Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription-polymerase chain reaction in chicken eggs

Abstract

Background: Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF) chicken eggs with Influenza virus (AIV) and Newcastle disease virus (NDV) demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for AIV and NDV to specifically detect the viral genomes in mixed infections.

Results: Data from this survey showed that when different doses of NDV (Lasota or F48E8) and AIV (F98 or H5N1) were simultaneously inoculated into embryonating chicken eggs (ECE), interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus.

Conclusion: AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Figure 1

The replication of NDV (Lasota of F48E8) in ECE after infection alone or as a mixture with AIV (F98 of H5N1) were measured by using the Real-time PCR. Supersript a represents an input multiplcitu pf 10³ EID₅₀ per chicken egg. Superscript b represents an input multiplicity of 10² EID₅₀ per chicken egg. Superscript c represents an input multiplicity of 10¹ EID₅₀ per chicken egg. Bars represents the standard deviations for two experiments carried out on three eggs. Statistical significance between the experimental group and control group was determined by the student’s t-test. *p <0.05, **p < 0.01,***p < 0.001.

Figure 2

The replications of NDV (Lasota of F48E8) in ECE after inoculated first with AIV (F98 of H5NI) were measured by using the Real-time PVR. Superscript 12 h represtnets 12 hours interval between two viruses. Superscripts 24 h represents 24 hours interval between two viruses. Bars represent the standard deviations for two experiments carried out on three eggs. Statistical significance between the experimental group and control group was determined by the student’s t-test. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

The replications of AIV (F98 or H5N1) in ECE after inoculated first with NDV (Lasota or F48E8) were measured by using the Real-time PCR. Supercript 12 h represtents 12 hours interval between two viruses. Superscript 24 hours interval between two viruses. Bars represents the standard deviations for two experiments carried out on three eggs. Statistical significance between the experimental group and control group was determined by the student’s t-test. *p<0.05, **p<0.01, ***p<0.001.