The pathogenesis of a 2022 North American highly pathogenic clade 2.3.4.4b H5N1 avian influenza virus in mallards (Anas platyrhynchos)

Figures & data

Table 1. Treatment group descriptions.

Dose^a	Exposure route	Description	Number of ducks
2	Intra-choanal	Infectious dose	5
2	Contact exposure^b	Evaluation of transmission	3
4	Intra-choanal	Infectious dose	5
4	Contact exposure	Evaluation of transmission	3
6	Intra-choanal	Infectious dose	5
6	Contact exposure	Evaluation of transmission	3
6	Intra-choanal	Pathogenesis	10
None	Not applicable	Controls	10

^a Log₁₀ 50% egg infectious doses per bird.

^b Contact exposure birds were housed with their respective dose group 24 h after inoculation.

Table 2. Number of mallards inoculated with A/AMWI/SC/345/22 H5N1 HPAIV shedding virus and mean titre equivalents based on real-time RT-PCR by dose and day post-challenge.

Dose	Swab type^a	Route of exposure^b	Day post-challenge
			2		4		7		10		14
			Number^c	Mean titre^d	Number	Mean titre	Number	Mean titre	Number	Mean titre	Number	Mean titre
1.8	OP	ICH	5/5 (100)	5.3 ± 0.6	5/5 (100)	4.8 ± 0.6	1/4 (25)	2.0 ± 1.3	1/4 (25)	2.1 ± 0.3	1/4 (25)	2.1 ± 0.3
		Contact	3/3 (100)	3.6 ± 0.5	3/3 (100)	5.6 ± 0.4	3/3 (100)	3.1 ± 0.7	0/2 (0)	N/A	0/2 (0)	N/A
	CL	ICH	5/5 (100)	4.7 ± 2.0	5/5 (100)	5.3 ± 0.7	2/4 (50)	2.3 ± 0.5	3/4 (75)	2.8 ± 0.8	1/4 (25)	2.1 ± 0.3
		Contact	2/3 (66.6)	2.5 ± 0.6	3/3 (100)	4.3 ± 0.5	2/3 (66.6)	2.8 ± 1.2	2/2 (100)	3.0 ± 1.0	2/2 (100)	2.5 ± 0.2
3.8	OP	ICH	5/5 (100)	5.4 ± 1.1	5/5 (100)	5.8 ± 0.4	2/3 (66.6)	2.6 ± 0.9	0/3 (0)	N/A	0/3 (0)	N/A
		Contact	3/3 (100)	5.4 ± 0.7	3/3 (100)	5.8 ± 0.4	1/2 (50)	2.8 ± 1.2	1/2 (50)	2.1 ± 0.3	1/2 (50)	2.1 ± 0.3
	CL	ICH	5/5 (100)	4.0 ± 1.0	5/5 (100)	5.5 ± 1.1	3/3 (100)	3.6 ± 0.5	2/3 (66.6)	2.5 ± 0.5	2/3 (66.6)	2.5 ± 0.8
		Contact	3/3 (100)	5.0 ± 0.9	3/3 (100)	5.8 ± 1.4	2/2 (100)	3.4 ± 0.5	1/2 (50)	2.1 ± 0.3	1/2 (50)	2.1 ± 0.3
5.8	OP	ICH	15/15 (100)	5.9 ± 0.6	13/13 (100)	5.1 ± 0.6	5/9 (55.5)	2.6 ± 0.7	1/9 (11.1)	2.0 ± 0.2	1/9 (11.1)	2.0 ± 0.2
		Contact	3/3 (100)	5.2 ± 0.1	3/3 (100)	5.8 ± 0.2	3/3 (100)	3.7 ± 1.3	1/3 (33.3)	2.3 ± 0.6	3/3 (100)	3.2 ± 0.7
	CL	ICH	15/15 (100)	5.0 ± 1.2	13/13 (100)	4.9 ± 1.2	4/9 (44.4)	2.6 ± 1.3	6/9 (66.6)	2.6 ± 0.6	6/9 (66.6)	2.5 ± 0.5
		Contact	3/3 (100)	4.6 ± 0.7	3/3 (100)	4.5 ± 0.6	2/3 (66.6)	3.8 ± 1.8	1/3 (33.3)	2.3 ± 0.6	3/3 (100)	3.4 ± 0.8
Total	OP	Both	34/34 (100)	5.7 ± 0.7	32/32 (100)	5.4 ± 0.6	15/24 (63)	2.8 ± 0.9	4/23 (17)	2.0 ± 0.3	6/23 (26)	2.2 ± 0.6
	CL	Both	29/34 (85)	4.8 ± 1.1	28/32 (88)	5.1 ± 1.2	15/24 (63)	3.1 ± 1.3	15/23 (65)	2.5 ± 0.5	15/23 (65)	2.6 ± 0.7

^a OP = oropharyngeal; CL = cloacal.

^b ICH = intrachoanal inoculation; contact = contact exposed by co-housing with ICH-inoculated ducks 24 h post-challenge.

^c Number positive/total ducks (percent positive).

^d Log₁₀ 50% egg infectious dose titre equivalent determined by quantitative real-time RT-PCR ± standard deviation. A value of 50% of the assay limit of detection (1.9log₁₀) was imputed for samples where no virus was detected as per the method of Cohen (Cohen, 1959).

Figure 1. Titre equivalents of A/American wigeon/SC/000345-01/2022 H5 HPAIV in oral or cloacal swabs by dose group, exposure route and day post-challenge or exposure as determined by quantitative real-time RT-PCR. Thick horizontal bars denote means, thin bars denote standard deviation. The horizontal dotted line is the assay limit of detection. Samples where no virus was detected were given a value of 50% of the assay limit of detection.

Table 3. Distribution of AIV antigen in tissues collected from mallards experimentally infected with A/American Wigeon/South Carolina/000345-001/2022 H5N1 HPAIV. Tissues were collected at 2 and 4 days post-challenge (DPC). The scoring is expressed in the form of bird 1/bird 2 for each DPC. No viral staining was found in the proventriculus, duodenum, caecal tonsils, kidney, and gonads.

Days post-challenge	Detection of AIV antigen in tissues
	Nasal epithelium	Salivary glands	Trachea	Lung	Eyelid	Brain	Eye	Liver	Adrenal gland	Pancreas	Spleen	Thymus	Cloacal bursa	Skeletal muscle
2	++/+++^a	−/−	+/+	++/+	++/+	+++/−	NA/NA	+/+	−/−	−/−	+/+	+/+	+/++	+/−
4	+++/+++	−/+++	++/na	++/+	++/++	+++/+++	+++/NA	++/+	++/−	+/+	+/+	+++/++	+/++	+++/+++

^a NA = not available; (+++) widespread staining, (++) common staining, (+) few stained cells, (−) no staining.

Table 4. Titre equivalents (50% egg infectious dose [EID₅₀]) by rRT-PCR of virus in tissues from ducks inoculated with A/American Wigeon/South Carolina/22-000345-001/2022 2 and 4 days post-challenge (DPC).

Tissue	Log10 EID50 titre equivalent per ml
	2 DPC		4 DPI
	Inoculate 1	Inoculate 2	Inoculate 3	Inoculate 4	Control 1	Control 2
Lung	ND^a	3.4	4.2	2.3	ND	ND
Spleen	5.9	4.9	5.0	4.9	ND	ND
Heart	6.5	ND	ND	6.2	ND	ND
Muscle	6.5	3.5	7.7	7.4	ND	ND
Brain	7.7	ND	ND	9.3	ND	ND

^a ND = not detected.

Figure 2. Immunohistochemical staining for AIV antigen in tissues of mallards infected with the A/American Wigeon/SC/000345-01/2022 H5N1 HPAI virus. Virus antigens are stained red. A, B, and C: tissues from ducks at 2 DPC. D-I: tissues collected from ducks at 4 DPC. A. Cerebellum. B. Lung. C. Nasal epithelium. D. Cerebrum. E. Eye ciliary process. F. Feather follicles. G. Thymus. H. Heart. I. Skeletal muscle. Magnification 20×.

Cohen, A.C. (1959). Simplified estimators for the normal distribution when samples are singly censored or truncated. Technometrics, 1, 217–237.

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