The Genome of a Very Virulent Marek's Disease Virus

Abstract

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the highly infectious cell-associated alphaherpesvirus MD virus serotype 1 (MDV1) (18). Yearly economic losses from MD total $1 billion worldwide (18). MDV1 infection results in a rapid onset of malignant T-cell lymphomas within several weeks of infection. Tumor infiltration results in a neural form of disease, which causes progressive paralysis, or a visceral form of disease, which is usually very acute and accompanied by high mortality. Productive virus replication in the skin and feather follicle epithelia with subsequent virus shedding is responsible for disease transmission (18).

MD is controlled by vaccination and good management practices (18). Naturally occurring nonpathogenic strains of MDV1, MDV2, and herpesvirus of turkey (HVT or MDV3) have been used individually or together in bivalent vaccines (18, 40). Recent increases in MD-related mortality and condemnations among vaccinated poultry have occurred in the United States. These increases in disease have occurred approximately 6 years after the introduction of new vaccines (99). In the late 1970s, following the introduction of HVT vaccines, and since 1992, after the introduction of bivalent MDV2-HVT-based vaccines, new MDV1 strains of greater virulence (very virulent [vv] and very virulent plus [vv⁺] MDV1) were isolated. These viruses are characterized by higher cytolytic activity, unusual tissue tropism, increased atrophy of lymphoid organs, immunosuppression, enhanced capacity to transform T cells, and earlier host death (7, 17, 99). It has been suggested that emergence of vv and vv⁺ MDV1 strains may be due to strong selective pressure generated by extensive vaccination and enhanced genetic resistance of commercial flocks (99).

To date, MDV1 genome characterization has involved partial sequencing of several different virus strains, accounting for approximately 40% of the complete genome (reviewed in reference 8). However, the genetic basis and molecular mechanisms underlying viral virulence and oncogenicity remain poorly understood. Genes encoding proteins involved in T-cell transformation (MEQ) and others with potential involvement in tumorigenicity, viral virulence, and host range (pp24, pp38, interleukin 8 [IL-8], SORF2) have been described (14, 24, 43, 57, 65, 66, 79, 89, 90, 102, 109). Additionally, virus attenuation has been associated with amplification of a 132-bp repeat within the long repeats (10–12, 34, 61, 78, 90). To improve understanding of MDV virulence and the mechanisms associated with enhanced viral virulence, more-complete information about the MDV genome and its gene complement is needed. Here we present the first complete genome sequence, with analysis, of a vv MDV1 isolate, Md5 (100).

MATERIALS AND METHODS

DNA isolation, cloning, and sequencing.

The Md5 strain of MDV was obtained from the American Type Culture Collection (Manassas, Va.) and passaged three times in primary chicken embryo fibroblast cell cultures. Viral DNA was extracted from the cytoplasm of infected cells as previously described (98). Random DNA fragments were obtained by incomplete enzymatic digestion with TaqI and AciI endonucleases (New England Biolabs, Beverly, Mass.). DNA fragments of 1.5 to 2.5 kbp were isolated after separation on agarose gels, cloned into the dephosphorylated AccI site of pUC19 plasmids, and grown in Escherichia coli DH10B cells (Gibco BRL, Gaithersburg, Md.). Plasmids were purified by alkaline lysis according to the manufacturer's instruction (Eppendorf 5 Prime, Boulder, Colo.). DNA templates were sequenced from both ends with M13 forward and reverse primers using dideoxy chain terminator sequencing chemistries (82) and the Applied Biosystem PRISM 377 automated DNA sequencer (PE Biosystems, Foster City, Calif.). ABI sequencing analysis software (version 3.3) was used for lane tracking and trace extraction. Bases were called from chromatogram traces with Phred (30), which also produced a quality file containing a predicted probability of error at each base position.

DNA sequence analysis.

DNA sequences were assembled with Phrap (29) using the quality files and default settings to produce a consensus sequence, which was manually edited with Consed (37). An identical sequence was assembled using the TIGR assembler with quality files and clone length constraints (95). Gap closure was achieved by primer walking of gap-spanning clones and sequencing of PCR products. The final DNA consensus sequence represented on average sixfold redundancy at each base position. The predicted restriction map for Md5 matched published data for the MDV1 GA strain (15). For descriptive purposes, we have presented Md5 in a linearized fashion as described by Dolan et al. (28). Genome DNA composition, structure, repeats, and restriction enzyme patterns were analyzed as previously described (2). Open reading frames (ORFs) encoding proteins of greater than or equal to 60 amino acids with a methionine start codon (92, 93) were evaluated for coding potential using the Hexamer (ftp.sanger.ac.uk/pub/rd) and Glimmer (81) computer programs. Other criteria included similarity to other herpesvirus or cellular proteins, published evidence for MDV proteins, and compact gene arrangement with little gene overlap (25, 96). Homology searches were conducted using Blast (3), PsiBlast (4), FASTA (70), and HMMER (16) programs with the following databases: PROSITE, Pfam, Prodom, Sbase, Blocks, Domo, and GenBank (16). GCG (26), MEMSAT (50), and SAPS (13) programs were used for gene analysis. Published mRNA and cDNA data were compared to the Md5 genomic sequence using the Est_genome (ftp.sanger.ac.uk/pub/EMBOSS) and Sim4 (33) alignment programs.

Nucleotide sequence accession number.

The MDV1 Md5 genome sequence has been deposited in GenBank under accession no. AF243438.

RESULTS AND DISCUSSION

Genome organization.

The Md5 genome is 177,874 bp long and contains a 44% G+C base composition. Md5 is organized in the same overall manner as other alphaherpesviruses (75). Long and short unique regions (UL and US regions, respectively) are 113,563 and 10,847 bp in length, respectively. Each unique region is bounded by identical inverted repeats. The terminal and internal UL repeats (TRL and IRL, respectively) are 13,065 bp, and the internal and terminal US repeats (IRS and TRS, respectively) are 12,264 bp. As with other herpesviruses, the G+C content in the repeat regions is higher than that in the unique regions (49 to 50% in repeats versus 41 to 42% in unique regions) (25, 36, 96). Md5 does not contain the retroviral long terminal repeat sequences previously reported at the US/short repeat boundary of cell culture-passaged MDV1 strains (44, 47, 48).

Alphaherpesvirus α-type sequences are located at the genomic termini and at the IRL/IRS junction of Md5. They consist of 7 tandem copies of a 60-bp repeat associated with the long direct repeat, a 43-bp unique spacer sequence, and 64 tandem copies of a 6-bp repeat associated with the short direct repeat (28, 52, 53, 96). As previously reported, the 6-bp repeat is identical to repetitive sequences in the direct repeats of human herpesvirus 6 and in eukaryotic telomeres (53).

Gene characterization.

Md5 contains 338 ORFs encoding proteins of 60 or more amino acids of which 103 are likely to be functional genes (Table 1). Seventy-three genes are present as single copies and initiate within unique regions. Thirty genes initiate and are partially or completely located within repeat regions, including two genes within α-sequence regions. MDV004, MDV007, MDV075, MDV077, MDV086, and MDV098 ORFs were annotated here because previous reports indicated that these ORFs were protein coding (41, 67, 72). Most Md5 genes are virtually identical (99% nucleotide and amino acid identity) to published genes from other MDV1 strains and less similar to those from MDV2 (40 to 86% amino acid identity) and HVT (38 to 81% amino acid identity). Among nonavian herpesviruses, EHV1 and EHV4 are generally the most similar to Md5 (25 to 61% amino acid identity).

TABLE 1.

Md5 ORFs

ORF	Position (nt) (length [aa])a	MDV1		MDV2			HVT			Closest non-MDV species				HSV-1 homologue	Predicted structure and/or functionc
		Accession no.b	Reference	Accession no.b	Blast score	Length (aa) (% identity)	Accession no.b	Blast score	Length (aa) (% identity)	Named	Accession no.b	Blast score	Length (aa) (% identity)
MDV001	1517–2173 (219)
MDV002	2478–1885 (198)														Arg-rich protein
MDV003	3555–3492 (134)	AF065430	57							Mus musculus	P10889	153	81 (41)		CxC chemokine
	3316–3181
	3082–2881
MDV004	5878–6285 (136)	U55025	72												23-kDa nuclear protein
MDV005	6753–5737 (339)	M89471	49												MEQ protein
MDV006c	10719–10508														14-kDa lytic phase protein, C-terminal exon
MDV006b	11821–11755 (93)	M77343	41												14-kDa lytic phase protein, alternate b N-term. exon
MDV006a	13147–13105 (85)	L26394	41												14-kDa lytic phase protein, alternate a N-term. exon
MDV007	13392–13048 (115)	M82861	78
MDV008	13833–14297 (155)	D21060	109	AB024414	169	91 (41)									24-kDa phosphoprotein, pp24
MDV009	14338–13340 (333)	S72466	9
MDV010	14430–14535 (756)	S72466	9	AB024414	1,985	757 (53)				FAdV	AF007578	271	205 (31)		Lipase
	14701–16872
MDV011	17431–17685 (85)	S72466	9
MDV012	17828–18979 (384)			AB024414	822	384 (48)
MDV013	19172–19756 (195)	U04994	105	AB024414	532	180 (57)				SVHV	AF108378	123	130 (29)	UL1	Virion surface glycoprotein L
MDV014	19641–20579 (313)	U04994	105	AB024414	1,070	318 (64)				BHV-1	AJ004801	683	228 (56)	UL2	Uracil-DNA glycosylase
MDV015	20607–21290 (228)	U04994	105	AB024414	754	215 (71)				EHV-4	AF030027	551	202 (61)	UL3	Nuclear phosphoprotein
MDV016	22615–21812 (268)			AB024414	768	268 (53)				EHV-1	P28943	282	137 (43)	UL4	Nuclear protein
MDV017	25245–22672 (858)			AB024414	3,617	848 (80)				EHV-4	AF030027	2,766	857 (60)	UL5	DNA helicase-primase-associated protein
MDV018	25313–27478 (722)			AB024414	2,544	724 (68)				EHV-1	P28944	1,591	729 (46)	UL6	Minor capsid protein, DNA packaging
MDV019	27318–28232 (305)			AB024414	966	303 (59)				DEV	AF043730	634	279 (46)	UL7
MDV020	30578–28272 (769)			AB024414	2,621	765 (63)				EHV-4	AF030027	1,127	744 (35)	UL8	DNA helicase-primase-associated protein
MDV021	33117–30595 (841)	U28785	101	AB024414	3,073	875 (69)				EHV-1	P28947	2,089	838 (50)	UL9	Ori binding protein
MDV022	33216–34487 (424)	AF118111		AB021169	1,719	424 (77)				EHV-4	AF030027	656	415 (34)	UL10	Virion membrane glycoprotein M
MDV023	34798–34547 (84)	AF118111		AB024414	176	76 (52)				HSV-2	P13294	81	28 (42)	UL11	Myristylated tegument protein
MDV024	36348–34777 (524)	AF118111		AB024414	1,487	507 (56)				EHV-4	AF030027	780	492 (38)	UL12	DNase
MDV025	37880–36342 (513)			AB024414	1,751	512 (65)				EHV-1	P28966	851	461 (39)	UL13	Serine/threonine protein kinase, tegument
MDV026	38329–37601 (243)			AB024414	585	237 (54)				VZV	P09295	227	148 (35)	UL14	Minor tegument protein
MDV027	38360–39403 (737)			AB024414	3,086	736 (80)				EHV-1	P28969	2,301	735 (61)	UL15	DNA packaging protein (terminase)
	42885–44051
MDV028	40525–39446 (360)			AB024414	1,257	352 (66)				EHV-4	AF030027	543	321 (37)	UL16	Tegument protein
MDV029	42741–40555 (729)			AB024414	2,093	716 (57)				EHV-4	AF030027	877	713 (35)	UL17	Tegument protein, DNA packaging
MDV030	45132–44176 (319)			AB024414	1,412	320 (83)				EHV-1	P28921	754	307 (46)	UL18	Capsid protein
MDV031	49439–45261 (1,393)			AB024414	6,508	1,393 (86)	Z54369	5,503	1,393 (75)	EHV-1	P28920	4,209	1,360 (58)	UL19	Major capsid protein
MDV032	50447–49746 (234)			AB018252	687	228 (56)				EHV-1	P28971	260	200 (31)	UL20	Membrane protein, virus egress
MDV033	50708–52345 (546)			AB018252	1,506	544 (58)				EHV-1	AF030027	525	544 (32)	UL21	Tegument protein
MDV034	54936–52498 (813)	P36336	85	AB024414	2,451	808 (57)	S62554	2,360	806 (56)	CeHV-9	U25806	587	642 (26)	UL22	Envelope glycoprotein H
MDV035	56132–57043 (304)	A04086		AB024414	727	303 (50)				BHV-1	L39072	366	183 (44)	UL24
MDV036	56177–55122 (352)	P17653	84	D85421	1,426	350 (74)	P25987	1,030	342 (58)	EHV-4	AF030027	510	334 (36)	UL23	Thymidine kinase
MDV037	57141–58889 (583)	A04086		AB012137	2,274	581 (74)				EHV-1	P28928	1,292	582 (48)	UL25	DNA packaging
MDV038	58933–60921 (663)			AB012137	1,834	661 (58)				EHV-1	P28936	695	365 (42)	UL26	Capsid maturation protease, minor capsid scaffold protein
MDV039	59887–60921 (345)													UL26.5	Minor capsid scaffold protein
MDV040	63652–61058 (865)	P18538	77	AB024414	3,786	865 (82)	U01887	3,749	865 (81)	CHV	S72091		834 (54)	UL27	Virion membrane glycoprotein B
MDV041	66108–63730 (793)			AB024414	2,917	790 (71)				EHV-1	P28973	1,795	756 (48)	UL28	DNA packaging protein
MDV042	69875–66303 (1,191)			AB024414	5,154	1,191 (80)				EHV-1	P28932	2,943	1,209 (46)	UL29	Single-stranded DNA binding protein
MDV043	70144–73803 (1,220)	L40431	94	AB024309	4,674	1,220 (74)				EHV-1	P28858	3,346	1,211 (56)	UL30	DNA polymerase catalytic subunit
MDV044	74632–73733 (300)			AB024309	1,254	304 (75)				EHV-4	AF030027	847	258 (62)	UL31	Nuclear phosphoprotein
MDV045	76573–76974 (134)			AB024309	442	134 (64)				HSV-1	P10217	240	132 (43)	UL33	Role in DNA packaging
MDV046	76574–74652 (641)			AB024309	2,235	641 (68)				EHV-1	AF030027	1,223	626 (45)	UL32	Role in DNA packaging
MDV047	77070–77900 (277)			AB024309	885	269 (64)				HSV-1	P10218	459	183 (50)	UL34	Membrane phosphoprotein
MDV048	77987–78379 (131)			AB024309	449	127 (70)				BHV-1	Z78205	135	94 (39)	UL35	Capsid protein
MDV049	88471–78446 (3,342)			AB024309	7,666	3,339 (51)				EHV-1	P28955	3,120	2,865 (31)	UL36	Large tegument protein
MDV050	91826–88689 (1,046)			AB024309	3,396	1,046 (62)				EHV-4	AF030027	1,292	1,033 (31)	UL37	Tegument protein
MDV051	92197–93606 (470)			AB024309	1,578	470 (62)				EHV-1	M86664	817	451 (40)	UL38	Capsid protein
MDV052	93831–96296 (822)			AB024309	3,162	792 (76)				PRV	X72087	2,002	729 (55)	UL39	Ribonucleotide reductase, large subunit
MDV053	96352–97380 (343)			AB024309	1,350	323 (79)				EHV-1	P28847	1,026	320 (62)	UL40	Ribonucleotide reductase, small subunit
MDV054	98756–97434 (441)			AB012572	1,425	438 (65)				BHV-1	Z54206	762	458 (39)	UL41	Virion host shutoff protein, tegument
MDV055	99413–100519 (369)			AB012572	1,451	365 (74)	A51521	1,407	369 (69)	HSV-1	P10226	1,407	270 (31)	UL42	DNA polymerase processivity subunit
MDV056	100682–101941 (420)			AB012572	1,116	420 (54)	A51521	1,040	422 (49)	BHV-1	Z54206	144	270 (24)	UL43	Probable membrane protein
MDV057	102164–103666 (501)	P22651	42	AB012572	1,939	501 (72)	P18535	1,999	488 (75)	EHV-1	P12889	468	444 (27)	UL44	Virion membrane glycoprotein C
MDV058	104532–105164 (211)	P22653	42	AB012572	788	211 (70)	P18536	861	212 (73)	EHV-4	AF030027	112	154 (31)	UL45	Envelope/membrane protein, cell fusion
MDV059	107006–105303 (568)	L10283	103	AB012572	1,035	553 (42)				EHV-4	AF030027	511	413 (32)	UL46	Tegument phosphoprotein
MDV060	109574–107151 (808)	L10283	103	AB012572	1,793	807 (50)				EHV-4	AF030027	299	491 (25)	UL47	Tegument phosphoprotein
MDV061	111096–109816 (427)	L10283	103	AB012572	1,211	409 (60)	Z54368	1,195	422 (55)	EHV-4	Q00028	743	406 (43)	UL48	Immediate-early gene transactivator, tegument
MDV062	111953–111207 (249)	L10283	103	AB012572	592	249 (52)				VZV	P09272	228	182 (32)	UL49	Tegument phosphoprotein
MDV063	112366–113673 (436)			AB012572	1,383	389 (65)				EHV-1	P28892	311	161 (42)	UL50	dUTPase
MDV064	112386–112102 (95)	L10283	103	AB012572	342	95 (70)				ILT	Y14300	101	59 (32)	UL49.5	Envelope/tegument protein
MDV065	114513–113767 (249)			AB012572	624	181 (69)				EHV-1	P28961	337	184 (42)	UL51	Virion phosphoprotein
MDV066	114515–117736 (1,074)			AB021976	3,786	1,071 (66)				VZV	P09270	1,859	1,072 (39)	UL52	DNA helicase-primase-associated protein
MDV067	117718–118779 (354)	U10040	74	AB016433	1,218	351 (64)				EHV-1	P28933	456	345 (31)	UL53	Glycoprotein K
MDV068	118929–120347 (473)	U10040	74	AB016433	1,119	476 (54)				PRV	X87246	461	352 (34)	UL54	Posttranslational gene regulation
MDV069	121289–120483 (269)			AB016433	619	192 (61)	A51541	742	259 (54)
MDV070	121457–121954 (166)			AB016433	476	162 (56)	A51541	423	164 (49)	EHV-4	AF030027	186	162 (29)	UL55
MDV071	122897–122316 (194)						A51541	337	143 (46)	VZV	P09267	143	112 (34)		VZV orf2, EHV-4 gene 3
MDV072	126241–123533 (903)	D13389	78	AB016433	2,455	897 (54)	A51541	1,255	771 (40)
MDV073	127787–126918 (290)	P30023	21	S70347	287	157 (40)	L37202	115	72 (38)						38-kDa phosphoprotein, pp38
MDV074	128228–128572 (115)	M82861	78
MDV075a	128473–128515 (85)	L26394	41												14-kDa lytic phase protein, alternate a N-term. exon
MDV075b	129799–129865 (93)	M77343	41												14-kDa lytic phase protein, alternate b N-term. exon
MDV075c	130901–131112														14-kDa lytic phase protein, C-terminal exon
MDV076	134867–135883 (339)	M89471	49												MEQ protein
MDV077	135742–135335 (136)	U55025	72												23-kDa nuclear protein
MDV078	138065–138128 (134)	AF065430	57							Mus musculus	P10889	153	81 (41)		CxC chemokine
	138304–138439
	138538–138739
MDV079	139142–139735 (198)														Arg-rich protein
MDV080	140103–139447 (219)
MDV081	141149–141634 (162)														a sequence
MDV082	143447–143124 (108)
MDV083	150610–150945 (112)	L29643	56												Antisense RNA protein
MDV084	150769–143807 (2,321)	Q02362	5							EHV-1	P17473	1,009	1,296 (30)	RS1	Immediate-early gene transactivator, ICP4
MDV085	151396–151001 (132)
MDV086	151988–152248 (87)	L13604	67												Cytoplasmic protein
MDV087	153443–153979 (179)	L22174	14							FPV	P14362	210	95 (42)		MDV1 SORF2
MDV088	154149–154685 (179)	M80595	80	AB016432	417	171 (50)	X68653	237	120 (40)	FHV-1	D42113	195	140 (34)	US1	Immediate-early phosphoprotein, ICP22
MDV089	154978–155616 (213)	M80595	80	AB016432	612	214 (57)	A18267	430	185 (43)	FHV-1	D42113	229	178 (32)	US10	Virion protein
MDV090	156785–155733 (351)	L22174	80	AB016432	948	345 (52)	X68653	757	345 (46)						MDV1 SORF3
MDV091	157824–157015 (270)	PRF1710264A	76	AB016432	1,061	265 (72)	X68653	908	259 (66)	EHV-1	P28964	317	172 (40)	US2
MDV092	157936–159141 (402)	PRF1710264C	76	AB016432	1,168	388 (59)	A18267	1,201	386 (59)	CHV	U84223	595	351 (37)	US3	Serine/threonine protein kinase
MDV093	159254–159694 (147)	PRF1710264D	76												MDV1 SORF4
MDV094	159865–161073 (403)	U60532	14	S83367	937	339 (56)	X68653	831	347 (48)	BHV-1	A25176	237	210 (30)	US6	Membrane glycoprotein D
MDV095	161183–162247 (355)	L22174	14	D85420	829	346 (49)	X68653	610	334 (40)	FHV-1	S72415	208	255 (28)	US7	Membrane glycoprotein I
MDV096	162389–163879 (497)	L22174	14	D86926	1,034	442 (47)	X68653	1,121	493 (44)	EHV-4	AF030027	305	410 (26)	US8	Membrane glycoprotein E
MDV097	165001–164555 (149)	M80595	80							FPV	P14362	90	47 (40)		N terminus MDV1 SORF2
MDV098	166456–166196 (87)	L13604	67												Cytoplasmic protein
MDV099	167048–167443 (132)
MDV100	167675–174637 (2,321)	Q02362	5							EHV-1	P17473	1,009	1,296 (30)	RS1	Immediate-early gene transactivator, ICP4
MDV101	167834–167499 (112)	L29643	56												Antisense RNA protein
MDV102	174997–175320 (108)
MDV103	177295–176810 (162)														a sequence

^aaa, amino acids; nt, nucleotides. 

^bAccession numbers are from GenBank, SwissProt, or PRF databases. 

^cFunction was deduced either from the degree of amino acid similarity to products of known genes or by the presence of Prosite signatures. term., terminal. 

^dFAdV, fowl adenovirus; SVHV, simian varicella herpesvirus; BHV-1, bovine herpesvirus 1, DEV, duck enteritis virus; CeHV-9, cercopithecine herpesvirus 9; CHV, canine herpesvirus; ILT, infectious laryngotracheitis virus; FPV, fowlpox virus; FHV-1, feline herpesvirus 1. 

UL region.

The UL region, extending from nucleotide positions 14029 to 127591, contains 64 genes of which 38 have not been previously described (Fig. 1, Table 1). MDV013 to MDV070 genes, which represent 57% of the Md5 genome, are colinear with UL1 to UL55 genes of herpes simplex virus type 1 (HSV-1). Proteins encoded by these Md5 genes are 22 to 61% identical to HSV-1 homologues and 42 to 86% identical to MDV2 homologues. Capsid proteins MDV030, MDV031, and MDV048, DNA replication proteins MDV017, MDV021, MDV042, MDV043, MDV055, and MDV066, nuclear proteins MDV015 and MDV044, and glycoproteins MDV022, MDV040, and MDV057 encoded in the UL are highly conserved with homologues in MDV2 (66 to 86% amino acid identity). Tegument proteins (MDV023, MDV033, MDV049, MDV059, MDV060, and MDV062) and membrane proteins MDV032 and MDV056 are less conserved (42 to 65% amino acid identity). Viral enzymes MDV025, MDV052, MDV055, and MDV063 contain notable insertions and deletions compared to MDV2 homologues. MDV009, MDV010, MDV011, MDV012, MDV069, and MDV072 genes, located at the ends of the UL region, are absent in nonavian herpesviruses such as HSV and equine herpesvirus (EHV), suggesting a possible role for these genes in avian host range.

FIG. 1.

Linear map of the Md5 genome. Genes (colored arrows) are numbered from left to right based on position of methionine initiation codons. Genes and RNA transcript regions (black-and-white arrows) are transcribed in the directions indicated. Genomic regions are defined in the color key. Yellow boxes, regions of 132-bp repeats. Nucleotide positions are indicated above the map.

The MDV010 gene encodes a 684-amino-acid protein that is similar to other viral proteins and to known eukaryotic lipases (Fig. 2). The gene for the MDV010 homologue in MDV1 strain GA (MDV1 GA) has previously been shown to be spliced (9). The predicted protein contains the serine active site within the lipase signature motif (Prosite PS00120) (Fig. 2) and conserved cysteines involved in disulfide bond formation (amino acid positions 416 and 438). The region between amino acids 216 and 374 is similar to those in eukaryotic lipases such as phospholipase A1 and triacylglycerol lipase. Similarity to predicted proteins from MDV2 and fowl adenovirus extends beyond this region, suggesting the presence of virus-specific domains. The presence of a signal peptide in the amino-terminal domain and a transmembrane domain at amino acid positions 540 to 553 suggest that MDV010 may be membrane localized.

FIG. 2.

Multiple amino acid sequence alignment of MDV010 with lipases. Asterisks, conserved sites at lipase Prosite signature (PS00120); boldface, serine active sites; shaded residues, amino acid identity to MDV010. Amino acid positions are indicated on the right. Avianadeno, avian adenovirus 8, accession no. AF021254; squirrel, Spermophilus tridecemlineatus, accession no. AF027293; pig, Sus scrofa domestica, accession no. P00591; wasp, Vespula vulgaris, accession no. L43561; MDV2, accession no. AB024414; MDV1, Md5 isolate, MDV010.

Type A1 phospholipases have been identified in many mammalian tissues (platelets, liver, and heart) as membrane-bound or cytosolic enzymes which catalyze transacylation reactions (39, 64, 83). Phospholipase A1 activity on phosphatidic acid substrates may modify intracellular second-messenger pathways (39). Modification of host cell second-messenger pathways has been observed with other virus infections (1, 27, 87). Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) encodes a G protein-coupled receptor which activates phospholipase C and which stimulates cell proliferation and transformation (35). Both cytomegalovirus and adenovirus affect arachidonic acid metabolism through pathways involving phospholipase A2 (1, 27). Arachidonic acid is a precursor to prostaglandins, leukotrienes, and lipoxins, molecules which modify inflammatory responses. Altered lipid metabolism has been observed both in vivo and in cell cultures during MDV infection (32, 38). MDV010 may perform host range functions involving alteration of host lipid metabolism and/or modification of second-messenger signaling pathways.

US region.

The US region, extending from positions 153799 to 164645 (10,847 bp), contains nine genes (encoding MDV088 to MDV097), which include homologues of the HSV-1 US1, US2, US3, US6, US7, US8, and US10 genes. The MDV1 GA US region has previously been completely sequenced (11,160 bp), and the MDV1 RB1B US region has been partially sequenced (14, 76). Md5 ORFs are colinear with and virtually identical (>99% nucleotide identity) to ORFs from these two MDV1 strains. Md5 contains sequences at the US/TRS boundary (nucleotide positions 164033 to 164518 and 164636 to 165464) that are absent in MDV1 GA. Due to the expansion of the TRS, the Md5 US region is 313 nucleotides shorter than the US region of MDV1 GA. As has been previously reported, the arrangement of genes in the MDV1 US region differs from those of other alphaherpesviruses. The MDV089 gene (US10 gene homologue) is inverted and translocated compared to the US10 gene in HSV-1, and no homologues of HSV-1 US genes are located in the short repeat regions as they are in pseudorabies virus (PRV), EHV, and varicella-zoster virus (VZV) (14, 25, 62, 76, 96, 108).

The arrangement of genes in the Md5 US region is similar to that in the US regions of MDV2 and HVT (46, 106). Proteins encoded in the Md5 US region are 47 to 72% and 40 to 66% identical to their homologues in MDV2 and HVT, respectively. MDV090, previously described as SORF3, is unique to the avian herpesviruses MDV1, MDV2, and HVT (106). MDV093 (SORF4) is unique to MDV1 (14, 46, 106). Absence of the MDV093 gene in nonpathogenic MDV2 and HVT suggests a possible role in viral virulence.

Long repeats.

The long repeat regions are 13,065 bp located at nucleotide positions 964 to 14028 and 127592 to 140656. These repeat regions contain 16 genes, all of which are unique to MDV1. Proteins from three of these genes associated with cellular transformation include the Marek's EcoRI Q fragment protein (MEQ), which is present in two copies (MDV005 and MDV076), pp38 (MDV073), and pp24 (MDV008) (24, 79, 90, 102, 109).

MDV005 and MDV076 genes encode MEQ, a 339-amino-acid basic region leucine zipper protein (49). MEQ is a transcriptional transactivator and potential oncoprotein detected in MDV-induced tumors and cell lines and has been shown to induce and maintain transformed cell phenotypes, protect transformed cells from apoptosis, and colocalize with cyclin-dependent kinase 2 in a cell cycle-dependent manner (49, 58, 59, 73, 102). Compared to MEQ from MDV1 GA, MDV005 and MDV076 contain amino acid substitutions at positions Ala 217 within the second full proline-rich repeat, Val 283, and Thr 320. The MDV004 and MDV077 genes are antisense to the MEQ gene and homologues of an ORF previously shown to encode a 23-kDa nuclear protein expressed in MDV-transformed lymphoblastoid cells (72).

The MDV008 and MDV073 genes encode oncogenicity-related phosphoproteins pp24 and pp38, respectively (21). pp24 and pp38 are among the first MDV proteins expressed in MDV-induced tumors and are part of a phosphorylated protein complex present in MDV-induced lymphoblastoid cell lines (43, 65, 66, 89). These genes span the long repeat/UL boundary (60, 109). The two proteins share 65 amino acids at their amino termini, which are encoded in the long repeats, while their carboxyl termini are encoded at either end of the UL region (60, 109). Interestingly, these two proteins have carboxyl-terminal amino acid similarity that has not been previously described. Conserved amino acids include a DLLVEAE motif (amino acid positions 85 to 91 in MDV008 and 163 to 169 in MDV073) and a region of 30% amino acid identity (amino acid positions 93 to 155 of MDV008 and 213 to 275 of MDV073). MDV2 pp24 and pp38 homologues and an HVT pp38 homologue do not contain the amino-terminal domains present in MDV1 proteins (68, 91). Given that MDV2 and HVT are nononcogenic, the novel amino-terminal regions present in MDV1 homologues may play some role in viral virulence and/or oncogenicity.

The MDV003 and MDV078 genes are spliced genes with homology to genes encoding mammalian CxC chemokines (Table 1). This gene was previously described as encoding an IL-8 homologue; however, IL-8 activity was not demonstrated (57). The MDV003 and MDV078 genes each comprise three exons, and the proteins share 41% amino acid identity with murine macrophage inhibitory protein 2 (MIP-2) and contain the four cysteine residues necessary for disulfide bonding. The amino-terminal region, which defines receptor-binding specificity, is less similar to those of MIP-2 and other CxC chemokines than is the carboxyl-terminal region (23). Chemokines mediate immune cell activation and migration during inflammation (6). Chemokine homologues encoded by other herpesviruses have been shown to function as either agonists or antagonists (54). An MDV-encoded chemokine may function in immune evasion by affecting host inflammatory responses. Chemokines have also been associated with vasculopathologies such as atherosclerosis and transplant vascular sclerosis (97). Atherosclerotic lesions including proliferative changes in arteries have been observed in MDV-infected chickens (31). Conceivably, the MDV-encoded chemokine may be involved in MDV-associated atherosclerosis.

A family of 1.8-kb RNAs mapping to the long repeat region has been associated with oncogenicity (11, 12, 51, 78) (Fig. 1). Transcripts originate from the same promoter/enhancer regions as the pp24 and pp38 genes but are transcribed in the opposite orientation (11, 22, 24, 88). Loss of oncogenicity and altered transcription have been associated with an expanded number of 132-bp repeats (4 to >35 units) within this 1.8-kb RNA region (10–12, 34, 61, 78, 90). Complex patterns of bidirectional transcription, which both initiate and terminate within the 132-bp repeats, have been previously reported (22). Md5 contains a single pair of 132-bp repeats located at nucleotide positions 12282 to 12546 and 129074 to 129338 (Fig. 1). This finding is consistent with the number of repeats (one to three copies) present in other pathogenic MDV strains (11, 61, 78). Although there are no readily identifiable genes in this region based on our criteria, there are four ORFs of greater than 60 codons present. Three of these ORFs have been previously found in cDNA clones (41, 45, 71). We have annotated the alternatively spliced MDV006 and MDV075 genes based on the work of Hong and Coussens (41), who identified a 14-kDa protein from this region. Given the complex transcriptional patterns and alternate splicing which occur within this region, additional protein-coding sequences which have not been annotated here may be present (12, 22, 41, 71).

Short repeats.

The short repeat regions are 12,264 bp at nucleotide positions 141535 to 153798 and 164646 to 176909 and contain 12 genes (Fig. 1). The MDV084 and MDV100 genes encode homologues of the HSV-1 major immediate-early transactivating protein ICP4. These proteins, which contain 2,321 amino acids and comprise over 57% of the short repeat regions, contain a 900-amino-acid amino-terminal extension compared with ICP4 homologues of other herpesviruses. A similarly sized ICP4 homologue is present in HVT and MDV1 GA (5, 107). Md5 encodes homologues of two additional immediate-early proteins found in HSV-1: ICP27 (MDV068), which is essential for HSV-1 replication, and ICP22 (MDV088). Md5 lacks homologues of immediate-early proteins ICP0, which is nonessential for the replication of HSV-1 in cell culture, and ICP47, a host range protein which blocks HSV-1 antigen presentation (104).

The region containing the US/short repeat junction is variable in MDV1, MDV2, and HVT (14, 46, 106). Expansion of the Md5 short repeat compared to those of the MDV1 Md11 strain and less-virulent JM and Cu-2 strains has been previously noted by restriction enzyme analysis (48). The MDV087 and MDV097 genes span the US/short repeat boundary (Fig. 1). The products of these two genes have 119 identical amino-terminal amino acids that are encoded within the short repeat region. MDV087 was previously described as SORF2 in MDV1 GA (14). Unlike MDV087, SORF2 is encoded entirely within the US region of MDV1 GA (14). A homologue of MDV097 is absent in MDV1 GA. SORF2 has been shown to be nonessential for replication of MDV1 in cell culture, and it is absent in the nonpathogenic MDV2 (46, 69). MDV087 and MDV097 are similar to putative proteins from fowlpox virus and fowl adenovirus, suggesting an avian host range function for these proteins (2, 14, 69, 86). Thus, differences at the US/short repeat junction, including the presence of a second gene (encoding MDV097) similar to the SORF2 gene, may affect viral virulence and contribute to Md5's enhanced virulence.

A family of latency-associated transcripts (LATs) antisense to the ICP4 homologue has been described in MDV1 (19, 20, 55, 56, 63). Although the role of LATs in viral latency and cell transformation is poorly understood, a gene homologue of the MDV086 and MDV098 genes has been shown to encode a protein from this region (67). The MDV083 and MDV101 genes also have been annotated here as potential genes. Given the complex splicing of LAT transcripts within this region, additional protein-coding sequences may be present.

Conclusions.

MDV1 genome analysis confirms the structural and functional relatedness of MDV1 to other alphaherpesviruses in gene complement and organization, particularly with regard to genes involved in basic replicative functions. Novel DNA sequences in direct repeat regions and near unique/repeat junctions contain genes likely involved in virulence and host range. The complete Md5 genome provides a basis from which comparisons with MDV strains of lesser or greater virulence may be made, thus contributing to our overall understanding of pathogen-host interactions and the evolution of MDV virulence. Additionally, this information will permit the engineering of novel MDV1 vaccine viruses and expression vectors with enhanced efficiency and greater versatility.