Plum Island Animal Disease Center, Agricultural Research Service, US Department of Agriculture, PO Box 848, Greenport, NY 11944-0848, USA1
Author for correspondence: John Neilan.Fax +1 516 323 2507. e-mail jneilan{at}asrr.arsusda.gov
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Abstract |
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An ASFV ORF with similarity to animal C-type lectin proteins has been described in the pathogenic African isolate Malawi Lil-20/1 (Borca et al., 1993 ) and in a cell culture-adapted European virus, BA71V (Yanez et al., 1995
). In animal cells, C-type lectins serve as extracellular cell-adhesion molecules that mediate specific recognition functions via Ca2+ carbohydrate recognition domains. For example, the hepatic lectins serve as membrane receptors mediating endocytosis (Hughes, 1992
) and lectin-like proteins mediate the early events involving attachment of migrating leukocytes to venule endothelium (Butcher, 1991
). They can also act as effectors in signal transduction pathways and/or gene regulation (Houchins et al., 1991
; Fujiwara et al., 1994
).
C-type lectin-like proteins have been described in poxviruses. The vaccinia virus gene A34R encodes a transmembrane protein similar to C-type animal lectins (Blasco et al., 1993 ) and it is suggested that A34R functions either in formation or stabilization of actin-containing microvilli that facilitate the cell-to-cell spread of virus (Wolffe et al., 1997
). An A34R homologue is present in smallpox virus (variola), where it is thought to play a role in virus infectivity (Massung et al., 1993
; McIntosh & Smith, 1996
). Other poxvirus lectin proteins have been described, e.g. fowlpox virus ORFs 2, 8 and 11 (Tomley et al., 1988
), vaccinia virus A40R (Goebel et al., 1990
) and molluscum contagiosum virus MC143R (Senkevich et al., 1996
); however, their functions have not yet been determined.
The Malawi Lil-20/1 ORF 8CR most closely resembles the fowlpox virus lec1 ORF (Tomley et al., 1988 ) and the vaccinia virus A40R ORF (Goebel et al., 1990
), with less similarity to the vaccinia virus A34R ORF, human NKG2 protein, hepatic lectins and Fc receptors. Here, we have characterized the ASFV 8CR ORF. Our data indicate that: (i) 8CR is transcribed late in the infection cycle; (ii) although conserved among pathogenic ASFV isolates, 8CR is non-essential for virus growth and spread in macrophage cell cultures in vitro; and (iii) 8CR does not affect disease course or virus virulence in domestic swine.
To assess the degree of conservation of the Malawi Lil-20/1 8CR ORF, DNA sequence analysis was performed on 12 pathogenic viruses, representing African, European and Caribbean isolates from both pig and tick sources. The ORF was amplified from purified virion DNA by PCR with oligonucleotide primers (forward primer 5' GTATAAGGATAACTTCGCCAC 3'; reverse primer 5' GGACTTTCTATTTCTTCAACCAC 3') and amplified products were cloned into the TA cloning vector pCR 2.1 (Invitrogen). Two independent PCR clones from each isolate were sequenced completely by using M13 forward and reverse primers and internal primers based on the derived sequence. The ASFV 8CR ORFs and other C-type lectin protein sequences in genetic databases were compared by using computer programs described elsewhere (Afonso et al., 1999 ; Neilan et al., 1997 a
).
Overall, 8CR was highly conserved among the viruses (Fig. 1 a), ranging in size from 153 to 166 amino acid residues with a median size of 158 residues and a predicted molecular mass of 18·5 kDa (pI 9·0). Pathogenic European isolates E70 and E75 and the Caribbean isolate Haiti 811 were identical at the nucleotide level to the cell culture- adapted European isolate BA71V (Yanez et al., 1995
). Similarity of the predicted 8CR amino acid sequence among the isolates ranged from 74 to 100% and there was no significant difference between isolates in overall relationships at the amino acid level (amino acid Poisson correction-distance estimate by using a neighbour- joining branch length test and a cluster test with 1000 bootstrap samples;
2=12·36, 12 degrees of freedom, P =0·42).
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Although 8CR was conserved in all isolates, the variability observed was greater than that seen with other well-characterized non-essential ASFV genes (Neilan et al., 1997a , b
; Zsak et al., 1996
). Analysis of this gene in conjunction with additional viral genes may prove useful for identifying and grouping ASFV strains.
To determine the transcriptional characteristics of 8CR, Northern blot analysis was performed on poly(A)+ RNA extracted from Malawi Lil-20/1-infected macrophage cell cultures using the Reagents Total RNA isolation system (Promega) and Poly(A) Quick mRNA isolation kit (Stratagene) according to the manufacturers' instructions. Northern blot hybridization analysis was performed by standard methods (Sambrook et al., 1989 ).
By using an 8CR-specific probe, an RNA transcript of 0·74 kb was detected late in the infection cycle. 8CR, like the late-gene control p72, was not transcribed in the presence of cytosine arabinoside, an inhibitor of ASFV late gene expression (Fig. 2 a). These data indicate that the 8CR ORF is transcribed at late times in virus infection.
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Sequence analysis of the six independent clones demonstrated that transcription initiated at an adenine residue either 165 (n=5) or 163 (n=1) bases upstream of the predicted 8CR translational initiation codon (Fig. 2b). This is unlike other ASFV late genes, where initiation of transcription has been located closer to the translational start site; l11l, A224L and I226R initiate 910, 1619 and 811 bases upstream of their respective translational start sites (Kleiboeker et al., 1998
; Chacon et al., 1995
; Rodriguez et al., 1996
). Analysis of the nucleotide sequence downstream of the 8CR ORF showed the presence of seven thymidylate residues (7T), a motif believed to function in 3'-end formation for ASFV mRNA (Almazan et al., 1992
, 1993
) (Fig. 2b
). The size of the 8CR transcript obtained by Northern blot analysis is consistent with the use of these nucleotides for initiation and termination of 8CR transcription (Fig. 2 a
).
To examine the role of the 8CR ORF in ASFV growth in vitro and in virulence in swine, an 8CR deletion mutant of Malawi Lil-20/1 was constructed by homologous recombination between parental virus and an engineered recombination transfer vector, p72GUS8CR, which was designed to remove all but 12 amino acid residues (one at the N terminus and 11 at the C terminus) of the 8CR ORF by using procedures described previously (Zsak et al., 1996
; Neilan et al., 1997 a
). Recombinant viruses representing independent primary plaques were purified to homogeneity by plaque assay and verified by Southern blot analysis and PCR as products of a double-crossover recombination event. One of these viruses,
8CR, was selected for further analysis.
Growth characteristics of 8CR were compared with those of the parental virus, Malawi Lil-20/1, by infecting primary macrophage cell cultures (m.o.i. of 0·01) in duplicate and then titrating extracellular and intracellular virus yields at various times p.i. In two independent experiments, the growth kinetics and maximum virus yields were:
8CR, intracellular yield 7·3±0·1 and extracellular yield 7·0±0·3 log10 TCID50/ml; parental virus, intracellular yield 7·3±0·1 and extracellular yield 7·0±0·3 log10 TCID 50/ml. Thus, no significant difference in growth kinetics or virus yield was observed, indicating that 8CR does not affect replication, release or spread of the virus in macrophage cell cultures. Thus, 8CR is not required for virus infectivity in porcine macrophages in vitro.
To examine the role of 8CR in ASFV pathogenesis and virulence, Yorkshire pigs were inoculated intramuscularly with 102 TCID 50 of the parental virus Malawi Lil-20/1 or the mutant, 8CR. A dose of 102 TCID50 of Malawi Lil- 20/1 represents a challenge dose of between 10 and 100 LD100 (Neilan et al., 1997 a)
. Clinical signs of ASFV infection, i.e. fever (a rectal temperature greater than 40 °C), anorexia, lethargy, shivering, cyanosis and recumbency, were monitored daily. Virus isolation and titration of ASFV in blood samples were performed as described previously (Onisk et al., 1994
).
Both groups of animals presented with clinical signs of ASF 34 days p.i. and these signs progressed until death in all cases (Table 1). No differences in the onset of clinical disease, viraemia or time to death were noted for the two groups, indicating that 8CR does not affect disease course or virus virulence in domestic pigs. Replication and spread of ASFV in cells of the mononuclearphagocytic system appears to be a critical factor in ASFV virulence in domestic swine (Colgrove et al., 1969
; Konno et al., 1971
; Mebus, 1988
; Moulton & Coggins, 1968
). Consistent with the observations made in vitro, the animal infection data indicate that 8CR is non-essential for replication in these cell types or for spread within the porcine mononuclearphagocytic system in vivo.
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References |
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Received 16 April 1999;
accepted 7 July 1999.