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Foot-and-mouth disease (FMD) vaccines must be produced in a biosafety level 3 facility, so the FMD virus (FMDV) must be completely inactivated after amplification. The inactivation kinetics of FMDV during vaccine antigen production were assessed by evaluating whether the viral titer dropped below 10−7 TCID50/mL within 24 h of binary ethyleneimine (BEI) treatment. This study dealt with four FMD vaccine candidate strains for the efficacy of BEI treatment at different concentrations and temperatures to determine the optimal inactivation condition of each virus. Two domestic isolates, O/SKR/Boeun/2017 (O BE) and A/SKR/Yeoncheon/2017 (A YC), and two recombinant viruses, PAK/44/2008 (O PA-2) and A22/Iraq/24/64 (A22 IRQ), were investigated. The O BE and A22 IRQ required 2 mM BEI at 26 °C and 0.5 mM BEI at 37 °C for complete inactivation. The O PA-2 and A YC required 2 mM BEI at 26 °C and 1 mM BEI at 37 °C. Crucially, the yield of FMD virus particles (146S) in the viral infection supernatant was higher (>4.0 µg/mL) than those previously reported; additionally, there was little antigen loss, even after 24 h of treatment with 3 mM BEI. Overall, it is considered economical to produce FMD vaccines using these four kinds of viruses; therefore, these candidate strains will be prioritized for the manufacture of FMD vaccines in South Korea.

Acanthamoeba is among the most ubiquitous protistan groups in nature. Knowledge of the biological diversity of Acanthamoeba comes in part from the use of strains maintained by the major microbial culture collections, ATCC and CCAP. Standard strains are vital to ensure the comparability of research. The diversity of standard strains of Acanthamoeba in the culture collections is reviewed, emphasizing the extent of genotypic studies based on DNA sequencing of the small subunit ribosomal RNA from the nucleus (18S rRNA gene; Rns) or the mitochondria (16S-like rRNA gene; rns). Over 170 different strains have been maintained at some time by culture centers. DNA sequence information is available for more than 70% of these strains. Determination of the genotypic classification of standard strains within the genus indicates that frequencies of types within culture collections only roughly mirror that from clinical or environmental studies, with significant differences in the frequency of some genotypes. Culture collections include the type of isolate from almost all named species of Acanthamoeba, allowing an evaluation of the validity of species designations. Multiple species are found to share the same Sequence Type, while multiple Sequence Types have been identified for different strains that share the same species name. Issues of sequence reliability and the possibility that a small number of standard strains have been mislabeled when studied are also examined, leading to potential problems for comparative analyses. It is important that all species have reliable genotype designations. The culture collections should be encouraged to assist in completing the molecular inventory of standard strains, while workers in the Acanthamoeba research community should endeavor to ensure that strains representative of genotypes that are missing from the culture collection are provided to the culture centers for preservation.

The Middle East respiratory syndrome (MERS) is a fatal acute viral respiratory disease caused by MERS-coronavirus (MERS-CoV) infection. To date, no vaccine has been approved for MERS-CoV despite continuing outbreaks. Inactivated vaccines are a viable option when developed using the appropriate inactivation methods and adjuvants. In this study, we evaluated the immunogenicity and protective effects of MERS-CoV vaccine candidates inactivated by three different chemical agents. MERS-CoV was effectively inactivated by formaldehyde, hydrogen peroxide, and binary ethylene imine and induced humoral and cellular immunity in mice. Although inflammatory cell infiltration was observed in the lungs four days after the challenge, the immunized hDPP4-transgenic mouse group showed 100% protection against a challenge with MERS-CoV (100 LD50). In particular, the immune response was highly stimulated by MERS-CoV inactivated with formaldehyde, and all mice survived a challenge with the minimum dose. In the adjuvant comparison test, the group immunized with inactivated MERS-CoV and AddaVax had a higher immune response than the group immunized with aluminum potassium sulfate (alum). In conclusion, our study indicates that the three methods of MERS-CoV inactivation are highly immunogenic and protective in mice and show strong potential as vaccine candidates when used with an appropriate adjuvant.

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanism.

Foot-and-mouth disease (FMD) vaccines must be produced in a biosafety level 3 facility, so the FMD virus (FMDV) must be completely inactivated after amplification. The inactivation kinetics of FMDV during vaccine antigen production were assessed by evaluating whether the viral titer dropped below 10 -7 TCID 50 /mL within 24 h of binary ethyleneimine (BEI) treatment. This study dealt with four FMD vaccine candidate strains for the efficacy of BEI treatment at different concentrations and temperatures to determine the optimal inactivation condition of each virus. Two domestic isolates, O/SKR/Boeun/2017 (O BE) and A/SKR/Yeoncheon/2017 (A YC), and two recombinant viruses, PAK/44/2008 (O PA-2) and A22/Iraq/24/64 (A22 IRQ), were investigated. The O BE and A22 IRQ required 2 mM BEI at 26 °C and 0.5 mM BEI at 37 °C for complete inactivation. The O PA-2 and A YC required 2 mM BEI at 26 °C and 1 mM BEI at 37 °C. Crucially, the yield of FMD virus particles (146S) in the viral infection supernatant was higher (>4.0 µg/mL) than those previously reported; additionally, there was little antigen loss, even after 24 h of treatment with 3 mM BEI. Overall, it is considered economical to produce FMD vaccines using these four kinds of viruses; therefore, these candidate strains will be prioritized for the manufacture of FMD vaccines in South Korea.

Acanthamoeba is among the most ubiquitous protistan groups in nature. Knowledge of the biological diversity of Acanthamoeba comes in part from the use of strains maintained by the major microbial culture collections, ATCC and CCAP. Standard strains are vital to ensure the comparability of research. The diversity of standard strains of Acanthamoeba in the culture collections is reviewed, emphasizing the extent of genotypic studies based on DNA sequencing of the small subunit ribosomal RNA from the nucleus (18S rRNA gene; Rns ) or the mitochondria (16S-like rRNA gene; rns ). Over 170 different strains have been maintained at some time by culture centers. DNA sequence information is available for more than 70% of these strains. Determination of the genotypic classification of standard strains within the genus indicates that frequencies of types within culture collections only roughly mirror that from clinical or environmental studies, with significant differences in the frequency of some genotypes. Culture collections include the type of isolate from almost all named species of Acanthamoeba , allowing an evaluation of the validity of species designations. Multiple species are found to share the same Sequence Type, while multiple Sequence Types have been identified for different strains that share the same species name. Issues of sequence reliability and the possibility that a small number of standard strains have been mislabeled when studied are also examined, leading to potential problems for comparative analyses. It is important that all species have reliable genotype designations. The culture collections should be encouraged to assist in completing the molecular inventory of standard strains, while workers in the Acanthamoeba research community should endeavor to ensure that strains representative of genotypes that are missing from the culture collection are provided to the culture centers for preservation.

The Middle East respiratory syndrome (MERS) is a fatal acute viral respiratory disease caused by MERS-coronavirus (MERS-CoV) infection. To date, no vaccine has been approved for MERS-CoV despite continuing outbreaks. Inactivated vaccines are a viable option when developed using the appropriate inactivation methods and adjuvants. In this study, we evaluated the immunogenicity and protective effects of MERS-CoV vaccine candidates inactivated by three different chemical agents. MERS-CoV was effectively inactivated by formaldehyde, hydrogen peroxide, and binary ethylene imine and induced humoral and cellular immunity in mice. Although inflammatory cell infiltration was observed in the lungs four days after the challenge, the immunized hDPP4-transgenic mouse group showed 100% protection against a challenge with MERS-CoV (100 LD 50 ). In particular, the immune response was highly stimulated by MERS-CoV inactivated with formaldehyde, and all mice survived a challenge with the minimum dose. In the adjuvant comparison test, the group immunized with inactivated MERS-CoV and AddaVax had a higher immune response than the group immunized with aluminum potassium sulfate (alum). In conclusion, our study indicates that the three methods of MERS-CoV inactivation are highly immunogenic and protective in mice and show strong potential as vaccine candidates when used with an appropriate adjuvant.

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level ( rtp3 , mx , mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanism.