A molecular based diagnosis of positive blood culture in the context of viral haemorrhagic fever: proof of concept.

OBJECTIVES: The aim of this study was to evaluate the possibility of using a PCR-based panel to identify bacterial and fungal bloodstream infections in the setting of suspected or confirmed viral haemorrhagic fever. METHODS: The accuracy of the FilmArray® Blood Culture Identification Panel (BCID) assay was assessed to identify the common bacterial and fungal pathogens associated with bloodstream infections after positive blood culture inactivation using a guanidinium thiocyanate containing buffer lysis that is commonly used for viral haemorrhagic fever molecular diagnostics. RESULTS: The FilmArray® BCID panel assay detected 95% (19/20) of the pathogens analysed in this study by using both protocols with and without inactivation. Absolute consistency (100%) was observed in all isolates with phenotypes compatible with the presence of the antibiotic resistance genes mecA, vanA, vanB and blaKPC. CONCLUSIONS: The FilmArray® BCID panel assay coupled to inactivation using a guanidinium thiocyanate containing buffer lysis represents a convenient, sensitive and specific diagnostic tool to detect some of the most pathogens associated with bloodstream infections in the context of a suspected or confirmed viral haemorrhagic fever.

Simultaneous outbreaks of dengue, chikungunya and Zika virus infections: diagnosis challenge in a returning traveller with nonspecific febrile illness.

Zika virus is an emerging flavivirus that is following the path of dengue and chikungunya. The three Aedes-borne viruses cause simultaneous outbreaks with similar clinical manifestations which represents a diagnostic challenge in ill returning travellers. We report the first Zika virus infection case imported to Switzerland and present a diagnostic algorithm.

Toscana virus meningitis case in Switzerland: an example of the ezVIR bioinformatics pipeline utility for the identification of emerging viruses.

Toscana virus (TOSV) represents a frequent cause of viral meningitis in the Mediterranean Basin that remains neglected in neighbouring countries. We report a documented TOSV meningitis case in a traveller returning from Tuscany to Switzerland. While routine serological and PCR assays could not discriminate between TOSV and Sandfly fever Naples virus infection, a high-throughput sequencing performed directly on the cerebrospinal fluid specimen and analysed with the ezVIR pipeline provided an unequivocal viral diagnostic. TOSV could be unequivocally considered as the aetiological agent, proving the potential of ezVIR to improve standard diagnostics in cases of infection with uncommon or emerging viruses.

[New viruses: myth, fantasy or reality?]. / Nouveaux virus: mythe, fantasme ou réalité?

Emerging viruses previously unknown or partially known that infect repeatedly the human population are more than ever in the medias actuality headlines. Multiple factors may explain this dynamic. The most important is certainly the rapid evolution and the adaptation capacity of these viruses. Note that the increase in travel and international trade or climate change also play an important role. On the other hand, laboratory tests and current surveillance systems are more efficient. Thus, transmission of virus from an animal reservoir to human are more easily detected, accentuating the feeling of increasing phenomenon. Virological predictions have very low reliability in epidemiology. It is a reality that we have to accept.

[Will avian influenza virus become a human virus?]. / Le viruS de la grippe aviaire sera-t-il humain ?

Since 1997, an Influenza virus of avian origin appears regularly in human causing severe respiratory infections leading to death in half cases. This Influenza A (H5N I) virus which is at the origin of this illness circulates in wild birds and in domestic birds. Million poultry have been regularly infected or slaughtered on 3 continents: Asia, Africa and Europe. H5NI virus, like any other Influenza virus, has the ability to adapt its genome and theoretically could easily jump from the avian animals to human directly. On the other hand, since 10 years it still did not acquire this capacity. This paper summarise our knowledge on the risk of a future pandemic.

[Emerging viral infections]. / Infections virales émergentes.

Emerging, re-emerging, rare or dangerous viruses are regularly citated in news. Most of theses viruses belong to the class 3 and 4. Clinical specimens must be handled with appropriate bio-security conditions, and, for some of them, high security facilities are required. In Geneva, a new P4D facility aiming to conduct diagnostic procedures targeting these viruses, fills a gap in Switzerland in this field. The goal of this review is to present some examples of past and ongoing viral outbreaks around the world, to present the virus classification according to the biological risk and to summarise basic knowledge concerning class 4 viruses.

An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Benzo[a]pyrene-induced mutagenesis of p53 hot-spot codons 248 and 249 in human hepatocytes.

Human tobacco-related cancers show a high frequency of G-to-T transversions in several mutation hot-spot regions of the p53 tumor suppressor gene, probably the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene. To gain insight into the mechanism of formation of these G-to-T transversions in tobacco-associated carcinogenesis, we studied the mutagenesis of p53 codons 247-250 by benzo[a]pyrene in human hepatocellular carcinoma cells by restriction fragment length polymorphism-polymerase chain reaction genotypic analysis. Benzo[a]pyrene preferentially induced G-to-T transversion in the second and third positions of codon 248 and C-to-A transversion in the first position of codon 248. However, benzo[a]pyrene did not induce base-pair changes in codon 249, which is a mutational hot-spot in aflatoxin-related hepatocarcinogenesis, in which predominantly G-to-T transversion in the third position of codon 249 is observed. The benzo[a]pyrene-induced G-to-T transversion in the middle position of codon 248, in which arginine is changed into leucine, is frequently observed in tumors of the lung. The other two benzo[a]pyrene-induced base-pair changes in codon 248, namely the C-to-A transversion in the first position and G-to-T transversion in the third position, do not lead to a change in the amino-acid composition of the p53 protein. These mutations are silent and therefore are not selected in tumors. It follows that benzo[a]pyrene-induced mutability on the DNA level in p53 codons 247-250 correlates well with the type of mutation found in tumors of the lung. Therefore, our results support the hypothesis that benzo[a]pyrene is the etiological agent in tobacco-related cancers.