The duck-derived isolates belonged to AIV H3 subtype
Of the 962 cloacal swabs only five samples were positive for influenza virus as confirmed by HI assay and RT-qPCR amplification. Three of the five isolates were identified as H3 subtypes based on RT-qPCR using H3-specific primers. RT-qPCR using the H5 and H9 specific primers came back negative (data not shown). Complete genome of the three H3 isolates (namely A/domestic duck/Iran/340/2017(H3N2), A/domestic duck/Iran/375/2017(H3N2), A/domestic duck/Iran/379/2017(H3N2)340, 375 and 379) were Sanger sequenced and deposited to GenBank (accession numbers shown in Table 1).
Table 1
Information of the Iranian avian influenza viruses of the current study
Isolate
|
Bird
|
Province
|
Date
|
MDT
|
Segment
|
Accession #
|
340
|
Domestic duck
|
Mazandran
|
2017/8/30
|
72 hours
|
PB2
|
• MW407061
|
PB1 and PB1-F2
|
MW407065
|
HA
|
• MW406901
|
NA
|
• MW406904
|
PA and PA-X
|
• MW407048
|
NP
|
• MW406925
|
M2 and M1
|
• MW406905
|
NEP and NS1
|
• MW406926
|
375
|
Domestic duck
|
Mazandran
|
2017/8/9
|
96 hours
|
PB1 and PB1-F2
|
• MW422783
|
PB2
|
• MW422785
|
HA
|
• MW422771
|
NA
|
• MW422775
|
PA and PA-X
|
• MW422784
|
NP
|
• MW422780
|
M2 and M1
|
MW422781
|
NEP and NS1
|
• MW422782
|
379
|
Domestic duck
|
Mazandran
|
2017/8/8
|
96 hours
|
PB1 and PB1-F2
|
• MW422893
|
PB2
|
• MW422891
|
HA
|
• MW422885
|
NA
|
• MW422888
|
PA and PA-X
|
• MW422890
|
NP
|
• MW422887
|
M2 and M1
|
MW422886
|
NEP and NS1
|
• MW422895
|
The Isolates Belonged To The Eurasian Lineage
The HA genes of the isolates were almost identical at a nucleotide distance of < 0.1% (Supplementary File 1). When BLAST was run on the HA nucleotide sequences, the top homolog isolates (as of January 2021) were some H3N8 isolates from North Kazakhstan (97.1% to MN945300 and MN945304, also see Table 2). The rest of the matching sequences belonged to the duck and other aquatic birds isolated from East Asia and Europe, better known as the Eurasian lineage. Phylogenetic tree was also constructed and the results showed that the isolates of this study formed a cluster with isolates mostly belonging to H3N8 subtype (Fig. 2). According to the tree, which was divided into American and Eurasian lineages, the H3 isolates of the current study clustered to the avian-Eurasion lineage, but were distinct from canine, feline, equine, swine, human or turkey influenza viruses. They also clearly differed from the North American lineage. Furthermore, the isolates showed 91% nucleotide similarity to Dk/Ukraine/1/63(H3N8), one of the earliest known avian H3 strains (Bean et al. 1992). The isolates showed distances of ~ 13%, 24–30%, 27% and > 34% with canine/feline, human, swine and equine isolates, respectively (Supplementary File 1).
Table 2
List of viruses with the highest BLAST identity to each segment of the isolates of the current study as of January 2021. Note that the isolates shared the same homologs.
Segment
|
Gene(s)
|
Highest homolog influenza virus
|
GenBank accession number
|
Percentage of homology
|
1
|
PB2
|
A/mallard duck/Netherlands/35/2015(H4N6)
|
MF693922
|
97.03%
|
2
|
PB1 and PB1-F2
|
A/mallard duck/Georgia/10/2016(H7N7)
|
MF694021
|
97.8%
|
3
|
PA and PA-X
|
A/duck/Bangladesh/37203/2019(H7N1)
|
MT090472
|
97.38%
|
4
|
HA
|
A/garganey/North-Kazakhstan/45/2018(H3N8)
|
MN945300
|
97.18%
|
5
|
NP
|
A/teal/Egypt/MB-D-621C/2016(H7N9)
|
MN208045
|
98.9%
|
6
|
NA
|
A/greater white-fronted goose/Netherlands/3/2011(H6N2)
|
KX978364
|
96.95%
|
7
|
M2 and M1
|
A/pintail/Egypt/MB-D-384C/2015(H3N6)
|
MN208008
|
98.9%
|
8
|
NEP and NS1
|
A/goose/Karachi/NARC-13N-969/2014(H14N3)
|
KX602672
|
98.71%
|
According to our protein analysis, the three H3 isolates shared the same amino acid sequence (PEKQTR/GLF) at the cleavage site between the HA1 and HA2, further indicating that they belong to low pathogenic strains. Moreover, all the three H3 isolates shared the same N-glycosylation sites at positions 24, 38, 54, 181, 301 and 499 aa. In addition, two amino acids of Q226 and T228 at the receptor binding site were identified, suggesting that the isolates bind to a-2,3-linked sialic acid receptors, which are generally recognized as the main receptors in avian species (Wiley and Skehel 1987; Matrosovich et al. 2000).
Similar analyses were performed for the segment 6 sequences. The NA genes of the three isolates were almost identical as their nucleotide distance was < 0.2% (Supplementary File 2). Furthermore, BLAST analyses showed highest homologies (max 96.5–96.9%) to a variety of aquatic bird-derived subtypes including H6N2, H4N2, H9N2, H1N2 and even H5N2, suggesting that the NA of the isolates belonged to N2 subtype. Figure 3 shows the location of the NA genes of the current study as compared to other NA selected as described in materials and methods. All the three isolates clustered to the avian-Eurasian lineage. As seen in the tree, the isolates did not cluster to canine, feline, swine and human influenza viruses. Interestingly, there was only 74–82% nucleotide similarity between the isolates of this study and chicken-derived H9N2 strains previously reported from Iran (Supplementary File 2). BLAST analysis also did not show any Iranian isolate in the top results.
Phylogenetic analysis showed all the internal protein genes were of avian origin
BLAST and phylogenic trees of the internal genes showed that the three isolates of this study belonged to the Eurasian lineage derived from aquatic birds (Supplementary File 3 to 8). For instance, BLAST analysis on the M gene (segment 7 including matrix M1 and M2 protein genes) showed that the three isolates had the highest nucleotide sequence homology with aquatic bird-origin influenza viruses of different combinations such as H3N6, H10N7, H3N8, H7N7 and H11N8 (at most 98.7%, see Table 2). The M segment also showed 12% distances with those of human H3N2 isolates from Iran as well as 9–14% distance with Iranian chicken H9N2 (Supplementary File 3). As for the NP gene (segment 5), there was high homology )98.9%) to A/teal/Egypt/2016(H7N3) and A/teal/Egypt/2016(H7N9) isolates. However, other combinations such as H4N6 and H10N4 and H3N6, all isolated from aquatic birds, were among the next matching sequences (98.5–98.7%). Also analyses on the NS gene (segment 8 containing nuclear export protein (NEP) and nonstructural protein (NS1) genes) demonstrated that the isolates of this study were closely related to A/goose/Karachi/NARC-13N-969/2014(H14N3) and A-mallard duck-Netherlands-2009(H5N3) (98.71%). Supplementary File 5 also shows the nucleotide distances compared with NS of several strains isolated from other vertebrate species including swine, canine and Homo sapiens. Similar to the other segments, the distance to homo sapiens-derived isolates were 16–18%.
Analyses were also run on the viral polymerase protein genes (PA, PB1 and PB2); PA gene (segment 3, including PA and PA-X genes) had the highest nucleotide sequence homology (97.38%) with to A/duck/Bangladesh/37203/2019(H7N1) followed by different subtype combinations (H4N6, H7N6, H2N2, etc.). The nucleotide differences are also available as Supplementary File 6. In addition, Isolates A/mallard duck/Netherlands/35/2015(H4N6) and A/tufted duck/Georgia/1/2012(H2N3) were the closest homologs to the PB2 gene (segment 1) of isolates of this study, both matching at 97.12%. Supplementary File 7 shows the nucleotide distance of PB2 compared to other strains, including those isolated from other vertebrates. The scores were distinct to those of Homo sapiens, swine and other vertebrates. The amino acids E627, D701 and S714, which are known to play crucial role in interspecies transmission (Subbarao et al. 1993; Manz et al. 2013; Czudai-Matwich et al. 2014) were also present on all the three PB2 proteins. Furthermore, the PB1 gene (segment 2 including PB1 and PB1-F2) of all the three isolates had the highest homology to A/mallard duck/Georgia/10/2016(H7N7) and A/garganey/Bangladesh/38920/2019(H7N4) at 97.8% (lowest nucleotide distance 1.95%-2.14%, Supplementary File 8).
Based on our findings, it was difficult to identify a particular geographical region, or a highly homologous isolate, as the original source of the AIV of this study. This was mainly because each genome segment showed homology to different strains (Table 2). Surprisingly however, no previously reported Iranian isolates showed up among the highly homologues BLAST isolates (> 90–95%), suggesting that the isolates of this study are the first of their kind from Iran.