Dengue vaccine candidates have been shown to improve vaccine safety and efficacy by altering the residues or accessibility of the fusion loop on the virus envelope protein domain II (DII(FL)) in an ex vivo animal study. The current study aimed to comprehensively investigate the impact of DII(FL) mutations on the antigenicity, immunogenicity, and protective efficacy of Japanese encephalitis virus (JEV) virus-like particles (VLPs) in mice. We found the DII(FL) G106K/L107D (KD) and W101G/G106K/L107D (GKD) mutations altered the binding activity of JEV VLP to cross-reactive monoclonal antibodies but had no effect on their ability to elicit total IgG antibodies in mice. However, JEV VLPs with KD or GKD mutations induced significantly less neutralizing antibodies against JEV. Only 46% and 31% of the KD and GKD VLPs-immunized mice survived compared to 100% of the wild-type (WT) VLP-immunized mice after a lethal JEV challenge. In passive protection experiments, naïve mice that received sera from WT VLP-immunized mice exhibited a significantly higher survival rate of 46.7% compared to those receiving sera from KD VLP- and GKD VLP-immunized mice (6.7% and 0%, respectively). This study demonstrated that JEV DII(FL) is crucial for eliciting potently neutralizing antibodies and protective immunity against JEV. IMPORTANCE: Introduction of mutations into the fusion loop is one potential strategy for generating safe dengue and Zika vaccines by reducing the risk of severe dengue following subsequent infections, and for constructing live-attenuated vaccine candidates against newly emerging Japanese encephalitis virus (JEV) or Japanese encephalitis (JE) serocomplex virus. The monoclonal antibody studies indicated the fusion loop of JE serocomplex viruses primarily comprised non-neutralizing epitopes. However, the present study demonstrates that the JEV fusion loop plays a critical role in eliciting protective immunity in mice. Modifications to the fusion loop of JE serocomplex viruses might negatively affect vaccine efficacy compared to dengue and zika serocomplex viruses. Further studies are required to assess the impact of mutant fusion loop encoded by commonly used JEV vaccine strains on vaccine efficacy or safety after subsequent dengue virus infection.

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.

Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.

Workers are at risk when entering (ingress) or exiting (egress) elevated scissor lifts. In this study, we recorded ground impact forces and postural sway from 22 construction workers while they performed ingress and egress between a scissor lift and an adjacent work surface with varying conditions: lift opening designs, horizontal and vertical gaps, and sloped work surfaces. We observed higher peak ground shear forces when using a bar-and-chain opening, with larger horizontal gap, with the lift surface more than 0.2 m below the work surface, and presence of a sloped (26) work surface. Similar trends were observed for postural sway, except that the influence of vertical distance was not significant. To reduce slip/trip/fall risk and postural sway of workers while ingress or egress of an elevated scissor lift, we suggest scissor lifts be equipped with a gate-type opening instead of a bar-and-chain design. We also suggest the lift surface be placed no more than 0.2 m lower than the work surface and the horizontal gap between lift and work surfaces be as small as possible. Selecting a non-sloped surface to ingress or egress a scissor lift is also preferred to reduce risk.

OBJECTIVE: The authors investigated the effect of boot weight and sole flexibility on spatiotemporal gait characteristics and physiological responses of firefighters in negotiating obstacles. BACKGROUND: Falls and overexertion are the leading causes of fire ground injuries and fatalities among firefighters. There have been few in-depth studies conducted to evaluate the risk factors of falls and overexertion associated with firefighter boots. METHOD: For the study, 13 female and 14 male firefighters, while wearing full turnout clothing and randomly assigned boots, walked for 5 min while stepping over obstacles. The independent variables included boot weight, sole flexibility, gender, and task duration. Spatiotemporal measures of foot trajectories and toe clearance were determined. Minute ventilation, oxygen consumption, carbon dioxide production, and heart rate were measured. RESULTS: Increased boot weight was found to significantly reduce trailing toe clearance when crossing the 30-cm obstacle. Significant increases in lateral displacement of the foot were found near the end of the 5-min walk compared with the beginning of the task Increased boot weight significantly increased oxygen consumption. There were significant decreases in oxygen consumption for more flexible soles. CONCLUSION: Firefighters were more likely to trip over obstacles when wearing heavier boots and after walking for a period of time. Boot weight affected metabolic variables (5% to 11% increases per 1-kg increase in boot weight), which were mitigated by sole flexibility (5% to 7% decrease for more flexible soles). APPLICATION: This study provides useful information for firefighters and boot manufacturers in boot selection and design for reducing falls and overexertion.

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.

Genotype I of Japanese encephalitis virus first appeared in Taiwan in 2008. Phylogenetic analysis of 37 viruses from pig farms in 2009-2010 classified these viruses into 2 unique subclusters of genotype I viruses and suggested multiple introductions and swift replacement of genotype III by genotype I virus in Taiwan.