Ionizing radiation is widely used to inactivate pathogens. It mainly acts by destroying nucleic acids but causes less damage to structural components like proteins. It is therefore highly suited for the sterilization of biological samples or the generation of inactivated vaccines. However, inactivation of viruses or bacteria requires relatively high doses and substantial amounts of radiation energy. Consequently, irradiation is restricted to shielded facilities—protecting personnel and the environment. We have previously shown that low energy electron irradiation (LEEI) has the same capacity to inactivate pathogens in liquids as current irradiation methods, but generates much less secondary X-ray radiation, which enables the use in normal laboratories by self-shielded irradiation equipment. Here, we present concepts for automated LEEI of liquids, in disposable bags or as a continuous process. As the electrons have a limited penetration depth, the liquid is transformed into a thin film. High concentrations of viruses (Influenza, Zika virus and Respiratory Syncytial Virus), bacteria (E. coli, B. cereus) and eukaryotic cells (NK-92 cell line) are efficiently inactivated by LEEI in a throughput suitable for various applications such as sterilization, vaccine manufacturing or cell therapy. Our results validate the premise that for pathogen and cell inactivation in liquids, LEEI represents a suitable and versatile irradiation method for standard biological research and production laboratories.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod-borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV-endemic regions co-circulation of both viruses is increasing. Flaviviruses are structurally highly similar, which leads to cross-reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false-positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross-reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV-and WNV-assays with improved specificities. K E Y W O R D Scross-reactivity, ELISA, serology, tick-borne encephalitis virus, West Nile virus, zoonoses

Zika virus (ZIKV) is a positive-stranded RNA virus within the Flaviviridae family. After decades of circulation in Asia, ZIKV was introduced to Brazil in 2014–2015, associated with a rise in congenital malformations. Unlike the genetically related dengue virus (DENV), ZIKV constitutes only one serotype. Although assumed that ZIKV infection may engender lifelong immunity, the long-term kinetics of ZIKV antibody responses are unclear. We assessed long-term kinetics of ZIKV NS1-IgG response in 144 individuals from 3 different subpopulations: HIV patients, tuberculosis patients and healthy individuals first tested in 2016 and retested 1.5–2 years after the 2015–2016 ZIKV epidemic in Salvador de Bahia, Brazil, using a widely distributed NS1-based commercial ELISA. The seropositivity in 2016 reached 59.0% (85/144, 95% confidence interval (CI) 50.7–66.7%), and decreased to 38.6% (56/144, CI 31.3–47.0%) 1.5–2 years later. In addition, the median ZIKV NS1-ELISA reactivity for individuals that remained positive in both timepoints significantly decreased from a ratio of 4.4 (95% CI 3.8–5.0) to 1.6 (95% CI 1.6–1.9) over the 2-year interval (Z: − 6.1; p < 0.001) irrespective of the subpopulation analyzed. Initial 2016 DENV antibody response was non-significant between groups, suggesting comparable DENV background. The high 20.6% seroreversion suggest that widely used serologic tests may fail to account a considerable proportion of past ZIKV infections in flavivirus endemic countries. In addition, ZIKV immunity might be shorter-lived than previously thought, which may contribute to local ZIKV resurgence once individual immune responses wane sufficiently to reduce community protective immunity in addition to birth and migration.

W est Nile virus (WNV) is a widely distributed arthropodborne fl avivirus transmitted predominantly by Culex mosquitoes (1). Among infected persons, ≈20% show clinical signs, such as mild fever, rash, joint pain, headache, vomiting, and diarrhea (1,2); ≈0.7% have severe illness, such as encephalitis, meningitis, acute fl accid paralysis, respiratory failure, and even death (1). Beyond vectorborne transmission, transfusion-transmitted WNV infections have endangered blood safety (3). Equids are susceptible to WNV and develop severe disease (fatality rate <30%), are exposed to WNV vectors outside and in stables, and are spatially distributed near human settlements. Thus, equids can be sentinels for early detection of regional WNV activity (4).In the Americas, WNV gained attention after its rapid spread in the United States beginning in 1999 (4). In South America, WNV dispersion is poorly understood. Seropositive horses were found in Colom-

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.