**Acknowledgements**

replication competence because of recombination or complementation between the left terminus end of the vector and the partially overlapping E1 sequence present in HEK293 cell genome or in adenoviral sequences previously acquired by the host (due to the general distribution of the Ad) [87]. Packaging cell lines with nonhomologous sequences with the vector or testing viral vector stocks for RC virus can be employed to reduce this risk [88]. Deletion of the E2A, E2B or E4 regions in the second-generation vectors reduces this risk but complicates the packaging of the recombinant adenoviral particle since specific packaging cells have to be designed to complement the missing adenoviral genome. Obtaining high titer stocks with these systems is more difficult, which often leads to reduced immunogenicity as only lower vaccine doses can be obtained [89]. These issues are even more pronounced with "gutless" adenoviral vectors, which are perfect in terms of safety, but can be problematic in terms of immunogenicity and ease of production.

The route of administration is also relevant in RDAd shedding. Intravenous (or systemic) administration results predominantly in liver adenovirus localization with minimal or no shedding to biologic fluids [6, 90]. When administered subcutaneously or intramuscularly, the point of inoculation should be disinfected to minimize the risk of vector propagation to the environment as leaks can sometimes be detected at the site. Nonetheless, no vector was detected in rodents 72 h after injection in tail swab, and the vectors were cleared from blood within 24 h [90]. As previously mentioned, RDAd vectors do not integrate efficiently into the host cell genome, the transgene expression is only transient and they do not produce infective particles, which inherently improves their biosafety [8]. Adenoviral vector survival in bedding or caging is also reduced compared to parent Ad [8]. Adenoviral vectors can, however, trigger episodes of inflammatory responses. This includes one death after a high dose direct injection of an adenoviral vector into the hepatic artery [91], which produced a fulminant immune reaction probably due to pre-existing vector immunity. It is, however, very difficult to detect vertical or germline transmission of adenovirus vectors in experimental animal models [92]. All measures (autoclave treatments for 30 min at 121°C under 1 atm pressure, 0.5% sodium hypochlorite, 5% phenol, or 2% glutaraldehyde) sufficient to eliminate the peril of adenoviral transmission have to be met to minimize risks (alcohol is not a good decontaminant for Ad), but we must not forget that RDAd do not replicate and should not, unless recombination and complementation occur, be able to shed from inoculated animals, and are thus even less likely to infect another organism. In the case of an RCAd, the risk is reduced to the range and tropism of the Ad; for example, human adenovirus is only known to replicate in two nonhu-

It is necessary to deepen in the knowledge of the biodistribution, dissemination, and *in vivo* transgene expression duration of these vectors in veterinary medicine to assess their risk more thoroughly. No standard procedures to monitor these risks exist, and thus, each independent

In the increasingly globalized world in which we live, animal health is of great importance and the prevention of animal diseases through vaccination is necessary for animal care, food production, food safety, food security, prevention of zoonotic and foodborne infections, reduction of

study analyses arbitrarily which biosafety parameters are evaluated.

man species: cotton rat and hamster [93].

40 Adenoviruses

**6. Conclusions and perspectives**

The work in the lab was funded by Grants RyC2010-06516, AGL2011-25025, AGL2012-33289, AGL2015-64290R, ADENONET-Redes de Excelencia (BIO2015-68990-REDT) from the Spanish Ministerio de Economía y Competitividad, and Grant S2013/ABI-2906-PLATESA from the Comunidad de Madrid and the European Union (Fondo Europeo de Desarrollo Regional, FEDER funds) and 731014-VetBioNet Project from European Union.
