Nathalie Desloges

Potency of a recombinant NDV-H5 vaccine against various HPAI H5N1 virus challenges in SPF chickens.

SUMMARY. For the past decade, several recombinant Newcastle disease viruses (rNDV) have been used as a vector to express native or modified avian influenza (AI) hemagglutinins (HA) in order to give preventive protection against highly pathogenic avian influenza (HPAI) H5N1 viruses. Obtained protections were dependent on the age of the chickens, on the constructs and, in particular, on the homology between the HA that was inserted and the challenge strains. The objective of this study was to investigate the vaccine efficacy of a recombinant NDV La Sota-vectored vaccine expressing an Asian clade 1 H5 ectodomain (rNDV-H5) vaccine expressing a modified H5 ectodomain from an HPAI clade 1 H5N1 isolate as vaccine for 1-day-old specific-pathogen-free chickens. The inoculation route (oculonasal vs. drinking water), the dose-effect, and the protective range of this rNDV-H5 vaccine were studied. Both routes of vaccination induced an H5 serologic response and afforded a high degree of clinical protection against an Asian clade 1 HPAI H5N1 (AsH5N1) challenge without a significant difference between inoculation routes. A clear dose-effect could be demonstrated. Furthermore, when evaluating the protective range against antigenically divergent descendants of the Asian clade 1 HPAI H5N1 lineage, namely two Egyptian clade 2.2.1 H5N1 strains, the vaccine efficacy was less satisfactory. The rNDV-H5 vaccine provided good clinical protection and reduced viral shedding against Egyptian 2007 challenge but was unable to provide a similar protection against the more antigenically divergent Egyptian 2008 strain.

Varicella-zoster virus infection induces the secretion of interleukin-8

Interleukin-8 (IL-8) is an important mediator in neutrophil-mediated acute inflammation but has also a wide range of actions on various cells types. We demonstrated that infection of melanoma cells and fibroblasts with cell-associated varicella-zoster virus (VZV) and infection of a T cell line with cell-free VZV resulted in an induction of IL-8 secretion in vitro. The inhibition of the VZV replication with a drug interfering with its DNA replication had no effect on the IL-8 release. Since the IL-8 promoter contains binding sites for NF-κB and AP-1, melanoma cells and the T cell line were treated with inhibitors of NF-κB, JNK/SAPK or p38/MAPK prior to infection. In melanoma cells, the JNK/SAPK pathway was shown to be important for the IL-8 secretion during the VZV replication, whereas in the T cell line, not only the JNK/SAPK but also the p38/MAPK pathways were required for IL-8 secretion. The neutralisation of the IL-8 bioactivity had no significant consequence on the VZV replication, suggesting that IL-8 acts neither as a proviral nor as an antiviral cytokine during the VZV replication in vitro.

Varicella-Zoster Virus Infection Influences Expression and Organization of Actin and α-Tubulin but Does Not Affect Lamin A and Vimentin

Objective: The aim of this study was to examine the effects of varicella-zoster virus (VZV) infection on the cytoskeletal components actin, lamin A, α-tubulin and vimentin. Methods: The expression patterns of these four proteins during VZV infection were studied by Northern and Western blotting. The filaments were also studied in their cellular environment by immunofluorescence using confocal microscopy. Treatment with nocodazole and cytochalasin B was performed to examine the effects of the destruction of actin or tubulin networks on the VZV replicative cycle. Results: The amounts of the mRNAs of actin, lamin A, α-tubulin and vimentin decreased slightly at 48 h post infection (p.i.) with VZV. The cellular content of the lamin A protein appeared to remain stable during the time period analyzed, whereas the amounts of actin, α-tubulin and vimentin decreased slightly at 24 h p.i. until the end of the viral cycle. Rearrangement of microfilaments and microtubules was observed at 24 h p.i. The addition of nocodazole or cytochalasin B decreased viral replication. Conclusions: During the VZV replicative cycle, tubulin and actin networks undergo significant changes including fiber elongation. If destroyed intentionally, viral replication is diminished, suggesting that these systems are vital for an efficient infection and viral replication.

The phosphorylation profile of protein kinase A substrates is modulated during Varicella-zoster virus infection

The cAMP-dependent protein kinase A (PKA) is a key enzyme for many cellular mechanisms. In this study, we investigated the importance of this kinase for the replication of the alphaherpesvirus Varicella-zoster virus (VZV). We report that the expression of the catalytic subunit of PKA was strongly increased at the beginning of the viral cycle. The presence of a peptide inhibitor of PKA had no consequence on viral replication in a melanoma cell line whereas in fibroblasts, it resulted in a drastic decrease of replication. An overall analysis of PKA substrates phosphorylation patterns during VZV replication showed that the phosphorylation of PKA substrates was modulated. These results were completed by investigating the accumulation and phosphorylation patterns of the PKA target cAMP response element binding protein (CREB). This transcription factor remained available throughout the VZV replication, but its phosphorylation decreased in the early phase of infection before it rose later on. These results indicate that the PKA signalling plays a cell-type dependent role for VZV replication and that the infection resulted in a regulated CREB-dependent gene expression.

Expression kinetics of the transcript and product of the UL28 homologue of bovine herpesvirus 1

We report that the bovine herpesvirus 1 (BHV1) UL28 ORF, a homologue of the herpes simplex virus (HSV) UL28 gene, represents a functional gene encoding a viral specific protein. The BHV1 UL28 ORF, located at positions 53058-->55538 of the viral genome, encodes a viral specific transcript of 3.4 kb detected at 6 h post-infection (p.i.) after which its levels accumulated up to 12 h p.i. and then remained constant up to 24 h p.i. Transcription of the BHV1 UL28 was determined to initiate 95 bases upstream from the ORFs initiating codon, which corresponds to 33 nucleotides downstream from a putative TATA box. A BHV1 UL28 specific antiserum, generated against a T7-Tag/UL28 fusion protein expressed in E. coli, specifically reacted with a 100 kDa protein in Western blots of BHV1-infected protein cell lysates. The expression kinetics of the protein was delayed by 6 h relative to that of its transcript suggesting that the gene is regulated at the translational level. In contrast to the HSV and pseudorabies virus UL28 genes, which belong to viral genes of the early (beta) class, that of BHV1 was unambiguously classified as a gamma2 gene. Further studies will be required to determine whether these kinetic differences have any functional implications.

Stronger Interference of Avian Influenza Virus–Specific Than Newcastle Disease Virus–Specific Maternally Derived Antibodies with a Recombinant NDV-H5 Vaccine

SUMMARY. Maternally derived antibodies (MDA) are known to provide early protection from disease but also to interfere with vaccination efficacy of young chicks. This interference phenomenon is well described in the literature for viral diseases such as infectious bursal disease, Newcastle disease (ND), and avian influenza (AI). The goal of this work was to investigate the impact of H5 MDA and/or ND virus (NDV) MDA on the vaccine efficacy of a recombinant NDV-H5–vectored vaccine (rNDV-H5) against two antigenically divergent highly pathogenic AI (HPAI) H5N1 challenges. In chickens with both H5 and NDV MDA, a strong interference was observed with reduced clinical protection when compared to vaccinated specific-pathogen-free (SPF) chickens. In contrast, in chickens from commercial suppliers with NDV MDA only, a beneficial impact on the vaccine efficacy was observed with full protection and reduced viral excretion in comparison with rNDV-H5–vaccinated SPF chickens. To distinguish between the respective effects of the H5 and NDV MDA, an SPF model where passive immunity had been artificially induced by inoculations of H5 and NDV hyperimmunized polysera, respectively, was used. In the presence of H5 artificial MDA, a strong interference reflected by a reduction in vaccine protection was demonstrated whereas no interference and even an enhancing protective effect was confirmed in presence of NDV MDA. The present work suggests that H5 and NDV MDA interact differently with the rNDV-H5 vaccine with different consequences on its efficacy, the mechanisms of which require further investigations.

Molecular Biology of Varicella-Zoster Virus

Varicella–zoster virus (VZV), also known as human herpesvirus 3 (HHV3) belongs to the herpesvirus family (Herpesviridae). This classification is based on the morphological characteristics of the virus and its physical and chemical properties. The Herpesvirus Study Group of the International Committee on the Taxonomy of Viruses (ICTV) divided the members of this family into three subfamilies: Alphaherpesvirinae, Betaherpesvirinea and Gammaherpesvirinea. Based on its host spectrum, the length of the replicative cycle, the cytopathic effect in vitro and the particularities in the establishment of latency, VZV together with herpes simplex virus type 1 (HSV1; HHV1) and type 2 (HSV2; HHV2) were grouped into the subfamily of Alphaherpesvirinae. Moreover, by its genome organisation, VZV was classified into the genus vari-cellovirus, whereas HSV was classified into the genus simplexvirus, for overview see [1]. Though symptoms of an infection with one of these herpesviruses differ strongly from each other, the morphology of the particles and the biological properties are very similar. VZV is characterised by a strongly limited spectrum of infectable host cells, which are, in fact, exclusive cells of human or simian origin. An important characteristic of herpesviruses is the architecture of the virion. Its size varies between 120 and 300 nm and is described to have a polygonal or round shape with a clearly visible central dot [2, 3]. Until now, it is not exactly known, how many polypeptides are involved in the assembly of the virion, but an average of 30–35 is reported. The virion is structured by four distinct components: envelope, tegument, capsid and core with the genome (fig. 1a).