Feline Model of Acute Nipah Virus Infection and Protection with a Soluble Glycoprotein-Based Subunit Vaccine

Bruce A. Mungall; Deborah Middleton; Gary Crameri; John Bingham; Kim Halpin; Gail Russell; Diane Green; Jennifer McEachern; L. Ian Pritchard; Bryan T. Eaton; Lin-Fa Wang; Katharine N. Bossart; Christopher C. Broder

doi:10.1128/JVI.01619-06

DOI: 10.1128/JVI.01619-06

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FIG. 1.
NiV susceptibility study body temperatures and respiration rates. (A) Rectal temperatures obtained at various days postinfection in cats inoculated with either 500 TCID₅₀ of NiV (circles) or 5,000 TCID₅₀ of NiV (squares). All cats became febrile 6 to 8 days postinfection. (B) Respiration rates determined for cats infected with either 500 TCID₅₀ NiV (circles) or 5,000 TCID₅₀ NiV (squares). Immediately prior to euthanasia, all cats exhibited a marked increase in respiration rate.
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FIG. 2.
Histopathology and immunohistopathology associated with NiV infection in cats. (A) Necrotizing alveolitis (arrows) in NiV-infected cat and endothelial syncytial cell (arrowhead) (HE). (B) Broncho-alveolitis in NiV-infected cat, with bronchiolar epithelial syncytial cells (arrows) and intraluminal debris (arrowhead) (HE). (C) Positive staining for NiV antigen in regions of alveolitis, including endothelial syncytial cells (arrows) (anti-NiV polyclonal antibody). (D) Positive staining for NiV antigen in bronchiolar epithelial cells (arrows) and intraluminal debris (arrowhead) (anti-NiV polyclonal antibody). Scale bar for all images = 50 μm.
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FIG. 3.
NiV genome in cats detected by TaqMan PCR. Normalized, relative NiV genome levels in samples collecting during NiV infection in cats and at necropsy. TaqMan PCR C_T values were determined in triplicate for the NiV genome and normalized by dividing this value by the 18S rRNA C_T values for each sample. The relative NiV genome was determined by linear regression of NiV cDNA standard curves for each assay. Values are expressed as the average of all replicates. The adrenal gland, liver, lung, spleen, and lymph nodes consistently displayed the highest relative NiV genome levels, while the brain and heart frequently revealed the lowest. The genome was detectable in the blood in all cats 1 day prior to euthanasia but only detectable in the urine from cats infected with 5,000 TCID₅₀ of NiV.
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FIG. 4.
Temperature recordings of naive and sG-immunized cats infected with NiV. (A) Two cats in the vaccine efficacy study were implanted with radiotelemetry temperature transmitters prior to NiV inoculation (arrow). Cat 10 became febrile (>39°C) 7 days postinfection, while cat 9 remained clinically normal until day 11, becoming febrile on day 12, and was euthanized on day 13. Rectal temperatures were recorded during sampling periods and are indicated for each cat (circles). (B) Three cats showed gradual but slight increases in temperature after NiV infection (arrow), while the fourth showed a gradual decrease over the course of the study. All immunized cats remained within normal physiological parameters up to 24 days p.i.

Tables

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TABLE 1.

Normalized relative NiV genome levels detected by TaqMan PCR in NiV-infected naive cats^a

Sample	Location	dpi	Relative NiV genome level in cat (inoculum size)^b:
Sample	Location	dpi	1 (500)	2 (5,000)	3 (5,000)	4 (500)
Adrenal gland		PM	1,083.866	0.282	369.042	3.461
Bladder		PM	0.067	32.387	NS	0.009
Blood		0	0.000	0.000	0.000	0.000
		2	0.000	0.000	0.000	0.000
		3	0.000	0.000	NS	NS
		4	NS	NS	0.000	0.000
		5	0.000	0.000	NS	NS
		6	NS	NS	0.854	0.000
		7	NS	NS	0.786	NS
		8	0.165	0.125	NS	0.628
		9	0.237	0.599	NS	0.768
Brain	Occipital lobe	PM	0.172	0.000	0.622	16.624
	Olfactory lobe	PM	0.000	0.411	0.578	0.251
Heart		PM	0.000	1.669	7.512	0.000
Kidney		PM	18.974	24.599	22.421	0.000
Liver		PM	0.842	0.001	0.334	18.679
Lung	Apical lobe	PM	1,094.862	NS	973.303	1,480.818
	Diaphragmatic lobe	PM	13.143	432.096	2,027.956	1,124.226
Lymph node	Thoracic	PM	1.695	NS	NS	NS
	Prescapular	PM	NS	NS	486.245	NS
Skeletal muscle		PM	NS	NS	0.001	NS
Spleen		PM	890.187	2,438.948	1,667.516	628.465
Swab		0	0.000	0.000	0.000	0.000
		2	0.000	0.000	0.000	0.000
		4	0.000	NS	0.000	0.000
		5	NS	0.000	NS	NS
		6	NS	NS	0.010	NS
		7	NS	NS	0.798	NS
		8	0.000	0.000	NS	NS
		9	0.000	0.311	NS	0.017
Urine		0	NS	0.000	NS	NS
		3	NS	0.000	NS	NS
		7	NS	NS	0.018	NS
		8	NS	0.000	NS	NS
		9	0.000	1.977	NS	0.000
Uterine horn		PM	NS	0.176	NS	NS

↵ a Relative values were determined by linear regression analysis using NiV cDNA standards and normalized using the ratio of NiV:18S rRNA C_T values. PM, postmortem tissue; dpi, days postinfection; NS, no sample. Values are expressed as the fold difference from 3.6 pg of NiV cDNA standard and are the average of all replicates. Values in boldface indicate samples from which virus was isolated.
↵ b The NiV inoculum (TCID₅₀) for each cat is indicated in parentheses.

TABLE 2.

Serum neutralization titers in naive and sG-immunized cats

Cat no. (immunogen)	SNT^a
	HeV at:		NiV at:
	2 wk	2 mo	2 wk	2 mo
5 (NiV sG)	1:1,280	1:640	1:20,480	1:20,480
6 (HeV sG)	1:20,480	1:20,480	1:20,480	1:2,560
7 (HeV sG)	1:20,480	1:20,480	1:20,480	1:10.240
8 (NiV sG)	1:2,560	1:1,280	1:20,480	1:20,480
9 (control)	ND	<1:20	ND	<1:20
10 (control)	ND	<1:20	ND	<1:20

↵ a Serum neutralization titers (SNT) were determined from serum samples at 2 weeks and 2 months after the final immunization with sG. SNTs were determined for both HeV and NiV. ND, not determined.

TABLE 3.

Normalized relative NiV genome levels in naive and sG-immunized cats^a

Sample	Location	dpi	Relative NiV genome level in cat (immunogen):
Sample	Location	dpi	5 (NiV sG)	6 (HeV sG)	7 (HeV sG)	8 (NiV sG)	9 (control)	10 (control)
Adrenal gland			0.000	0.003	0.000	0.000	10.084	709.758
Bladder			0.000	0.000	0.000	0.000	18.265	1.218
Blood		0	0.000	0.000	0.000	0.000	0.000	0.000
		6	0.000	0.000	0.000	0.000	0.000	1.146
		8	0.000	0.000	0.000	0.000	0.000	0.706
		10	0.000	0.000	0.000	0.000	0.108	NS
		13	NS	NS	NS	NS	0.561	NS
		24	0.000	0.000	0.000	0.000	NS	NS
Brain	Occipital lobe	PM	0.000	0.001	0.000	0.000	0.087	1.286
	Olfactory lobe	PM	0.005	0.000	0.000	0.000	0.018	1.095
Heart		PM	0.000	0.003	0.000	0.000	0.061	0.033
Kidney		PM	0.000	0.000	0.000	0.000	12.957	43.692
Liver		PM	0.000	0.000	0.000	0.000	41.321	2.419
Lung	Apical lobe	PM	0.000	0.000	0.000	0.000	851.193	2,158.535
	Diaphragmatic lobe	PM	0.000	0.075	0.000	0.000	868.615	1,874.764
Lung exudate		13	NS	NS	NS	NS	1.465	NS
Lymph node	Thoracic	PM	0.000	0.000	0.000	0.000	2,030.773	NS
	Retropharyngeal	PM	NS	NS	NS	NS	NS	31.537
Ovary		PM	0.000	0.000	0.000	0.000	51.300	NS
Serum		6	0.000	0.000	0.000	0.000	0.000	0.000
		8	0.000	0.000	0.000	0.000	0.000	0.068
		10	0.000	0.000	0.000	0.000	0.000	NS
		13	NS	NS	NS	NS	0.254	NS
		24	0.000	0.000	0.000	0.000	NS	NS
Spleen		PM	0.000	0.000	0.000	0.000	1,630.840	890.055
Swab		6	NS	NS	NS	NS	0.000	0.000
		8	NS	NS	NS	NS	0.000	0.311
		10	0.000	0.000	0.000	0.000	0.000	NS
		13	NS	NS	NS	NS	0.055	NS
		24	0.000	0.000	0.000	0.000	NS	NS
Urine		6	NS	NS	NS	NS	0.000	NS
		8	NS	NS	NS	NS	0.000	4.708
		10	0.000	0.000	0.000	0.000	2.897	NS
		13	NS	NS	NS	NS	13.914	NS
		24	0.000	0.000	0.000	0.000	NS	NS
Uterine horn		PM	0.000	0.000	0.000	0.000	33.063	NS

↵ a Relative values were determined by linear regression analysis using NiV cDNA standards and normalized using the ratio of NiV:18S rRNA C_T values. PM, postmortem tissue; dpi, days postinfection; NS, no sample. Values are expressed as the fold difference from 3.6 pg of NiV cDNA standard and are the average of all replicates. Values in boldface indicate samples from which virus was isolated.