It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10 4 min ؊1 . We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis.

Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2 ′ -OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to the fully conjugated protoporphyrin IX. Eukaryotes and Gram-positive bacteria possess an oxygen-dependent, FAD-containing enzyme for this step while the majority of Gram-negative bacteria lack this oxygen-dependent PPO. In E. coli, PPO activity is known to be linked to respiration and the quinone pool. In E. coli SASX38, the knockout of hemG causes loss of measurable PPO activity. HemG is a small soluble protein typical of long chain flavodoxins. Herein purified recombinant HemG was shown to be capable of a menadione-dependent conversion of protoporphyrinogen IX to protoporphyrin IX. Electrochemical analysis of HemG revealed similarities to other flavodoxins. Interestingly, HemG, a member of a class of the long chain flavodoxin family that is unique to the γ-proteobacteria, possesses a 22 residue sequence that, when transferred into E. coli flavodoxin A, produces a chimera that will complement an E. coli hemG mutant, indicating that this region confers PPO activity on the flavodoxin. These findings reveal a previously unidentified class of PPO enzymes that do not utilize oxygen as an electron acceptor thereby allowing γ-proteobacteria to synthesize heme in both aerobic and anaerobic environments.