[Rapid differential diagnosis of Orthopoxviruses and Herpesviruses based upon multiplex real-time PCR]. / Un nuovo metodo rapido, basato su real-time PCR multiplex, per la diagnosi differenziale di infezioni da Orthopoxvirus e Herpesvirus.

OBJECTIVE: Variola virus, belonging to Orthopoxviridae family, is one of the most dangerous human pathogens that could be used as biological weapon. We have developed a new rapid assay, based upon Real-time PCR and melting temperatures analysis of amplicons, for the contemporary detection of Orthopoxvirus, VZV and HSV1-2, that are the most important infectious agents to be considered for differential diagnosis. METHODS: The target for detection of orthopoxvirus DNA has been a region of the crmB gene which is common to Variola virus and to other old world orthopoxviruses pathogenic for humans. The targets for VZV and HSV1-2 have been ORF 29 and DNA polymerase, respectively. Suitability of the amplified fragments to RFLP or sequencing analysis, to recognize the involved viral species, has been also tested. RESULT: The selected primers have showed high sensitivity, specificity and compatibility with common amplification conditions. A mean melting temperature difference of 8.7 degree C was observed between the amplicons from the two virus types. Further identification of individual pathogens was made using RFLP analysis. CONCLUSION: The PCR-based protocol set up in this study for presumptive differential diagnosis of variola and herpesviral infections is rapid and specific and it can be used also to detect other orthopoxviral infections, like monkeypox.

Antagonistic antiviral activity between IFN-lambda and IFN-alpha against lethal Crimean-Congo hemorrhagic fever virus in vitro.

BACKGROUND AND AIMS: Crimean Congo Hemorrhagic fever virus (CCHFV) is the causative agent of Crimean-Congo hemorrhagic fever, a severe disease with a mortality rate of around 30% in humans. Previous studies demonstrate that pre-treatment with type I IFNs have an antiviral effect against CCHFV, while established CCHFV infection is almost insensitive to subsequent IFN-α treatment. No data concerning type III IFNs antiviral activity against CCHFV are available so far. The aim of the present study was to explore the capability of IFN-λ1 to inhibit the replication of CCHFV and the possible synergism/antagonism between IFN-α and IFN-λ1 both in the inhibition of CCHFV replication and in the activation of intracellular pathways of IFN response. METHODS: Human A549 and HuH7 cells were treated with increasing amounts of IFN-λ1, or IFN-α or a combination of them, infected with CCHF; the extent of virus yield inhibition and the induction of MxA and 2'-5'OAS mRNA was measured. RESULTS AND CONCLUSIONS: Our study pointed out that type III IFN possess an antiviral activity against CCHFV, even if lower than type I IFN. Moreover, a clear antagonism between IFN-λ and IFN-α was observed in both cell lines (A549 and HuH7 cells), in terms of antiviral effect and activation of pivotal ISGs, i.e. MxA and 2'-5'OAS. Elucidating the interplay between type I and III IFNs will help to better understand innate defence mechanisms against viral infections and may provide novel scientific evidence for a more rational planning of available and future treatments, particularly against human diseases caused by high concern viruses.

Design and clinical application of a molecular method for detection and typing of the influenza A/H1N1pdm virus.

In March/April 2009, Mexico experienced an outbreak of respiratory illness, due to a new influenza of swine origin virus, which spread rapidly via human-to-human transmission, and became pandemic (A/H1N1pdm). Because of its unique genome composition, which includes gene segments of swine, avian and human origin, and to the considerable differences to the human influenza A viruses that have circulated so far, the currently used molecular methods proved inadequate. Based on published sequences, a primer set targeting the nucleoprotein gene was designed, which provided enhanced sensitivity for the new strain and proved suitable for sequence-based strain identification. The novel nucleoprotein reverse-transcription-PCR showed higher sensitivity for A/H1N1pdm than a commercial test for influenza A, and was comparable to the real-time-based method developed by the Centers for Disease Control and Prevention. It was used to screen 177 clinical samples referred to the laboratory for suspected A/H1N1pdm infection, detecting 17 (9.6%) infections that were confirmed by sequence analysis (100% sensitivity as compared to the real-time kit). The novel method is suitable for the diagnosis of A/H1N1pdm, and is also suitable, at least in the screening phase, for laboratories not equipped with the real-time PCR technology.