Molecular detection and isolation of avian metapneumovirus in Mexico

Figures & data

Table 1. Seroprevalence of aMPV in pullets and breeder hens in the state of Jalisco, Mexico.

Type of chicken	Age (weeks)	Number of sera positive/number of samples examined (%)	Mean antibody titre (log2) ± standard deviation
Pullet	2	1/14 (7.1)	0.78 ± 2.93
Pullet	17	27/27 (100)	4.59 ± 1.27
Pullet	19	29/29 (100)	6.13 ± 3.05
Pullet	23	40/45 (88.8)	6.68 ± 1.10
Pullet	24	29/29 (100)	5.68 ± 0.89
Pullet	26.5	20/21 (95.2)	10.95 ± 2.67
Breeder hen	31	41/41 (100)	8.42 ± 2.60
Breeder hen	33	34/37 (91.8)	9.45 ± 3.15
Breeder hen	37 to 48	138/138 (100)	9.58 ± 3.10
Breeder hen	50 to 73	169/169 (100)	10.74 ± 2.58
Total		528/550 (96)

Table 2. Seroprevalence of aMPV in pullets and breeder hens in the state of Puebla, Mexico.

Type of chicken	Age (weeks)	Number of sera positive/ number of samples examined (%)	Mean antibody titre (log2) ± standard deviation
Pullet	3	0/16 (0)	0
Pullet	5	3/16 (18.7)	2 ±4.30
Pullet	7	6/14 (42.8)	5.64 ±6.77
Pullet	10	32/45 (71.1)	9.34 ±6.18
Pullet	12	15/15 (100)	11.86 ±0.80
Pullet	20 to 29	163/163 (100)	11.66 ±3.25
Breeder hen	30 to 36	97/97 (100)	11.37 ±3.21
Breeder hen	40 to 53	52/52 (100)	12.15 ±0.80
Total		368/418 (88.03)

Figure 1. Fitted linear and quadratic effects of week of age on the log₂ antibody titres of chickens analysed from the states of Jalisco and Puebla.

Figure 2. Biological properties in Vero cell cultures of prototype strain aMPV/A/chicken/Mx/Jal/2008. 2a: Mock-infected cells observed by phase contrast microscopy. 2b: Cultures infected with aMPV at 96 h post infection. 2c: Syncytia observed in cultures infected with aMPV at 96 h post infection. 2d: Mock-infected cells stained with crystal violet. 2e: Observation of large syncytia in cultures infected with aMPV at 96 h. 2f: Syncytia in cultures infected with aMPV at 96 h. 2g: Immunofluorescence of mock-infected cultures. 2h: Intracytoplasmic granular fluorescent staining in cultures infected with aMPV at 48 h post infection. 2i: Intracytoplasmic granular fluorescent staining in cultures infected with aMPV at 48 h post infection.

Table 3. Number of aMPV nRT-PCR-positive samples and positive virus isolates (identified by cytopathic effect and indirect immunofluorescence) in samples collected from chicken frocks in Jalisco and Puebla states, Mexico.

		Passage number in primary cell cultures				Followed by a passage number in Vero cell cultures
Test used	21 pooled samples of trachea examined	1	2	3	4	1	2	3	4
Number of positive samples by nRT-PCR	5/21^a							5/6^b	5/6^b
Number of positive samples by CPE		0/21	0/21	0/21	0/21	3/21	5/21	6/21^c	6/21^c
Number of positive samples by IF						5/21			5/21

Empty cells indicate that values were not determined.

^a Number of samples positive/number tested.

^b In these passages, only samples that were positive for CPE and IF were analysed by nRT-PCR.

^c One of the samples presented abnormal CPE in these passages and was not examined further.

Figure 3. Molecular phylogenetic analysis by the neighbour-joining method among aMPV G gene nucleotide sequences from Mexico (•), Brazil, China, Korea, France, the UK, Germany and the USA. Sequences include subtypes A, B, C, and D of aMPV. The name indicates the GenBank accession number and subtype, geographic origin, isolate number and year of isolation. The analysis involved 21 nucleotide sequences. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).

Table 4. Nucleotide sequences of the partial G gene (6352 to 6364 nucleotides) of different aMPV subtype A strains demonstrating that the virus isolated in this work is not a vaccine strain or a virulent strain derived from a vaccine strain.

aMPV strain	Sequence (5′ to 3′)
IT/Ty/A/259-01/03 (virulent strain from vaccine)	UGGUUUCGAUUGU
Vaccine 1^a	UGGUUUCGAUUGU
Vaccine 2^a	UGGUUUUGAUUGU
UK#8544 (vaccine progenitor strain)	UGGUUUUGAUUGU
TRTV/UK/CVL14/1/1988	UGGUUUUGAUUGU
TRTV/UK/1992	UGGUUUUGAUUGU
aMPV/A/chicken/BR/119/1995	UGGUUUUGAUUGU
aMPV/A/chicken/Mx/Jal/02/2008^b	UGGUUUUGAUUGU
aMPV/A/chicken/Mx/Jal/03/2008^b	UGGUUUUGAUUGU

^a Nucleotide sequences obtained from Lupini et al. (2011).

^b aMPV strains isolated in the present study.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 128, 2731–2739. 10.1093/molbev/msr121

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Lupini, C., Cecchinato, M., Ricchizzi, E., Naylor, C.J. & Catelli, E. (2011). A turkey rhinotracheitis outbreak caused by the environmental spread of a vaccine-derived avian metapneumovirus. Avian Pathology, 40, 525–530. 10.1080/03079457.2011.607428

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