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.

Diversity and genetic characterization of orthohantavirus from small mammals and humans during 2012-2022 in Hubei Province, Central China.

Hemorrhagic fever with renal syndrome (HFRS) is a significant public health problem in Hubei Province, China, where a novel strain of orthohantavirus, HV004, was reported in 2012. However, no systematic study has investigated the prevalence and variation of orthohantavirus in rodents and humans. Herein, 2137 small mammals were collected from ten HFRS epidemic areas in Hubei Province from 2012 to 2022, and 143 serum samples from patients with suspected hemorrhagic fever were collected from two hospitals from 2017 to 2021. Orthohantavirus RNA was recovered from 134 lung tissue samples from five rodent species, with a 6.27 % prevalence, and orthohantavirus was detected in serum samples from 25 patients. Genetic analyses revealed that orthohantavirus hantanense (HTNV), orthohantavirus seoulense (SEOV), and orthohantavirus dabieshanense (DBSV) are co-circulating in rodents in Hubei, and HTNV and SEOV were identified in patient serum. Phylogenetic analysis showed that most of the HTNV sequences were clustered with HV004, indicating that HV004-like orthohantavirus was the main HNTV subtype in rodents. Two genetic reassortments and six recombination events were observed in Hubei orthohantaviruses. In summary, this study identified the diversity of orthohantaviruses circulating in Hubei over the past decade, with the HV004-like subtype being the main genotype in rodents and patients. These findings highlight the need for continued attention and focus on orthohantaviruses, especially concerning newly identified strains.

Pathogenicity of novel hantavirus isolate and antigenicity and immunogenicity of novel strain-based inactivated vaccine.

BACKGROUND: Hantaan virus (HTNV, Orthohantavirus hantanensae species, Hantaviridae family) is the main etiological agent responsible for hemorrhagic fever with renal syndrome (HFRS). The novel HTNV may pose a potential danger to the control and prevention of HFRS in China, which highlights the importance of vaccine development in public health management. In previous studies, our laboratory discovered and successfully isolated a new HTNV strain, HV004 strain, from Apodemus agrarius captured in an epidemic area in Hubei, China. METHODS: An initial biological and pathogenicity characterization of HTNV 76-118 (standard train), HV114 strain (a clinical isolate from Hubei province in 1986), and the novel isolate HV004 strain from the epidemic areas of Hubei province were performed in susceptible cells and in vivo. An experimental HV004 strain inactivated vaccine was prepared, and its corresponding immunogenicity was analyzed in BALB/c mice. RESULTS: HV004 strain had a similar but higher pathogenicity than HTNV 76-118 and HV114 in suckling mice. A subcutaneous vaccination (s.c.) with the inactivated HTNV vaccine adjuvanted with aluminum, followed by a challenge intraperitoneally with 106 FFU/ml HTNV, afforded full protection against an HTNV challenge. All immunized mice in every group elicited serum neutralizing antibodies with increasing dosages, which may protect mice from HTNV infection. A dose-dependent stimulation index of splenocytes was also observed in immunized mice. The percentage of IFN-γ-producing CD3+CD8+ T cells was significantly higher in the spleens of immunized mice than in those of control mice. CONCLUSIONS: These findings suggest that the inactivated HTNV vaccine may stimulate mice to produce high levels of antibodies with neutralization activity and elicit specific anti-HTNV humoral and cellular immune responses in BALB/c mice against the prevalent strain of HTNV in south central China.

Extensive genetic diversity of severe fever with thrombocytopenia syndrome virus circulating in Hubei Province, China, 2018-2022.

Severe fever with thrombocytopenia syndrome virus (SFTSV), an etiological agent causing febrile human disease was identified as an emerging tick-borne bunyavirus. The clinical disease characteristics and case fatality rates of SFTSV may vary across distinct regions and among different variant genotypes. From 2018 to 2022, we surveyed and recruited 202 severe fever with thrombocytopenia syndrome (SFTS) patients in Hubei Province, a high-incidence area of the epidemic, and conducted timely and systematic research on the disease characteristics, SFTSV diversity, and the correlation between virus genome variation and clinical diseases. Our study identified at least 6 genotypes of SFTSV prevalent in Hubei Province based on the analysis of the S, M, and L genome sequences of 88 virus strains. Strikingly, the dominant genotype of SFTSV was found to change during the years, indicating a dynamic shift in viral genetic diversity in the region. Phylogenetic analysis revealed the genetic exchange of Hubei SFTSV strains was relatively frequent, including 3 reassortment strains and 8 recombination strains. Despite the limited sample size, SFTSV C1 genotype may be associated with higher mortality compared to the other four genotypes, and the serum amyloid A (SAA) level, an inflammatory biomarker, was significantly elevated in these patients. Overall, our data summarize the disease characteristics of SFTSV in Hubei Province, highlight the profound changes in viral genetic diversity, and indicate the need for in-depth monitoring and exploration of the relationship between viral mutations and disease severity.

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.

Incidence of Orthohantavirus infection in humans follows observed changes in rodent reservoirs, Hubei Province, China (1984-2010).

OBJECTIVE: Orthohantaviruses (genus Orthohantavirus, family Hantaviridae of order Bunyavirales) are rodent-borne viruses causing 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which are mainly prevalent in Eurasia and the Americas, respectively. We initiated this study to investigate and analyze the Orthohantaviruses infection in rodent reservoirs and humans in the Hubei Province of China from 1984 to 2010. SAMPLE: The study included 10,314 mouse and 43,753 human serum samples. PROCEDURES: In this study, we analyzed the incidence of Orthohantavirus infection in humans and observed changes in rodent reservoirs in Hubei Province. RESULTS: The results indicated that although the incidence of HFRS declined from the 1990s, the human inapparent infection did not decrease dramatically. Although elements of the disease ecology have changed over the study period, Apodemus agrarius and Rattus norvegicus remain the major species and a constituent ratio of Rattus norvegicus increased. Rodent population density fluctuated between 16.65% and 2.14%, and decreased quinquennially, showing an obvious downward trend in recent years. The average orthohantaviruses-carrying rate was 6.36%, of which the lowest rate was 2.92% from 2006 to 2010. The analysis of rodent species composition showed that Rattus norvegicus and Apodemus agrarius were the dominant species over time (68.6% [1984 to 1987] and 90.4% [2000 to 2011]), while the composition and variety of other species decreased. The density of rodents was closely related to the incidence of HFRS (r = 0.910, P = .032). CLINICAL RELEVANCE: Our long-term investigation demonstrated that the occurrence of HFRS is closely related to rodent demographic patterns. Therefore, rodent monitoring and rodent control measures for prevention against HFRS in Hubei are warranted.

Reversal of P-glycoprotein overexpression by Ginkgo biloba extract in the brains of pentylenetetrazole-kindled and phenytoin-treated mice.

The purpose of this study was to investigate the combined effects of Ginkgo biloba extract and phenytoin (PHT) sodium as a dose regimen simulating the clinical treatment of patients with epilepsy, on P-glycoprotein (P-GP) overexpression in a pentylenetetrazole-kindled mouse model of epilepsy. Epilepsy was induced by intraperitoneal administration of pentylenetetrazole (40 mg/kg) for 7 days followed by intragastric administration of PHT (40 mg/kg) for 14 days. Thirty mice that developed seizures were randomly divided into three groups and administered PHT as well as the following treatments: saline (negative control); verapamil (20 mg/kg, positive control); and G. biloba (30 mg/kg). Seizure severity was recorded 30 minutes after treatment on Day 4 of drug administration, after which the mice were euthanized, and their brains isolated. Western blots and immunohistochemistry were performed to analyze the expression of P-GP and caspase-3, respectively, in the brain tissue. High-performance liquid chromatography was used to measure the concentrations of PHT in the brains of the treated mice. After 4 consecutive days of treatment, the seizure severity in the mice in the G. biloba extract group was more significantly reduced than the seizure severity in the saline control group, and a significant difference was observed between the G. biloba extract and verapamil control groups (p < 0.05). P-GP expression in the brain more significantly decreased in the mice treated with G. biloba extract and verapamil than it did in the saline-treated control group (p < 0.05). Compared with the saline-treated control group, the mice treated with G. biloba extract and verapamil showed significantly increased brain PHT concentrations (p < 0.05). Furthermore, caspase-3 expression in the brain tissue of the G. biloba extract group was significantly lower than that in the vehicle control group (p < 0.05); this finding demonstrated the neuroprotective effects of G. biloba. Therefore, this study showed that treatment with G. biloba extract in combination with PHT prevented the upregulation of P-GP expression in mice. Moreover, G. biloba extract decreased seizure severity in pentylenetetrazole-kindled/PHT-treated mice through a mechanism that might be related to the reduction of P-GP expression in the brain.

Nuclear expression of dynamin-related protein 1 in lung adenocarcinomas.

Dynamin-related protein 1 (DRP1), an 80 kDa GTPase, is involved in mitochondrial fission and anticancer drug-mediated cytotoxicity, which implicate an association with disease progression of cancer. In this study we investigated the prognostic value of DRP1 in lung adenocarcinomas. Using immunohistochemistry, we measured the expression of DRP1 in 227 patients with lung adenocarcinomas. Expression of DRP1 was confirmed by immunoblotting. The correlation between DRP1 expression and clinicopathological parameters was analyzed by statistical analysis. Difference of survivals between different groups was compared by a log-rank test. The results showed that DRP1 expression was detected in 202 patients with lung adenocarcinomas. Among these, nuclear DRP1 (DRP1(nuc)) was detected in 184 patients. A significant difference was found in cumulative survival between patients with high DRP1(nuc) levels and those with DRP1(cyt) levels (P<0.001). In vitro, hypoxia increased DRP1(nuc) levels and cisplatin resistance. Antibodies specific to DRP1 co-precipitated a human homologue of yeast Rad23 protein A (hHR23A) and silencing of hHR23A decreased the nuclear DRP1 level and cisplatin resistance. In conclusion, DRP1(nuc) is highly expressed in lung adenocarcinomas, and correlates with poor prognosis. Nuclear DRP1 may increase drug resistance during hypoxia, and hHR23A is essential for nuclear transportation of DRP1. Our results suggest that other than the protein level alone, intracellular distribution of the protein is critical for determining the protein function in cells.