Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-;, interleukin 2 (IL-2), or tumor necrosis factor A, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment. [Cancer Res 2007;67(17):8285-92]

Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest that Ban/AF can provide better protection than commercial vaccines and is a promising vaccine candidate against NDV strains circulating in Indonesia.

Complete genome sequences were determined for two strains of avian paramyxovirus serotype 6 (APMV-6): the prototype Hong Kong (HK) strain and a more recent isolate from Italy (IT4524-2). The genome length of strain HK is 16236 nucleotide (nt), which is the same as for the other two APMV-6 strains (FE and TW) that have been reported to date, whereas that of strain IT4524-2 is 16230 nt. The length difference in strain IT4524-2 is due to a 6-nt deletion in the downstream untranslated region of the F gene. All of these viruses follow the “rule of six”. Each genome consists of seven genes in the order of 3’N-P-M-F-SH-HN-L5’, which differs from other APMV serotypes in containing an additional gene encoding the small hydrophobic (SH) protein. Sequence comparisons revealed that strain IT4524-2 shares an unexpectedly low level of genome nt sequence identity (70%) and aggregate predicted amino acid (aa) sequence identity (79%) with other three strains, which in contrast are more closely related to each other with nt sequence 94–98% nt identity and 90–100% aggregate aa identity. Sequence analysis of the F-SH-HN genome region of two other recent Italian isolates showed that they fall in the HK/FE/TW group. The predicted signal peptide of IT4524-2 F protein lacks the N-terminal first 10 aa that are present in the other five strains. Also, the F protein cleavage site of strain IT4524-2, REPR↓L, has two dibasic aa (arginine, R) compared to the monobasic F protein cleavage site of PEPR↓L in the other strains. Reciprocal cross-hemagglutination inhibition (HI) assays using post infection chicken sera indicated that strain IT4524-2 is antigenically related to the other APMV-6 strains, but with 4- to 8-fold lower HI tiers for the test sera between strain IT4524-2 and the other APMV-6 strains. Taken together, our results indicated that the APMV-6 strains represents a single serotype with two subgroups that differ substantially based on nt and aa sequences and can be distinguished by HI assay.