No abstract

Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3 % DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1i 10 5 viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol ; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHApositive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC50 of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O’nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host–pathogen interactions and the rational design of vaccines against CHIKV.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop “groove” as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.