Recombinant Adeno-associated Viral Vectors Serotypes 6 and 9 are Able to Transduce Human Tracheal Epithelial Cells but Not Human Induced Pluripotent Stem Cells.

Recombinant adeno-associated viruses (rAAVs) may be useful for the development of gene therapy for hereditary diseases. Patient-specific human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of cells which are difficult or impossible to obtain by biopsy. To date, few research on the efficiency of rAAV transduction of hiPSCs has been published, but the obtained data are very contradictory and do not answer the actual question: how effective are rAAVs for the delivery of transgenes into hiPSCs. In this work, we used rAAV serotypes 5, 6, and 9 carrying the GFP transgene. The transduction efficiency of rAAV2/9-GFP and rAAV2/6-GFP for the immortalized tracheal epithelial cell line derived from a patient with cystic fibrosis (CFTE29o-) was relatively high. At the same time, the efficiency of transduction of iPSCs from a healthy donor and a cystic fibrosis (CF) donor was extremely low. Thus, our results show that the efficiency of hiPSC transduction by rAAV serotypes 5, 6, and 9 is not suitable for the delivery of transgenes.

Monitoring of rabies in wild animals in the Kirov region after oral immunization.

This work presents the results of the molecular genetic research on genomes of field isolates of the rabies virus circulating in the territory of the Kirov region in order to analyze the phylogenetic relationship between the wild isolate genomes and to determine the possible reversion of the vaccine strain of the rabies virus used in the oral vaccine to virulent variant. We studied 24 brain samples from wild carnivores shot after oral immunization of the area with Rabivak-O/333. A bait with the vaccine provided by the Veterinary Service of the Kirov was also studied. All samples were found to be positive for the presence of the rabies virus as established by FAT and RT-PCR techniques. Phylogenetic analysis of N genome fragments of the rabies virus showed that the field isolates from the Kirov regions were genetically close to the field isolates from Buryatia 2012. Analysis of G genome fragments showed that the Kirov field isolates were close to the isolates from Lipetsk (2011), as well as to the Ukrainian isolates (2006 and 2010). Molecular genetic analysis of the gene fragments N and G for the field isolates and fragments of the genome of the rabies virus vaccine did not reveal any reversion to the virulent vaccine strain.

[Molecular genetic characteristics of the virus isolated from patients with human acute encephalomyelitis and multiple sclerosis].

The study of the antigenic and molecular genetic structure of human acute encephalomyelitis virus (HAEV) showed a high similarity of the HAEV N gene with the homologous gene of the fixed rabies virus strain. The results of the nucleotide sequence analysis indicate that HAEV belongs to the lyssavirus genotype 1. The N gene sequence is the closest to those of the ERA-CB20-M and RV-97 strains of the rabies virus. The need for further research into the role of the human acute encephalomyelitis virus in human pathology stems from past surveys that revealed the presence of the VNAs against this virus in 6 per cent of the blood received from donors in the USA and in each third among the patients with multiple sclerosis in the former USSR.

Post-reassortment amino acid change in the hemagglutinin of a human-avian influenza H5N1 reassortant virus alters its antigenic specificity.

It was shown earlier that the reassortant influenza virus having hemagglutinin (HA) gene of A/Duck/Primorie/2621/2001 (H5N2) virus and 7 genes of A/Puerto Rico/8/34 (H1N1) virus produced low yields in embryonated chicken eggs. We found that a variant reassortant selected by serial passages in eggs produced higher yields than the initial reassortant. The variant reassortant had an amino acid substitution in the hemagglutinin N244D (H3 numbering). In this report we demonstrated that the post-reassortment amino acid substitution N244D altered the antigenic specificity of HA as revealed by the loss of reactivity with an anti-H5 monoclonal antibody in hemagglutination-inhibition (HI) test. The results are discussed in association with the evolution of H5 hemagglutinin.

Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants.

Reassortants between a low-pathogenic avian influenza virus strain A/Duck/Primorie/2621/2001 (H5N2) and a high-yield human influenza virus strain A/Puerto Rico/8/34 (H1N1) were generated, genotyped and analyzed with respect to their yield in embryonated chicken eggs, pathogenicity for mice, and immunogenicity. A reassortant having HA and NA genes from A/Duck/Primorie/2621/2001 virus and 6 genes from A/Puerto Rico/8/34 virus (6:2 reassortant) replicated efficiently in embryonated chicken eggs, the yields being intermediate between the yields of the avian parent virus and those of the A/Puerto Rico/8/34 parent strain. The reassortant having the HA gene from A/Duck/Primorie/2621/2001 virus and 7 genes from A/Puerto Rico/8/34 virus (7:1 reassortant) produced low yields. A variant of the 7:1 reassortant selected by serial passages in eggs had an amino acid substitution in the hemagglutinin (N244D, H3 numbering). The variant produced yields similar to those of the 6:2 reassortant. A 5:3 reassortant generated by a back-cross of the 6:2 reassortant with the avian parent and having PB1, HA and NA genes of A/Duck/Primorie/2621/2001 virus produced higher yields than the 7:1 or 6:2 reassortants, although still lower than the yields of A/Puerto Rico/8/34 virus. The 7:1, 6:2 and 5:3 reassortants were pathogenic for mice, with the level of virulence close to A/Puerto Rico/8/34 virus, in contrast to the extremely low pathogenicity of the A/Duck/Primorie/2621/2001 parent strain. Immunization of mice with an inactivated 6:2 H5N2 reassortant provided efficient immune protection against a reassortant virus containing the HA and NA genes of a recent H5N1 isolate. The results are discussed in connection with the problem of the improvement of vaccine strains against the threatening H5N1 pandemic.