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Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is the leading cause of pediatric viral encephalitis in Asia. Japanese encephalitis virus is transmitted byCulexspecies mosquitoes that also vector several zoonotic flaviviruses. Despite the knowledge that mosquito saliva contains molecules that may alter flavivirus pathogenesis, whether or not the deposition of viruses by infected mosquitoes has an impact on the kinetics and severity of JEV infection has not been thoroughly examined, especially in mammalian species involved in the enzootic transmission. Most JEV pathogenesis models were established using needle inoculation. Mouse models for West Nile (WNV) and dengue (DENV) viruses have shown that mosquito saliva can potentiate flavivirus infections and exacerbate disease symptoms. In this study, we determined the impact of mosquito salivary components on the pathogenesis of JEV in pigs, a species directly involved in its transmission cycle as an amplifying host. Interestingly, co-injection of JEV and salivary gland extract (SGE) collected fromCulex quinquefasciatusproduced milder febrile illness and shortened duration of nasal shedding but had no demonstrable impact on viremia and neuroinvasion. Our findings highlight that mosquito salivary components can differentially modulate the outcomes of flavivirus infections in amplifying hosts and in mouse models.

Background. Disinfection of contaminated or potentially contaminated surfaces has become an integral part of the mitigation strategies for controlling coronavirus disease 2019. Whilst a broad range of disinfectants are effective in inactivating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), application of disinfectants has a low throughput in areas that receive treatments. Disinfection of large surface areas often involves the use of reactive microbiocidal materials, including ultraviolet germicidal irradiation, chlorine dioxide, and hydrogen peroxide vapor. Albeit these methods are highly effective in inactivating SARS-CoV-2, the deployment of these approaches creates unacceptable health hazards and precludes the treatment of occupied indoor spaces using existing disinfection technologies. Deployment of dry hydrogen peroxide (DHP) is an emerging pathogen reduction technology, which produces hydrogen peroxide in the ambient atmosphere at 5 and 25 parts per billion using a commercially available catalytic unit. The low concentration of hydrogen peroxide released using DHP technology has been found to be tolerated by humans in indoor spaces and is effective in inactivating bacterial pathogens responsible for nosocomial infections. In this study, the feasibility of using DHP in inactivating SARS-CoV-2 on contaminated surfaces in large indoor spaces was evaluated. Methods. Glass slides were inoculated with SARS-CoV-2 and treated with DHP for up to 24 hours. Residual infectious virus samples were eluted and titrated in African green monkey VeroE6 cells. Results. In comparison with the observed relatively high stability of SARS-CoV-2 on contaminated glass slides in the control group, residual infectious titers of glass slides inoculated with SARS-CoV-2 were significantly reduced after receiving 120 minutes of DHP treatment. Conclusions. The accelerated decay of SARS-CoV-2 on contaminated glass slides suggests that treatment with DHP can be an effective surface disinfection method for occupied indoor spaces.

The research addresses public speculation that SARS-CoV-2 might be transmitted by mosquitoes. The World Health Organization has stated “To date there has been no information nor evidence to suggest that the new coronavirus could be transmitted by mosquitoes”. Here we provide the first experimental data to the capacity of SARS-CoV-2 to infect and be transmitted by mosquitoes. Using three species of mosquitoes from both genera involved in transmission of most arboviruses that infect humans we demonstrate that SARS-CoV-2 virus is unable to replicate in mosquitoes and therefore cannot be transmitted to people.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne flavivirus endemic in the Asia-Pacific region. Maintenance of JEV in nature involves enzootic transmission by competent Culex mosquitoes among susceptible avian and swine species. Historically, JEV has been regarded as one of the most important arthropod-borne viruses in Southeast Asia. Oronasal shedding of JEV from infected amplification hosts was not recognized until the recent discovery of vector-free transmission of JEV among domestic pigs. In this study, oral shedding of JEV was characterized in domestic pigs and miniature swine representing the feral phenotype. A rope-based sampling method followed by the detection of viral RNA using RT-qPCR allowed the collection and detection of JEV in oral fluid samples collected from intradermally challenged animals. The results suggest that the shedding of JEV in oral fluid can be readily detected by molecular diagnostic assays at the acute phase of infection. It also demonstrates the feasibility of this technique for the diagnosis and surveillance of JEV in swine species.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus endemic in the Asia Pacific region. Despite use of several highly effective vaccines, it is estimated that up to 44,000 new cases of Japanese encephalitis (JE) occur every year including 14,000 deaths and 24,000 survivors with permanent sequelae. Humoral immunity induced by vaccination is critical for effective protection. Potently neutralizing antibodies reactive with the JEV envelope (E) protein are important since protective immune responses induced by both live-attenuated and inactivated JE vaccines target the E protein. Our understanding of how vaccine-induced humoral immunity protects vaccinees from morbidity and mortality is, however, limited and largely obtained from in vitro studies. With the exception of neurovirulence mouse models, very few platforms are available for evaluating the protective efficacy of neutralizing antibodies against JEV in vivo. Swine are a major amplifying host in the natural JEV transmission cycle and develop multiple pathological outcomes similar to humans infected with JEV. In this study, prophylactic passive immunization was performed in a miniature swine model, using two vaccination-induced monoclonal antibodies (mAb), JEV-31 and JEV-169. These were selected as representatives for antibodies reactive with the major antigenic structures in the E protein of JEV and related flaviviruses. JEV-31 recognizes the lateral ridge of E protein domain III (EDIII) whilst JEV-169 has a broad footprint of binding involving residues throughout domains I (EDI) and II (EDII) of the E protein. Detection of neutralizing antibodies in the serum of immunized animals mimics the presence of neutralizing antibodies in vaccinated individuals. Passive immunization with both mAbs significantly reduced the severity of diseases that resemble the symptoms of human JE including fever, viremia, viral shedding, systemic infection, and neuroinvasion. In contrast to the uniformed decrease of viral loads in lymphoid and central nervous systems, distinct kinetics in the onset of fever and viremia between animals receiving JEV-31 and JEV-169 suggest potential differences in immune protection mechanisms between anti-EDI and anti-EDIII neutralizing antibodies elicited by vaccination. Our data demonstrate the feasibility of using swine models in characterizing the protective humoral immunity against JEV and increase our understanding of how clonal populations of anti-E mAbs derived from JE vaccination protect against infection in vivo.