Sequence analysis of fusion protein gene of Newcastle disease virus isolated from outbreaks in Egypt during 2006
© Mohamed et al; licensee BioMed Central Ltd. 2011
Received: 8 April 2011
Accepted: 18 May 2011
Published: 18 May 2011
Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. The F protein cleavage site sequence is a well-characterized determinant of NDV pathogenicity in chickens. In this study, the sequences of fusion protein (F) gene of three Newcastle disease virus (NDV) strains isolated from outbreak in chickens in the Al-Sharkia province of Egypt in 2006 were determined.
The viral genomic RNAs were extracted from the infective allantoic fluid and F gene is amplified using primer sets designed from the available sequences of NDV strains from GenBank. The pathogenicity of NDV strains was determined by three internationally recognized tests mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index. The phylogenetic analysis showed that the Egypt isolates are closely related with the genotype II of class II NDV strains.
The sequences of the F genes of the 2006 Egypt isolates are closely related to that of the 2005 Egypt isolate from the same province suggesting that these strains are probably circulating in the vaccinated bird population in Egypt until development of an outbreak.
Newcastle disease (ND) is a highly contagious and fatal disease of chickens. In many developing countries ND is endemic and the disease has the greatest impact on villages where the livelihood of people depends on poultry farming. The causative agent Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. NDV isolates display a spectrum of virulence in chickens ranging from inapparent to fatal infection. Based on their pathogenicity in chickens, NDV isolates are categorized into three main pathotypes: lentogenic (low virulence), mesogenic (intermediate virulence) and velogenic (high virulence). It may appear that continents having warm climates are reservoirs of virulent NDV strains . NDV is a well-studied paramyxovirus and complete genome sequences of many North American NDV strains are available; however, very little is known about the genome sequences of NDV strains isolated from different parts of Africa with in isolated cases [2–4].
Pathogenicity index tests of Egyptian strains of NDV.
2.1 ± 0.24
1.6 ± 0.15
60 ± 1.5
2.2 ± 0.29
1.8 ± 0.19
50 ± 1.8
2.25 ± 0.23
1.7 ± 0.18
55 ± 2.3
102 ± 2.5
The F gene was chosen for sequencing because this gene is a major determinant of virulence and NDV isolates are grouped into genotypes based on the sequences of this gene [7–9]. To determine the F gene sequence of all the Egyptian strains, the viruses were grown in the allantoic cavities of 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Plaque purification of the viruses was not performed to avoid the genetic selection, which may not represent the actual viral genome. The viral genomic RNAs were extracted from the infective allantoic fluid using RNeasy mini kit (QIAGEN). Primer sets were designed from the available sequences of NDV strains Beaudette C (accession numbers X04719) and Texas GB (accession numbers GU978777). Three primers namely F-1for 5' ACGGGTAGAAGATTCTG 3', F-655for 5'GTTGACTAAGTTAGGTG 3'and F-1780rev 5' CTCTCCGAATTGACAGAC 3' (number corresponds to NDV F gene nucleotide sequence) were used to sequence the entire F gene. Reverse transcription and PCR were done using virus specific primers by Superscript-II reverse transcriptase and high fidelity Platinum Pfx polymerase (both from Invitrogen), respectively. The PCR-amplified products were directly sequenced using BigDye terminator v 3.1 matrix standard kit and 3130xl genetic analyzer data collection software v3.0 (Applied Biosystems Inc). The entire F gene was sequenced at least three times from independent RNA preparations to ensure a consensus sequence. All experiments were carried out in an enhanced BSL3 containment facility certified by the USDA to work with highly virulent NDV strains with the investigators wearing appropriate protective equipment and compliant with all protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland and under Animal Welfare Association (AWA) regulations.
Percent amino acid sequence identity of the Egyptian strains with other strains of NDV.
Our results indicate that high levels of nucleotide and amino acid sequence identity exist between the African and North American NDV strains. It is possible that NDV strains isolated from Egypt may represent the strains circulating in the poultry population in that region. These results also suggest that more than one genotype of NDV is circulating in the African subcontinents. However, determination of the complete genome sequences of Egyptian strains isolated in different geographic regions of Africa is necessary to understand the genetic relatedness among NDV strains circulating in different parts of the world.
The close genetic relatedness of the NDV strains isolated in 2005 and 2006 in the same province in Egypt suggest that these strains are endemic in the bird population in this province. Furthermore, these results suggest that NDV vaccine used in this province may not be very effective in stopping viral shedding, which will allow unnoticed circulation of the virulent virus in the vaccinated bird population until development of an outbreak. Therefore, it may be necessary to evaluate the effectiveness of the current vaccine used in the Al-Sharkia province against circulating NDV strains.
We thank Daniel Rockemann and all our laboratory members for their excellent technical assistance.
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