Interference of AIV (H5N1 and F98) with NDV (Lasota and F48E8), and vice versa, was studied in the ECE system for simultaneous and relatively short intervals between inoculations. In this system, a number of variables have been measured, e.g. the effects of varying the dose of interfering and challenge virus or the time interval between the two inoculations and the use of different virus strains. We report here for the first time an evaluation of the interference between AIV and NDV in terms of viral replication using real-time RT-PCR. The results of the experiment clearly show that AIV inhibited the growth of NDV, and that the degree of interference depended upon the quantity and relative virulence of the virus strains used. The studies also indicate that NDV can’t suppress the growth of AIV even when the NDV was given a growth advantage in the ECE by being inoculated at a higher EID50 than AIV. With the virus doses and intervals between inoculations described, significant reduction of the genome titer of the AIV only occurred when NDV was inoculated prior to AIV. However, although infection of ECE by AIV uniformly interfered with subsequent infection by NDV, a primary infection by NDV was not always able to prevent later infection by AIV. For instant, even when Lasota was inoculated 24 hours prior to H5N1, there was still no interference on H5N1 (Figure 3c). Therefore, it seems clear that Lasota did not induce interference against H5N1 multiplication either with simultaneous or preoculation with Lasota.
Comparison of the results obtained from this study pointed to a similar qualitative conclusion with other studies, viz. that AIV was found to be a far more powerful agent than NDV in causing interference between these viruses. Additionally, the virulence of the virus strains was another important factor that affects interference in the reciprocal interference. In other words, the virulent strain of NDV is more puissant than avirulent strain in resistanting interference induced by AIV. It can be explained by the theory that the NDV replication is directly associated with the virulence of virus . The above findings in general agree with those reported by Shortridge and King, who explored co-cultivation of AIV and NDV in ECE , and also by Wenbo Liu et al., who studied H9-NDV interference in chicken embryo . However, a controversial finding was reported by Zowalaty et al that in the presence of mixed infection with NDV and AIV in cloacal swabs, NDV could be recovered from ECE but not AIV . This different discovery may be due to the presence of inactivated AIV or large quantitative difference between AIV and NDV in cloacal swabs. In our study, the NDV was added into ECE at an input multiplicity of 1000 EID50 per chicken egg, whereas the AIV was used at a multiplicity of 1000,100 and 10 EID50 per chicken egg. If the AIV was used at a multiplicity of 1 EID50 or less, we may find the similar result as describe by Zowalaty.
Interference could have taken place either on or within the available cells. Different mechanisms have been proposed to explain viral interference. These have been classified broadly into two categories: (i) attachment interference, and (ii) intracellular interference. The former is mediated through blockade or destruction of available receptor sites for the superinfecting virus. The latter involves virus-induced interferon interference, or competition for replication sites or essential factors of viral replication, or formation of defective interfering (DI) particles, etc. .
Virologist’s enormous interest in the observation of viral interference phenomena led to the discovery of interferon. In the early 1940s, Henle & Henle  and Ziegler & Horsfall  discovered that an inactivated influenza virus particle was capable of interfering with the multiplication of live virus added later. This finding opens the door to study viral interference mediated by the interferon system. Subsequent intensive research proved that interferon production was a common event and it inhibits the growth of many viruses, certainly including AIV and NDV . Other experiments additionally found that in order to establish interferon-mediated interference, several hours was required to create interferon . AIV and NDV are capable interferon inducers and their ability to induce the formation of interferon is closely related with their virulence . The basic characteristics of interferon-mediated interference mentioned above agree with the observation of experiment 2 in this study. That is, the pre-inoculated virus always inhibited the growth of superinfection virus. However, in experiment 2, an exception occurred in the Lasota inoculation prior to inoculation with H5N1. This is probably due to the fact that Lasota is a weak interferon-inducer. 24 hours is not sufficient time for Lasota to produce inhibition-level interferon to suppress the growth of H5N1. This fits precisely with one of the properties of interferon, viz that the time of induction of maximum interferon depends on the virulence of the strain . However, for experiment 1 in this research, when AIV and NDV inoculations were made at the same time, interference always occurred in NDV. This phenomenon is not easily explained by interferon-mediated interference because, according to the literature, in order to establish interferon-mediated interference, several hours are required to establish interference . That is also the reason why most virus interference phenomena were not interpreted by physical blockade of receptors.
A simple but likely explanation for the interference between AIV and NDV in experiment 1 could be the competition for the same receptor. It has been clearly established that the cell surface receptor for AIV is sialic acid-containing glycoconjugates , whereas the cellular receptors for Newcastle disease virus have been proposed as Gangliosides and N-glycoproteins, both of which contain sialic acid . These findings imply the existence of a common receptor site on permissive host cells shared by AIV and NDV , and raise the possibility that when these two viruses are inoculated into the same egg, they could compete for shared virus receptor-sialic acid which is essential for virus adherence. Homologous and heterologous viral interference induced by blocking or destruction of viral receptors among NDV or AIV has long been described [31, 32]. Therefore, theoretically, when AIV and NDV were simultaneously inoculated into the ECE, there was obvious interference, probably due to direct competition for the same viral receptors on the cell surfaces.
In addition, AIV and NDV are both (−)sense RNA viruses, grouped under the term myxovirus, due largely to their property to adsorb onto the erythrocytes cell-surface receptors of fowl, causing their agglutination, a striking biologic property common to all of myxovirus . Also to be considered is that AIV and NDV do exhibit certain similarities. In aves, the clinical symptoms of highly pathogenic avian influenza A (HPAI) are very similar to those of a severe attack of virulent NDV strain.