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A series of novel 2-fluoro ketolide antibiotics with 11,12-quinoylalkyl side chains derived from telithromycin and cethromycin were designed and synthesized. The corresponding targets 2a-o were tested for their in vitro activities against a series of macrolide-sensitive and macrolide-resistant pathogens. Some of them showed a similar antibacterial spectrum and comparable or slightly better activity to telithromycin. Among them, compounds 2g and 2k, displayed excellent activities against macrolide-sensitive and macrolide-resistant pathogens.

In China, cats cause about 5% of human rabies cases. Rabies control in cats plays a role in achieving the ultimate goal of elimination of dog rabies-mediated human deaths. However, there is no cat-specific rabies vaccine in China yet. In this study, we constructed a recombinant rabies vaccine by using a felid herpesvirus 1 (FHV-1) isolate, and deleted the gI/E in the FHV-1 and replaced the region with a glycoprotein (G) of rabies virus (RABV) strain BD06 through homologous recombination. The recombinant virus FHV-RVG was recovered and purified, and the expression of RABV glycoprotein was verified by indirect immunofluorescent assay. For potency in cats, each animal was inoculated intranasally with 1 ml FHV-RVG at 106.5 TCID50. Blood samples were collected at defined intervals for antibody titration. The animals were challenged by herpes and rabies after completion of vaccination on day 180 and day 194, respectively. Our results demonstrated all vaccinated cats generated antibodies against both FHV-1 and RABV, and reached an arbitrary protective titer > 0.5 IU/ml for rabies viral neutralizing antibody (VNA) by day 14 post inoculation (dpi) and titer peaked on 30 dpi with VNA at 24.5 ± 10.23 IU/ml. All vaccinated cats presented no clinical signs of FHV-1 infection and survived rabies challenge, while the control cats had severe rhinotracheitis and died from rabies after challenge. All this demonstrated that the FHV-based recombinant vaccine is effective in protection against both FHV-1 and RABV infections.

The NIH potency test for human rabies vaccines has disadvantages for use, especially in developing countries where rabies is endemic and prophylaxis needs ample, rapid, and reliable vaccine supplies. In China, 60–75 million doses of human rabies vaccines are administered each year. Vaccine quality control is of paramount importance, as is the release of potency-validated vaccines. We intended to design an alternative to the NIH in vivo method, and developed a relative potency test using an ELISA. Using Pearson's correlation analysis, we found a close relationship between the rabies vaccine glycoprotein content in vitro and the potency values in vivo. We suggest the relative potency test developed here as a simplified method for human rabies vaccine quality control in China and a possible alternative to the NIH method.

Plasmid DNA (pDNA) vaccines have the potential for protection against a wide range of diseases including rabies but are rapid in degradation and poor in uptake by antigen-presenting cells. To overcome the limitations, we fabricated a pDNA nanoparticulate vaccine. The negatively charged pDNA was adsorbed onto the surface of cationic PLGA (poly ( d , l -lactide-co-glycolide))-chitosan nanoparticles and were used as a delivery vehicle. To create a hydrogel for sustainable vaccine release, we dispersed the pDNA nanoparticles in poloxamer 407 gel which is liquid at 4 °C and turns into soft gels at 37 °C, providing ease of administration and preventing burst release of pDNA. Complete immobilization of pDNA to cationic nanoparticles was achieved at a pDNA to nanoparticles ratio (P/N) of 1/50. Cellular uptake of nanoparticles was both time and concentration dependent and followed a saturation kinetics with Vmax of 11.389 µg/mL h and Km of 139.48 µg/mL. The in vitro release studies showed the nanoparticulate vaccine has a sustained release for up to 24 days. In summary, pDNA PLGA-chitosan nanoparticles were non-cytotoxic, their buffering capacity and cell uptake were enhanced, and sustained the release of pDNA. We expect our pDNA vaccine’s potency will be greatly improved in the animal studies.

Low molecular weight heparins (LMWHs) are heterogeneous mixtures of glycosaminoglycan chains composed of mixture of different lengths and substitution patterns. Structural characterization and quality control of LMWHs have always been challenging. The Chinese drug regulatory authorities have been committed to improve the supervision standards of LMWHs to better regulate the quality and safety of LMWHs in current Chinese market. In the present paper, 80 batches of three types LMWHs (dalteparin, enoxaparin and naldroparin) marketed in China from different manufacturers were studied by 1H NMR experiments and chemometric analysis. The method can be used not only to monitor impurities and contaminants, but also to check the batch-to-batch consistency of each manufacture. Moreover, for the biosimilar LMWHs from different manufactures, they can be differentiated and clustered according to their slightly different structural compositions originated from production process. By using this method, the quality and safety of LMWHs marketed in China were initially assessed.

While current rabies post-exposure prophylaxis (PEP) is highly effective, it is costly and the vaccination regimen is complicated, requiring both inactivated vaccines and immunoglobulins. A one-dose rabies vaccine for human PEP remains a long-term goal. Here, we describe development of a highly attenuated rabies virus ERAg3m, with a mutation in the glycoprotein (G) gene and a switch of the G gene with the matrix protein gene in the viral genome. After a one-dose intramuscular vaccination, the ERAg3m virus protected 100% of mice and hamsters from lethal challenge. In co-infections, using a lethal dose of street rabies virus mixed with ERAg3m, 100% of hamsters and 90% of mice survived and were protected against subsequent infection. A mock co-infection, using inactivated commercial human rabies vaccine and a lethal dose of street rabies virus, protected 100% and 40% of hamsters and mice, respectively. In co-infections, when vaccine was administrated in the left leg and challenge virus in the right leg, the ERAg3m virus protected 40% of mice, while the inactivated vaccine showed no protection. Therefore, live attenuated rabies virus when given pre-exposure or co-infected with street rabies virus, is capable of preventing rabies in two different animal models. Overall, this highly attenuated live rabies virus offered better protection than the inactivated vaccine.

Rabies in Asia is emerging as a serious public health issue. To explore the possible origin, phylogenetic relationships, and evolutionary dynamics of Asian Rabies viruses (RABV), we examined 200 complete nucleoprotein (N) gene sequences from RABV isolates in the region. Phylogeny supported the classification of Asian RABVs into five distinct clusters in lyssavirus genotype 1. Our geospatial and temporal analyses demonstrated that China appears to be the prime source of Asian RABVs. Understanding of rabies transmission and associated human activities, such as dog translocation, can help rabies control and elimination in Asia through collaborative efforts or programs.

Background Rabies is a serious reemerging zoonosis in China. The molecular evolution and transmission patterns of rabies virus inferred from historical data can provide guidelines for better disease control and prevention in the future. Objectives To investigate the epidemiology and evolutionary dynamics of the rabies virus in China. Study design The molecular evolution of 132 viral glycoprotein gene sequences of Chinese rabies viruses collected in 17 provinces and 3 municipalities between 1969 and 2009 was analyzed. Results Phylogenetic analysis revealed that Chinese rabies viruses are subdivided into 6 lineages (A–F) within Lyssavirus genotype 1. Lineage A represents the widely dispersed cosmopolitan lineage while lineage B is closely related to Arctic-like rabies viruses. The remaining lineages (C–F) are typical of those circulating across much of Southeast Asia. The evolutionary rate for Chinese rabies virus was 1.532×10 −4 substitutions per site per year, and the corresponding common ancestor was in about 1115. Conclusions The phylogeographic structure demonstrated Chinese rabies viruses have been transmitted intra-provincially and extra-provincially due to human-related activities.

Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (P0.01). Based on these preliminary results, a modified glycoprotein gene from the ERA rabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

Rabies prevention and appropriate population management of free-ranging animals have an important role to play in the eventual elimination of rabies in dogs. An effective sterilant based on rabies vaccines has the potential to create a supportive measure of public acceptability and to reduce associated clinic visit costs. We inserted the coding sequence of gonadotropin-releasing hormone (GnRH) into different locations within the rabies virus ERA glycoprotein (G) gene, and demonstrated that the amino terminus (N), antigenic site IIa, and the junction between the ecto- and cytoplasmic domains (C) of the G were suitable sites for GnRH insertion. The rescued recombinant rabies viruses ERA-N-GnRH and ERA-C-GnRH grew as well as the parental ERA virus, reaching 1x10(9)ffu/ml in cell culture. Insertion and expression of the GnRH were stable in the viruses after multiple passages in vitro. To increase immunogenicity of the GnRH peptide, two copies of GnRH, aligned in tandem, were fused to the N terminus of the G. The recombinant rabies virus ERA-N-2GnRH was recovered and grown to high titers in cell culture. All GnRH-carrying rabies viruses induced antibodies against GnRH in immunized mice and protected 100% of the animals after rabies virus challenge. The recombinant viruses reacted strongly with the serum from a GonaCon-immunized animal. The GnRH-carrying rabies viruses have significant potential in rabies and animal population control.

Despite progress in vaccine development in the past century the mechanisms behind immune responses elicited by rabies biologics or via natural infection remain largely unknown. In this study, we compared protection elicited by standard, early, or delayed prophylaxis with a reduced number of vaccine doses using inactivated and live-attenuated vaccines. Two-month-old Syrian hamsters, 4-week-old ICR mice or adult rhesus macaques were inoculated with canine rabies virus variants. Thereafter, prophylaxis was initiated 6 h, 1, 2, 3, 4, 5, 6 or 7 days post-exposure (p.e.). One or several doses of inactivated (HDCV), or reverse genetically attenuated (live), or gamma-irradiated (inactivated)-ERAG333 vaccines were administered intramuscularly. The dynamics of virus spread were measured over time in the rodent models. Rabies virus reached the spinal cord at day 4 and brain at day 6 p.e. All hamsters succumbed in groups in which live ERAG333 was delayed until days 5 and 6 p.e. However, 78%, 44%, 56% and 22% of hamsters survived when one dose of live ERAG333 was administered 6 h, 1, 2, 3, and 4 days p.e., respectively. Similarly, 67% survived when inactivated ERAG333 was administered at 24 h p.e. All hamsters succumbed when standard prophylaxis (the Essen regimen) was delayed until days 3–6, but 67% and 33% of hamsters survived when PEP began 1 or 2 days p.e., respectively. Macaques were protected by one dose of attenuated ERAG333 at 24 h p.e. The highly attenuated (live) and inactivated ERAG333 vaccines elicited potent protective immune responses, even when prophylaxis initiation was delayed. When 2–5 doses of commercial vaccine and HRIG were administered according to the Essen scheme, 89–100% of the animals survived. Reduced vaccine schedules provided efficacious intervention, regardless of the total number of vaccine doses administered.

Individual lyssavirus genes were evaluated for phylogenetic studies from available full genome sequences. The full genome of the ERA rabies virus was sequenced and its accuracy was confirmed through virus recovery by reverse genetics. The full length of the ERA is 11,931 nucleotides (nt), with a leader sequence of 58 nt, the nucleoprotein (N) gene of 1350 nt, phosphoprotein (P) gene of 891 nt, matrix protein (M) gene of 606 nt, glycoprotein (G) gene of 1572 nt, RNA-dependent RNA polymerase (L) gene of 6384 nt, Psi-region (or G–L intergenic region) of 400 nt, and a trailer region of 70 nt. The five mono-cistrons are separated by intergenic regions of 2, 5, 5 and 24 nt, respectively. One obvious difference between the ERA and SAD-B19 rabies virus strains was the putative stop/polyadenylation signal of the G gene, with a poly(A 8 ) tract for ERA, and a poly(A 5 ) for SAD-B19. The TGpoly(A 8 ) sequence tract was identified to be a leaky termination signal in the ERA strain. Through analyses of nt diversity, protein co-variations, structural and functional constraints, and reconstruction of phylogenetic trees from comprehensive datasets, we propose lyssavirus genes probably are of similar value for phylogenetic analyses.

It has been reported that phosphorylation of rabies virus N plays an important role in the process of viral transcription and replication. Rabies virus N is phosphorylated when expressed alone, indicating that cellular kinase phosphorylates rabies virus N. To identify what cellular kinase phosphorylates rabies virus N, the N was expressed in Escherichia coli and purified by metal affinity chromatography. The recombinant N was phosphorylated by BHK cellular extracts and by purified CK-II. In addition, the phosphorylation of the recombinant N in vitro can be blocked by a CK-II inhibitor, heparin. Furthermore, N phosphorylation in the virus-infected cells can be inhibited by a CK-II specific inhibitor, 5,6-dichloro-beta-D-ribofuranosyl benzimidazole. However, PKC did not phosphorylate the recombinant N in vitro; nor did staurosporine, a PKC and other kinase inhibitor, prevent rabies virus N from phosphorylation. Thus, our data demonstrate that cellular CK-II phosphorylates rabies virus N.