescence staining of primary neuron cells demonstrated the furin-S2' site expanded the cell tropism of the mutant avian coronavirus to neuron cells. TNF-α, which has a key role in blood-brain barrier permeability, was highly induced by avian coronavirus with a furin-S2' site compared with the parent avian coronavirus. We demonstrated the process involved in mutant avian coronavirus-induced disease and that the addition of a furin-S2' site changed the virus cell tropism.IMPORTANCECoronaviruses have broken out three times in two decades. Spike (S) protein plays a key role in the process of infection. To clarify importance of furin cleavage site in spike protein for coronavirus, we investigated the pathogenesis of neurotropic avian coronavirus whose spike protein contains an extra furin cleavage site (furin-S2' site). By combining real-time quantitative polymerase chain reaction and immunohistochemistry we demonstrated that infectious bronchitis virus (IBV) infects brain instead of trachea when its S protein contains furin-S2' site. Moreover, the virus was shown to increase the permeability of blood-brain barrier, infect neuron cells and induce high expression of TNF-α. Based on these results we further show that furin cleavage site in S protein plays an important role in coronavirus pathogenicity and cell tropism. Our study extends previous publications on function of S protein of coronavirus, increasing the understanding of researchers to coronavirus.