Evaluation of a BE-inactivated whole virus preparation using an encephalomyocarditis virus strain that was isolated from fatal infection in orangutans

A novel Encephalomyocarditis virus (EMCV) of the group 3 cluster (EMCV-3) was first reported in 2002 to be responsible for the deaths of orang-utans in an outbreak in the Singapore Zoo. After this first outbreak, sporadic infections among the primate population caused by EMCV-3 continued to be reported, suggesting that the virus remains prevalent in Singapore. To prevent future infections, we constructed an experimental vaccine using binary ethylenimine (BE) to inactivate the EMCV-3 virus. The immunological performance of the BE-inactivated (BEI) virus was analysed in mice and the neutralising titre of the immune sera measured against the wild-type EMCV-3. The BEI virus showed a strong immunological response in BALB/c mice at 1: 40,960 titre, suggesting that it can be used as a promising experimental vaccine candidate to prevent EMCV-3 infections.

infections are now documented worldwide in primate and non-primate species, and in 55 domesticated and wild animals (Lee GKW, 2015). EMCV has also been reported in 56 humans with either asymptomatic or mild symptoms (Oberste et al., 2009), suggesting 57 that the virus is involved in zoonotic episodes. Rats and bats found in various locations 58 have been implicated as the virus reservoir (Doysabas et al., 2019;Liu et al., 2017). 59 We previously reported an outbreak of EMCV infections in orang-utans in the 60 Singapore zoo caused by a novel EMCV-3 in 2002 (Yeo et al., 2013). The outbreak 61 was caused by a novel EMCV strain designated SING-M105, and was associated with 62 fatalities of the orang-utan population. Since the first outbreak reported in Singapore, 63 EMCV continues to be sporadically detected (Lee et al., 2015), suggesting that the 64 virus persists in the natural environment. There is an urgent need to implement 65 effective control strategies for EMCV and one of these strategies could be the use of 66 an effective vaccine to protect susceptible animals from EMCV infection. The 67 persistence of this virus in the natural environment is therefore the motivation behind 68 the current study that was to develop a specific vaccine candidate with the Singapore 69 EMCV-3 strain. Given that the local circulating EMCV strains are likely to have specific 70 immunogenic properties that are regional-specific, the preparations of vaccine 71 candidates using local circulating EMCV strains is desirable. In this current study we 72 therefore describe the development of an inactivated vaccine candidate using the 73 EMCV-3 strain SING-M105 that was isolated from infected orang-utans during the first 74 outbreak reported in 2002.

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The virus used in this study was the EMCV strain SING-M105 isolated from an orang- Culture Collection (ATCC, USA) as described in (Yeo et al., 2013). Cells were 83 maintained in a 37°C in a humidified chamber with 5% CO2 in cell growth medium 84 (DMEM supplemented with 10% heat-inactivated FCS and penicillin/streptomycin).

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The antigen-coated plates were blocked with 1% Bovine Serum Albumin for 1 hr, Results and discussion. 158 An EMCV vaccine has been previously described for use in Australia candidates; however BPL is also a suspected carcinogen (Information., 2022; 164 Španinger & Bren, 2020), which may limit its general use in Singapore. In this context, 165 BE has been used for the inactivation of various veterinary viruses to prevent livestock 166 diseases, and we therefore evaluated BE inactivation of EMCV-3 isolate in our study. 167 Promising virus vaccine candidates have been prepared using BE and described for    in parallel with the micro-neutralisation but in the absence of EMCV strain SING-M105.

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Although the BEI-EMCV preparation was well-tolerated in the inoculated mice with no

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The immune serum of mice immunised using the recombinant VP1 was performed 312 using micro-neutralisation assay method described above. This analysis indicated that 313 although the recombinant VP1 was able to elicit a neutralising antibody response in 314 an immunised mouse, the neutralising antibody responses recorded was significantly 315 lower than in mice immunised with the BEI-EMCV (SFigure 2). Interestingly, a panel 316 of monoclonal antibodies was generated from the mouse immunised with the 317 recombinant VP1, and one of these antibodies (MAb 5C2) exhibited virus neutralising 318 activity that was higher than recorded for the VP1 immune serum (SFigure 3).

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Although the neutralising titre for the VP1 monoclonal antibody was lower than that 320 recorded for the serum obtained from the BEI-EMCV immunised animals, these data  Our current finding has shown that the BEI-EMCV is able to produce a strong

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The authors declared no conflict of interest.