Kevin P. Dalton

Relative Neurotropism of a Recombinant Rhabdovirus Expressing a Green Fluorescent Envelope Glycoprotein

ABSTRACT A new recombinant vesicular stomatitis virus (rVSV) that expresses green fluorescent protein (GFP) on the cytoplasmic domain of the VSV glycoprotein (G protein) was used in the mouse as a model for studying brain infections by a member of the Mononegavirales order that can cause permanent changes in behavior. After nasal administration, virus moved down the olfactory nerve, first to periglomerular cells, then past the mitral cell layer to granule cells, and finally to the subventricular zone. Eight days postinoculation, rVSV was eliminated from the olfactory bulb. Little sign of infection could be found outside the olfactory system, suggesting that anterograde or retrograde axonal transport of rVSV was an unlikely mechanism for movement of rVSV out of the bulb. When administered intracerebrally by microinjection, rVSV spread rapidly within the brain, with strong infection at the site of injection and at some specific periventricular regions of the brain, including the dorsal raphe, locus coeruleus, and midline thalamus; the ventricular system may play a key role in rapid rVSV dispersion within the brain. Thus, the lack of VSV movement out of the olfactory system was not due to the absence of potential for infections in other brain regions. In cultures of both mouse and human central nervous system (CNS) cells, rVSV inoculations resulted in productive infection, expression of the G-GFP fusion protein in the dendritic and somatic plasma membrane, and death of all neurons and glia, as detected by ethidium homodimer nuclear staining. Although considered a neurotropic virus, rVSV also infected heart, skin, and kidney cells in dispersed cultures. rVSV showed a preference for immature neurons in vitro, as shown by enhanced viral infection in developing hippocampal cultures and in the outer granule cell layer in slices of developing cerebellum. Together, these data suggest a relative affinity of rVSV for some neuronal types in the CNS, adding to our understanding of the long-lasting changes in rodent behavior found after transient VSV infection.

Variant Rabbit Hemorrhagic Disease Virus in Young Rabbits, Spain

Outbreaks of rabbit hemorrhagic disease have occurred recently in young rabbits on farms on the Iberian Peninsula where rabbits were previously vaccinated. Investigation identified a rabbit hemorrhagic disease virus variant genetically related to apathogenic rabbit caliciviruses. Improved antivirus strategies are needed to slow the spread of this pathogen.

cis-Acting Sequences Required for Coronavirus Infectious Bronchitis Virus Defective-RNA Replication and Packaging

ABSTRACT The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5′ terminus was replicated; the 5′ untranslated region (UTR) comprises 528 nt. Region I of the 3′ UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3′ UTR, was replicated. Thus, the 5′-terminal 544 nt and 3′-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5′ end of region II of the 3′ UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

Targeting Norovirus Infection—Multivalent Entry Inhibitor Design Based on NMR Experiments

Noroviruses attach to their host cells through histo blood group antigens (HBGAs), and compounds that interfere with this interaction are likely to be of therapeutic or diagnostic interest. It is shown that NMR binding studies can simultaneously identify and differentiate the site for binding HBGA ligands and complementary ligands from a large compound library, thereby facilitating the design of potent heterobifunctional ligands. Saturation transfer difference (STD) NMR experiments, spin-lock filtered NMR experiments, and interligand NOE (ILOE) experiments in the presence of virus-like particles (VLPs), identified compounds that bind to the HBGA binding site of human norovirus. Based on these data two multivalent prototype entry-inhibitors against norovirus infection were synthesized. A surface plasmon resonance based inhibition assay showed avidity gains of 1000 and one million fold over a millimolar univalent ligand. This suggests that further rational design of multivalent inhibitors based on our strategy will identify potent entry-inhibitors against norovirus infections.

Spread of new variant RHDV in domestic rabbits on the Iberian Peninsula

Rabbit hemorrhagic disease outbreaks in young rabbits have been recently observed in Spain. In this study we have tracked the spread of variant RHDV in samples collected from rabbit farms over a period of more than one year using RT-PCR and antigen-capture ELISA. The isolates were sequenced and compared to classic and variant RHDV strains and phylogenetic analyses were conducted. Mortalities have been observed in kits as young as 11 days. More than 50% of the dead rabbits had been previously vaccinated against RHDV using commercially available inactivated vaccines indicating a putative lack of protection against the variant RHDV. The large majority of the studied outbreaks (94.5%) in Spanish farms during 2012 were due to variant RHDV and only 3 out of the 55 farms were affected by classic RHDV. The data demonstrates that the variant RHDV has spread through a large number of Spanish provinces in a relatively short period of time, largely replacing the previously predominant G1 RHDV genotypes. Considering the lack of efficient vaccines against the variant RHDV strains strict disease control and greater vigilance measures should be put in place.

Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toads (Alytes obstetricans) in Northern Spain: a comparative pathological study of an emerging ranavirus.

This report describes the isolation and characterisation of the common midwife toad virus (CMTV) from juvenile alpine newts (Mesotriton alpestris cyreni) and common midwife toad (CMT) tadpoles (Alytes obstetricans) in the Picos de Europa National Park in Northern Spain in August 2008. A comparative pathological and immunohistochemical study was carried out using anti-CMTV polyclonal serum. In the kidneys, glomeruli had the most severe histological lesions in CMT tadpoles, while both glomeruli and renal tubular epithelial cells exhibited foci of necrosis in juvenile alpine newts. Viral antigens were detected by immunohistochemical labelling mainly in the kidneys of CMT tadpoles and in ganglia of juvenile alpine newts. This is the first report of ranavirus infection in the alpine newt, the second known species to be affected by CMTV in the past 2 years.

Development of a Novel Surrogate Virus for Human T-Cell Leukemia Virus Type 1: Inhibition of Infection by Osteoprotegerin

ABSTRACT To develop a high-titer surrogate virus for human T-cell leukemia virus type 1 (HTLV-1), we generated recombinant vesicular stomatitis viruses (VSVs) in which the gene encoding the single transmembrane glycoprotein (G) was deleted. Genes encoding HTLV-1 envelope glycoproteins (HTEnv) or HTEnvG hybrid proteins were then inserted into either of two different sites in the VSV genome. The viruses also encoded a green fluorescent protein. With this surrogate virus, we found that a soluble protein, osteoprotegerin (OPG), or an OPG/Fc chimeric protein inhibited the infection of various cell lines. Our experiments indicate that this inhibition resulted from binding of heparan sulfate by OPG.

Detection of RHDVa on the Iberian Peninsula: isolation of an RHDVa strain from a Spanish rabbitry

Rabbit haemorrhagic disease virus (RHDV), genus Lagovirus, family Caliciviridae, causes a large number of deaths in wild and domestic adult European rabbits (Oryctolagus cuniculus). The first documented outbreak dates from 1984 in China, but the virus rapidly dispersed worldwide. In 1997, an antigenic variant was detected in Italy and designated RHDVa. Despite causing symptoms similar to those caused by classic RHDV strains, marked antigenic and genetic differences exist. In some parts of Europe, RHDVa is replacing classic strains. Here, we report the presence of RHDVa on the Iberian Peninsula, where this variant was thought not to contribute to viral diversity.

Role of annexin A2 in cellular entry of rabbit vesivirus.

The mechanisms of calicivirus attachment and internalization are not well understood, mainly due to the lack of a reliable cell-culture system for most of its members. In this study, rabbit vesivirus (RaV) virions were shown to bind annexin A2 (ANXA2) in a membrane protein fraction from HEK293T cells, using a virus overlay protein-binding assay and matrix-assisted laser desorption/ionization time-of-flight analysis. A monoclonal anti-ANXA2 antibody and small interfering RNA-mediated knockdown of ANXA2 expression in HEK293T cells reduced virus infection significantly, further supporting the role of ANXA2 in RaV attachment and/or internalization.

Proposal for a unified classification system and nomenclature of lagoviruses

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970-1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.