In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors.

Newcastle disease is an economically important disease of poultry for which vaccination is applied as a preventive measure in many countries. Nevertheless, outbreaks have been reported in vaccinated populations. This suggests that either the vaccination coverage level is too low or that vaccination does not provide perfect immunity, allowing the virus to spread in partially vaccinated populations. Here we study the requirements of an epidemiologically effective vaccination program against Newcastle disease in poultry, based on data from experimental transmission studies. The transmission studies indicate that vaccinated birds with low or undetectable antibody titres may be protected against disease and mortality but that infection and transmission may still occur. In fact, our quantitative analyses show that Newcastle disease virus is highly transmissible in poultry with low antibody titres. As a consequence, herd immunity can only be achieved if a high proportion of birds (>85%) have a high antibody titre (log(2) haemagglutination inhibition titre > or =3) after vaccination. We discuss the implications for the control of Newcastle disease in poultry by vaccination.

Phylogenetic studies have largely contributed to better understand the emergence, spread and evolution of highly pathogenic avian influenza during epidemics, but sampling of genetic data has never been detailed enough to allow mapping of the spatiotemporal spread of avian influenza viruses during a single epidemic. Here, we present genetic data of H7N7 viruses produced from 72% of the poultry farms infected during the 2003 epidemic in the Netherlands. We use phylogenetic analyses to unravel the pathways of virus transmission between farms and between infected areas. In addition, we investigated the evolutionary processes shaping viral genetic diversity, and assess how they could have affected our phylogenetic analyses. Our results show that the H7N7 virus was characterized by a high level of genetic diversity driven mainly by a high neutral substitution rate, purifying selection and limited positive selection. We also identified potential reassortment in the three genes that we have tested, but they had only a limited effect on the resolution of the inter-farm transmission network. Clonal sequencing analyses performed on six farm samples showed that at least one farm sample presented very complex virus diversity and was probably at the origin of chronological anomalies in the transmission network. However, most virus sequences could be grouped within clearly defined and chronologically sound clusters of infection and some likely transmission events between farms located 0.8–13 Km apart were identified. In addition, three farms were found as most likely source of virus introduction in distantly located new areas. These long distance transmission events were likely facilitated by human-mediated transport, underlining the need for strict enforcement of biosafety measures during outbreaks. This study shows that in-depth genetic analysis of virus outbreaks at multiple scales can provide critical information on virus transmission dynamics and can be used to increase our capacity to efficiently control epidemics. Outbreaks of highly pathogenic avian influenza (HPAI) viruses have affected poultry worldwide in the last decades, resulting in vast socioeconomic damages and many human infections. It is important to determine the route of transmission between poultry farms to be able to implement efficient control measures. Here, we investigate possible use of sequence data to unravel the route of virus transmission during an HPAI H7N7 epidemic that took place in 2003 in the Netherlands. We obtained virus sequence data from most of the outbreaks during the epidemic, and found a high level of genetic diversity driven by a rapid evolutionary rate of HPAI H7N7 virus. The phylogenetic inference of the inter-farm transmission network turned out to be difficult due to the presence of potential reassortant virus strains, multiple mutations at highly variable sites and within farm virus diversity. However, most virus samples could be grouped within clearly defined and chronologically sound clusters of infection, giving us valuable insights on the diffusion of the virus during the outbreak. We discuss the implications of the results obtained for the evolutionary and epidemiological dynamics of avian influenza viruses and disease control.

Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.

Reassortments in HPAI A(H5N1) viruses, Indonesia

The tissue tropism of non-virulent and virulent Newcastle disease virus (NDV) was investigated using 8-day-old and 14-day-old embryonating chicken eggs (ECE), inoculated with an infectious clone of the non-virulent La Sota strain (NDFL-GFP) or its virulent derivative (NDFLtag-GFP). Both strains expressed the gene encoding jellyfish green fluorescence protein (GFP) as a marker. The GFP was readily expressed in chicken embryo cells infected with the NDV strains indicating virus replication. Whereas both strains replicated in the chorioallantoic membrane (CAM) and infected the skin of 8-day-old ECE, only the virulent strain (NDFLtag-GFP) spread to internal organs (pleura/peritoneum). In 14-day-old ECE, the initial target organs appeared to be the CAM and the lungs for both strains. At 48 h after inoculation, the virulent strain (NDFLtag-GFP) had also spread to the spleen and heart and was detected in a wide-range of embryonic cell types. The kinetics of virus replication and spread in the CAM closely resembled each other in both the 8-day-old and 14-day-old ECE. Infection of 8-day-old and 14-day-old ECE forms a convenient model to investigate tissue tropism of NDV, as well as the kinetics of viral infection. The advantage of using GFP is that samples can be easily screened by direct fluorescence microscopy without any pre-treatment.