*4.4. Adenovirus*

Lactoferrin-mediated anti-HAdV activity occurs at multiple stages during infection. In one study LF inhibited HAdV replication during different phases of infection; when the peptide was introduced before infection, after viral adsorption, and when the peptide was present throughout the experiment, indicating more than one mechanism of action may be involved [87]. Bovine LF has also demonstrated anti-HAdV properties in a dose-dependent manner similar to human LF [87–89]. Bovine LF demonstrated the greatest inhibition when virus was pre-incubated with LF (1 mg/mL), which was validated by electron microscopy imaging the binding of LF to HAdV, thus suggesting direct LF inhibition as a mechanism of action [89], In contrast, human LF in tear fluids was implicated in promoting HAdV binding to epithelial cells, whereby HAdV hijacks human LF and utilizes the protein in order to bind to host cells [90].

#### *4.5. Herpes Simplex Viruses 1 and 2*

Studies assessing inhibition of HSV-1 have revealed the potency of LF at inhibiting replication of the viruses. LF (0.5–1 mg/mL) inhibited HSV-1 replication in human embryo lung cells and prevented virus adsorption and entry [91]. Marchetti et al. also demonstrated the ability of bovine and human LF to inhibit HSV-1 replication and adsorption in Vero cells independent of iron-binding [92,93]. Furthermore, intracellular trafficking of virions that gained entry was delayed [94]. Similar inhibitory properties were displayed by Lfcin when the peptide was tested against HSV-1 [94]. In addition, cell-to-cell viral spread was inhibited by LF as well as Lfcin [95]. The data presented by these studies demonstrate LF's antiviral properties against HSV.

## *4.6. Hepatitis C and B Virus*

Lactoferrin has demonstrated efficacy against HCV and HBV. Bovine LF can prevent HCV infection in human hepatocytes and in turn was tested as a measure to control HCV viremia in chronic hepatitis C patients [96]. However, LF only reduced HCV RNA in patients with previously low HCV RNA serum concentration. Conversely, a randomized trial of bovine LF in patients with chronic hepatitis C reported that orally-administered LF did not demonstrate significant efficacy against HCV when compared to a placebo control [97]. Combination therapy using the antiviral molecule ribavirin, LF, and interferon therapy, on the other hand, suggested that LF contributed to decreased HCV RNA titers [98]. These contradictory results suggest that while orally-administered LF may not be a promising stand-alone therapeutic, it can enhance the effectiveness of currently used therapeutics. Studies on the interaction of HCV and LF reported the direct interaction and binding of human and bovine LF with the HCV envelope proteins E1 and E2 [99]. A more recent study concluded that LFs of various species directly prevented HCV cell entry by binding to virions. Furthermore, pre-incubation of virions or post-treatment of infected cells with human, bovine, sheep, or camel LF inhibited HCV replication in human hepatoma (HepG2) cells, suggesting that LF may be used prophylactically as well as therapeutically, particularly in combined therapies [100].

Pre-incubation of HBV with LF had no effect on viral replication; however, pre-treatment of cells with human or bovine LF prevented infection [101]. Bovine LF did not interact with HBV but indirectly protected cells from HBV infection [102]. In addition, iron or zin-saturated LF on HBV-infected HepG2 cells inhibited HBV-DNA amplification suggesting its use as a possible candidate for the treatment of HBV infections [103].
