[Crimean-Congo hemorrhagic fever: basics for general practitioners]. / La fièvre hémorragique de Crimée-Congo: l'essentiel pour le praticien.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease described in more than 30 countries in Europe, Asia and Africa. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV) that is a member of the genus Nairovirus of the family Bunyaviridae. CCHFV that is characterized by a high genetic variability is transmitted to humans by tick bites or contact with fluids from an infected individual or animal. The initial symptoms of CCHF are nonspecific and gradually progress to a hemorrhagic phase that can be lethal (case-fatality rate: 10 to 50%). Characteristic laboratory findings of CCHF are thrombocytopenia, elevated liver and muscle enzymes, and coagulation defects. The pathogenesis of CCHF remains unclear but might involve excessive pro-inflammatory cytokine production and dysfunction of the innate immune response. Diagnosis of CCHF is based mainly on isolation of the virus, identification of the viral genome by molecular techniques (RT-PCR), and serological detection of anti-CCHFV antibodies. There is currently no specific treatment for CCHFV infection and the efficacy of ribavirin is controversial. In absence of an effective vaccine, prevention is based mainly on vector control, protection measures, and information to increase the awareness of the population and of healthcare workers.

Standardization of needlestick injury and evaluation of a novel virus-inhibiting protective glove.

Rubber surgical gloves worn as a barrier to prevent contamination from body fluids offer relative protection against contamination through direct percutaneous injuries involving needles, scalpel blades or bone fragments. To determine the main experimental parameters influencing the volume of blood transmitted by a hollow-bore needle (worst case scenario) during an accidental puncture, we designed an automatic puncture apparatus. Herpes simplex type 1 virus (HSV1), a model for enveloped viruses, was used as a 'marker' in an in-vitro gelatine model. Of the experimental parameters studied, the most critical influences were found to be needle diameter and puncture depth, whereas puncture speed, puncture angle and glove-stretching feature appeared to be less influential. A single glove reduced the volume of blood transferred by 52% compared with no glove, but double gloving offered no additional protection against hollow-bore needle punctures. Using 'standardized' puncture conditions, the virus-inhibiting surgical glove G-VIR elicited an 81% reduction in the amount of HSV1 transmitted as compared with single or double latex glove systems.

Bioterrorism: management of major biological agents.

Bioterrorism is defined by the intentional or threatened of microorganisms or toxins derived from living organisms to cause death or diseases in humans, animals or plants on which we depend. The other major point is to generate fear in the population. More than 180 pathogens have been reported to be potential agents for bioterrorism. The following is an overview of several agents that could be involved in a biological attack.

Evaluation of a Crimean-Congo hemorrhagic fever virus recombinant antigen expressed by Semliki Forest suicide virus for IgM and IgG antibody detection in human and animal sera collected in Iran.

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by ticks or by direct contact with infected blood. It causes severe, often fatal, hemorrhagic diseases in humans but infection in animals is asymptomatic. CCHFV can spread from person to person and has caused many nosocomial outbreaks. Because the virus is very pathogenic for humans it must be manipulated in a biosafety level 4 (BSL4) laboratory, rendering the production of antigen for serological diagnosis difficult. To replace the native antigen, we produced a recombinant nucleoprotein expressed in mammalian cells via the recombinant Semliki Forest alphavirus replicon and developed an indirect immunofluorescence assay (IFA) as well as an enzyme-linked immunosorbent assay (ELISA) by immunocapture to detect IgM and IgG in human and animal serum. Using these methods, we analyzed clinical samples from human patients and sera from domestic animals collected in Iran and we show that this novel antigen provides a novel, sensitive and specific tool for CCHF diagnosis.

[Update on smallpox vaccines]. / Actualités sur la vaccination antivariolique.

Smallpox is among the most dangerous pathogens that could be used by bioterrorists. The former vaccines produced by scarification on the flanks of calves or sheep could be used to protect the whole French population when used with bifurcated needles. They should be replaced by a second-generation vaccine grown in cell culture and, eventually later by new and safer third-generation vaccines using non-replicative viral strains.

Rapid inactivation of vaccinia virus in suspension and dried on surfaces.

A bioterrorist attack with smallpox virus would be disastrous with a 30% disease fatality rate. Such an outbreak would require biomedical laboratories for diagnosis and analyses and extensive use of clinical care facilities for patient quarantine. Safe decontamination procedures will have to be in place in order to limit the spread of the disease. In order to fulfil this need, Sanytex, a new non-corrosive commercial solution containing quaternary ammonium, aldehydes, alcohol and detergent, was tested with a view to using it in decontamination procedures. Vaccinia virus was used in this investigation as a model for smallpox virus. We determined exposure time and the concentration of Sanytex required to inactivate the virus in suspension and dried on surfaces in the presence of protein (up to 70 mg/mL). After 3 min incubation, Sanytex at a concentration of 3% led to a complete inactivation (virus titre reduction >10(4)-fold of vaccinia virus in suspension containing protein up to 30 mg/mL. A virus suspension containing 70 mg protein/mL, simulating biological fluids, was decontaminated with 10% Sanytex after 3 min. After 10 min, Sanytex at a concentration of 30%, applied on to a dried vaccinia virus contaminated surface in the presence of protein (10 mg/mL before desiccation), led to complete decontamination of the surface. Thirty minutes exposure with 30% Sanytex was necessary for a virus titre reduction of >10(4)-fold on a surface contaminated with a dried suspension of vaccinia virus in the presence of protein at 70 mg/mL. Sanytex is not corrosive, not toxic to environment and stable for up to three months even diluted. Its virucidal effect was preserved when used under pressure in a fire-hose nozzle. These results support the use of Sanytex for decontamination of biological fluids and surfaces contaminated by the smallpox virus.

In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination.

Chikungunya virus (CHIKV) and Semliki Forest virus (SFV) were used in our laboratory to screen active antiviral compounds against viruses of the Alphavirus genus. Antiviral activity was estimated by the reduction of the cytopathic effect of each alphavirus on infected Vero cells and by virus titer reduction. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion in confluent cell cultures and by the evaluation of the inhibitory effect on cell growth. With CHIKV and SFV, the selectivity indices of human recombinant interferon-alpha and iota-carrageenan were much higher than that of ribavirin, which has been previously investigated for its inhibitory effect on alphavirus infections. Compared to ribavirin, 6-azauridine was more effective against CHIKV and showed a similar antiviral activity against SFV. IFN-alpha2b, glycyrrhizin, 6-azauridine, and ribavirin caused a concentration-dependent reduction in the virus yield with CHIKV and SFV. Moreover, the combination of IFN-alpha2b and ribavirin had a subsynergistic antiviral effect on these two alphaviruses and should be evaluated for the treatment of these infections.

Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds.

The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.

Highly sensitive Taqman PCR detection of Puumala hantavirus.

An increasing number of clinical cases of Hantavirus infections have been reported from various regions in Asia, Europe and North America. Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped and possess a single-stranded trisegmented RNA genome of negative polarity. Rodents or insectivores are natural hosts of hantaviruses and transmit the virus to humans chiefly by aerosolisation. These viruses are the causative agents of haemorrhagic fever with renal and pulmonary syndromes. In the northeast of France, Puumala hantavirus causes, every year, more than 150 mild forms of haemorrhagic fever with a renal syndrome known as nephropathia epidemica. Serological tests may lack sensitivity for diagnosing early stages of infection and virus isolation is limited because it grows poorly in cell culture. Since reverse transcription (RT)-PCR amplification is an efficient method for detecting viral genomes in patient specimens, we developed an assay using a Taqman probe and compared it with the classical RT-PCR amplification. To achieve this goal, a Puumala strain was grown in Vero E6 cells and RNA extracted from the culture supernatant. We found that the semi-nested RT-PCR detected a minimal amount of 300 TCID(50) mL(-1), while the Taqman PCR allowed detection of less than 10 TCID(50) mL(-1 )and provided a quantitative analysis.

[Hepatitis C virus culture systems]. / Les systèmes de culture du virus de l'hépatite C.

Hepatitis C virus pathogenesis and cycle are difficult to study because of the lack of culture system able to replicate efficiently the virus. Furthermore such a system will permit screen new antiviral drugs. Studies were realized to select cell culture system able to allow hepatitis C virus replication. Primary cell cultures and cell lines were used to performed HCV culture. Most of these works used lymphocyte and hepatocyte primary cultures or cell lines because of HCV tropism in these cells in vivo. Animals and arthropods cell lines were used as well for their capacity to bind and replicate HCV. The aim of this review is to present the different cell systems used to replicate HCV in culture and the results obtained.

Mosquito cells bind and replicate hepatitis C virus.

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.

Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences.

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3' NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10(5) 50% tissue culture infectious doses (TCID(50)s) ml(-1) with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10(5) TCID(50)s ml(-1). Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml(-1) with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.

Persistence of infectious hepatitis A virus and its genome in artificial seawater.

The stability of the hepatitis A virus (HAV) genome detectable by RT-PCR in artificial sterile seawater seeded with HAV has been compared to that of HAV detectable in cell culture. The HAV genome was detectable by RT-PCR for 232 days while virus particles were detectable in cell culture for only 35 days. This difference in stability indicates that detection of the HAV genome by RT-PCR is not a reliable indicator of the survival of HAV detectable in cell cultures. However, before these results can be extrapolated to stability in natural seawater, the effect of additional elements in the natural environment, such as bacteria, fungi and suspended matter, on the stability of the HAV genome and cell culture infectious HAV particles, will have to be examined.

Inhibition of sandfly fever Sicilian virus (Phlebovirus) replication in vitro by antiviral compounds.

Sandfly fever Sicilian virus (SFSV) was used in our laboratory to screen antiviral substances active toward viruses of the Bunyaviridae family. Antiviral activity was estimated by the reduction of the cytopathic effect of SFSV on infected Vero cells. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion. The specificity of action of each tested compound was estimated by the selectivity index (CD50/ED50). Selectivity indices of human recombinant interferon-alpha (IFN alpha) (Roferon and Introna), iota-, kappa- and lambda- carrageenans, fucoidan and 6-azauridine were much higher than that of ribavirin, the only antiviral substance which has been previously investigated for its inhibitory effects on Phlebovirus infections. Other compounds showed significant antiviral activity: glycyrrhizin, suramin sodium, dextran sulphate and pentosan polysulphate. All these compounds caused a concentration-dependent reduction in the virus yield. Ribavirin, 6-azauridine and IFN alpha have been shown to inhibit a late step of the virus replicative cycle, whereas glycyrrhizin and suramin sodium were active at an early step and the sulphated polysaccharides inhibited adsorption of SFSV on the cells. The antiviral compounds selected in this study as specific inhibitors of in vitro replication of SFSV are promising candidates for the chemotherapy of haemorrhagic fevers caused by viruses of the Bunyaviridae family. The combination of IFN alpha and ribavirin, which showed a synergistic antiviral effect, should be evaluated for the treatment of these infections.

Inhibition of the expression of hepatitis A and B viruses (HAV and HBV) proteins by interferon in a human hepatocarcinoma cell line (PLC/PRF/5).

AIMS/METHODS: PLC/PRF/5 is a continuous human hepatocarcinoma cell line whose genome contains integrated HBV DNA and which secretes two of the hepatitis B virus envelope proteins (HBs and PreS2). This line is also susceptible to infection by hepatitis A virus and was therefore used to compare the effects of interferon on protein synthesis of these two viruses and to assess the interactions which occur between them during infection. RESULTS: Results showed that recombinant interferon alpha 2-a inhibited the expression of the two hepatitis B virus envelope antigens (HBs and PreS2) and of the only hepatitis A virus antigen in a dose-dependent fashion. Comparison of the effect of interferon on antigenic protein production of these two viruses, showed stronger inhibition of hepatitis A virus capsid antigen than of hepatitis B virus envelope antigens. Infection with hepatitis A virus also downregulates the expression of the two hepatitis B virus proteins. CONCLUSIONS: Considering the absence of cytotoxic effects from the doses used, this study confirms the relevance of this cellular model for the study of antiviral cytokines in vitro. It also provides a further rationale for the clinical evaluation of the therapeutic potential of interferons in severe hepatitis cases due either to hepatitis A virus alone or to superinfection of hepatitis B virus carriers by hepatitis A virus.

Antiviral activity of recombinant interferon-alpha on hepatitis A virus replication in human liver cells.

Human recombinant interferon-alpha (IFN-alpha) was assayed for its antiviral effect on hepatitis A virus (HAV) replication in the human hepatoma cell line PLC/PRF/5. IFN-alpha resulted in concentration-dependent reduction of HAV antigen expression and HAV replication. IFN-alpha had a prophylactic effect, but was still effective when it was added after the infection, even at the end of the first replication cycle. An important increase in 2',5'-oligoadenylate synthetase activity in the IFN-treated human liver cells was observed. The antiviral effect of IFN-alpha could be attributed to the induction of this enzyme. Moreover we have shown that IFN-alpha and glycyrrhizin were synergistic in their antiviral actions against HAV. IFN-alpha emerged, from the present study, as a promising candidate for chemotherapy of severe forms of hepatitis A.