Fan Luo

In Vitro and in Vivo Studies of the Inhibitory Effects of Emodin Isolated from Polygonum cuspidatum on Coxsakievirus B4

The lack of effective therapeutics for Coxsackievirus B4 (CVB4) infection underscores the importance of finding novel antiviral compounds. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is one of the natural anthraquinone derivatives obtained from the root and rhizome of Polygonum cuspidatum. In the present study, the possibility of using emodin as a potential antiviral to treat CVB4 infection was explored in vitro and in mice. Emodin reduced CVB4 entry and replication on Hep-2 cells in a concentration- and time-dependent manner, with a 50% effective concentration (EC50) of 12.06 μM and selectivity index (SI) of 5.08, respectively. The inhibitory effect of emodin for CVB4 entry and replication was further confirmed by a quantitative real time PCR (qPCR) assay. The results further showed that the mice orally treated with different dosages of emodin displayed a dose dependent increase of survival rate, body weight and prolonged mean time of death (MTD), accompanied by significantly decreased myocardial virus titers and pathologic scores/lesions. Moreover, emodin could inhibit CVB4-induced apoptosis in vitro and in vivo. Our results indicated that emodin could be used as potential antiviral in the post-exposure prophylaxis for CVB4 infection.

The Epidemic Characteristics and Changing Trend of Hemorrhagic Fever with Renal Syndrome in Hubei Province, China

Background Hemorrhagic fever with renal syndrome (HFRS) is caused by different hantaviruses within the Bunyaviridae family. HFRS is a fulminant, infectious disease that occurs worldwide and is endemic in all 31 provinces of China. Since the first HFRS case in Hubei Province was reported in 1957, the disease has spread across the province and Hubei has become one of the seriously affected areas in China with the greatest number of reported HFRS cases in the 1980s. However, the epidemic characteristics of HFRS in Hubei are still not entirely clear and long-term, systematic investigations of this epidemic area have been very limited. Methods The spatiotemporal distribution of HFRS was investigated using data spanning the years 1980 to 2009. The annual HFRS incidence, fatality rate and seasonal incidence between 1980 and 2009 were calculated and plotted. GIS-based spatial analyses were conducted to detect the spatial distribution and seasonal pattern of HFRS. A spatial statistical analysis, using Kulldorffs spatial scan statistic, was performed to identify clustering of HFRS. Results A total of 104,467 HFRS cases were reported in Hubei Province between 1980 and 2009. Incidence of and mortality due to HFRS declined after the outbreak in 1980s and HFRS cases have been sporadic in recent years. The locations and scale of disease clusters have changed during the three decades. The seasonal epidemic pattern of HFRS was characterized by the shift from the unimodal type (autumn/winter peak) to the bimodal type. Conclusions Socioeconomic development has great influence on the transmission of hantaviruses to humans and new epidemic characteristics have emerged in Hubei Province. It is necessary to reinforce preventative measures against HFRS according to the newly-presented seasonal variation and to intensify these efforts especially in the urban areas of Hubei Province.

Efficacy of arbidol on lethal hantaan virus infections in suckling mice and in vitro

AbstractAim:Arbidol is an immunomodulator that was first developed in Russia. In this study, we report the antiviral activity of arbidol against Hantaan virus (HTNV) in vitro and in vivo.Methods:The antiviral activity of arbidol in vitro was determined by plaque-forming assay, ranging from 0.5 to 8 μg/mL. To investigate whether arbidol has an antiviral effect in vivo, suckling BALB/c mice infected with HTNV were treated with arbidol at 24 h before infection with a 5, 10 or 20 mg·kg−1·d−1, once per day, for 10 days. On day 12 and 28 post infection (pi), histopathological changes and viral antigen were detected. On days 4, 8, 12, and 16 pi, the viral load of target organs and serum TNF-α levels of arbidol-treated animals were determined.Results:Arbidol was found to have potent inhibitory activity against HTNV when added in vitro before or after viral infection, with a 50% inhibitory concentration (IC50) of 0.9 and 1.2 μg/mL, respectively. The 50% lethal dose (LD50) of arbidol for suckling mice was 78.42 mg·kg−1·d−1. Oral administration of arbidol increased both survival rate and mean time to death (MTD). Treatment with arbidol reduced histopathological changes, decreased viral load and viral antigen levels, and modulated the level of serum TNF-α.Conclusion:Arbidol has the ability to elicit protective antiviral activity against HTNV in vivo and in vitro.

Antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza A (H1N1) virus infection

Aim:To investigate the effects of arbidol hydrochloride (ARB), a widely used antiviral agent, on the inflammation induced by influenza virus.Methods:MDCK cells were infected with seasonal influenza A/FM/1/47 (H1N1) or pandemic influenza A/Hubei/71/2009 (H1N1). In vitro cytotoxicity and antiviral activity of ARB was determined using MTT assay. BALB/c mice were infected with A/FM/1/47 (H1N1). Four hours later the mice were administered ARB (45, 90, and 180 mg·kg−1·d−1) or the neuraminidase inhibitor oseltamivir (22.5 mg·kg−1·d−1) via oral gavage once a day for 5 d. Body-weight, median survival time, viral titer, and lung index of the mice were measured. The levels of inflammatory cytokines were examined using real-time RT-PCR and ELISA.Results:Both H1N1 stains were equally sensitive to ARB as tested in vitro. In the infected mice, ARB (90 and 180 mg·kg−1·d−1) significantly decreased the mortality, alleviated virus-induced lung lesions and viral titers. Furthermore, ARB suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10 in the bronchoalveolar lavage fluids and lung tissues. However, ARB did not significantly affect the levels of IFN-α and IFN-γ, but reduced the level of IFN-β1 in lung tissues at 5 dpi. In peritoneal macrophages challenged with A/FM/1/47 (H1N1) or poly I:C, ARB (20 μmol/L) suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10. Oseltamivir produced comparable alleviation of virus-induced lung lesions with more reduction in the viral titers, but less effective modulation of the inflammatory cytokines.Conclusion:ARB efficiently inhibits both H1N1 stains and diminishes both viral replication and acute inflammation through modulating the expression of inflammatory cytokines.

Amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea

Aim:To study whether epigallocatechin gallate (EGCG), a green tea-derived polyphenol, exerted anti-influenza A virus activity in vitro and in vivo.Methods:Madin-Darby canine kidney (MDCK) cells were tested. The antiviral activity of EGCG in the cells was determined using hemagglutination assay and qPCR. Time of addition assay was performed to determine the kinetics of inhibition of influenza A by EGCG. The level of reactive oxygen species (ROS) were determined with confocal microscopy and flow cytometry. BALB/c mice were treated with EGCG (10, 20 or 40 mg·kg−1·d−1, po) for 5 d. On the 3rd d of the treatment, the mice were infected with influenza A virus. Histopathological changes, lung index and virus titers in the lungs were determined.Results:Treatment of influenza A-infected MDCK cells with EGCG (1.25–100 nmol/L) inhibited influenza A replication in a concentration-dependent manner (the ED50 value was 8.71±1.11 nmol/L). Treatment with EGCG (20 nmol/L) significantly suppressed the increased ROS level in MDCK cells following influenza A infection. In BALB/c mice infected with influenza virus, oral administration of EGCG (40 mg·kg−1·d−1) dramatically improved the survival rate, decreased the mean virus yields and mitigated viral pneumonia in the lungs, which was equivalent to oral administration of oseltamivir (40 mg·kg−1·d−1), a positive control drug.Conclusion:The results provide a molecular basis for development of EGCG as a novel and safe chemopreventive agent for influenza A infection.

Genetic analysis of hantaviruses and their rodent hosts in central-south China

Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs.

The Inhibitory Effect of Rheum palmatum Against Coxsackievirus B3in Vitro and in Vivo

Coxsackievirus B(3)(CVB(3)) infection is the major cause of viral myocarditis, as well as dilated cardiomypathy. Rhubarb is one of the oldest and best-known traditional Chinese medicines. We initiated this study to determine the antiviral effect of an ethanol extract from the roots and rhizoma of Rheum palmatum (R. palmatum, one of the Chinese Rhubarbs), against CVB(3) in tissue culture cells and in a mouse model. The ethanol extract from R. palmatum showed significant inhibitory activity against CVB(3) on HEp-2 cells when added after infection, with IC(50) of 4 μg/ml, TI of 10. The medicated mouse serum still contained the pharmaceutical compound 24 h after intraperitoneal injection, and exhibited an antiviral effect on CVB(3)-infected cells, especially in the 0.3 and 0.5 g/kg/day treatment groups. Furthermore, the CVB(3)-infected mice were treated with the extract solution with dosages of 0.3 g/kg/day beginning 24 h post-CVB(3) exposures. The ethanol extract treated mice showed alleviated clinical signs, better survival rate, prolonged MTD and decreased viral titers compared to the virus control group. Our results indicate that the ethanol extract from R. palmatum has the anti-CVB(3) activity in vitro and in vivo and thus provides a re-evaluation of this old remedy with a broad therapeutic potential.

Expression of HIV-encoded microRNA-TAR and its inhibitory effect on viral replication in human primary macrophages

A number of virus-encoded microRNAs have been shown to play important roles in virus replication and virus-host interactions, although the expression and function of miR-TAR-3p derived from the human immunodeficiency virus type 1 (HIV-1) TAR element remain controversial. In this study, miR-TAR-3p was detected in human peripheral blood monocyte-derived macrophages (MDMs) infected by HIV-1. Overexpression of miR-TAR-3p impaired viral replication, while inhibition of miR-TAR-3p enhanced it. Additionally, miR-TAR-3p repressed viral transcription and replication by targeting the TAR element in the HIV-1 5’-LTR in a sequence-specific manner. These results confirm the presence of miR-TAR-3p in HIV-1-infected MDMs and suggest that its function might be used as a mechanism to modulate HIV-1 replication through the expression of a negative regulatory factor.

Influence of HLA-DRB alleles on haemorrhagic fever with renal syndrome in a Chinese Han population in Hubei Province, China.

Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequence-specific primer–polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLA-DRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*1101-1105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls demonstrated that the HLA-DRB1*0401-0411, HLA-DRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.

An Efficient Method for Isolation of Hantaan Virus through Serial Passages in Suckling Mice

Objective: Hantaan virus (HTNV) is one of the main etiologic agents for hemorrhagic fever with renal syndrome in China. However, it is very difficult to isolate the virus from its original host, which hampers the viral characterization. This study describes an efficient method for isolating HTNV in suckling mice. Methods: Hantavirus-infected Apodemus agrarius were screened by quantitative real-time PCR. The homogenates of one positive rodent lung tissue were inoculated into suckling mice for virus propagation through serial passages. Results: During the three passages in suckling mice, the number of viral RNA copies/nanogram of GAPDH mRNA increased significantly ranging from 477 to 7,278 and 46 to 4,898 in the tissues of brain and lung, respectively. Hantaviral antigens could be detected by indirect immunofluorescence assay and around 100-nm virion-like structures were also observed in brain tissue by transmission electron microscopy. No nucleotide exchange was found except for one in the 3′-non-coding domain of S segment when comparing the complete genome sequences from hantavirus in the first and the third passages. Conclusion: These results suggest inoculation of suckling mice with suspected hantavirus-infected rodent samples is an efficient method for isolation and maintenance of HTNV.