Newcastle disease (ND) is a highly contagious and fatal disease of chickens leading to huge economic losses in the poultry industry worldwide [1]. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae [1, 2]. NDV isolates display a spectrum of virulence in chickens, ranging from inapparent infection to 100% mortality. Based on their pathogenicity in chickens, NDV isolates are categorized into three main pathotypes, lentogenic (low virulence), mesogenic (intermediate virulence), and velogenic (high virulence) [1]. The velogenic strains cause severe outbreaks in poultry. Based on the intracloacal inoculation test, velogenic strains are further classified into viscerotropic, which induce mortality with hemorrhagic lesions in the intestinal tract and neurotropic, when nervous signs predominate without hemorrhagic lesions in the intestine [1, 3, 4].

NDV strain Texas GB is considered as the prototype neurotropic velogenic strain and is widely used as the standard challenge virus in vaccine efficacy studies in the U.S. Though, strain Texas GB is biologically one of the well characterized NDV strains to date [3–8], the complete genome sequence of this strain has not been reported. Only partial or full sequences of few genes dating from 1988 to 2004 are available [9–15]. The complete sequences of phosphoprotein and large polymerase genes, the intergenic sequences between N/P, P/M, HN/L genes, and the trailer sequence are not available. Since the current sequences of strain Texas GB were determined by different groups at different times, it is necessary to determine the complete genome sequence of strain Texas GB to understand its pathogenicity and neurovirulence. In this study, we report the complete genome sequence of the NDV strain Texas GB (APMV-1/chicken/U.S.(TX)/GB/1948).

The NDV strain Texas GB was isolated from a fatal ND outbreak in chicken with severe neurological involvement, in Texas, U.S.A., in 1948 [3, 4]. We received this strain from National Veterinary Services Laboratory, Ames, Iowa, U.S.A. We passaged the virus three times in 9-day-old specific pathogen free (SPF) embryonated chicken eggs. The pathogenicity of NDV strain Texas GB was determined by mean death time (MDT) in 9-day-old embryonated SPF chicken eggs and by intracerebral pathogenicity index (ICPI) in 1-day-old SPF chicks. The MDT and ICPI values of strain Texas GB were 50 h and 1.925, respectively, consistent with a velogenic NDV strain. The work was performed in our USDA approved enhanced biosafety level 3 (BSL-3+) facility.

To determine the complete genome sequence of NDV strain Texas GB, the virus was grown in the allantoic cavities of 9-day-old embryonated SPF chicken eggs. Plaque purification of the virus was not performed to avoid the likely chance of picking up a minor variant that may not represent the actual viral genome. The viral genomic RNA was extracted from the infective allantoic fluid using RNeasy mini kit (Invitrogen). Primer sets were designed from the available sequences of strain Texas GB (accession numbers, AF144730, AF138899, AY505057 and U22293) and from the complete genome sequence of BC [16]. 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 3′-leader and 5′-trailer sequences of the strain Texas GB were determined using 3′-rapid amplification of cDNA ends (3′-RACE) and the 5′-RACE, respectively [17, 18]. 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 genome was sequenced at least four times from four independent RNA preparations to ensure a consensus sequence.

The genome of NDV strain Texas GB is 15,186 nt (GenBank accession number GU978777), a length that is present in most NDV strains. There are two other genome size classes reported among strains of NDV, which are 15,192 nt and 15, 198 nt [19–22]. The longer genome sequences are due to the presence of 6 and 12 additional nt in the 3′ untranslated region (3′-UTR) of N gene and in the open reading frame (ORF) of P gene, respectively (Fig. 1b). Similar to other NDV strains, the genome of strain Texas GB consists of six genes encoding six different proteins in the order of a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), an attachment protein called the hemagglutinin-neuraminidase (HN), and a large polymerase protein (L) (3′leader-N-P-M-F-HN-L-5′trailer). The length, position, and characteristics of the six genes and their intergenic sequences (IGS) are summarized in Table 1, and the comparison of its proteins with other NDV strains in Table 2.

Fig. 1
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a Comparison of nucleotide (nt) sequences of gene-end of N and L genes and IGS between N/P genes (genome sense), b gene map of NDV strains Texas GB, BC, Fontana and Alaska 415 (numbers on the top of gene map of strain Texas GB indicate nt sequence lengths of leader, intergenic sequences (IGS) between N/P, P/M, M/F, F/HN, HN/L genes and trailer, respectively; numbers at the bottom of the gene map of Alaska 415 indicate putative nt length differences in N genes, IGS between N/P, P genes, L genes and trailer sequences of the four NDV strains described; numbers given in the parenthesis at the end of each gene map reflects total genome length of each NDV strain; numberswithin the N, P, L gene-boxes of genomes BC, Fontana and Alaska 415 indicate putative additions in length compared to N, P and L genes of strain Texas GB), c nt sequence complimentarity between 3′-leader and 5′-trailer (genome sense) sequences of strain Texas GB (stars indicate base pairs) and d phylogenetic tree of NDV strain Texas GB with 78 other available genome sequences of NDV strains was constructed using maximum parsimony method with bootstrap values calculated for 1000 replicates (strain Texas GB is indicated by a soliddiamond)

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Table 1 Genomic features and protein characteristics of NDV strain Texas GB
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Table 2 Percent nucleotide sequence identity of the complete genome sequence and percent amino acid sequence identity of different proteins of strain Texas GB with other NDV strains
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The 3′-leader sequence of strain Texas GB consists of 55 nt, a length present in all NDV strains [5]. The leader sequence of strain Texas GB showed 100% identity with the leader sequence of strain BC, but there are 2, 3, 7, 8, 7, and 6 nt differences between the leader sequence of strain Texas GB and leader sequences of strains LaSota, B1, Anhinga, Fontana, Largo and Alaska 415, respectively. Comparison of the sequences of NDV strains Texas GB with those of NDV strains BC and LaSota showed differences in the stretch of 6 or 7 Uracil residues in the gene-ends of N and L genes and that alters the putative IGS between N/P genes to 1 or 2 residues and the putative trailer sequence to 113 or 114 nt, respectively. This leads to the putative length of N gene to 1,746 or 1,747 nt (in case of genomes with 15,186 nt) or 1,753 nt (in case of genomes with 15,192 nt) and the putative length of L gene to 6,703 or 6,704 nt (Fig. 1a, b). The putative length of P gene consists 1,451 (in genome lengths 15,186 and 15, 192 nt) or 1,463 nt (in genome length of 15,198 nt). The P gene of strain Texas GB contains a putative editing site 2280UUUUUCCC2287 (genome sense) that is identical in position to other NDV strains [2]. The 5′-trailer sequence of the strain Texas GB is 114 nt long and showed 5 nt differences from the trailer sequence of strain BC.

All the six major proteins of strain Texas GB invariably showed higher amino acid sequence identity with the cognate proteins of other NDV strains that are well characterized (Table 2). The comparison of complete genome sequences showed that strain Texas GB is more related to strain BC (99.1% nt identity) than to other NDV strains (Table 2; Fig. 1d). Comparison of the amino acid (aa) sequences between strains Texas GB and BC showed differences in all six proteins; one aa in N (K421E), four aa in P (P30S, K36R, A98T V104A and D336E), four aa in M (M132V, D152N, P216S and A336T), eight aa in F (S10P, A11V, S265G, E304G, T457I, T510I, A520V and A550T), 11 aa in HN (Q7R, V9A, A34V, M35V, I191V, N228S, V271A, S310G, E332G, L454P and A571V), and 14 aa in L (T75A, I89V, N265D, C698R, K889N, F1379Y, D1531N, N1643S, R1668K, F1706L, L1734P, S1758A, M1785I and I2067V). There was no aa difference between the unique C-terminal regions of V and W proteins of strains Texas GB and BC.

The F protein cleavage site is a well-characterized determinant of NDV pathogenicity in chickens [23, 24]. Virulent NDV strains typically contain a polybasic cleavage site (R-X-K/R-R↓F), which is recognized by intracellular proteases of most cells. The cleavage site of strain Texas GB is 112 R-R-Q-K-R↓F117, which contained four basic amino acids at positions 112–116, corresponding to those of virulent NDV strains [1, 2].

Phylogenetic tree analysis of complete genome sequence of strain Texas GB with 78 full length genomes (or genome sequences >15 kb) of other NDV strains was performed by maximum parsimony method using MEGA 4 software (Molecular Evolutionary Genetics Analysis) [25, 26]. Based on the available genotype classification system for NDV, strain Texas GB is classified under genotype II of class II viruses. Phylogenetic clustering of strain Texas GB with other genotype II strains including BC, LaSota, B1, VG/GA, was evident (Fig. 1d). It was also observed that strain Texas GB is more closely related to strain BC and viruses isolated from China [27] and Egypt [28] than to other NDV strains. The recently sequenced NDV isolates from a northern pintail, a mottled duck, and mallards in the U.S. [29] showed phylogenetic relatedness to strain Texas GB at the genome level than the virulent and avirulent NDV isolates from cormorants [29] (Fig. 1d). The high degree of nt identity and phylogenetic relatedness of velogenic strain Texas GB with a mesogenic strain BC suggest that subtle differences at the genome level may have a profound effect on the pathogenicity of a NDV strain. Availability of the complete genome sequence of strain Texas GB will be useful in further understanding of the NDV pathogenesis and neurovirulence.