*4.10. Rotavirus*

Rotavirus is one of the world's major causes of gastroenteritis in children. As an effort to develop anti-rotavirus strategies, milk proteins including apo-LF (25 μM) and Fe-LF were assessed for inhibitory properties against rotavirus [113]. Results demonstrated apo-LF to be the most potent inhibitor of rotavirus by binding to viral particles and hindering virus attachment as well as preventing rotavirus hemagglutination [113]. A randomized trial; however, concluded that orally-administered LF did not

provide protection against rotavirus infection [114]. This serves as an example that the pronounced inhibition in vitro does not necessarily recapitulate the effects that occurs in vivo.

#### *4.11. Other Viruses*

Lactoferrin antiviral activity against other viruses including poliovirus, alphaviruses, DENV, and Japanese encephalitis virus (JEV) has been studied. In Vero cells, iron-, manganese-, and zinc-saturated LFs have demonstrated anti-poliovirus properties in a dose-dependent manner when LFs were present either during the entire viral life cycle or during viral adsorption [115]. Against alphaviruses, human LF (200 μg/mL) inhibited infection of BHK-21 cells by Sindbis virus (SINV) and Semliki Forest viruses (SFV) adapted to bind heparan sulfate (HS) upon pre-treatment of cells prior to infection [116]. The LF-mediated inhibition was thought to occur by binding of LF to heparan sulfate moieties, thereby preventing virus–receptor interactions [116]. Bovine LF was tested against JEV and DENV, and was capable of inhibiting replication of both viruses. In JEV, similar to alphaviruses, HS-adapted JEV strains were inhibited by bovine LF, while wild-type strains were not [117]. Bovine LF inhibited binding of DENV to HS by directly interacting with HS. Additionally, morbidity of DENV-infected suckling mice was reduced upon administration of a mixture of DENV and LF as compared to DENV-only-infected mice [118].

#### **5. Human Antimicrobial Proteins–Eosinophil Proteins**

#### *5.1. Expression*

Eosinophils contain large cytoplasmic granules that play a critical role in innate immune responses. These granules are storage hotspots for major cationic antimicrobial proteins including eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Both EDN and ECP are human antimicrobial proteins that are active ribonucleases and members of the human RNase A superfamily [119,120]. The antimicrobial properties of RNases were mapped to the N-terminal domain that is conserved among ribonucleases [3]. While both proteins contain characteristic RNase A superfamily structures and catalytic residues, they exhibit antimicrobial activity against both bacteria and viruses (Table 1).

#### *5.2. Respiratory Syncytial Virus*

Eosinophil-derived neurotoxin (EDN), also known as RNase 2, has demonstrated inhibitory activity against RSV in several studies. In one study, RSV infectivity decreased in a dose-dependent manner when eosinophils were introduced to a suspension of RSV [121]. This property was abolished upon addition of a ribonuclease inhibitor suggesting that an RNase was responsible for the antiviral activity. At 50 nM, a plasmid-constructed recombinant human EDN exhibited a 40-fold reduction in RSV infectivity [121]. A decrease in RSV genomic RNA copies as quantified by RT-PCR upon incubation of EDN with virions suggests EDN ribonuclease activity directly targets extracellular viral particles. EDN may contain unique features that help mediate its antiviral activity as ribonuclease A alone has no effect on RSV infectivity.

Additionally, ECP, which is also known as RNase 3, has been detected in infants with acute bronchiolitis following RSV infection [122]. ECP levels were significantly higher in infants who developed persistent wheezing compared to those that do not after a five-year follow-up [122]. Additionally, while ECP was able to reduce RSV infectivity in the same suspension as EDN, ECP is less effective when used to inhibit RSV alone, demonstrating only a six-fold reduction as compared to EDN at a concentration of 50 nM [121]. Hence while ECP exhibits some inhibitory activity against RSV, it is not sufficient in controlling infection as RSV infection itself induces the production of ECP with no evident antiviral effects. Instead, the production of ECP in infants with bronchiolitis can be used as tool in prediction the risk of wheezing cough development in those infants as higher ECP levels correlate with wheezing cough development.
