Conditional reprogrammed human limbal epithelial cell model for anti-SARS-CoV-2 drug screening.

To minimize the global pandemic COVID-19 spread, understanding the possible transmission routes of SARS-CoV-2 and discovery of novel antiviral drugs are necessary. We describe here that the virus can infect ocular surface limbal epithelial, but not other regions. Limbal supports wild type and mutant SARS-CoV-2 entry and replication depending on ACE2, TMPRSS2 and possibly other receptors, resulting in slight CPE and arising IL-6 secretion, which symbolizes conjunctivitis in clinical symptoms. With this limbal model, we have screened two natural product libraries and discovered several unreported drugs. Our data reveal important commonalities between COVID-19 and ocular infection with SARS-CoV-2, and establish an ideal cell model for drug screening and mechanism research.

Direct blue 53, a biological dye, inhibits SARS-CoV-2 infection by blocking ACE2 and spike interaction in vitro and in vivo.

COVID-19 is a global health problem caused by SARS-CoV-2, which has led to over 600 million infections and 6 million deaths. Developing novel antiviral drugs is of pivotal importance to slow down the epidemic swiftly. In this study, we identified five azo compounds as effective antiviral drugs to SARS-CoV-2, and mechanism study revealed their targets for impeding viral particles' ability to bind to host receptors. Direct Blue 53, which displayed the strongest inhibitory impact, inhibited five mutant strains at micromole. In vitro, mechanism study demonstrated Direct Blue 53 inhibited viral infection through interaction with the spike of SARS-CoV-2. And 25 mg/kg/d compound treatment showed 50% or 60% survival protection against lethal Delta or Omicron BA.2 infection in vivo. Taken together, our results demonstrate that azo compounds with dimethyl-biphenyl-diyl-bis(azo)bis structure may be promising anti-SARS-CoV-2 drug candidates, which provide practicable therapies with the aid of structural optimizations and further research.

The effect of influenza A (H1N1) pdm09 virus infection on cytokine production and gene expression in BV2 microglial cells.

Acute influenza infection has been reported to be associated with neurological symptoms such as influenza-associated encephalopathy (IAE). Although the pathophysiology of this condition remain unclear, neuroinflammation and associated alterations in the central nervous system (CNS) are usually induced. Microglia (MGs), CNS-resident macrophages, are generally the first cells to be activated in response to brain infection or damage. We performed reverse transcriptase droplet digital PCR (RT-ddPCR) and luminex assays to investigate virus proliferation and immune reactions in BV2 MGs infected with influenza A(H1N1)pdm09 virus. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to investigate the dynamic change in the protein expression profile in BV2 MGs to gain insight into the CNS response to influenza A (H1N1) pdm09 infection. Our results showed that the influenza A(H1N1)pdm09 virus was replicative and productive in BV2 MG cells, which produced cytokines such as interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. The expression of osteopontin (OPN) in the influenza A (H1N1) pdm09-infected BV2 MGs was upregulated at 16 and 32 h post-infection (hpi) compared to that in the control group, resulting in aggravated brain damage and inflammation. Our study indicates that OPN signalling might provide new insights into the treatment of CNS injury and neurodegenerative diseases in IAE.

RP1, a RAGE antagonist peptide, can improve memory impairment and reduce Aß plaque load in the APP/PS1 mouse model of Alzheimer's disease.

Amyloid-ß (Aß) accumulation is a pathological hallmark of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is involved in the production and accumulation of Aß. RP1, a peptide antagonist of RAGE, was screened by phage display technology in our previous studies, and its neuroprotective effects on an AD cell model have been confirmed. However, its efficacy in vivo remains unclear. Here, the intranasal delivery of RP1 to APPSwe/PS1dE9 (APP/PS1) mice significantly improved memory impairment and relieved the Aß burden by decreasing the expression of amyloid precursor protein and ß-secretase. RNA-sequencing (RNA-seq) was utilized to identify differentially expressed genes (DEGs) in APP/PS1 mice after RP1 administration. Several DEGs in RAGE downstream signalling pathways were downregulated. Some transcription factors (such as Fos) and the pathways enriched in the remarkable modules may also be related to the efficacy of RP1. In conclusion, RP1 significantly improves the AD symptoms of APP/PS1 mice, and the RNA-seq results provide new ideas for elucidating the possible mechanisms of RP1 treatment.

Forecasting influenza epidemics from multi-stream surveillance data in a subtropical city of China.

BACKGROUND: Influenza has been associated with heavy burden of mortality and morbidity in subtropical regions. However, timely forecast of influenza epidemic in these regions has been hindered by unclear seasonality of influenza viruses. In this study, we developed a forecasting model by integrating multiple sentinel surveillance data to predict influenza epidemics in a subtropical city Shenzhen, China. METHODS: Dynamic linear models with the predictors of single or multiple surveillance data for influenza-like illness (ILI) were adopted to forecast influenza epidemics from 2006 to 2012 in Shenzhen. Temporal coherence of these surveillance data with laboratory-confirmed influenza cases was evaluated by wavelet analysis and only the coherent data streams were entered into the model. Timeliness, sensitivity and specificity of these models were also evaluated to compare their performance. RESULTS: Both influenza virology data and ILI consultation rates in Shenzhen demonstrated a significant annual seasonal cycle (p<0.05) during the entire study period, with occasional deviations observed in some data streams. The forecasting models that combined multi-stream ILI surveillance data generally outperformed the models with single-stream ILI data, by providing more timely, sensitive and specific alerts. CONCLUSIONS: Forecasting models that combine multiple sentinel surveillance data can be considered to generate timely alerts for influenza epidemics in subtropical regions like Shenzhen.

[Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus].

OBJECTIVE: To develop a novel sensitive duplex real-time PCR assay for accurately identifying B/Yamagata and B/Victoria lineages of influenza virus type B. METHODS: 50 HA (hemagglutinin) gene sequences coding for B/Yamagata and B/Victoria lineage, respectively, were randomly downloaded for GenBank and analyzed by software MEGA. Primers and probes specific for HA gene of B/Yamagata and B/Victoria lineages were designed by Primer Primer and then applied in the duplex real-time RT-PCR method that was followed developed. Influenza virus B type and A type isolated in our laboratory and typing-confirmed by HAI method were used as reference strains to determine the specificity of this assay and the sensitivity of the duplex amplification was evaluated by viral load testing in terms of in vitro transcribed RNA copy number. RESULTS: In 2006-2010, 793 influenza virus type B strains were isolated from 17 765 throat swab samples, among which 152 strains were differentiated as By lineage and 641 as Bv lineage by this assay. These results was agreement with that determined by HAI assay. This developed assay allows to accurately identify approximately 10(2) copies/microl for Bv and By lineage virus with intra- and inter-coefficient of variation (CV) < 3.5% and nearly 100% specificity. CONCLUSIONS: This method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination of influenza virus, which could be applied in influenza surveillance laboratories for rapid molecular diagnosis.

[Compare real-time RT-PCR with two culture methods for influenza virus detection].

OBJECTIVE: Real-time RT-PCR, cell culture and embryonated eggs culture for influenza detection were compared by analyzing the data of influenza surveillance in Shenzhen in second half of 2009. METHODS: 1092 clinical samples (throat swabs) collected during second half of 2009 were tested by real-time RT-PCR, cell culture and embryonated eggs culture, and the results were analyzed by statistical methods. RESULTS: The positive rate were 54.21%, 27.11% and 16.21% using real-time RT-PCR, cell culture and embryonated eggs culture, and the sensitive were 100%, 50% and 29.9%. The lowest dilutions of virus detected by real-time RT-PCR were 10(-2) TCID50/ml. CONCLUSION: The sensitive of real-time RT-PCR was higher than culture and the specificity was also very high. It was more suitable for emergency detect. The sensitive of cell culture for H3N2 subtype was higher, and sensitive of embryonated eggs culture for type B was higher.

Three fatal cases of pandemic 2009 influenza A virus infection in Shenzhen are associated with cytokine storm.

China had taken strict measures for pandemic 2009 H1N1 infection with enhanced surveillance and hospital isolation since April 2009. In Shenzhen, over 1200 confirmed cases of H1N1 infection were identified. Three young patients died of severe pneumonia. Among them, two boys developed neurological complications. Cytokine storm seemed an important cause.

[Association of cooking oil fumes exposure and oxidative DNA damage among occupational exposed populations].

BACKGROUND: Previous investigations indicate that cooks are exposed to polycyclic aromatic hydrocarbons (PAH) from cooking oil fumes (COF). However, Emission of PAH and their carcinogenic potencies from cooking oil fumes sources have not been investigated among cooks. AIMS: To investigate the urinary excretion of a marker for oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), in different groups of cooks and different exposure groups, and to study the association between 8-OHdG and 1-hydroxypyrene (1-OHP), a biological marker for PAH exposure. METHODS: Urine samples were collected from different groups of cooks (n = 86) and from unexposed controls (n = 36), all are male with similar age and smoking habits. The health status, occupational history, smoking, and alcohol consumption 24 hours prior to sampling was estimated from questionnaires. The urinary samples were frozen for later analyses of 8-OHdG and 1-OHP by high performance liquid chromatography. RESULTS: Excretion in urine of 8-OHdG were similar for controls (mean 1.2 µmol/mol creatinine, n = 36), and for those who had been in the kitchen room with exhaust hood operation (mean 1.5 µmol/mol creatinine, n = 45). COF exposed cooks without exhaust hood operation had increased excretion of 8-OHdG (mean 2.3 µmol/mol creatinine, n = 18). The difference between this group and the unexposed controls was significant. The urinary levels of ln 1-OHP and ln 8-OHdG were still significantly correlated in a multiple regression analysis. CONCLUSION: Results indicate that exposure to PAH or possibly other compounds in COF may cause oxidative DNA damage.

[The preparation of P particle of the norovirus strain SZ9711 from China and its affinity analysis with human histo-blood group antigens in saliva].

OBJECTIVE: To study the binding profile of NV strain SZ9711 (GII-4) with human histo-blood group antigens (HBGAs). METHODS: The P domain-encoding fragment was amplified by RT-PCR from the stain SZ9711 and cloned into the pGEX-4T-1 vector. The recombinant fusion protein was expressed in E. coli and purified using the column Sepharose 4B. The P protein was released by thrombin cleavage. The binding of P particles of SZ9711 and VA387 with the HBGAs were measured by saliva-based EIA method. RESULTS: The expression of the recombinant fusion protein was shown by the SDS-PAGE, in which a 38 x 10(3)-P protein was obtained. Saliva-based EIA revealed that the P particle of SZ9711 bound to HBGAs in saliva similar to that of the strain VA387 reported previously. It bound strongly to saliva of type A, B and O(secretor) but did not interact with saliva of type O(non-secretor). Noteworthy, binding ability of SZ9711 P particle to type A saliva was lower than that of the VA387 P particle. CONCLUSION: This is the first time that a P particle was prepared from a norovirus strain isolated in China and the binding ability of the P particle with HBGAs was analyzed. The result indicated the binding profile of the SZ9711 P particle was similar to that of VA387 reported previously. These data may be valuable in studying the relationship between noroviruses and their bindings to HGBA receptors.

[Analyses of serological and genetic characteristics on novel H1N1 influenza A virus from the infected patient in Shenzhen].

Analysis of serological and genetic characteristics on 2009 swine-origin influenza A (H1N1) virus (S-OIV) isolated from four patients with severe disease in Shenzhen were performed. Microneutralization assay showed that the neutralizing antibody titers of the infected patients did not exceed 1 : 20 in a short term post infection, which could not neutralize the viruses efficiently. Hemagglutination inhibition (HI) tests confirmed that the antigenicity of S-OIV from the patients was distinct from the seasonal influenza A virus, but similar to the reference strains of S-OIV. Phylogenetic and molecular analysis showed that S-OIV from the patients still belonged to the classical swine lineages and did not have the genetic characteristics of highly pathogenic influenza virus. Several amino acid residue mutations on HA protein were detected, which seemed not to affect the virulence and pathogenicity of the viruses. Further, A His 275 Tyr mutation on NA protein of a virus strain was detected, which induced the oseltamivir resistance of the virus.

[Epidemiological and molecular characterization of seasonal influenza A/H3N2 viruses circulating in Shenzhen, 2005 - 2007].

OBJECTIVE: To determine the epidemiological characteristics of seasonal influenza in Shenzhen from 2005 to 2007 and the molecular variation of HA1 domain of influenza H3N2 viruses. METHODS: The consultation rate for influenza-like illness (ILI) were calculated weekly for indicating the influenza activities (the Shenzhen Influenza Surveillance System mainly consisted of 16 institutions with 9 hospitals, 6 districts and one municipal centers of disease control and prevention). Pharyngeal swabs from the cases of ILI, which were collected during 2005 to 2007 from the city-wide and quality-controlled surveillance network, were used to propagate the viruses. The HA1 region of the influenza A/H3N2 viruses were detected by RT-PCR and sequenced subsequently. The analyses of pairwise amino acid variations, genetic clustering and phylogenetics was performed. RESULTS: The activity levels of influenza showed certain changes during each year from 2005 to 2007, and there were summer peaks from May to July in 2006 and 2007. The positive rates of influenza virus were 4.78% (114/2385), 5.77% (212/3674) and 12.12% (343/2831) from 2005 to 2007 respectively. The weekly isolating rates changed accordingly with the trend of the percentages of ILI. The proportions of influenza H3N2 virus were 25.46% (28/114) and 2.83% (6/212) in 2005 and in 2006 respectively, but the proportion increased to 62.68% (215/343), which indicated that H3N2 virus became the predominant strain in 2007. Phylogenetic clustering analysis of influenza H3N2 virus revealed that there were 5 clades. The viruses which were isolated in 2005 contained in the clade I and II, the viruses in 2006 were comprised in clade III, and clade IV and V included the viruses isolated in 2007. Although the stem of cladogram developed with one accord of the time isolated viruses, the viruses which were similar to vaccine strains had circulated in Shenzhen before a given strain was determined as vaccine strain by WHO. It was also noticed that more amino acid changes at antigenic sites, especially at sites A and B in the H3N2 viruses, were found in 2007 than that in 2005 and in 2006. But the sequences at the receptor-binding sites and disulphide bond sites were conserved and no new circulating strain for genetic reassortment had been found in the period. CONCLUSION: Shenzhen might be one of areas where the ongoing genetic drift of influenza H3N2 viruses appeared earlier in China. The changes of influenza H3N2 virus showed the active status in the population. The results suggested that monitoring seasonal influenza viruses by sequence analysis could provide important and timely information on the appearance of strains with epidemiologic significance.

[Study on molecular epidemiological characteristics of influenza H1N1 viruses circulating in Shenzhen, China from 2005 to 2007].

OBJECTIVE: To study the genetic and epidemiological characteristics of HA1 of influenza H1N1 viruses circulating in Shenzhen from 2005 to 2007. METHODS: The HA1 region was analyzed by RT-PCR and subsequently sequenced to analyze the HA1 genetic evolution. Phylogenetic analysis was confirmed on the homology of nucleotide comparing with the reference viruses of vaccines recommended by WHO and representative virus confirmed by China CDC. Relationship between isolation rates and genetic evolutions was explored. RESULTS: The average isolation rate from 2005 to 2007 was 7.16%. Of the isolates, the proportions of influenza H1N1 viruses in 2005, 2006 and 2007 were 56.14%, 66.03%, 3.61%, respectively. Data from HA1 phylogenetic analysis showed that there were at least three clades circulated in Shenzhen. Different viruses isolated during January to April were clustered with A/New Caledonia/20/1999 viruses isolated in the latter months of 2005 clustered with A/Solomon Island/3/2006 and viruses from 2006 to 2007 were in the same clade with A/GDLH/219/2006. Results showed that most viruses had a deletion of lysine at position 130. Compared with A/New Caledonia/20/1999, the virus isolated after May of 2005 occurred T82K, Y94H, R146K, R209K, T267N amino acid substitution, while some virus isolated after May 2006 took place the amino acid substitutions of A190T, H193Y, E195D (located at antigenic site B) and R146K (antigenic site A). The sequences at the receptor-binding sites and glycosylation sites were conserved. Compared with referring viruses, A/SZ/68/2007 had 50 amino acid substitutions in the HA1 region. Of these, eleven and six were located at antigenic sites and receptor-binding sites, respectively. Four amino acid substitution resulted in the deletion of glycosylation site. CONCLUSION: Three different genetic lineages of influenza H1N1 virus were circulated in the population in Shenzhen during 2005 - 2007. The special virus named A/SZ/68/2007 should be paid further attention on its antigenic and epidemiological characteristics.

[Laboratory diagnosis and molecular characterization analysis of the H5N1 influenza virus isolated from the first human case in Shenzhen, China].

The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza (firstly found in Shenzhen, China) were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient's tracheal aspirates on MDCK cell and was named A/Guangdong/2/06(H5N1). The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1 : 160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.

[Rapid detection of influenza virus A by fluorescence quantitative RT-PCR].

OBJECTIVE: To develop a method to rapidly detect influenza virus type A by fluorescence real-time quantitative RT-PCR. METHODS: According to conservative sequence of influenza virus type A M2 gene, a pair primers and Taqman probe were designed, respectively. After the quantitative curve of the assay were established using tenfold serial dilution of TCID50, the sensitivity and the specificity of clinic samples were determined. RESULTS: The detection sensitivity of the assay was 2.56 x 10(-5) TCID50 and the regression coefficient of the quantitative curve was 0.999. The specificity was determined by testing five other specimens, all of which yielded negative results. CONCLUSION: The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.

[Chemical modification of chymopapain with monomethoxypolyethylene glycol and its effect on enzymic activity and antigenicity].

OBJECTIVE: To study the effect of chemical modification of chymopapain with monomethoxypolyethylene glycol on enzymic activity and antigenicity. METHODS: Under the substrate protecting and non-substrate protecting, the chymopapain (Cp) was modified with two types of mPEG derivatives mPEG1 and mPEG2. The average ratio of modified-NH2 was tested by trinitrobenzenesulfonic acid (TNBS) method, the enzymic activity was tested with macromolecular casein and ATEE as substrates, the antigenicity of modified enzyme was determined by ELISA method. RESULTS: (1) Both mPEG1 and mPEG2 can reduce and eliminate antigenicity of Cp, the mPEG2 was better than mPEG1. (2) The enzymic activities of modified Cp were reduced, the enzymic activities of mPEG1-modified Cp were higher than that of mPEG2-modified Cp (especially macromolecular protein as its substrate). (3) The enzymic activities in present of ATEE were obviously higher than that in absent of substrate. CONCLUSION: When mPEG1 as the modifier and in present of ATEE, the antigenicity of Cp was completely eliminated, and the enzymic activities were still higher.

[Detection of SARS-coronavirus in both human and animals by RT-PCR].

OBJECTIVE: To find the most suitable RT-PCR detection method for SARS-Coronavirus (SARS-CoV) detection in both human and animals, and to study the source of SARS virus by investigating the condition of virus carried by wild, domestic animals and animals sold in market. METHODS: 350 throat washes of confirmed, suspected and observed SARS cases were tested by TaqMan and molecular beacon fluorescence RT-PCR methods. 386 animals with 442 nasal-throat swabs, fecal swabs of animals and cell cultured were detected by TaqMan method and conventional RT-PCR method. RESULTS: 10 positive were detected from 41 SARS clinical confirmed cases (24.39%). The results of TaqMan and molecular beacon are basically coincident with the coincident rate of 88.89%. 18 cell cultures with CPE were detected and find 16 positive, among which, 14 were civet cats (87.5%). The detecting results of animal samples from Dongmen Market (Shenzhen) and other markets and farms in peripheral areas show that: the total positive rate of 4 kinds of wild animal (civet cat, raccoon dog, hog-badge and Chinese ferret-badge) is 39.02% , which has an significant difference (P < 0.01) compared with the positive rate of other animals as 0. The positive rate of PCR for nasal and fecal swabs from 109 major wild animals is 44.04%, but the positive rate of 145 wild live-animals is 0. CONCLUSION: TaqMan and molecular beacon PCR methods both can be used in SARS detection.The results could support the hypothesis that animals (especially civet cat) could carry SARS virus.

[Evaluation on construction and expression in vitro of nucleic acid vaccine of nucleoprotein of influenza virus A].

OBJECTIVE: Constructing nucleic acid vaccine of influenza virus A to study the protective effects. METHODS: NP gene was reverse transcripted from influenza virus A and linked with pSECTAG 2/Hygro A to constructed nucleic acid vaccine of influenza virus A. The constructed nucleic acid vaccine was transinfected into VERO cell resorting to lipofectamineTM2000. NP gene of influenza virus A was expressed in VERO cell by the method of ELISA. RESULTS: The results showed that the NP gene was expressed to the highest level at 36h after transinfection with the A40 value of 0.382. From 36 h to 60 h after transinfection, the expression of NP gene was stable (the A45, value at 48h and 60h was 0.385 and 0.387, respectively) . CONCLUSION: The nucleic acid vaccine of influenza virus A was successfully constructed, which sets up groudworks for the research of effection of nucleic acid vaccine in vivo.

[Expression and assay in vitro of phage single chain antibody against nucleoprotein of influenza virus A type].

OBJECTIVE: To expression the phage single chain antibody against nucleoprotein of influenza virus A type in E. coli strain HB2151 by screening the positive clone from the phage antibody library against nucleoprotein, which will prepare for the construction of fast leak kit of Influenza virus A type. METHODS: The positive clone ratio in the phage antibody library was enriched by three-round continual solid panning, ELISA, SDS-PAGE and Western-blot methods were used to analyze the protein secreted into the supemants and periplasmic. Affinity chromatography was used to purified the protein expressed in periplasmic and the titer was also analyzed using ELISA method. RESULTS: 10 clones were screened from96 clones and among the 10, there were 3 stronger positive with OD450nm value of 0.469, 0.582 and 0.507, respectively. The phage single chain antibody against Influenza virus A type was primary expressed in periplasmic. The ELISA results were positive even if the single chain antibody purified by affinity chromatography was diluted 4096 folds. CONCLUSION: Using phage antibody library technique, phage single antibody against nucleoprotein of influenza virus A type was preliminary expressed in E. coli.

[Clone expression and purification of influenza A virus nucleoprotein gene].

OBJECTIVE: To clone the influenza A virus NP gene into expression vector and to purify the target protein, which was used to study the preparation of monoclonal antibody. METHODS: The RNA of influenza A virus was extracted and primers were designed according the NP gene sequence, then the NP gene of influenza A virus was expressed in E.coli DH5alpha and the NP protein was purified by affinity chromatography. RESULTS: The recombinant expression vector-pGEX-4T-2-NP was successfully constructed and relatively pure target protein was obtained. CONCLUSION: Through reasonably controlling the fermentation time, growth temperature and induction concentration, satisfactory soluble target product was obtained.