Virus Genes (2014) 49:497–501 DOI 10.1007/s11262-014-1109-1
Full genome sequence of a peste des petits ruminants virus (PPRV) from Ghana W. G. Dundon • C. Adombi • A. Waqas • H. R. Otsyina • C. T. Arthur • R. Silber • A. Loitsch • A. Diallo
Received: 2 July 2014 / Accepted: 14 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The full genome of a peste des petits ruminants virus (PPRV) isolated from a sheep lung sample collected in Ghana, Western Africa, in 2010, has been sequenced. Phylogenetic analysis demonstrated that the virus clustered within the lineage II clade while comparison of its full genome with those of other PPRV strains revealed the highest identity (96.6 %) at a nucleotide level with the PPRV strain Nigeria/76/1. This is the first full genome sequence generated for a PPRV lineage II isolated since 1976. Keywords Peste des petits ruminants Ghana Full genome
Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1109-1) contains supplementary material, which is available to authorized users. W. G. Dundon C. Adombi A. Waqas A. Diallo Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria W. G. Dundon (&) APHL Joint FAO/IAEA Division, IAEA Laboratories, 2444 Seibersdorf, Austria e-mail: [email protected] H. R. Otsyina School of Veterinary Medicine, College of Agriculture and Consumer Sciences, University of Ghana, Legon, Ghana C. T. Arthur CSIR-Animal Research Institute, P.O. Box AH 20, Achimota, Accra, Ghana R. Silber A. Loitsch Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety, Moedling, Austria
Peste des petits ruminants (PPR) is a highly infectious transboundary viral animal disease that affects mainly sheep, goats and small wild ruminants. With morbidity and mortality rates that can be as high as 70–80 %, PPR is classified within the group of animal diseases that are notifiable to the OIE (World Organization for Animal Health). Sheep and particularly goats contribute considerably to the cash income and nutrition of small farmers in many countries so the control of a disease such as PPR is considered an essential element in the fight for global food security and poverty alleviation [1]. The causative agent of PPR, the peste des petits ruminants virus (PPRV), is a member of the genus Morbillivirus within the family Paramyxoviridae [1]. It is a non-segmented, negative, single-stranded RNA virus that encodes eight proteins. These are the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein, (F), haemagglutinin protein (H), polymerase (L) and two nonstructural proteins (V and C). PPRV strains have been classified into four genetic lineages based on the comparison of a sequence fragment for the N (351 bp) and/or F (370 bp) genes [2, 3]. To date Lineage I and II isolates have been confined to Western Africa (e.g. Mali, Senegal, Burkina Faso, Guinea, Cote d’Ivoire and Nigeria), while Lineage III has been detected in Eastern Africa (e.g. Ethiopia, Uganda, Tanzania and Kenya) Yemen and Oman [1, 4]. Lineage IV is presently the predominant PPRV lineage globally. Viruses of this lineage were first detected in Asia, Turkey and the Middle East and for this reason it has been referred to as the Asian lineage. Several reports have now indicated the spread of lineage IV viruses within the African continent [1, 4]. To date, only eight full genome sequences are available in public databases representing three of the four lineages [5–9]. It is evident that more, full genome sequence data need to be generated in
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order to completely understand the evolution, pathogenesis and transboundary movement of this important virus. The samples analysed in this study were collected in Ghana between September 2009 and March 2010 and sent on dry ice to the Austrian High Security Laboratory, Vienna, for processing. They included ocular (n = 7), nasal (n = 10) and buccal (n = 6) swabs from 6 animals and 12 tissue samples from four other animals [lung (n = 4), lymph node (n = 4) and spleen (n = 4)]. They were collected from sheep and goats suspected of being infected with the PPRV as they were manifesting the following symptoms: fever, ocular and nasal discharges, diarrhoea and erosive lesions in the buccal mucous membrane. Upon arrival in Vienna, the tissue samples were ground with sterile quartz beads to make a 10 % homogenate in Dulbecco’s Modified Eagle’s Medium High Glucose medium (DMEM-HG medium). The cotton buds of the swabs were squeezed into 1 ml of DMEM-HG to extract the contents. Homogenates and swab extracts were clarified at 1,200 9 g for 5 min. The supernatants were collected and aliquots were submitted for total RNA extraction using the RNeasy kit (Qiagen). The extracted RNA samples were analysed by RT-PCR using the OneStep RT-PCR kit (Qiagen) to amplify a 351 bp fragment of the PPRV N gene with primers NP3 and NP4 according to Couacy-Hymann et al. [2]. All tested samples were positive for PPRV. Attempts were then made to isolate the virus from the 12 tissue homogenates and four swabs extracts. For this, 500 ll of each sample was used to infect CHS-20 cells in 25 cm2 flask following the procedure described by Adombi et al. [10]. These cells, which are derived from monkey CV1 cells, express the goat SLAM, a cell surface protein used by morbilliviruses as a cell receptor [10]. CHS-20 cells have been shown to be very sensitive in isolating PPRV from pathological samples. Between 2 to 6 days post infection, a cytopathic effect was detected for 11 out of the 16 samples tested. Total RNA was extracted directly from the CHS-20 cells infected with 4 of the positive samples (i.e. lung tissue isolate for each animal) using the RNAeasy Mini Kit (Qiagen) following three free/thawing cycles. RT-PCR reactions were performed as described above. PCR amplicons (351 bp) were purified directly using the WizardÒ SV gel and PCR Clean-up system (Promega) and sent for sequencing using standard Sanger methods at LGC genomics (Berlin, Germany). The generated sequences have been submitted to Genbank under accession numbers (KJ676597 to KJ676600). Phylogenetic analysis of the sequences generated using the MEGA4 programme [11] revealed that they all belonged to lineage II (Fig. 1). One of the positive isolates (i.e. KJ676597) was then chosen for full genome sequencing. This isolate (PPRV Ghana/NK1/2010) originated from the lung of a female
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sheep collected on the 14-01-2010 in Accra. Thirty pairs of primers were designed to cover the full genome (Supplementary Table S1). The 30 and 50 terminal sequences were determined using a protocol as described by Li et al. [12] and the 50 RACE System for Rapid Amplification of cDNA Ends kit (Life Technologies), respectively. The genome was sequenced twice from two separate RNA extractions resulting in a coverage of a minimum of fourfold per nucleotide. The Staden Package (http://staden.sourceforge. net/) was used to assemble the generated sequences. The full genome sequence of PPRV Ghana/NK1/2010 is available from GenBank under accession number KJ466104. The sequencing of the full genome of the virus generated 15,948 bp of sequence which is the same length for all of the other PPRV genomes sequenced to date and is in agreement with the ‘‘rule of six’’ for paramyxoviruses [13]. A Blast analysis revealed that the PPRV Ghana/NK1/2010 genome showed the highest identity (96.6 %) with the lineage II PPRV Nigeria/76/1 genome (EU267274) and the lowest (90.1 %) with the lineage I virus isolated in Coˆte d’Ivoire in 1989 (PPRV CI/89) [6] (Table 1). The organization of the genome is the same as those described previously with a 107 nt genome promoter region at the 30 end followed by the transcription units for the N, P, M, F. H and L proteins and the antigenome promoter at the 50 end [5–9]. There is an unusually long (1,080 bp) and highly GC-rich untranslated region (UTR) between the M and F open-reading frames in all PPRV sequenced to date. The sequence of this UTR is not conserved between PPR viruses and its function is unclear. The GC content of the UTR for PPRV Ghana/NK1/2010 is 66.2 % which is less than that for any of the other PPRV genomes presently available in the public databases (Table 1). A recent study of the proximal 85 nt of the PPRV F 50 UTR by Chulakaisan et al. [14] has indicated the presence of translational enhancing activity within this region based on the binding of host 18S RNA and the formation of stable RNA structures. They identified a 9 bp sequence (GCCCGCCAG) with perfect complementarity to human, murine, bovine and canine 18 sRNA. This 9 bp sequence is also present in PPRV Ghana/NK1/2010. The N protein of PPRV Ghana/NK1/2010 showed the highest identity (97.7 %) with Nigeria/76/1 and the lowest (94.3 %) with the lineage IV virus Sungri 96 (Table 1). The N protein of PPRV has been shown to contain both a nuclear export signal (NES) and a nuclear localization signal (NLS) similar to other morbilliviruses [6, 15]. PPRV Ghana/NK1/ 2010 possesses both the NES (4-LLKSLALF-11) and the NLS (70-TGVMISML-77) motifs. The P protein has a highly variable N-terminus but a conserved C-terminus. Despite its variability, the N-termini of all Paramyxovirinae P (except respiroviruses) have
Virus Genes (2014) 49:497–501
499 KC594074(08-Morocco) DQ840195(99-Saudi Arabia)
91
DQ840187(98-Iran) DQ840178(95-India) DQ840184(96-Turkey) Lineage IV
DQ840191(98-Israel) DQ840198(04-Tajikistan) 95
JX217850(08-Tibet)
73
99 JF939201(07-Tibet)
KF727981(96-Sungri) 98 AJ849636(00-Turkey)
EU267274(76/1-Nigeria) HQ197753(75/1-Nigeria)
74
DQ840166(78-Ghana) DQ840194(99-Mali)
98
Lineage II KJ676598
77
KJ676600
95 99
KJ676597 KJ676599
DQ840165(68-Senegal) EU267273(89-Ivory Coast)
100
DQ840172(88-Burkina Faso)
99
Lineage I
DQ840171(89-Guinea-Bissau)
90 90
DQ840174(94-Senegal)
DQ840175(94-Ethiopia) 100
DQ840168(83-Oman)
Lineage III
100 DQ840169(86-UAE) 0.01
Fig. 1 Phylogenetic analysis using the MEGA4 software of the nucleotide sequence generated using primers NP3 and NP4 from 4 PPRV positive samples (Genbank KJ676597 to KJ676600; filled
triangles). The numbers indicate the bootstrap values calculated from 1,000 bootstrap replicates
recently been shown to possess a 16 aa motif referred to as soyuz1 with experimental data indicating that it may bind the nucleoprotein and prevent its self-assembly [16]. The soyuz 1 sequence for PPRV China/Tibet/07, as studied by Karlin and Belshaw [16], is EQAYHVNKGLECIKSL (aa 4–20) and is conserved in PPRV Ghana/NK1/2010. Phosphorylation of the P protein of paramyxoviruses by cellular casein kinase II is known to be required for transcription of the viral genome [17]. For the measles
virus (MV) and rinderpest virus (RPV), three serine residues (Ser 86, Ser 151 and Ser 180) and (Ser 49, Ser 88 and Ser 151), respectively, have been shown to be involved in the regulation of viral transcription through alterations in its phosphorylation status [17–19]. Interestingly, only the Ser 151 phosphorylation sites are present in PPRV Ghana/NK1/2010 and so further characterization of phosphorylation sites within the PPRV P protein is required.
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Table 1 Comparison of genome characteristics of PPRV Ghana/NK1/2010 and other PPRV genome sequences available in public databases Genome
Accession number
Lineage
Nucleotide identity with PPRV Ghana/NK1/2010 (%)
GC % (M-F) UTR
Protein identity (%) N
P
V*
C
M
F
H
L
Ghana/NK1/2010
KJ466104
II
100
66.2
100
100
100
100
100
100
100
100
Nigeria 76/1
EU267274
II
96.6
67.6
97.7
95.8
92.3
92.4
99.4
99.1
96.8
98.9
Nigeria 75/1 Turkey 2000
HQ197753 AJ849636
II IV
95.8 91.6
67.6 68.9
97.3 95.5
95.2 88.6
92.3 83.6
92.4 88.0
97.9 97.0
98.3 96.3
96.5 93.2
98.6 97.3
Morocco 2008
KC594074
IV
91.4
68.1
95.9
89.2
84.4
85.4
99.1
96.1
92.1
97.0
Tibet 2007
JF939201
IV
91.5
69.3
95.1
88.3
77.1
86.7
97.9
96.5
92.5
97.4
Tibet 2008
JX217850
IV
91.4
69.4
95.1
87.9
82.4
85.4
97.9
96.5
92.7
97.2
Sungri 96
KF727981
IV
90.5
68.8
94.3
85.9
n/a
n/a
94.8
93.6
89.8
94.3
ICV89
EU267273
I
90.1
67.9
95.5
85.7
78.4
85.4
98.5
94.9
92.7
96.8
* The V-protein sequence in a number of the Genbank submissions has been incorrectly annotated or is not available (n/a). For this study, the nucleotide sequence of the genomes available in Genbank has been re-annotated according to Mahapatra et al. [26]
The V protein was the least conserved of the proteins (92.3 %) with 20 amino acid differences between PPRV Nigeria/76/1 spread over the length of the 298 aa protein. A tyrosine at position 110 of the V protein has been shown in both MV and canine distemper viruses (CDV) to be responsible for specific binding of the signal transducer and activator of transcription I (STAT1) binding [20, 21]. Activated STAT1 is involved together with other transcription and regulatory factors in the expression of proteins responsible for the antiviral effect of interferon [22]. This Y110 is present in PPRV Ghana/NK1/2010 suggesting that a similar mechanism may occur for PPRV, but this would need to be confirmed experimentally. The Matrix (M) protein was the most conserved (99.4 %) of the proteins between PPRV Ghana/NK1/2010 and PPRV Nigeria/76/1 with an Isoleucine to Valine change at position 31. Both V and I are aliphatic amino acids, so no functional difference would be expected with this change highlighting the conservation of this protein over the 34 years that separates the isolation of PPRV Nigeria/76/1 and PPRV Ghana/NK1/2010. The F-protein cleavage site GRRTRR (aa position 108) as described by Meyer and Diallo [23] for PPRV Nigeria 75/1 is present in PPRV Ghana/NK1/2010. This is the site where host cell proteases cleave the F0 inactive precursor protein into two highly conserved disulphide-linked subunits F1 (438-546) and F2 (1-108). The F2 of PPRV Ghana/NK1/2010 possesses three potential glycosylation sites (NLS 25-27, NIT 57-59 and NCT 63-65) that are conserved among all morbilliviruses [6]. PPRV Ghana/NK1/2010 was isolated on a Monkey CV1 cell line expressing the sheep/goat SLAM receptor [10]. In MV, the binding of the virus to SLAM is mediated by an asparagine residue at position 481 of the H protein [24]. However, the H protein of PPRV Ghana/NK1/2010 has a histidine residue at position 481 as do all of the other PPRV
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H proteins in Genbank suggesting that PPRV binds in a different manner to its receptor. Zipperle et al. [25] identified three more residues in the globular head domain of the H protein that control SLAM binding activity in CDV. These three residues (Y525, D526 and R529) are also found in PPRV Ghana/NK1/2010 but at aa positions 529, 530 and 533, respectively. Finally, the L protein, composed of 2,183 aa, is the largest of the PPRV proteins. The QGDNQ motif (aa position 771–775) and the GDDD (aa position 1464–1467) associated with RNA polymerase activity for negative single-stranded viruses are conserved in PPRV Ghana/ NK1/2010 and are identical to all available L-protein sequences from other isolates. In conclusion, this is the first genome sequence from a PPRV isolated in Ghana and the most recent lineage II sequence available. The other sequences of lineage II viruses in Genbank are from viruses isolated in 1975 and 1976, respectively, so this study provides important data on the evolution of lineage II viruses over a 35-year period. As the sequences of more PPRV genomes become available, particularly in combination with data on experimental infections in animals, a clearer understanding of the genetic influences on host specificity and viral pathogenicity and transmission of PPRV will become apparent. Acknowledgements This work was supported by funding from the IAEA PPR project CRP D32026 and the tripartite FAO/OIE/WHO IDENTIFY Project of the USAID Emergent Pandemic Threats Program.
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501 15. H. Sato, M. Masuda, R. Miura, M. Yoneda, C. Kai, Virology 352, 121–130 (2006) 16. D. Karlin, R. Belshaw, PLoS One 7, e31719 (2012) 17. T. Das, A. Schuster, S. Schneider-Schaulies, A.K. Banerjee, Virology 211, 218–226 (1995) 18. P. Saikia, M. Gopinath, M.S. Shaila, Arch. Virol. 153, 615–626 (2008) 19. A. Sugai, H. Sato, M. Yoneda, C. Kai, FEBS Lett. 586, 3900–3907 (2012) 20. P. Devaux, V. von Messling, W. Songsungthong, C. Springfeld, R. Cattaneo, Virology 360, 72–83 (2007) 21. A. Ro¨thlisberger, D. Wiener, M. Schweizer, E. Peterhans, A. Zurbriggen, P. Plattet, J. Virol. 84, 6328–6343 (2010) 22. R.E. Randall, S. Goodbourn, J. Gen. Virol. 89, 7836–7839 (2008) 23. G. Meyer, A. Diallo, Virus Res. 37, 23–35 (1995) 24. S. Vongpunsawad, N. Oezgun, W. Braun, R. Cattaneo, J. Virol. 78, 302–313 (2004) 25. L. Zipperle, J.P. Langedijk, C. Orvell, M. Vandevelde, A. Zurbriggen, P.J. Plattet, Virology 84, 9618–9624 (2010) 26. M. Mahapatra, S. Parida, B.G. Egziabher, A. Diallo, T. Barrett, Virus Res. 96(85–98), 14 (2003)
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Full genome sequence of a peste des petits ruminants virus (PPRV) from Ghana W. G. Dundon • C. Adombi • A. Waqas • H. R. Otsyina • C. T. Arthur • R. Silber • A. Loitsch • A. Diallo
Received: 2 July 2014 / Accepted: 14 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The full genome of a peste des petits ruminants virus (PPRV) isolated from a sheep lung sample collected in Ghana, Western Africa, in 2010, has been sequenced. Phylogenetic analysis demonstrated that the virus clustered within the lineage II clade while comparison of its full genome with those of other PPRV strains revealed the highest identity (96.6 %) at a nucleotide level with the PPRV strain Nigeria/76/1. This is the first full genome sequence generated for a PPRV lineage II isolated since 1976. Keywords Peste des petits ruminants Ghana Full genome
Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1109-1) contains supplementary material, which is available to authorized users. W. G. Dundon C. Adombi A. Waqas A. Diallo Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria W. G. Dundon (&) APHL Joint FAO/IAEA Division, IAEA Laboratories, 2444 Seibersdorf, Austria e-mail: [email protected] H. R. Otsyina School of Veterinary Medicine, College of Agriculture and Consumer Sciences, University of Ghana, Legon, Ghana C. T. Arthur CSIR-Animal Research Institute, P.O. Box AH 20, Achimota, Accra, Ghana R. Silber A. Loitsch Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety, Moedling, Austria
Peste des petits ruminants (PPR) is a highly infectious transboundary viral animal disease that affects mainly sheep, goats and small wild ruminants. With morbidity and mortality rates that can be as high as 70–80 %, PPR is classified within the group of animal diseases that are notifiable to the OIE (World Organization for Animal Health). Sheep and particularly goats contribute considerably to the cash income and nutrition of small farmers in many countries so the control of a disease such as PPR is considered an essential element in the fight for global food security and poverty alleviation [1]. The causative agent of PPR, the peste des petits ruminants virus (PPRV), is a member of the genus Morbillivirus within the family Paramyxoviridae [1]. It is a non-segmented, negative, single-stranded RNA virus that encodes eight proteins. These are the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein, (F), haemagglutinin protein (H), polymerase (L) and two nonstructural proteins (V and C). PPRV strains have been classified into four genetic lineages based on the comparison of a sequence fragment for the N (351 bp) and/or F (370 bp) genes [2, 3]. To date Lineage I and II isolates have been confined to Western Africa (e.g. Mali, Senegal, Burkina Faso, Guinea, Cote d’Ivoire and Nigeria), while Lineage III has been detected in Eastern Africa (e.g. Ethiopia, Uganda, Tanzania and Kenya) Yemen and Oman [1, 4]. Lineage IV is presently the predominant PPRV lineage globally. Viruses of this lineage were first detected in Asia, Turkey and the Middle East and for this reason it has been referred to as the Asian lineage. Several reports have now indicated the spread of lineage IV viruses within the African continent [1, 4]. To date, only eight full genome sequences are available in public databases representing three of the four lineages [5–9]. It is evident that more, full genome sequence data need to be generated in
123
498
order to completely understand the evolution, pathogenesis and transboundary movement of this important virus. The samples analysed in this study were collected in Ghana between September 2009 and March 2010 and sent on dry ice to the Austrian High Security Laboratory, Vienna, for processing. They included ocular (n = 7), nasal (n = 10) and buccal (n = 6) swabs from 6 animals and 12 tissue samples from four other animals [lung (n = 4), lymph node (n = 4) and spleen (n = 4)]. They were collected from sheep and goats suspected of being infected with the PPRV as they were manifesting the following symptoms: fever, ocular and nasal discharges, diarrhoea and erosive lesions in the buccal mucous membrane. Upon arrival in Vienna, the tissue samples were ground with sterile quartz beads to make a 10 % homogenate in Dulbecco’s Modified Eagle’s Medium High Glucose medium (DMEM-HG medium). The cotton buds of the swabs were squeezed into 1 ml of DMEM-HG to extract the contents. Homogenates and swab extracts were clarified at 1,200 9 g for 5 min. The supernatants were collected and aliquots were submitted for total RNA extraction using the RNeasy kit (Qiagen). The extracted RNA samples were analysed by RT-PCR using the OneStep RT-PCR kit (Qiagen) to amplify a 351 bp fragment of the PPRV N gene with primers NP3 and NP4 according to Couacy-Hymann et al. [2]. All tested samples were positive for PPRV. Attempts were then made to isolate the virus from the 12 tissue homogenates and four swabs extracts. For this, 500 ll of each sample was used to infect CHS-20 cells in 25 cm2 flask following the procedure described by Adombi et al. [10]. These cells, which are derived from monkey CV1 cells, express the goat SLAM, a cell surface protein used by morbilliviruses as a cell receptor [10]. CHS-20 cells have been shown to be very sensitive in isolating PPRV from pathological samples. Between 2 to 6 days post infection, a cytopathic effect was detected for 11 out of the 16 samples tested. Total RNA was extracted directly from the CHS-20 cells infected with 4 of the positive samples (i.e. lung tissue isolate for each animal) using the RNAeasy Mini Kit (Qiagen) following three free/thawing cycles. RT-PCR reactions were performed as described above. PCR amplicons (351 bp) were purified directly using the WizardÒ SV gel and PCR Clean-up system (Promega) and sent for sequencing using standard Sanger methods at LGC genomics (Berlin, Germany). The generated sequences have been submitted to Genbank under accession numbers (KJ676597 to KJ676600). Phylogenetic analysis of the sequences generated using the MEGA4 programme [11] revealed that they all belonged to lineage II (Fig. 1). One of the positive isolates (i.e. KJ676597) was then chosen for full genome sequencing. This isolate (PPRV Ghana/NK1/2010) originated from the lung of a female
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Virus Genes (2014) 49:497–501
sheep collected on the 14-01-2010 in Accra. Thirty pairs of primers were designed to cover the full genome (Supplementary Table S1). The 30 and 50 terminal sequences were determined using a protocol as described by Li et al. [12] and the 50 RACE System for Rapid Amplification of cDNA Ends kit (Life Technologies), respectively. The genome was sequenced twice from two separate RNA extractions resulting in a coverage of a minimum of fourfold per nucleotide. The Staden Package (http://staden.sourceforge. net/) was used to assemble the generated sequences. The full genome sequence of PPRV Ghana/NK1/2010 is available from GenBank under accession number KJ466104. The sequencing of the full genome of the virus generated 15,948 bp of sequence which is the same length for all of the other PPRV genomes sequenced to date and is in agreement with the ‘‘rule of six’’ for paramyxoviruses [13]. A Blast analysis revealed that the PPRV Ghana/NK1/2010 genome showed the highest identity (96.6 %) with the lineage II PPRV Nigeria/76/1 genome (EU267274) and the lowest (90.1 %) with the lineage I virus isolated in Coˆte d’Ivoire in 1989 (PPRV CI/89) [6] (Table 1). The organization of the genome is the same as those described previously with a 107 nt genome promoter region at the 30 end followed by the transcription units for the N, P, M, F. H and L proteins and the antigenome promoter at the 50 end [5–9]. There is an unusually long (1,080 bp) and highly GC-rich untranslated region (UTR) between the M and F open-reading frames in all PPRV sequenced to date. The sequence of this UTR is not conserved between PPR viruses and its function is unclear. The GC content of the UTR for PPRV Ghana/NK1/2010 is 66.2 % which is less than that for any of the other PPRV genomes presently available in the public databases (Table 1). A recent study of the proximal 85 nt of the PPRV F 50 UTR by Chulakaisan et al. [14] has indicated the presence of translational enhancing activity within this region based on the binding of host 18S RNA and the formation of stable RNA structures. They identified a 9 bp sequence (GCCCGCCAG) with perfect complementarity to human, murine, bovine and canine 18 sRNA. This 9 bp sequence is also present in PPRV Ghana/NK1/2010. The N protein of PPRV Ghana/NK1/2010 showed the highest identity (97.7 %) with Nigeria/76/1 and the lowest (94.3 %) with the lineage IV virus Sungri 96 (Table 1). The N protein of PPRV has been shown to contain both a nuclear export signal (NES) and a nuclear localization signal (NLS) similar to other morbilliviruses [6, 15]. PPRV Ghana/NK1/ 2010 possesses both the NES (4-LLKSLALF-11) and the NLS (70-TGVMISML-77) motifs. The P protein has a highly variable N-terminus but a conserved C-terminus. Despite its variability, the N-termini of all Paramyxovirinae P (except respiroviruses) have
Virus Genes (2014) 49:497–501
499 KC594074(08-Morocco) DQ840195(99-Saudi Arabia)
91
DQ840187(98-Iran) DQ840178(95-India) DQ840184(96-Turkey) Lineage IV
DQ840191(98-Israel) DQ840198(04-Tajikistan) 95
JX217850(08-Tibet)
73
99 JF939201(07-Tibet)
KF727981(96-Sungri) 98 AJ849636(00-Turkey)
EU267274(76/1-Nigeria) HQ197753(75/1-Nigeria)
74
DQ840166(78-Ghana) DQ840194(99-Mali)
98
Lineage II KJ676598
77
KJ676600
95 99
KJ676597 KJ676599
DQ840165(68-Senegal) EU267273(89-Ivory Coast)
100
DQ840172(88-Burkina Faso)
99
Lineage I
DQ840171(89-Guinea-Bissau)
90 90
DQ840174(94-Senegal)
DQ840175(94-Ethiopia) 100
DQ840168(83-Oman)
Lineage III
100 DQ840169(86-UAE) 0.01
Fig. 1 Phylogenetic analysis using the MEGA4 software of the nucleotide sequence generated using primers NP3 and NP4 from 4 PPRV positive samples (Genbank KJ676597 to KJ676600; filled
triangles). The numbers indicate the bootstrap values calculated from 1,000 bootstrap replicates
recently been shown to possess a 16 aa motif referred to as soyuz1 with experimental data indicating that it may bind the nucleoprotein and prevent its self-assembly [16]. The soyuz 1 sequence for PPRV China/Tibet/07, as studied by Karlin and Belshaw [16], is EQAYHVNKGLECIKSL (aa 4–20) and is conserved in PPRV Ghana/NK1/2010. Phosphorylation of the P protein of paramyxoviruses by cellular casein kinase II is known to be required for transcription of the viral genome [17]. For the measles
virus (MV) and rinderpest virus (RPV), three serine residues (Ser 86, Ser 151 and Ser 180) and (Ser 49, Ser 88 and Ser 151), respectively, have been shown to be involved in the regulation of viral transcription through alterations in its phosphorylation status [17–19]. Interestingly, only the Ser 151 phosphorylation sites are present in PPRV Ghana/NK1/2010 and so further characterization of phosphorylation sites within the PPRV P protein is required.
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500
Virus Genes (2014) 49:497–501
Table 1 Comparison of genome characteristics of PPRV Ghana/NK1/2010 and other PPRV genome sequences available in public databases Genome
Accession number
Lineage
Nucleotide identity with PPRV Ghana/NK1/2010 (%)
GC % (M-F) UTR
Protein identity (%) N
P
V*
C
M
F
H
L
Ghana/NK1/2010
KJ466104
II
100
66.2
100
100
100
100
100
100
100
100
Nigeria 76/1
EU267274
II
96.6
67.6
97.7
95.8
92.3
92.4
99.4
99.1
96.8
98.9
Nigeria 75/1 Turkey 2000
HQ197753 AJ849636
II IV
95.8 91.6
67.6 68.9
97.3 95.5
95.2 88.6
92.3 83.6
92.4 88.0
97.9 97.0
98.3 96.3
96.5 93.2
98.6 97.3
Morocco 2008
KC594074
IV
91.4
68.1
95.9
89.2
84.4
85.4
99.1
96.1
92.1
97.0
Tibet 2007
JF939201
IV
91.5
69.3
95.1
88.3
77.1
86.7
97.9
96.5
92.5
97.4
Tibet 2008
JX217850
IV
91.4
69.4
95.1
87.9
82.4
85.4
97.9
96.5
92.7
97.2
Sungri 96
KF727981
IV
90.5
68.8
94.3
85.9
n/a
n/a
94.8
93.6
89.8
94.3
ICV89
EU267273
I
90.1
67.9
95.5
85.7
78.4
85.4
98.5
94.9
92.7
96.8
* The V-protein sequence in a number of the Genbank submissions has been incorrectly annotated or is not available (n/a). For this study, the nucleotide sequence of the genomes available in Genbank has been re-annotated according to Mahapatra et al. [26]
The V protein was the least conserved of the proteins (92.3 %) with 20 amino acid differences between PPRV Nigeria/76/1 spread over the length of the 298 aa protein. A tyrosine at position 110 of the V protein has been shown in both MV and canine distemper viruses (CDV) to be responsible for specific binding of the signal transducer and activator of transcription I (STAT1) binding [20, 21]. Activated STAT1 is involved together with other transcription and regulatory factors in the expression of proteins responsible for the antiviral effect of interferon [22]. This Y110 is present in PPRV Ghana/NK1/2010 suggesting that a similar mechanism may occur for PPRV, but this would need to be confirmed experimentally. The Matrix (M) protein was the most conserved (99.4 %) of the proteins between PPRV Ghana/NK1/2010 and PPRV Nigeria/76/1 with an Isoleucine to Valine change at position 31. Both V and I are aliphatic amino acids, so no functional difference would be expected with this change highlighting the conservation of this protein over the 34 years that separates the isolation of PPRV Nigeria/76/1 and PPRV Ghana/NK1/2010. The F-protein cleavage site GRRTRR (aa position 108) as described by Meyer and Diallo [23] for PPRV Nigeria 75/1 is present in PPRV Ghana/NK1/2010. This is the site where host cell proteases cleave the F0 inactive precursor protein into two highly conserved disulphide-linked subunits F1 (438-546) and F2 (1-108). The F2 of PPRV Ghana/NK1/2010 possesses three potential glycosylation sites (NLS 25-27, NIT 57-59 and NCT 63-65) that are conserved among all morbilliviruses [6]. PPRV Ghana/NK1/2010 was isolated on a Monkey CV1 cell line expressing the sheep/goat SLAM receptor [10]. In MV, the binding of the virus to SLAM is mediated by an asparagine residue at position 481 of the H protein [24]. However, the H protein of PPRV Ghana/NK1/2010 has a histidine residue at position 481 as do all of the other PPRV
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H proteins in Genbank suggesting that PPRV binds in a different manner to its receptor. Zipperle et al. [25] identified three more residues in the globular head domain of the H protein that control SLAM binding activity in CDV. These three residues (Y525, D526 and R529) are also found in PPRV Ghana/NK1/2010 but at aa positions 529, 530 and 533, respectively. Finally, the L protein, composed of 2,183 aa, is the largest of the PPRV proteins. The QGDNQ motif (aa position 771–775) and the GDDD (aa position 1464–1467) associated with RNA polymerase activity for negative single-stranded viruses are conserved in PPRV Ghana/ NK1/2010 and are identical to all available L-protein sequences from other isolates. In conclusion, this is the first genome sequence from a PPRV isolated in Ghana and the most recent lineage II sequence available. The other sequences of lineage II viruses in Genbank are from viruses isolated in 1975 and 1976, respectively, so this study provides important data on the evolution of lineage II viruses over a 35-year period. As the sequences of more PPRV genomes become available, particularly in combination with data on experimental infections in animals, a clearer understanding of the genetic influences on host specificity and viral pathogenicity and transmission of PPRV will become apparent. Acknowledgements This work was supported by funding from the IAEA PPR project CRP D32026 and the tripartite FAO/OIE/WHO IDENTIFY Project of the USAID Emergent Pandemic Threats Program.
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