Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus.

Recombinant Newcastle disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene, and was related to deamination of adenosines in the negative strand viral genome RNA. The biased hypermutation was found both after virus rescue in chicken cell line DF-1 followed by passaging in embryonated chicken eggs, and after direct virus rescue and subsequent passaging in Vero E6 cells. Levels of biased hypermutation were higher in constructs containing codon-optimized as compared to native S gene sequences, suggesting potential association with increased GC content. These data show that deep sequencing of candidate recombinant vector vaccine constructs in different phases of development is of crucial importance in the development of NDV-based vaccines.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. METHODS: To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. RESULTS: Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. CONCLUSIONS: Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence surrounding the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. Nevertheless, comparison of NDV strains that carry exactly the same F protein cleavage site shows that significant differences in virulence still exist. For instance, virulent field strain Herts/33 with the F cleavage site 112RRQRRF117 had an intracerebral pathogenicity index of 1.88 compared with 1.28 for strain NDFLtag, which has the same cleavage site. This implies that additional factors contribute to virulence. After generating an infectious clone of Herts/33 (FL-Herts), we were able to map the location of additional virulence factors by exchanging sequences between FL-Herts and NDFLtag. The results showed that, in addition to the F protein cleavage site, the haemagglutinin-neuraminidase (HN) protein also contributed to virulence. The effect of the HN protein on virulence was most prominent after intravenous inoculation. Interestingly, both the stem region and the globular head of the HN protein seem to be involved in determining virulence.

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.