Study of rabies virus by Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.     

Molecular epidemiology and sequencing of the G-L intergenic region of rabies viruses isolated in China

A group of 25 rabies viruses (RABVs),recovered from 24 dogs and one human case,were collected from various areas in China between 2004 and 2006.Genetic and phylogenetic analyses of the G-L intergenic region were carried out in 25 street RABV isolates and CTN vaccine strains of 7 generations.The study was based on the comparison of a 519 bp nucleotide sequence,encompassing the G-L intergenic region.The nucleotide sequence homologies of Chinese street strains were from 95.5% to 100%.The phylogenetic analysis showed that all Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and they were distributed according to their geographical origins.All of the Chinese strains were closely related but they could still be divided into two groups:group of street strains and group of CTN strains.This study presents details about the molecular epidemiology of rabies viruses based on the sequences of the G-L Intergenic region.     

Novel small RNA-encoding genes in the intergenic regions of Bacillus subtilis

Small, non-coding RNAs (ncRNAs) perform diverse functions in a variety of organisms, but few ncRNAs have been identified in Bacillus subtilis. To search the B. subtilis genome for genes encoding ncRNAs, we focused on 123 intergenic regions (IGRs) over 500 bp in length and analyzed expression from these regions. Seven IGRs termed bsrC, bsrD, bsrE, bsrF, bsrG, bsrH and bsrI expressed RNAs smaller than 380 nt. All small RNAs except BsrD RNA were expressed in transformed Escherichia coli cells harboring a plasmid with PCR-amplified IGRs of B. subtilis, indicating that their own promoters independently express small RNAs. Under the non-stressed condition, depletion of the genes for the small RNAs did not affect growth. Although their functions are unknown, gene expression profiles at several time points showed that most of the genes except for bsrD were expressed during the vegetative phase (4-6 h), but undetectable during the stationary phase (8 h). Mapping the 5' ends of the 6 small RNAs revealed that the genes for BsrE, BsrF, BsrG, BsrH, and BsrI RNAs are preceded by a recognition site for RNA polymerase sigma factor sigma(A). These small RNAs might lack an SD sequence and exert their actions as ncRNAs.     

The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avian or human influenza virus internal genes. Twenty-four reassortant viruses were isolated and first evaluated for infectivity in avian (primary chick kidney [PCK]) and mammalian (Madin-Darby canine kidney [MDCK]) tissue culture lines. Reassortant viruses with two specific constellations of viral polymerase genes exhibited a significant host range restriction of replication in mammalian (MDCK) tissue culture compared with that in avian (PCK) tissue culture. The viral polymerase genotype PB2-avian (A) virus, PB1-A virus, and PA-human (H) virus was associated with a 900-fold restriction, while the viral polymerase genotype PB2-H, PB1-A, and PA-H was associated with an 80,000-fold restriction of replication in MDCK compared with that in PCK. Fifteen reassortant viruses were subsequently evaluated for their level of replication in the respiratory tract of squirrel monkeys, and two genetic determinants of attenuation were identified. First, reassortant viruses which possessed the avian influenza virus nucleoprotein gene were as restricted in replication as a virus which possessed all six internal genes of the avian influenza A virus parent, indicating that the nucleoprotein gene is the major determinant of attenuation of avian-human A/Pintail/79 reassortant viruses for monkeys. Second, reassortant viruses which possessed the viral polymerase gene constellation of PB2-H, PB1-A, and PA-H, which was associated with the greater degree of host range restriction in vitro, were highly restricted in replication in monkeys. Since the avian-human influenza reassortant viruses which expressed either mode of attenuation in monkeys replicated to high titer in eggs and in PCK tissue culture, their failure to replicate efficiently in the respiratory epithelium of primates must be due to the failure of viral factors to interact with primate host cell factors. The implications of these findings for the development of live-virus vaccines and for the evolution of influenza A viruses in nature are discussed.     

Intracerebral administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus

Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

Differential amplification of rRNA genes by polymerase chain reaction.

The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes.     

Evolution of the noncoding regions inDrosophila mitochondrial DNA: Two intergenic regions

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to theDrosophila immigrans species group and compared to the corresponding segments ofDrosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between thetRNA ^gln and thetRNA ^ile genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between thetRNA ^f-met and thetRNA ^ile genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in thenasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.     

Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species.

The nucleotide sequence of segment 1 of the double stranded RNA genome of bluetongue virus serotype 10 (BTV-10), encoding the largest viral core protein, VP1, has been determined. Linear sequence analysis of the predicted amino acid sequence of the 149-K Da protein, a putative component of the viral RNA-directed RNA polymerase, revealed extensive homology with the vaccinia virus 147K Da DNA-directed RNA polymerase subunit. Similar homologies were detected between the VP1 polypeptide and the beta chain subunit of Escherichia coli and common tobacco chloroplast RNA polymerases, yeast RNA polymerase II and III and fruit fly polymerase II.