During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 microM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.
Cytopathogenic Effect, Viral ; Fermentation* ; Fungi ; Inhibitory Concentration 50 ; Madin Darby Canine Kidney Cells ; Neuraminidase*

BACKGROUND: Acyclovir is a highly effective antiviral agent specifically inhibiting the replication of members of the herpes virus group, in particular the has been used extensively herpes simplex virus types 1 and 2 and the varicella zoster virus. Although acyclovir it has not caused for the treatment or prevention of herpes simplex and varicella zoster virus infections, significant changes in virus sensitivity. OBJECTIVE: The purpose of this study was to evaluate the sensitivity of HSV to acyclovir. METHODS: A total of 80 strains were used 43 strains of non-genital herpes and 37 strains of genital gerpes. These were isolated from 80 patients and were studied to evaluate their sensitivities to acyclovir by the plaque reduction assay. The methods employed to monitor the sensitivity of virus isolates rely on simple dose-response experiments, looking at the effects of increasing concentrations of acyclovir on infected cell culture specimen. The assay is based on quantified plaque counting. The sensitivity of virus strains are then expressed as ID50(concentrations of drug reducing viral cytopathic effect by 50%) and MIC(minimum inhibitory concentration). RESULTS: The results were summarized as follows. 1. The ID50 values of acyclovir for HSV ranged between 0.0625 - 4.0 microgram / ml. For non-genital herpes isolates the mean and median values were 0.459 microgram / ml (SD = 0.624) and 0.250 microgram / ml ; for genital herpes isolates these values were 0.649 microgram / ml (SD = 0.746) and 0.50 microgram / ml . 2. The MIC values of acyclovir for HSV ranged between 0.250 - 32 microgram / ml . For non-genital herpes isolates the mean and median values were 2.605 microgram / ml (SD = 5.270) and 1.00 microgram / ml ; for genital herpes isolates these values were 2.716 microgram / ml (SD = 3.015) and 2.00 microgram / ml . 5. 93.75%(75 strains) of HSV isolates were within the ranges of sensitive HSV strains for acyclovir. CONCLUSION: We are concerned about the resistance of viruses to antiviral drugs, but so far, this has not been documented to be a big problem. With the increasing interest and ability to measure sensitivity of viruses to antiviral drugs we will find out more about viral resistance and its clinical significance.
Acyclovir* ; Antiviral Agents ; Cell Culture Techniques ; Cytopathogenic Effect, Viral ; Herpes Genitalis ; Herpes Simplex* ; Herpesvirus 3, Human ; Humans ; Simplexvirus*

To investigate biological characteristics of the IVpi-189 progeny virus derived from the culture of influenza A virus as a live-attenuated vaccine candidate. Persistent infection of a cultured cell line with influenza A virus (MDCK-IVpi) was established by incubating continuously influenza virus-infected cells at a lower temperature. The infectious progeny virus derived from MDCK-IVpi cells at the 189rd subculture was designated as the IVpi-189 strain of influenza virus. The cytopathic effect induced by IVpi-189 virus was observed under different temperature conditions. The production of infectious progeny virus was examined at 38 and 32 degrees C by plaque titration of cell-associated and released virus. IVpi-189 virus showed cytopathic effect as strong as that of IVwt in infected cell line of MDCK at 32 degrees C. However, when culture temperature was raised to 38 degrees C, the cytopathic effect induced by IVpi-189 virus was delayed and less pronounced. Virus growth in IVpi-189 virus-infected cells at 38 degrees C was significantly reduced as compared with that of IVwt virus, although both viruses yielded nearly equivalent high titers of cell-associated and released virus at 32 degrees C. The reasons of the decreased proliferative ability of IVpi-189 virus at high culture temperature were unrelated with virus inactivation or the release of progeny virus, but associated with the decreased replication of infectious progeny virus in the infected cells. IVpi-189 virus derived from MDCK cells infected persistently with influenza A virus showed biological characteristics as a potential live-attenuated vaccine candidate.
Animals ; Cell Line ; Cytopathogenic Effect, Viral ; Dogs ; Humans ; Influenza A virus ; genetics ; physiology ; Temperature ; Virus Cultivation ; methods ; Virus Replication

We have previously described the development of a onetube SYBR Green real-time RT-PCR assay for the detection and quantitation of infectious salmon anemia virus (ISAV) in various biological samples. The twofold aim of the present study was to verify that the optimized SYBR Green real-time RT-PCR conditions could detect ISAV isolates of different geographic origins, and to analyze the growth patterns of the selected ISAV isolates in the Chinook head salmon embryo (CHSE) -214 cells by this assay to better characterize their CHSE-phenotypes. A total of 24 ISAV isolates were used in this study. The results indicated that the SYBR Green real-time RT-PCR could detect ISAV of different geographic origins or laboratory sources. The capacity of ISAV isolates to cause cytopathic effects (CPE) in the CHSE-214 cell line, viral titration of the infected CHSE-cell harvests, and analysis of viral RNA levels in CHSE-214 cells at post-infection day zero, 7 and 14 by SYBR Green real-time RT-PCR confirmed the existence of three CHSE-phenotypes of ISAV: replicating cytopathic, replicating non-cytopathic, and non-replicating non-cytopathic. The identification of these three CHSE- phenotypes of ISAV has important implications from diagnostic and biological points of view.
Animals ; Cell Line ; Cytopathogenic Effect, Viral ; Isavirus/genetics/*metabolism ; Phenotype ; RNA, Viral/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Salmon/*embryology/*virology ; Time Factors ; Virus Replication

This research aims to evaluate the application of Real - time cell assay (RTCA) based on microelectronics sensor technology in the detection of HEV71 induced cell lesion. Growth indexes of RD cells at different stages were observed dynamically, appropriate cell concentration was selected to test HEV71 infectivity and to determine the HEV71 neutralizing antibody titer in serum. The traditional microplate test was used as methodology comparison and results validation at the same time. Cell impedance was transformed to cell index (CI) value and visual dynamic curve through software, and the result showed that the observation of HEV71 infectivity was more than 5d when the RD cells concentration was 1. 5 X 10(4) hole on the 96 electronic orifice plate. Compared with the traditional cytopathic effect (CPE) through microscope observation method, the end point judgment results were consistent between these two methods at 132h (about 5. 5d) post virus inoculation. In the neutralization tests, three CI values of neutralizing antibody titers against HEV71 in human serum were correspond to those obtained from traditional 96 microplate microscopy. RTCA also suggested that the presentation time of CPE induced by the i virus could be different even the end point judgment was the same with the same neutralization antibody titer. Compared with the traditional microplate monitoring method, RTCA can save labor and eliminate the hands-on error in the monitoring HEV71 infectivity and antibody titer detection in serum. RTCA can be served as one of the supplementary methods of traditional detection method, with the advantages of dynamically observing the occurrence and development of cell pathological changes, and the variation of virus infectivity and serum neutralizing antibodies.
Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Cell Line, Tumor ; Cytopathogenic Effect, Viral ; Electric Impedance ; Enterovirus A, Human ; immunology ; pathogenicity ; Enterovirus Infections ; virology ; Humans ; Microelectrodes ; Neutralization Tests ; methods

OBJECTIVETo study the relationship between late mRNA and the cytopathic effect(CPE) and ultrastructural features after human cytomegalovirus (HCMV) infection in vitro.

METHODSHuman embryo fibroblast cells(HEL) were infected with HCMV AD169 strain. The expression of the HCMV late mRNA was measured by semiquantitative RT-PCR, the cytopathic effect (CPE) and the cell ultrastructure were observed by means of light microscopy and electron microscopy, respectively.

RESULTSThe HCMV late mRNA could be detected 12 hours postinfection and increased gradually, but the CPE appeared 48 hours postinfection in HEL cells. The HCMV infected cells exhibited significant mitochondrial enlargement and the number of mitochondrial ridge deletion, the cisternae lumen of endoplasmic reticulum (ER) dilation and vacuolization (at the end age). The mature nucleocapsid could be observed 96 hours postinfection.

CONCLUSIONThe ultrastructural changes have an intimate correlation with the expression of HCMV late mRNA and play an important role in the life circle of the virus. HCMV late mRNA may serve as a indicator of the clinical effect of treatment in active HCMV infection.

Cytomegalovirus ; genetics ; physiology ; Cytopathogenic Effect, Viral ; Embryo, Mammalian ; Endoplasmic Reticulum ; pathology ; virology ; Fibroblasts ; metabolism ; ultrastructure ; virology ; Humans ; Inclusion Bodies ; pathology ; virology ; Mitochondrial Swelling ; RNA, Messenger ; metabolism

Akabane, Aino and Chuzan virus are arthropod-borne (arbo)viruses mainly associated with reproductive failures in cattle. We investigated apoptosis in Vero cells (C-1586) infected with Akabane, Aino and Chuzan virus. The fragmentation of chromosomal DNA was simultaneously detected with the progress of cytopathic effect from 48 hr to 72 hr post infection, depending on viruses. Although the treatment of cycloheximide blocked apoptosis in Vero cells infected with three viruses, actinomycin D did not prevent DNA oligomerization, thus indicating that de novo viral protein synthesis is critical for viral apoptosis. In addition, the activation of caspase-3 was also detected in Vero cells by indirect fluorescent assay. From the present results, it is of future interest whether apoptotic characteristics of these viruses are related to pathogenecity in vivo.
Animals ; Apoptosis/*physiology ; Bunyaviridae/*physiology ; Caspase 3 ; Caspases/metabolism ; Cercopithecus aethiops ; Cytopathogenic Effect, Viral/*physiology ; DNA Fragmentation/physiology ; Dactinomycin ; Enzyme Activation ; Orbivirus/*physiology ; Vero Cells

OBJECTIVETo evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA).

METHODSAntiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR.

RESULTSEight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection.

CONCLUSIONSiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

Coxsackievirus Infections ; therapy ; virology ; Cytopathogenic Effect, Viral ; drug effects ; Enterovirus ; genetics ; physiology ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; Virus Replication ; drug effects

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.
Animals ; Cytopathogenic Effect, Viral ; Enterovirus A, Human ; genetics ; pathogenicity ; Enterovirus Infections ; pathology ; virology ; Gene Expression Regulation, Viral ; Mice ; Mice, Inbred ICR ; Mutation ; Viral Load ; Viral Proteins ; genetics ; metabolism ; Virulence ; Virus Replication

To study the proliferation characteristics of PPV in differently infected way and the variance of concentrations in different cells. A strain of porcine parvovirus(PPV) was adapted to PK-15 cells, and a Real-time fluorescent quantitative PCR (FQ-PCR) assay was developed based on the specific region of the NS1 gene of PPV to quantify the PPV. The FQ-PCR was used to measure the viral concentration of virus-infected cells by simultaneous or step by step inoculation and plot one-step growth curves. The proliferation characteristics of PPV strain in different cells lines (HeLa, MDBK, PK-15 ,ST, F81, BHK-21 and Marc-145) was also compared. The results showed the PK-15 cell -adapted strain of PPV produced CPE after 12 passages, and maintained stable CPE at the following 10 messages. The one-step growth curve showed that the virus concentration of simultaneous inoculation was higher than that of the step-by-step inoculation, and the proliferation cycle of step-by-step inoculation was shorter. The proliferation ability of PPV strain in different cells showed that CPE appeared first inPK-15, followed by ST, HeLa and MDBK, and the virus concentration was highest in ST, followed byPK-15, MDBK and HeLa. NO proliferation was observed in F81, BHK-21 and Marc-145 cells. These findings lay a material foundation for the basic researches on PPV and the development of vaccine.
Animals ; Cell Line ; Cricetinae ; Cytopathogenic Effect, Viral ; DNA, Viral ; genetics ; Female ; Haplorhini ; Humans ; Male ; Parvoviridae Infections ; virology ; Parvovirus, Porcine ; genetics ; physiology ; Real-Time Polymerase Chain Reaction ; Reproducibility of Results ; Sensitivity and Specificity ; Swine ; Viral Proteins ; genetics ; Virus Replication