Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

Biophotons, hallucinogens, and fluorescence

Several experiments show that there is a cell to cell communicaton by light in different cell types. The most convincing experiment shows that baby hamster kidney cells can communicate their spatial orientation through a glass film, this can only happen by photon signals. If so, it can be assumed that the cells with the highest differentiation, the neurons also use this mechanism. The nervous system would have excellent conditions for a cell to cell communication by light. Neurons are large, metabolically very active (lightproducing) cells with wide arborisation, contain little pigment and are protected from ambient light by bone and connective tissue. Signal to noise ratio should be high for photon signals. It has been shown that light can be propagated along the axis tracts. Also the hollow microtubules (neurofibrillae) could act as light guiding structures. According to Jibu et al. their inner diameter of 15 nm is ideal for light guidance free of thermal noise and loss. Other findings that may be of importance in this context, are the strong flurescence properties of the major hallucinogens: LSD, bufetonine, dimethyl-tryptamine, psilocybine, psilocin, iboguanin, harmine, cannabidinol and mescaline. Furthermore it has been shown that hallucinogenic properties of these substances have a direct correlation to their fluorescence properties and their readyness to donate electrons. As hypothesis we propose that the fluorescence interacts physically with the proposed Biophoton mediated cell to cell communication thus producing hallucinations. This would be an easy and plausible explanation for the strong hallucinogenic properties of these fluorescent substances.

Effects of multiple amino acids of the parainfluenza virus 5 fusion protein on its haemagglutinin–neuraminidase-independent fusion activity

The fusion (F) protein of parainfluenza virus 5 (PIV-5) strain W3A is able to induce cell fusion when it is expressed alone in baby hamster kidney cells, whilst the F protein of PIV-5 strain WR induces cell fusion only when co-expressed with the haemagglutinin–neuraminidase (HN) protein. It has been shown previously that when Leu-22 of the WR F protein is replaced with the W3A F counterpart (Pro-22), the resulting mutant L22P exhibits HN-independent fusion activity. Furthermore, previous chimeric analysis between L22P and the F protein of PIV-5 strain T1 has suggested that Glu-132 also contributes to the HN-independent fusion activity of L22P. It was shown here that substitution of Glu-132 of L22P with various amino acids including the T1 F protein counterpart (Lys-132) resulted in a reduction in fusion activity, whereas substitution with Asp was the exception in being tolerated. Interestingly, reduced fusion activity of an L22P mutant that harboured the E132K substitution could be restored by an additional D416K substitution but not by a D416E mutation, suggesting that the presence of the same charge at positions 132 and 416 is important for the HN-independent fusion activity. In contrast, substitution of Leu-22 of the WR F protein with various amino acids except those with aliphatic side chains resulted in acquisition of fusion activity, suggesting that the HN dependence of the WR F protein in the induction of cell fusion is attributable to the hydrophobicity of Leu-22. These results indicate that at least three amino acids are involved in the HN-independent fusion activity of the PIV-5 F protein.