Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection.

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV.

Long-term circulation of Zika virus in Thailand: an observational study.

BACKGROUND: Little is known about the historical and current risk of Zika virus infection in southeast Asia, where the mosquito vector is widespread and other arboviruses circulate endemically. Centralised Zika virus surveillance began in Thailand in January, 2016. We assessed the long-term circulation of Zika virus in Thailand. METHODS: In this observational study, we analysed data from individuals with suspected Zika virus infection who presented at hospitals throughout the country and had biological samples (serum, plasma, or urine) tested for confirmation with PCR at the National Institute of Health laboratories in Bangkok. We analysed the spatial and age distribution of cases, and constructed time-resolved phylogenetic trees using genomes from Thailand and elsewhere to estimate when Zika virus was first introduced. FINDINGS: Of the 3089 samples from 1717 symptomatic individuals tested between January, 2016, and December, 2017, 368 were confirmed to have Zika virus infection. Cases of Zika virus infection were reported throughout the year, and from 29 of the 76 Thai provinces. Individuals had 2·8 times (95% CI 2·3-3·6) the odds of testing positive for Zika virus infection if they came from the same district and were sick within the same year of a person with a confirmed infection relative to the odds of testing positive anywhere, consistent with focal transmission. The probability of cases being younger than 10 years was 0·99 times (0·72-1·30) the probability of being that age in the underlying population. This probability rose to 1·62 (1·33-1·92) among those aged 21-30 years and fell to 0·53 (0·40-0·66) for those older than 50 years. This age distribution is consistent with that observed in the Zika virus epidemic in Colombia. Phylogenetic reconstructions suggest persistent circulation within Thailand since at least 2002. INTERPRETATION: Our evidence shows that Zika virus has circulated at a low but sustained level for at least 16 years, suggesting that Zika virus can adapt to persistent endemic transmission. Health systems need to adapt to cope with regular occurrences of the severe complications associated with infection. FUNDING: European Research Council, National Science Foundation, and National Institutes of Health.

Highly sensitive EGFR mutation detection by specific amplification of mutant alleles.

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene predict benefit from tyrosine kinase inhibitors in patients suffering from non-small-cell lung cancer. In this study, we developed a fast, simple, cost-effective and highly sensitive assay for detection of five clinically important EGFR mutations in exon 19 (2235_2249del and 2236_2250del), exon 20 (C2369T) and exon 21 (T2573G and c.2573_2574 TG > GT). We designed EGFR mutation detection assays by combining allele-specific PCR amplification with the detection of SYBR Green I fluorescence, and optimized PCR conditions to specifically amplify mutant alleles. These one-step assays were able to detect the mutations at levels as low as 1.5 mutant copies in a DNA sample. Commercially available probe-based allele-specific PCR exhibited relatively poor performance when detecting very low copies of mutated DNA, especially in exon 19 and 20. Our assays offered dramatically less reagent cost than that of the commercial kit and generated results in less than 90 min after DNA extraction. These protocols can also be applied to conventional thermal cyclers followed by gel electrophoresis detection.