Efficient Marek's disease virus (MDV) and herpesvirus of turkey infection of the QM7 cell line that does not contain latent MDV genome

Figures & data

Figure 1. Immunofluoresence and CPE of MDV (652)-infected QM7 foci. QM7 cells were infected with MDV 652 for 7 days in DMEM/F12 with 3% FBS. 1a: CPE of 652/QM7-infected foci. 1b: Infected cells were then fixed and stained with a mixture of monoclonal antibodies H19 (anti-pp38) and 1AN86 (anti-gB). The negative control, QM7 co-cultivated with DEF, stained completely negative.

Table 1. Titration of viral titres for 652/QM7.

FBS (%)	Split ratio	PFU per 2 × 10^6cells
3	1:2	1 × 10^4
3	1:5	1.3 × 10^4
3	1:10	2.5 × 10^4
10	1:2	2.3 × 10^2
10	1:5	6.3 × 10^2
10	1:10	2.5 × 10^2

Infected 652/QM7 cells were passaged once under two serum concentrations (3%, 10%) and three culture split ratios (1:2, 1:5, 1:10), and were titrated on fresh QM7 cells. Viral titres were determined by both CPE and indirect immunofluorescent antibody analysis (staining with a mixture of anti-pp38 and anti-gB monoclonal antibodies).

Figure 2. Immunofluoresence and CPE of MDV (652)-transfected QM7 cells. 2a: QM7 cells were transfected with DNA isolated from 652/DEF. Infected foci were seen 7 days post transfection. 2b: Infected cells were then fixed with 80% acetone and stained with a mixture of monoclonal antibodies H19 (anti-pp38) and 1AN86 (anti-gB).

Figure 3. Immunofluoresence and CPE of HVT-infected QM7 foci. QM7 cells were infected with HVT vaccine (MD-Vac; Zoetis, formerly Fort Dodge Animal Health, Fort Dodge, IA, USA) for 7 days in DMEM/F12 with 3% FBS. 3a: CPE of HVT/QM7-infected foci. 3b: Infected cells were then fixed and stained with monoclonal antibody L78. The negative control, QM7, stained completely negative.

Table 2. Different doses of 652/QM7 and 652/DEF that cause in vivo pathogenicity.

Sample	PFU	Mortality	Lesions
DEF+QM7	–	0/30 (0%)	0/30 (0%)
652/QM7	1250	26/30 (87%)	29/30 (97%)
	125	22/30 (73%)	25/30 (83%)
	12.5	8/30 (27%)	20/30 (67%)
652/DEF	2000	27/30 (90%)	30/30 (100%)
	200	28/30 (93%)	28/30 (93%)
	20	23/30 (77%)	29/30 (97%)

Various doses of 652/QM7 and 652/DEF infected cells were inoculated intraperitoneally into 1-day-old chicks. Mortality and MDV-related key clinical signs were recorded. All birds including birds surviving at 14 days post inoculation were necropsied and examined for signs of MDV infection, including microscopic nerve lesions and nodules on internal organs.

Table 3. Virus re-isolation and pathogenicity following in ovo injection.

Viral re-isolation
Virus	Cells	Day 3	Day 6	Day 13^a	Pathogenicity
HVT	QM7	+	+	−	−
MDV (652)	QM7	+	+	+	+
MDV (652)	DEF	+	+	+	+

Commercial broiler chicken eggs were injected on embryonic day 18 with 5000 PFU of either MDV (652) or HVT grown in QM7 or DEF cells. On days 3, 6, and 13 post hatch, blood and spleen cells were collected from the birds. On day 13, all remaining birds were euthanized and examined for lesions characteristic of Marek's disease to assess pathogenicity. +, re-isolation of virus in both PBMCs and spleen cells, or the presence of Marek's disease lesions at necropsy; −,no virus was found in either PBMCs or the spleen sample, or the absence of visible MDV lesions. ^aDay 13 viral re-isolation sensitivity might have been compromised by multiple freeze/thaw cycles prior to testing.

Figure 4. PCR analysis of potential presence of latent MDV genome in avian cells. PCR primers for various MDV gene sequences were used in PCR analysis: ICP4, LAT, ICP22, ICP27, meq, pp38, VP16, ORF L1, and gB. PCR was carried out with 2x JumpStart RED Taq (Sigma). The amplified fragments were resolved by electrophoresis in 2% agarose E-Gels (Invitrogen).