Identification of an effective fraction from Ampelopsis Radix with anti-dengue virus activities in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) infection is a global public health issue without effective therapeutic interventions. Chinese medicine with heat-clearing and detoxifying properties has been frequently used in the treatment of viral infection. Ampelopsis Radix (AR) is a traditional Chinese medicine for clearing heat and detoxification that has been widely used in the prevention and treatment of infectious diseases. However, no studies on the effects of AR against viral infection have been reported, thus far. AIM OF THE STUDY: To explore the anti-DENV activities of the fraction (AR-1) obtained from AR both in vitro and in vivo. MATERIALS AND METHODS: The chemical composition of AR-1 was identified by liquid chromatography-tandem MS (LC‒MS/MS). The antiviral activities of AR-1 were studied in baby hamster kidney fibroblast BHK-21 cells, ICR suckling mice and induction of interferon α/ß (IFN-α/ß) and IFN-γ R-/- (AG129) mice. RESULTS: Based on LC‒MS/MS analysis, 60 compounds (including flavonoids, phenols, anthraquinones, alkaloids and other types) were tentatively characterized from AR-1. AR-1 inhibited the cytopathic effect, the production of progeny virus and the synthesis of viral RNA and proteins by blocking DENV-2 binding to BHK-21 cells. Moreover, AR-1 significantly attenuated weight loss, decreased clinical scores and prolonged the survival of DENV-infected ICR suckling mice. Critically, the viral load in blood, brain and kidney tissues and the pathological changes in brain were remarkably alleviated after AR-1 treatment. Further study on AG129 mice showed that AR-1 obviously improved the clinical manifestations and survival rate, reduced viremia, attenuated gastric distension and relieved the pathological lesions caused by DENV. CONCLUSIONS: In summary, this is the first report that AR-1 exhibits anti-DENV effects both in vitro and in vivo, which suggests that AR-1 may be developed as a therapeutic candidate against DENV infection.

A simple and effective aerosol pathogen disinfection test for a flowing air disinfector.

Aerosol transmission is an important disease transmission route and has been especially pertinent to hospital and biosafety laboratories during the SARS-CoV-2 pandemic. The thermal resistance of airborne SARS-CoV-2 is lower than that of Bacillus subtilis spores, which are often used to test the effectiveness of SARS-CoV-2 and other pathogen disinfection methods. Herein, we propose a new method to test the disinfection ability of a flowing air disinfector (a digital electromagnetic induction air heater) using B. subtilis spores. The study provides an alternative air disinfection test method. The new test system combined an aerosol generator and a respiratory filter designed in-house and could effectively recover spores on the filter membrane at the air outlet after passing through the flowing air disinfector. The total number of bacterial spores used in the test was within the range of 5 × 105-5 × 106 colony-forming units (CFUs) specified in the technical standard for disinfection. The calculation was based on the calculation method in Air Disinfection Effect Appraisal Test in Technical Standard for Disinfection (2002 Edition). At an air speed of 3.5 m/s, we used a digital electromagnetic induction air heater to disinfect flowing air containing 4.100 × 106 CFUs of B. subtilis spores and determined that the minimum disinfection temperature was 350 °C for a killing rate of 99.99%. At 400 °C, additional experiments using higher spore concentrations (4.700 × 106 ± 1.871 × 105 CFU) and a higher airspeed (4 m/s) showed that the killing rate remained>99.99%. B. subtilis spores, as a biological indicator for testing the efficiency of dry-heat sterilization, were killed by the high temperatures used in this system. The proposed method used to test the flowing air disinfector is simple, stable, and effective. This study provides a reference for the development of test systems that can assess the disinfection ability of flowing air disinfectors.

Enterovirus A71 utilizes host cell lipid ß-oxidation to promote its replication.

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD), which is an infectious disease that endangers children's health. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Metabolism and stress are known to play critical roles in multiple stages of the replication of viruses. Lipid metabolism and ER stress is an important characterization post viral infection. EV-A71 infection alters the perturbations of intracellular lipid homeostasis and induces ER stress. The characterizations induced by viral infections are essential for optimal virus replication and may be potential antiviral targets. In this study, we found that the addition of the chemical drug of ER stress, PKR IN, an inhibitor, or Tunicamycin, an activator, could significantly reduce viral replication with the decrease of lipid. The replication of viruses was reduced by Chemical reagent TOFA, an inhibitor of acetyl-CoA carboxylase (ACC) or C75, an inhibitor of fatty acid synthase (FASN), while enhanced by oleic acid (OA), which is a kind of exogenous supplement of triacylglycerol. The pharmacochemical reagent of carnitine palmitoyltransferase 1 (CPT1) called Etomoxir could knock down CPT1 to induce EV-A71 replication to decrease. This suggests that lipid, rather than ER stress, is the main factor affecting EV-A71 replication. In conclusion, this study revealed that it is the ß-oxidation of lipid that plays a core role, not ER stress, which is only a concomitant change without restrictive effect, on virus replication.

Murine diabetic models for dengue virus infection.

Dengue virus (DENV) is a critical public health concern in tropical and subtropical regions worldwide. Thus, immunocompetent murine models of DENV infection with robust viremia are required for vaccine studies. Diabetes is highly prevalent worldwide, making it frequent comorbidity in patients with dengue fever. Therefore, murine models are needed to understand viral pathogenesis and disease progression. Acquired-induced and inherently diabetic C57BL/6 and db/db mice were inoculated with DENV-3 via the tail vein. After infection, both the diabetic C57BL/6 and db/db mice showed obvious weight loss with clinical manifestations. Quantitative reverse-transcription polymerase chain reaction revealed robust and replicable viremia in the two types of diabetic mice. Immunohistochemical detection showed persistent DENV-3 infection in the liver. Enzyme-linked immunosorbent assay for cytokine detection revealed that diabetic mice showed more severe inflammatory responses than did nondiabetic mice, and significant histological alterations were observed in diabetic mice. Thus, the diabetic mice were more susceptible to DENV infection than the nondiabetic mice. Taken together, we established two types of immunocompetent diabetic mice for DENV infection, which can be used to further study the mechanisms of dengue pathogenesis in diabetes and to develop antiviral pharmaceuticals and treatments.

Dengue virus is involved in insulin resistance via the downregulation of IRS-1 by inducing TNF-α secretion.

During the epidemic, the individuals with underlying diseases usually have a higher rate of mortality. Diabetes is highly prevalent worldwide, making it a frequent comorbidity in dengue fever patients. Therefore, understanding the relationship between dengue virus (DENV) infection and diabetes is important. We first demonstrated that DENV-3 infection down-regulated the expression of IRS-1. In vitro, treatment of HepG2 cells with TNF-α inhibitors and siRNA proved that after DENV-3 infection in HepG2 cells, cellular TNF-α secretion was increased, which negatively regulated IRS-1, thereby leading to an insulin-resistant state. In vivo, DENV-3 induced insulin resistance (IR) in hepatocytes by promoting the secretion of TNF-α and inhibiting the expression of IRS-1 was proved. In vivo approaches also showed that after DENV-3 infection, TNF-α levels in the serum of C57BL/6 mice with insulin resistance increased, and upon TNF-α antagonist III treatment, IRS-1 expression in the liver, reduced by infection, was upregulated. In addition, transcriptomic analysis revealed more negative regulatory events in the insulin receptor signaling pathway after DENV-3 infection. This is the first report of a link between DENV-3 infection and insulin resistance, and it lays a foundation for further research.

Pulmonary tuberculosis screening in emigrants and travellers: A retrospective analysis of Guangzhou Port in China.

BACKGROUND: China is beginning to transform from a migrant exporting country to a migrant importing country. Our study aimed to assess risks of imported tuberculosis among travellers and to determine risk factors, to tailor institutional guidelines. METHODS: We conducted an observational, retrospective, population-based cohort study. Molecular epidemiology surveillance methods were used to screen travellers for cases of pulmonary tuberculosis (PTB) at Guangzhou Port in China from January 2010 to December 2016. RESULTS: A total of 165,369 travellers from 190 countries and regions were screened for PTB. The rate of suspected PTB, laboratory confirmed rate, and the total detection rate in emigrants were significantly higher than those in travellers (p<0.01). There were four differences in the PTB screening process between emigrants and travellers. According to the transmission risk degree of the tuberculosis, forty high-risk PTB importing countries were divided into five levels. The travellers diagnosed with PTB were significantly younger than the emigrants (p<0.01). The distribution of genotypes differed significantly between the travellers and emigrants (p<0.001). CONCLUSIONS: PTB screening process in travellers at ports should include a risk assessment of high-risk groups. It should reduce diagnosis time by rapid molecular detection methods and strengthen drug resistant (DR) transmission and monitoring of imported PTB strains through molecular genotyping at ports.

High-specificity targets in SARS-CoV-2 N protein for serological detection and distinction from SARS-CoV.

Numerous serological detection kits are being rapidly developed and approved for screening and diagnosing suspected coronavirus disease 2019 (COVID-19) cases. However, cross-reactivity between pre-existing antibodies against other coronaviruses and the captured antigens in these kits can affect detection accuracy, emphasizing the necessity for identifying highly specific antigen fragments for antibody detection. Thus, we performed a conservation and specificity analysis of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein. We also integrated various B-cell epitope prediction methods to obtain possible dominant epitope regions for the N protein, analyzed the differences in serological antibody levels for different epitopes using ELISA, and identified N protein epitopes for IgG and IgM with high-specificity. The SARS-CoV-2 N protein showed low mutation rates and shared the highest amino acid similarity with SARS-CoV; however, it differed substantially from other coronaviruses. Tests targeting the SARS-CoV-2 N protein produce strong positive results in patients recovering from SARS-CoV. The N18-39 and N183-197 epitopes for IgG and IgM detection, respectively, can effectively overcome cross-reactivity, and even exhibit good specificity between SARS-CoV-2 and SARS-CoV. The antibody levels detected with these were consistent with those detected using the complete N protein. These findings provide a basis for serological diagnosis and determining the kinetics of SARS-CoV-2 antibody detection in patients.

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2.

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

SARS-CoV-2: characteristics and current advances in research.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly across the world and become an international public health emergency. Both SARS-CoV-2 and SARS-CoV belong to subfamily Coronavirinae in the family Coronaviridae of the order Nidovirales and they are classified as the SARS-like species while belong to different cluster. Besides, viral structure, epidemiology characteristics and pathological characteristics are also different. We present a comprehensive survey of the latest coronavirus-SARS-CoV-2-from investigating its origin and evolution alongside SARS-CoV. Meanwhile, pathogenesis, cardiovascular disease in COVID-19 patients, myocardial injury and venous thromboembolism induced by SARS-CoV-2 as well as the treatment methods are summarized in this review.

The effects of Japanese encephalitis virus antibodies on Zika virus infection.

Recently, Zika virus (ZIKV) has become more widespread, thus attracting global attention. The vaccine against Japanese encephalitis virus (JEV) is currently used in China, being included in planned immunisation regimes. Although ZIKV and JEV are closely related mosquito-borne Flaviviruses, and a complex cross-immune response within flaviviruses has been demonstrated, the effect of JEV vaccination on ZIKV infection has not been well described. Thus, this study aimed to explore the impact of different titres of anti-JEV antibodies (Abs) against ZIKV infection using sera from healthy human donors in Guangzhou and anti-JEV rabbit polyclonal antibodies (pAbs) in vitro and vivo. Human anti-JEV Ab titres were tested at decreasing concentrations as the age increased. A neutralising effect on ZIKV infection was observed when anti-JEV Ab titres in human sera or rabbit pAbs were high (the corresponding age was under 30 years). Even though a lower titre in human sera showed no apparent effect, whereas rabbit pAbs had an antibody-dependent enhancement(ADE)effect, we proved an ADE effect in vivo for the first time. This study suggests that individuals over 60 years of age are at high risk for JEV and ZIKV infection, and screening this age group for infection should strengthen. Furthermore, a deep exploration of the relationship between anti-JEV Abs and ZIKV infection is needed.

Research advancements in the neurological presentation of flaviviruses.

Owing to the large-scale epidemic of Zika virus disease and its association with microcephaly, properties that allow flaviviruses to cause nervous system diseases are an important area of investigation. At present, although potential pathogenic mechanisms of flaviviruses in the nervous system have been examined, they have not been completely elucidated. In this paper, we review the possible mechanisms of blood-brain barrier penetration, the pathological effects on neurons, and the association between virus mutations and neurotoxicity. A hypothesis on neurotoxicity caused by the Zika virus is presented. Clarifying the mechanisms of virulence of flaviviruses will be helpful in finding better antiviral drugs and optimizing the treatment of symptoms.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus.

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 101 to 108 copy/µL, with a standard curve R2 of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/µL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 101-104 copy/µL and was able to detect concentrations as low as 1 copy/µL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 101 copy/µL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Effective Suckling C57BL/6, Kunming, and BALB/c Mouse Models with Remarkable Neurological Manifestation for Zika Virus Infection.

Since 2015, 84 countries and territories reported evidence of vector-borne Zika Virus (ZIKV) transmission. The World Health Organization (WHO) declared that ZIKV and associated consequences especially the neurological autoimmune disorder Guillain-Barré syndrome (GBS) and microcephaly will remain a significant enduring public health challenge requiring intense action. We apply a standardization of the multi-subcutaneous dorsal inoculation method to systematically summarize clinical neurological manifestation, viral distribution, and tissue damage during the progress of viremia and systemic spread in suckling mouse models. We found that C57BL/6 and Kunming mice (KM) both showed remarkable and uniform neurologic manifestations. C57BL/6 owned the highest susceptibility and pathogenicity to the nervous system, referred to as movement disorders, with 100% incidence, while KM was an economic model for a Chinese study characterized by lower limb weakness with 62% morbidity. Slight yellow extraocular exudates were observed in BALB/c, suggesting the association with similar ocular findings to those of clinical cases. The virus distribution and pathological changes in the sera, brains, livers, kidneys, spleens, and testes during disease progression had strong regularity and uniformity, demonstrating the effectiveness and plasticity of the animal models. The successful establishment of these animal models will be conducive to expound the pathogenic mechanism of GBS.

Molecular characterization and phylogenetic analysis of dengue virus type 1 in Guangdong in 2014.

BACKGROUND: Dengue is one of the most important emerging diseases of humans, with no preventive vaccines or antiviral cures available currently. In 2014, the Southeast Asian region experienced an unprecedented outbreak of dengue, especially in Guangdong, China. RESULTS: The nucleotide sequences of the E gene from 23 patients sera of dengue virus type 1 (DENV-1) from Guangzhou, China, were determined. One isolate that was recovered from a patient with serious liver damage was designated GZ02. The whole genome sequence of GZ02 was amplified, and confocal microscopy and plaque reduction neutralization test were performed to investigate the replication kinetics in liver L02 cells. In the study, assembly and genetic comparisons showed 11 of those E gene nucleotide sequences were absolutely accordant, and the nucleic acid sequence divergence among the other strains had no marked difference. CONCLUSIONS: Phylogenetic analysis based on the E gene indicated that the 23 new strains were closely related to strains from Malaysia or Singapore. Two different genotypes (genotype I and III) of DENV-1 were co-circulating in Guangdong, Malaysia, and Singapore from 2013 to 2014. However, no recombination event was found after 2005 between DENV strains from Guangdong and Malaysia or Singapore. GZ02 had a significant replicative advantage over DG14 and the DV1 standard strain. Importation of DENV-1 from Southeast Asian countries may have been an important contributing factor to the 2014 outbreak in Guangdong.