Genetic and serologic surveillance of canine (CIV) and equine (EIV) influenza virus in Nuevo León State, México

Ethics statement

All animal experiments were approved by the Animal Research and Welfare Ethics Committee (CEIBA-2018-024) of the Laboratory of Immunology and Virology of the College of Biological Sciences (FCB), Universidad Autónoma de Nuevo León (UANL) and the sampling was made under the indications of the NOM-062-ZOO-1999. Informed consent was obtained from the owners of the animals for the collection of additional information.

Study area and collection of samples

The samples were obtained in the period between March of 2013 and December of 2015 from nine municipalities (Apodaca, Cadereyta Jimenez, Escobedo, Guadalupe, Juarez, Montemorelos, Monterrey, San Nicolas de los Garza and Zuazua) of Nuevo Leon, Mexico. The College of Veterinary Medicine and Zootechnics (FMVZ), UANL sampled domestic dogs with acute respiratory symptoms. Of this dog population, 58 nasal swabs, five lung necropsies and five conjunctival swabs were obtained. For the canine serological study, 123 samples were collected from stray dogs in Guadalupe and Juarez, Nuevo Leon. None of the dogs had a travel history and none had been vaccinated against CIV. Samples (123 sera and eight nasal swabs) were also collected from trash service horses. Swabs and necropsies were kept in Minimum Essential Medium additioned with antibiotic–antimycotic Gibco^® (Thermo Fisher Scientific, Waltham, MA, USA) 2% and stored at −70 °C until use. Blood samples were obtained from venipuncture and centrifuged at 3,000 rpm to obtain the serum, which was stored at −20 °C until use.

Sample collection

Samples of necropsies and nasal and conjunctival swabs were used to obtain viral RNA. The sampling frame was the presence of respiratory symptoms characteristic of CIV and EIV in the populations analyzed. Analysis of seroprevalence in stray dogs and trash service horses was performed on randomly selected samples.

RNA isolation and M gene amplification

Viral RNA was purified from lung necropsies, nasal and conjunctival swabs using AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit (AxygenBioscience, Union City, CA, USA) following the manufacturer’s protocol. Coding region of 756 bp of M gene was obtained by SuperScript^® III One-Step RT-PCR system with Platinum^® Taq High Fidelity (Thermo Fisher Scientific, Waltham, MA, USA) using M specific primers (5′-CACGGATCCAAGATGAGTCTTCTAACCGAC-3′ and 5′-CACGTCGACAGGATCACTTGAATCGCTGCA-3′). Subsequently, a second PCR was performed using oligonucleotides recommended by the World Health Organization (2009) for Influenza A virus detection (5′-ATGAGYCTTYTAACCGAGGTCGAAACG-3′ and 5′-TGGACAAANCGTCTACGCTGCAG-3′) obtaining a 244 bp PCR product. RNA from the vaccines Fluvac Innovator Pfizer Equine Influenza Vaccine (A2/Kentucky/97) and Vanguard Plus 5 L4 Vaccine were used as controls. Amplification product was visualized on a 1.5% agarose gel and then sequenced to confirm its genetic identity.

Sequence analysis and phylogenetic tree

Products of the expected size (244 bp), were purified using Wizard^® SV Gel and PCR clean-up system (Promega, Madison, WI, USA) and directly sequenced by an automated system (Perkin Elmer/Applied Biosystem, Foster City, CA, USA) at the Institute of Biotechnology of the Universidad Nacional Autónoma de México (IBT-UNAM). The sequence analysis was done using CLUSTAL W/BioEdit sequence alignment v7.0 (Hall, 1999) and MEGAX software (Kumar et al., 2018). The partial sequences of M1 obtained in this study were submitted to the GenBank database under accession numbers MH463596, MH463597, MH463598, MH463599, MH463600, MH463601 and MH463602. The phylogenetic tree contains sequences corresponding to the M1 matrix gene of Influenza A virus obtained from the Influenza Virus Resource (NCBI) database. Zoonotic reassortants: MK690088, CY050848, MG254077, GU471689; Contemporary isolates: AY737298, HQ237603, MF173290, MH363692, MK690096 and Ancestral strains of CIV and EIV: CY109269, GU433346, DQ124160, CY067361, DQ222916, CY028837, Outgroup: CY125948 and KR077935. Maximum likelihood Jones–Taylor–Thornton model was performed to the construction of the phylogenetic tree.

M1 recombinant matrix protein

A feces sample of a wild bird positive for Influenza A virus (kindly provided by Dr. Jose Ignacio Gonzalez Rojas of the Laboratory of Ornithology, College of Biological Sciences, UANL) was processed to obtain the coding sequence of the M1 gene. The sequence whose identity coincided 100% with the matrix gene present in subtype H1N1/2009 was subcloned into the expression vector pET-28a (+) (Promega, Madison, WI, USA). Protein expression was carried out in competent Escherichia coli BL21 bacteria with pET-28 (+) and IPTG expression system. Bacteria with 0.4 OD (600 nm) of biomass without IPTG was taken before induction as negative control. Subsequently, the expression was induced and after 5 h of incubation the protein was purified with an OD of 1.6. Bacteria lysis was carried out by lysozymes and sonication. Purification by HisTrap™ HP columns (GE Healthcare, Chicago, IL, USA) under denaturing conditions with urea (4 M) was performed. The quantification of the purified protein fractions was performed by a semi quantitative–qualitative method comparing band intensity in the SDS-Page, using bovine albumin fractions as control. The purified protein was analyzed by Western blot to confirm its identity using a monoclonal antibody against influenza virus M1 protein (Abcam, Cambridge, UK).

Polyclonal antibodies

New Zealand White rabbits were immunized subcutaneously with recombinant matrix protein at six different points. For the primary immunization, 1 mg of matrix protein was administered with Freund’s complete adjuvant. For booster immunizations, 0.5 mg of protein was administered with Freund’s incomplete adjuvant 15 days after the primary immunization and once more 2 weeks after that (Leenaars & Hendriksen, 2005). Blood was collected 15 days after the final immunization and sera was obtained. The conditions of animal protection were followed by NOM-062-ZOO-1999.

Sandwich ELISA assay

Microtiter plates were coated with dog/horse antibodies diluted in carbonate–bicarbonate buffer pH 9.6 and incubated overnight at 4 °C. After two washes with phosphate-buffered saline (PBS), the plates were blocked with 5% nonfat dry milk in PBS at 37 °C for 1 h. After two washes, recombinant matrix protein was added at a concentration of 40 µg/mL and incubated at 37 °C for 1 h. The anti-M1 antibody (1:7,000) produced in rabbit was added and incubated at 37 °C for 1 h after two washes. Peroxidase-conjugated protein A (Amersham, Buckinghamshire, UK) was added and incubated at 37 °C for 1 h, after two washes. The plates were then washed two times and the ABTS (2,2′-azino-bis (3-ethylbenzothuazoline-6-sulphonic acid)) peroxidase substrate (KPL, Gaithersburg, MD, USA) was added and incubated at 37 °C for 30 min. The absorbance at 405 nm was measured using an automatic microplate reader (Digital and Analog Systems, Roma, Italy). Human serum positive for Influenza H1N1/2009 and fetal bovine serum were used as a positive and negative control, respectively. The cutoff value was defined as the mean of the negative control OD 405 nm values plus three standard deviations.

Matrix gene amplification of Influenza A virus

Of the total samples, the matrix gene was amplified in 13 (19.11%) nasal swabs, two (2.94%) conjunctival swabs and five (7.35%) lung necropsies, giving a total of 20 (29.41%) positive samples in dog population. Meanwhile, six (75%) positive samples of equine nasal swab were amplified. All viruses detected were isolated from unvaccinated animals (Table 1).

Sequence analysis of matrix gene

Amplicons of 244 bp corresponding to the partial sequence of the matrix gene (M) from canines and equine samples were sequenced. Nucleotide alignments of the analyzed sequences showed a 96–99% identity with sequences of M1 gene of Influenza A virus. These isolates were compared with matrix sequences of representative strains of clade 1 from American lineage (Florida): A/eq/Miami/63, A/eq/Wisconsin/03 and A/canine/Colorado/06, presents 2–6 amino acid substitutions (97.18–99.15% identity). This comparison showed genetic variability in the coding region between amino acids 10 and 80 of M1, observing greater genetic variability in the canine sequences than in the equine sequences (Fig. 1).

The phylogenetic analysis of the sequences shows proximity with zoonotic and potentially pandemic strains such as the H3N2, H1N2 and H1N1/2009. Sequences of equine, canine and avian origin were included to determine the genetic divergence in the partial sequence of the M gene. In this analysis, it is evidenced how the sequences obtained in this study (Mexican isolates) possess a genetic identity of 95.78–97% with matrix sequences of H3N2 and H1N1 isolates obtained from domestic cats, as well as matrix sequences from isolates of the pandemic strain H1N1/2009 from swine and humans (Zoonotic reassortants). Mexican isolates also have an identity of 92.96–98.6% with avian, equine and canine contemporary strains. Partial sequences of the M gene from the ancestral strain of H3N2 and H3N8 of CIV and EIV were included from GenBank, obtaining an identity of 91.55–97.19% (Fig. 2).

Seroprevalence and symptomatology

A total of 123 sera from canines and 123 sera from equines obtained from nine municipalities were tested by Sandwich enzyme-linked immunosorbent assay (ELISA) system. Polyclonal antibodies against recombinant Influenza A matrix protein were founded in the 123 (100%) sera in canines. At least 21.46% of canine population had been vaccinated against other respiratory viral agents. Meanwhile, 114 (92.68%) of equines presented polyclonal antibodies against Influenza virus. Vaccine information in equines was only reported against tetanus toxoid.

The population of canines resulting positive for CIV had at least three predominant symptoms: conjunctivitis, runny nose and sneezing. Conjunctivitis and runny nose were presented in 27.94% of the sampled population, whereas sneezing was observed in at least 10 dogs positive for CIV. The equines reported fewer symptoms, predominantly runny nose and sneezing, both in the sampled population and in the positive samples for EIV (Table 2).