Molecular Changes of the Fusion Protein Gene of Chicken Embryo Fibroblast–Adapted Velogenic Newcastle Disease Virus: Effect on Its Pathogenicity

Abstract

Molecular changes of cell culture-adapted Newcastle disease virus (NDV) were studied by adapting a velogenic NDV isolated from commercial layer chicken-to-chicken embryo fibroblast (CEF) cells. The isolate was passaged 50 times in CEF cells. At every 10th passage the virus was characterized conventionally by mean death time analysis, intracerebral pathogenicity index, and virus titration. As the passage level increased, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription-polymerase chain reaction. Sequence analysis revealed a total of 134 nucleotide substitutions, which resulted in the change of 41 amino acids between the parent and the 50th passage virus. Pathogenicity studies conducted in 20-wk-old seronegative chickens revealed gross and histopathologic changes in the chickens injected with the parent virus and absence of the lesions in chickens injected with the adapted virus. The 50th passage cell culture virus was back-passaged five times in susceptible chickens and was subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor differences between them.

Isolation and Detection of Newcastle Disease Virus From Field Outbreaks in Broiler and Layer Chickens by Reverse Transcription–polymerase Chain Reaction

“…The failure of virus isolation from the blood samples at day 3 post-infection might be due to the lodgment of the virus in different tissues of the infected birds at day 3 of post infection. The findings of NDV isolation using CEF cell culture system has close agreement with the findings of Mohan et al (2005) and Takehara et al (1987). In their study, the isolation rate of NDV was higher in CEF culture system compared to that of avian embryo.…”

Isolation and Characterization of a Pathogenic Newcastle Disease Virus from a Natural Case in Indonesia

“…(Table 2), and is identical to the motif found in the virulent isolates from Indonesia, Vietnam, Taiwan, Germany and U.S.A. [7,23]. Changes in the F protein cleavage site motif have been shown to alter the virulence of NDVs and changing virulent clones to avirulent ones [14] and vice versa [6,24,27]. In particular, the LaSota strain, which is used as a vaccine in Indonesia, was shown to acquire virulence by a change in the F cleavage site motif [6,27].…”

Complete Genome Sequencing and Genetic Characterization of Alkhumra Hemorrhagic Fever Virus Isolated from Najran, Saudi Arabia

“…When compared to the AHFV NC004355 and AHFV JF 416949 strains, a total of 139-203 nucleotides were missing from the AHFV/997/NJ/09/SA strain primarily in the UTR, likely due to passage of the virus in cell culture. Previous studies reported such deletions and even insertions and/or duplications of nucleotides of viruses when passaged in cultures [16,17,18,19,20,21]. Several studies have shown that nucleotide deletion/insertion/duplication occur in the 3′ UTR (formerly known as a noncoding region or NCR) among various members of flaviviruses and even among strains of the same viral species [16,17], as well as in the 5′ UTR of hepatitis A virus [18] and the Newcastle disease virus genome [19].…”

“…Previous studies reported such deletions and even insertions and/or duplications of nucleotides of viruses when passaged in cultures [16,17,18,19,20,21]. Several studies have shown that nucleotide deletion/insertion/duplication occur in the 3′ UTR (formerly known as a noncoding region or NCR) among various members of flaviviruses and even among strains of the same viral species [16,17], as well as in the 5′ UTR of hepatitis A virus [18] and the Newcastle disease virus genome [19]. In another study it was observed that size variation between yellow fever virus strains was due to duplications and/or deletions of repeated nucleotide sequence elements that occurred in the 3′ UTR by passaging the virus in cell culture [20].…”