Caprine Arthritis Encephalitis Virus (CAEV) of goats and Maedi Visna Virus
(MVV) of sheep belong to Small Ruminant Lentiviruses (SRLVs). Caprine arthritis
encephalitis virus infection may lead to chronic disease of the joints and on
rare occasion's encephalitis in goat kids <6 months of age. The disease was
first diagnosed in goats in 1974 (Cork et al., 1974).
Since that time, it has been diagnosed in goats in North America, Europe, Kenya,
Peru, Australia, and New Zealand (Knoweles, 1997). CAEV
is an RNA virus from the Lentivirus genus of the Retroviridae family (Cork
et al., 1974). Their genomes have the typical proviral genomic organization
of lentiviruses consisting of Long Terminal Repeats (LTR), the gag, pol and
env genes open reading frames which encode for proteins with regulatory functions
in viral replication (Clements and Zink, 1996). Gag
and pol genes encode for core proteins and viral enzyme (i.e., reverse transcriptase),
respectively and relatively conserved (Pepin et al.,
1998). Env gene encodes for the surface and transmembrane glycoproteins
and exhibits high heterogeneity (Pepin et al., 1998).
Complete nucleotide sequences of CAEV and MVV strains, i.e., CAEV-Co from United
States (Saltarelli et al., 1990), K1514 from
Iceland (Sonigo et al., 1985), SA-OMVV from South
Africa (Querat et al., 1990) and EV-1 from Scotland
(Sargan et al., 1991) in addition to some partial
sequences have been published (Leroux et al., 1995;
Chebloune et al., 1996).
SRLVs have previously been classified phylogenetically into at least six clades
with no clear separation according to host species or geographical origin (Zanoni,
1998; Rolland et al., 2002). According to
a recently proposed phylogentic organization based on gag and pol sequences
(Shah et al., 2004), SRLVs can be subdivided
genetically into groups A-D with subgroups present in groups A and B (Shah
et al., 2004). Group A corresponds to the heterogenous MVV type and
can further be subdivided into seven subtypes, designated A1-7. The second group
(Group B) refers to the genetically less complex CAEV type and comprises only
two distinct lineages termed subtypes B1 and 2.
Two additional SRLV Groups C and D have been recently identified on basis of
their great genetic divergence with the two previous groups but are not yet
fully described because they are either represented by few isolates or recognized
based only on pol sequence (Shah et al., 2004). Sheep SRLVs prototypes
such as South Africa SA-OMVV strain, the British EV-1 strain and Icelandic strain
visna K1514 were all assigned to the A1 group which contained only sheep SRLV
(Shah et al., 2004), goats prototypes such as CAEV-Co and CAEV-1GA (Gjerset
et al., 2006) belonged to the B1 and C groups, respectively both
containing only goat SRLV sequences. Groups A2 and D appeared to contain only
sheep SRLV sequences, Groups A5 and 7 only contained goat SRLV sequences. However,
groups A3, A4, A6 and B2 contained both sheep and goat SRLV sequences (Shah et al., 2004).
Infection with CAEV in goats from Sudan was recently reported (Elfahal
et al., 2010). Infected goats develop arthritis and mainly exhibit
no clinical signs. Infected goats were diagnosed by the presence of CAEV-specific
antibodies and/or detection of CAEV-proviral DNA. In this study we describe
the first sequence analysis of SRLVs of goats in Sudan. A fragment was sequenced
from the gag gene covering partial part of the coding sequences for the Capsid
(CA) P25 protein. Resulting sequences were aligned with those from other ovine/caprine
MATERIALS AND METHODS
Animals and blood samples: Eight goats from different sites in Khartoum State proven positive for CAEV infection by ELISA were analysed in this study. Whole blood (5 mL in EDTA vacutainer tubes) was collected from each of these animals.
DNA extraction: DNA was extracted according to Reina
et al. (2006) using phenol-chloroform-isoamylic acid alcohol solution.
In brief; Peripheral Blood Mononuclear Cells (PBMC) were separated on Ficoll-Paque
(GE Healthcare, Sweden). PBMC were then lysed in buffer (100 mM Tris-HCl Ph
7.5; 12.5 mM EDTA-Na2; 150 mM NaCl; 0.5% SDS) containing proteinase
K at 55°C for 2 h or 37°C overnight. Genomic DNA was subsequently extracted
twice with phenol-chloroform-isoamylic acid alcohol (25:24:1) and again extracted
twice with chloroform-isoamylic acid alcohol (24:1). Following treatment with
96% ethanol and centrifugation, the precipitate was resuspended in distilled
water (50 μL) and stored at -20°C till the day of the PCR testing.
PCR and sequencing: The PCR reaction was carried out using a Primus
96 thermal cycler (MWG-Biotech, Germany). Primers that target the CAEV gag gene
of the CAEV-Co prototype strain (GenBank accession number M33677) were used
to amplify 433 bp of the gag gene (Clavijio and Thorsen,
1996). DNA fragment were amplified in 25 μL reaction mixtures containing:
2.5 μL of 10xPCR buffer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide
triphosphate (dNTPs), 400 nM of each primer, 2.5 U of Taq DNA polymerase (Promga,
USA), 5% DMSO and 3 μL of DNA template.
The PCR amplification was achieved by 35 cycles each including a denaturation step at 94°C for 1 min, annealing step at 50°C for 30 sec and extension step at 72°C for 1 min. PCR amplified DNA fragments were analysed by electrophoresis on 1.5% agrose gels stained with ethidium bromide and visualized on a UV transilluminator.
PCR products were sent for commercial sequencing at Macrogen Company (Seoul, Republic of Korea).
Sequence analysis, GenBank accession numbers, alignment and phylogenetic
tree: Nucleotide sequences obtained from sequencing the resulting 354-nucleotide
fragments of the gag gene of the eight CAEV strains were submitted to GenBank
and the accession numbers FJ619565-FJ619572 were assigned to them. Other SRLVs
sequences used in this analysis were downloaded from GeneBank which include
Cork-co, EV1, SA-OMVV and K1514 (Table 1). Further sequences
were used in the tree to determine the group of the isolates (Table
1). The sequences obtained from the samples and their deduced amino acids
were compared to sequences and amino acids in the GenBank database using the
BLAST program and multiple sequences and amino acids alignment was generated
using Clustal W (Thompson et al., 1994). Percentage
divergence value-obtained from multiple sequence alignment were used to construct
the phylogenetic tree.
For this purpose, nucleotide sequences were first aligned along with reference
sequences obtained from GenBank using ClustalW program at DDBJ website. Thereafter,
a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method (Saitou
and Nei, 1987) and genetic distances were calculated with the Kimura 2 parameter
model. Phylogenetic tree was constructed using the nucleotide sequences and
was performed by using DDJB website. Phylogenetic tree was finally drawn with
Treeview software (Page, 1996).
Sequeces comparisons: The PCR products from the gag gene obtained from
eight seropositive goats were sequenced (Table 1) and the
sequences were submitted to the GenBank. Because a direct PCR product sequencing
was carried out, an average number of 354 nucleotides out of the 433 nucleotides
were obtained. These were translated and their 117 derived amino acids sequences
were aligned using Clustal W software along with the sequences of well-known
prototypic strains of CAEV and MVV.
|| SRLVs nucleotide sequences used in the study
|| Nucleotide and amino acid sequence diversity of Sudanese
|Percentage divergence values were obtained by comparisons
of nucleotide sequences (plain number) and deduced amino acid sequences
(number in bold) between the Sudanese strains
The percentage of nucleotide and amino acid substitution were determined by
pairwise comparisons between the eight Sudanese isolates (Table
Interestingly, all sequences were found to be closely related to each other at both nucleotide and amino acid residues except for sample number 8 which displayed a lesser identity number to the rest of the samples but still closer to them, than to strains from other geographical areas. Divergences among the Sudanese samples range from 0.29-2% at the nucleotide level and from 0-1.7% at amino acid level (Table 2). Sample number 8 differed from the rest of the sequences in that it showed divergences from 2.8-3.4% at nucleotide level and 3.4-4.3% at the amino acid level.
Comparison with known Ovine/Caprine SRLV strains suggested that Sudanese isolates are divergent from the ovine strains, closely related to the CAEV-Co and revealed the following differences at the nucleotide level: 26% with SA-OMVV, 27.4% with K1514, 27.4% with EV1. Likewise at amino acid level, the average pairwise divergences were 6.1% with SA-OMVV, 6% with K1514, 8.8% with EV1 ovine strains. On the other hand, comparison with CAEV-Co caprine prototypic strain revealed an average 0.95 and 1.1% differences at nucleotide and amino acid sequence, respectively (Table 3).
Sequence alignment was performed to clarify the position of differences at nucleotide and amino acid sequences between the Sudanese isolates and some known isolates of Ovine/Caprine SRLV. In these alignments, 354 residues of the sequence were used and the CAEV-Co was chosen as a reference sequence. The alignment has shown an overall identity of 60.45 and 66.67% at nucleotide and amino acid sequences respectively (Fig. 1 and 2) and revealed a total of 27 polymorphism sites (Fig. 1).
All sequences from Sudan were found to be closely related to each other and to the CAEV-Co strain and showed more divergence from the sheep isolates at nucleotide and amino acid level (Fig. 1 and 2). The alignment displayed three gaps at positions 324, 338, 344 of the sequenced product (Fig. 1).
|| Nucleotide and amino acid sequence diversity between Sudanese
and other SRLV strains
|Percentage diversity values were obtained by comparisons of
nucleotide sequences (plain numbers) and deduced amino acid sequences (number
in bold) between Sudanese and known SRLV strains (GenBank accession numbers
are in Table 1)
||Nucleotide sequence alignment of the gag gene from eight Sudanese
isolates of CAEV with the sequences of prototype CAEV and ovine prototypes.
Only nucleotides differing from the CAEV-Co sequence are shown. Dots indicate
identity with CAEV-Co whereas gaps are represented by dashes
Phylogenetic analysis: Phylogenetic analysis of the Sudanese isolates
compared to those from known prototypic strains of ovine/caprine lentiviruses
was performed using the neighbor-joining method. The additional sequences used
were some isolates from the GenBank that belong to Group B1 (Shah et al., 2004) and the Norwegian isolate that belong to group C (Gjerset
et al., 2006) (Table 1).
||Alignment of amino acid sequences from the gag gene encoding
capsid protein. Only amino acids differing from the CAEV-Co sequences are
shown. Dots indicate identity with CAEV-Co, whereas gaps represented by
||Phylogenetic tree obtained using neighbour joining method
with the Kimura program showing the relationship between Sudanese isolates
(No. 01-08) and other known SRLV strains
The derived phylogenetic tree is shown in Fig. 3. The analysis
revealed that Sudanese isolates were closely related to the CAEV-Co, both clustering
in one group that also contained sequences of Group B1 of Shah et al. (2004). This result confirmed that the Sudanese sequences actually fall within
group B subgroup B1 which contain isolates from goats only (Shah
et al., 2004).
The ovine strains K1514, EV1, SA-OMVV were in a separate group (Group A) and the Norwegian isolate in a third separate group (Group C) (Fig. 3).
Small Ruminant Lentiviruses (SRLVs) infections including Caprine Arthritis
Encephalitis Virus (CAEV) and Maedi-Visna Virus (MVV) are widespread in many
countries all over the world and they cause substantial economic damage. SRLV
infections persist for life and carriers are considered a continuous potential
source of virus for transmission. A few complete genomes and many partial sequences
of SRLV strains have been isolated and characterized in many European countries,
South Africa and in USA (Angelopoulou et al., 2005).
In the Sudan SRLV strains have never been isolated or characterized.
In the present study, we described the analysis of 354 nucleotide residues
and their deduced 117amino acids of gag gene of SRLV strains from 8 goats in
Khartoum State. The sequences of gag gene for the comparison and phylogenetic
analysis was focused because this region is well known to be specific for the
group of lentiviruses and have less divergences compared to those of the envelope
gene (Rolland et al., 2002).
The data indicated that the isolates (except for SUD08) which have been collected
from different areas in Khartoum state are very similar and closely related
to each other differing by an average of 1.1% at the nucleotide sequence and
by 0.97% at the amino acid sequence. This is in line with Kuzmak
et al. (2007) who reported that isolates which were extracted from
Polish goats were found to be closely related to each other and to the CAEV-Co
based on gag gene. One isolate, SUD08, differed more from all other Sudanese
samples and showed differences of 3.1% at nucleotide sequence and 4.2% at the
amino acid sequence but still closely related to them. The nucleotide sequences
in the gag region of the Sudanese isolates appeared to be more similar to the
CAE-Co caprine prototypic (0.95% divergence) than to the ovine prototypic strains
(27% divergence) at nucleotide sequence as we further confirmed by the phylogenetic
Phylogenetic analysis showed that Sudanese isolates lied within the CAEV-Co group and closely related to this prototypic isolate. All of the isolates lied among the isolates introduced from the GenBank and this confirmed that sequences are in the group B in subgroup B1 which contained isolates from goats only and this is in line with Shah et al. (2004).
Sudanese isolates investigated here in this study are related to each other, similar to the CAEV-Co prototypic and clustered in group B1. The isolates are more similar to the CAEV-Co than to Shah Isolates of the same group and most divergent from sheep isolates.
The study showed that CAE viruses infecting goats in Sudan might have arisen from the same source as the isolates nucleotide and amino acid sequences were closely related to one another and related to those isolated from goats in the other parts of the world.
We are grateful to Prof. Stephen Valas (National Reference Laboratory for Small-Ruminant Lentiviruses, France) for kindly providing DNA positive control.