Poliomyelitis, caused by poliovirus, is on the verge of eradication, and the world is preparing to shift from live to inactivated polio vaccine. In view of the requirement of non-infectious reagents, especially protein antigens, for surveillance during the final phase of poliovirus eradication, we have attempted to generate reagents that may be of use for the development of diagnostic tests. Polioviral proteins VP0, VP3, VP1, and 3AB were expressed in Escherichia coli using the autoinduction system, purified, and the proteins were used to raise antisera in rabbits. All antisera detected all three serotypes of PV from infected cell lysates in enzyme-linked immunosorbent assay, immunofluorescence and western blotting.

Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two β-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

No abstract

Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.

Mycobacterium avium infects human macrophages causing opportunistic infections. A steady increase of these infections over the past four decades and resistance to common anti-mycobacterial drugs, create an urgent need for new treatments; however, drug discovery is held back by a lack of knowledge about how M. avium replicates or persists in host cells. We implemented an image-based assay using a fluorescence dilution (FD) system to measure M. avium replication and persistence. M. avium strain 104 carrying a plasmid encoding GFP and TurboFP635 under constitutive and inducible promoters, respectively, is induced prior to infection of THP1 macrophages and the fluorescent signals are tracked over time in the absence of the inducer. Loss of the TurboFP635 signal while GFP signal is maintained, identifies replicating and retention of both signals non-replicating bacteria. In the absence of inducer, the M. avium 104 FD strain replicated in the macrophages, leading to increasing numbers of GFP-expressing intracellular bacteria and concomitant loss of TurboFP635 signal in >90% of the infected cells after 24 hours. Upon re-induction, these bacteria expressed TurboFP635, suggesting they are metabolically active and alive. We observed the presence of a small non replicating population that persisted over 96 hours pi. We applied our assay to compare the effect of a panel of anti-mycobacterial drugs, revealing different effects on killing, intracellular replication and induction of persisting, non-replicating bacteria, illustrating the power of this system to facilitate the dissection of the biology of persistence and anti-mycobacterial drug discovery in the future.