Using reverse genetics, we generated a recombinant low-pathogenic LaSota NDV that expresses GP of EBOV, designated rLa-EBOVGP, and evaluated its biological characteristics in vivo and in vitro. The introduction and expression of the EBOV GP gene did not increase the virulence of the NDV vector in poultry or mice, which is consistent with the results of a previous study in monkeys . EBOV GP was incorporated into the viral particles of rL-EBOVGP and allowed the NDV vector to infect mammalian cells independently of exogenous trypsin.
The restriction of NDV replication in mammalian host cells is one of the most attractive properties of lentogenic NDV in terms of its safety when used as a live vaccine vector in animals and humans, as is also the case for fowlpox virus [54, 55] and a modified vaccinia virus Ankara [56, 57]. The V protein encoded by the NDV P gene functions as an interferon antagonist and is usually less efficient in mammalian cells [58–60]; NDV is usually a strong inducer of the interferon response in mammalian cells and is highly sensitive to the interferon induced in these cells [61, 62]. The limited replication in mammalian cells of low-pathogenic NDV, like the LaSota strain, is also determined by its trypsin-dependent infectivity. The trypsin-independent infectivity acquired by rLa-EBOVGP means that this virus behaves like a velogenic NDV in mammalian host cells. Its restricted replication may have to depend on the only defense line in host, the native immunity.
The ability of foreign envelope proteins to function as new cell-entry proteins has also been demonstrated in other enveloped negative-strand RNA viruses . The function of EBOV GP in mediating the cell entry of VSV when the G gene had been deleted from the VSV genome could be compensated by several foreign envelope glycoproteins from different viruses in trans or with recombinant expression, including Ebola virus, Marburg virus, Lassa fever virus, Hantaan virus, and Nipah virus [4, 6, 7, 64–67]. Therefore, it is reasonable to infer that the incorporation of EBOV GP into the viral particle may cause the trypsin-independent infectivity of rL-EBOVGP.
So far, very few studies have examined the biological functions of native and foreign envelope glycoproteins when they are incorporated into the same viral particle. Our previous study showed that the G proteins of the rabies virus were incorporated on the surface of a recombinant NDV LaSota particle. An anti-rabies virus antiserum did not reduce the infectivity of the recombinant NDV . The G proteins on the virion surface of the rabies virus did not mediate the infection of cells by the recombinant NDV particle . The reasons for these phenomena are unclear. Another study showed that EBOV GP was incorporated into the viral particle of recombinant human parainfluenza virus 3 (hPIV3) and that the recombinant hPIV3 became more sensitive to neutralizing antibody directed against EBOV than to neutralizing antibody directed against hPIV3 . Because the biological functions of the hPIV3 envelope glycoproteins could not be abolished, it was difficult to clarify whether EBOV GP on the virion surface functioned independently to mediate the infection of cells by the recombinant hPIV3 particle. In the present study, the recombinant NDV rLa-EBOVGP-cell was prepared in BHK-21 cells in the absence of trypsin, so the membrane fusion function of the F protein, essential for viral entry, was completely abolished. Therefore, we can confidently conclude that the EBOV GP that was incorporated into the viral particle independently mediated the cell entry of the recombinant NDV.
As a prototype member of the paramyxoviruses, NDV usually enters host cells by direct fusion at the plasma membrane via a pH-independent mechanism [68–70]. However, NDV can also enter host cells by an endocytic pathway . It had been shown that the cellular entry of EBOV involves a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes [46, 47, 71]. Recently, Niemann-Pick C1, a protein involved in the endocytic pathways, has been identified as an important host factor in the cell-entry process [72, 73]. In this study, the neutralization assay showed that the mixture of anti-NDV antiserum and anti-EBOV GP antiserum completely blocked the infectivity of rLa-EBOVGP, whereas either anti-NDV antiserum or anti-EBOV GP antiserum only partially blocked the infectivity of this virus. Therefore, we infer that both the NDV envelope proteins F/HN and EBOV GP contribute to the cell entry of rLa-EBOVGP. It is surprising that the inhibition of macropinocytosis almost completely abolished the infectivity of rLa-EBOVGP, whereas it did not markedly reduce the infectivity of rLa. These results indicate that the direct fusion between rLa-EBOVGP and the plasma membrane facilitated by F/HN may not allow viral entry into the cell. EBOV GP plays a major role in the cell-entry process of rLa-EBOVGP, and may predominate over NDV F/HN in the cell-entry function of this recombinant virus. The exact mechanism underlying this predominance requires further investigation. The recombinant virus rLa-EBOVGP provides an interesting model with which to explore the functional interactions between native and foreign envelope glycoproteins in one viral particle.
Although animal tests have shown that recombinant NDV expressing EBOV GP is safe for monkeys  and for poultry and mice (this study), safety concerns remain. Because the incorporation of EBOV GP protein into NDV particles significantly alters the behavior of the vector virus, the use of an NDV-vectored EBOV vaccine should be investigated with caution and evaluated rigorously.