Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants.

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

Axonopathy and Reduction of Membrane Resistance: Key Features in a New Murine Model of Human GM1-Gangliosidosis.

GM1-gangliosidosis is caused by a reduced activity of ß-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1-/- mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1-/- mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1-/- mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon-glial interactions and therapy trials of lysosomal storage diseases.

Evolutionary evidence for multi-host transmission of cetacean morbillivirus.

Cetacean morbillivirus (CeMV) has emerged as the pathogen that poses the greatest risk of triggering epizootics in cetacean populations worldwide, and has a high propensity for interspecies transmission, including sporadic infection of seals. In this study, we investigated the evolutionary history of CeMV by deep sequencing wild-type viruses from tissue samples representing cetacean species with different spatiotemporal origins. Bayesian phylogeographic analysis generated an estimated evolutionary rate of 2.34 × 10-4 nucleotide substitutions/site/year and showed that CeMV evolutionary dynamics are neither host-restricted nor location-restricted. Moreover, the dolphin morbillivirus strain of CeMV has undergone purifying selection without evidence of species-specific mutations. Cell-to-cell fusion and growth kinetics assays demonstrated that CeMV can use both dolphin and seal CD150 as a cellular receptor. Thus, it appears that CeMV can readily spread among multiple cetacean populations and may pose an additional spillover risk to seals.

Comparison of Different In Situ Hybridization Techniques for the Detection of Various RNA and DNA Viruses.

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.

Axonopathy in the Central Nervous System Is the Hallmark of Mice with a Novel Intragenic Null Mutation of Dystonin.

Dystonia musculorum is a neurodegenerative disorder caused by a mutation in the dystonin gene. It has been described in mice and humans where it is called hereditary sensory autonomic neuropathy. Mutated mice show severe movement disorders and die at the age of 3-4 weeks. This study describes the discovery and molecular, clinical, as well as pathological characterization of a new spontaneously occurring mutation in the dystonin gene in C57BL/6N mice. The mutation represents a 40-kb intragenic deletion allele of the dystonin gene on chromosome 1 with exactly defined deletion borders. It was demonstrated by Western blot, mass spectrometry, and immunohistology that mice with a homozygous mutation were entirely devoid of the dystonin protein. Pathomorphological lesions were restricted to the brain stem and spinal cord and consisted of swollen, argyrophilic axons and dilated myelin sheaths in the white matter and, less frequently, total chromatolysis of neurons in the gray matter. Axonal damage was detected by amyloid precursor protein and nonphosphorylated neurofilament immunohistology. Axonopathy in the central nervous system (CNS) represents the hallmark of this disease. Mice with the dystonin mutation also showed suppurative inflammation in the respiratory tract, presumably due to brain stem lesion-associated food aspiration, whereas skeletal muscles showed no pathomorphological changes. This study describes a novel mutation in the dystonin gene in mice leading to axonopathy in the CNS. In further studies, this model may provide new insights into the pathogenesis of neurodegenerative diseases and may elucidate the complex interactions of dystonin with various other cellular proteins especially in the CNS.

Novel canine bocavirus strain associated with severe enteritis in a dog litter.

Bocaviruses are small non-enveloped viruses with a linear ssDNA genome, that belong to the genus Bocaparvovirus of the subfamiliy Parvovirinae. Bocavirus infections are associated with a wide spectrum of disease in humans and various mammalian species. Here we describe a fatal enteritis associated with infection with a novel strain of canine bocavirus 2 (CaBoV-2), that occurred in a litter of German wirehaired pointers. Necropsy performed on three puppies revealed an enteritis reminiscent of canine parvovirus associated enteritis, accompanied with signs of lymphocytolytic disease in bone marrow, spleen, lymph nodes and thymus. While other major causes of enteritis of young dogs, including canine parvovirus, were excluded, by random PCR in combination with next-generation sequencing, a novel CaBoV-2 strain was detected. Phylogenetic analysis of the genome of this novel canine bocavirus strain indicated that this virus was indeed most closely related to group 2 canine bocaviruses. Infection with canine bocavirus was confirmed by in situ hybridization, which revealed the presence of CaBoV-2 nucleic acid in the intestinal tract and lymphoid tissues of the dogs. In a small-scale retrospective analysis concerning the role of CaBoV-2 no additional cases were identified. The findings of this study provide novel insights into the pathogenicity of canine bocaviruses.

Novel divergent nidovirus in a python with pneumonia.

The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin-neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.