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Article

Avian Influenza a H9N2 Viruses in Morocco, 2018–2019

by
Fatima-Zohra Sikht
1,2,
Mariette Ducatez
2,
Charifa Drissi Touzani
1,
Adam Rubrum
3,
Richard Webby
3,
Mohammed El Houadfi
1,
Nour-Said Tligui
4,
Christelle Camus
2,*,† and
Siham Fellahi
1,†
1
Avian Pathology Unit, Department of Veterinary Pathology and Public Health, Agronomy and Veterinary Institute Hassan II, Rabat B.P. 6202, Morocco
2
IHAP, Toulouse University, INRAE, ENVT, 31300 Toulouse, France
3
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
4
Anatomo-Pathology Unit, Department of Veterinary Pathology and Public Health, Agronomy and Veterinary Institute Hassan II, Rabat B.P. 6202, Morocco
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this article.
Viruses 2022, 14(3), 529; https://doi.org/10.3390/v14030529
Submission received: 26 January 2022 / Revised: 28 February 2022 / Accepted: 2 March 2022 / Published: 4 March 2022
(This article belongs to the Section Animal Viruses)
Browse Figures
Versions Notes

Abstract

:
Low pathogenic H9N2 avian influenza (LPAI H9N2) is considered one of the most important diseases found in poultry (broiler, laying hens, breeding chickens, and turkeys). This infection causes considerable economic losses. The objective of this work was to monitor and assess the presence of avian influenza virus (AIV) H9N2 in eight different regions of Morocco using real-time RT-PCR, and to assess the phylogenetic and molecular evolution of the H9N2 viruses between 2016 and 2019. Field samples were collected from 108 farms suspected of being infected with LPAI H9N2 virus. Samples were analyzed using H9N2-specific real-time RT-PCR. Highly positive samples were subjected to virus isolation and seven isolates were fully sequenced. Low pathogenic H9N2 avian influenza virus was introduced in Morocco in 2016. We show that in 2018–2019, the virus was still present irrespective of vaccination status. Phylogenetic and molecular analyses showed mutations related to virulence, although our viruses were related to 2016 Moroccan viruses and grouped in the G1 lineage. Specific amino acid substitutions were identified in Moroccan H9N2 viruses that are believed to lead to increased resistance to antiviral drugs.

1. Introduction

Low pathogenic H9N2 avian influenza is an emerging disease that causes important economic losses in the poultry sector and is considered a threat to both poultry farms and public health.
Being a member of the genus Alphainfluenzavirus, and of the family Orthomyxoviridae, avian influenza viruses are enveloped RNA virus, with a genome composed of eight negative-sense RNA segments [1]. They are classified as low or highly pathogenic, on the basis of their virulence and hemagglutinin (HA) and neuraminidase (NA) sequences [2]. There are currently18 HA and 11 NA identified, including the bat-specific H17–H18 and N10–N11 [3]. Avian influenza virus subtype H9N2 is pathotyped as a low pathogenic virus (LPAI). However, co-infections with other pathogens can lead to severe outbreaks with high mortality rates and severe economic losses [4,5].
H9N2 LPAIV had first been described in 1966 in a turkey farm in the USA [6]. Since then, it has been reported in numerous countries around the world. Between 1992 and 1994, an H9N2 outbreak occurred in Guangdong Province, China, and affected chicken farms with a mortality rate of 10% to 40%, with a reduction in egg-laying rate of 14% to 75% [7].
In 1996, H9N2 LPAIV was reported in South Korea [8]. In 1998, it was isolated from most provinces in China and, as a result, it was considered to be one of the most widespread avian influenza virus in China [9]. Other countries in the Middle East and North Africa have been affected by this virus including Iran [10], Saudi Arabia [11], Jordan [12], the United Arab Emirates [13], Tunisia [14], Egypt [15], Sultanate of Oman [16], and Libya [17].
Phylogenetic analysis of the genome of LPAI H9N2 strains allowed to classify this virus into two distinct lineages: Eurasian and American. Though the Eurasian lineage contains several clades, most of the strains detected were classified in two clades (G1 and Y280) [18]. The G1 clade is represented by the A/Quail/Hong Kong/G1/1997 prototype virus, which mainly circulates in South China, Central Asia and the Middle East, while the Y280 clade viruses circulate throughout China and are represented by the A/Duck/Hong Kong/Y280/1997 prototype.
The main sources of LPAI H9N2 infections are the domestic and wild avian species. Wild birds are considered one of the reservoirs of the virus, and can transmit it over long distances. Transmission of the H9N2 virus can occur through direct contact with infected animals, and the infection can spread between farms through the movement of infected birds, vehicles, contaminated equipment or people with contaminated shoes or clothing [19]. In Pakistan, sparrows were shown to play a very important role in the transmission of the virus between farms [20]. In general, the sensitivity and receptivity of H9N2 is strongly dependent on the avian species (chicken and turkey). However, other species raised for consumption and/or hunting, such as guinea fowl, quail, pheasant, partridge, duck, goose, and ostrich are also considered sensitive. LPAI H9N2 virus has also been reported to be transmissible to mammals including dogs and cats [21] as well as humans [22,23,24,25].
The LPAIV H9N2 strain (SF1, GenBank accession number SCA48100) introduced in Morocco in January 2016, belongs to the G1 lineage, and is closely related to viruses circulating in the Middle East. As a response, the competent authorities authorized vaccination of any type of chicken as the best choice to limit the rapid spread of this disease [26]. However, in order to guide veterinarians to a rational choice of vaccines, it is important to determine and to phylogenetically analyze the circulating strains.
The aim of this study was to monitor the presence of LPAI H9N2 viruses in farms where animals with respiratory signs are reported using real-time reverse transcription PCR. Sequencing of isolates was performed in order to detect potential mutations that might affect the efficacy of commercial vaccines.

2. Materials and Methods

2.1. Specimen Collection

In collaboration with private veterinarians, a total of 151 samples, which included organs (trachea, lungs) and tracheal swabs, were collected from 108 commercial broilers farms (vaccinated and non-vaccinated) in eight regions of Morocco. Our sampling was based on chickens suspected of being infected with LPAI H9N2 virus, and presenting respiratory signs (rales, sneezing), associated with a decrease in food consumption and a drop in production. The specimens were collected in a period of 11 months, from 28/06/2018 to 31/05/2019.

2.2. Samples Processing

2.2.1. RNA Extraction and Real Time RT-PCR

RNA extraction was performed using the NucleoSpin® RNA Virus Kit (Macherey-Nagel, Düren, Germany, No. 740956.250), following the manufacturer’s instructions. In order to detect the H9N2 virus, the extracted RNA was amplified on the 7500 Fast Real-Time PCR System thermal cycler (Applied Biosystems, Foster City, CA, USA), using the primers and probe for generic detection of H9 subtypes described by [27], which target a conserved region in the HA2 subunit of the HA gene sequence.

2.2.2. Virus Isolation

In order to obtain a maximum viral load detectable by conventional RT-PCR and for full genome sequencing purposes, 17 samples among those with the highest Ct in RT-qPCR, from 17 different farms, were grown on 10-day-old, specific pathogen free (SPF) embryonated eggs. Briefly, the eggs were mirrored and the air chamber was delimited. The viral inoculums were prepared by mixing 0.2 mL of the viral suspension, 0.6 mL of sterile PBS and 0.2 mL of antibiotic OXY-Kel 20 L.A (oxytetracycline) and injected via allantoic cavity route using a sterile needle into the air chamber of the embryonated eggs. After viral inoculation, the eggs were incubated at 37 °C and examined daily for five days to assess the viability of the embryos. After the death of the embryo, eggs were refrigerated at 4 °C for 4 h. Then, the lesions on the embryos were observed and the allantoic fluids were collected, clarified, and stored at −80 °C until use.

2.2.3. Full Genome Amplification and Sequencing of H9N2 Moroccan Isolates

Viral RNA was extracted from allantoic fluids harvested from the 7 SPF embryonated eggs with the highest viral load, using the Macherey Nagel kit (Duren, Germany, No. 740956.250). Whole genome sequencing of 2018–2019 Moroccan isolates was performed with an Illumina MiSeq System (Illumina, San Diego, CA, USA) as previously described [28]. The preparation of libraries was performed using an Illumina Nextera XT library prep kit (FC-131-1096) (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. A tape station was used to verify the library quantity and quality. CLC genomic workbench was used for genomes assembly. The nucleotides sequences of all characterized strains in this study are submitted in the GenBank database under accession numbers summarized in Table 1.

2.3. Sequences and Phylogenetic Analyses

Bioedit 7.2.5 software [29] and ClustalW (version 1.83) [30] were used to compare and align nucleotide sequences of the complete genomes of seven Moroccan H9N2 isolates.
The phylogenetic tree was constructed by the maximum likelihood method, using the Mega 6.06 software [31]. The Blast [32] and Bioedit programs [30] were used to determine the sequence identity and compare the Moroccan strains with those selected from Genbank.

2.4. Statistical Analysis

Statistics describing the correlation between H9N2 positivity and the different factors: regions and vaccination status were calculated for each variable, including the mean and percentage distribution of frequencies. A non-parametric test (chi-squared test) was used to calculate the correlation between the H9N2 frequency in farms and their vaccination status.

3. Results

3.1. Case History and H9N2 Detection

One hundred and fifty-one samples from respiratory tissues and tracheal swabs were collected between June 2018 and May 2019 from different areas of Morocco: Fes-Meknes, Rabat-Sale-Kenitra, Casablanca-Settat, Draa-Tafilalet, Benimellal-Khenifra, Souss-Massa, Marrakech-Safi, and the eastern region. The samples were tested by real time RT-PCR to detect the presence of influenza virus. A total of 83 were positive for AIV with cycle threshold (Ct) values varying from 12 to 39 (Table A1), of which 40%, 56%, and 4% of the samples had a Ct below or equal to 25, between 25 and 35, and above 35, respectively. The epidemiological survey resulted in a positivity rate of the disease of 58% (63 positive farms out of 108 sampled farms) (Table 2).

3.2. Vaccination Status

The positivity rate (relative prevalence) of LPAI H9N2 positive farms was estimated to be 50% in unvaccinated farms (32 positive farms out of 64 chicken unvaccinated farms tested), while it was 70% in vaccinated farms (31 positive farms out of 44 chicken vaccinated farms tested). The overall vaccination rate reached 41% (44 out of 108 farms tested against H9N2) (Table 2).
The presence of LPAIV H9N2 was detected differently between the groups of vaccinated and unvaccinated farms, but the difference was not statistically significant (95% CI, p value: 0.9).

3.3. Molecular Characterization and Phylogenetic Analysis of the Eight Viral Segments

Viruses from highly positive samples were isolated. The genome of 7 of them was fully sequenced with an IlluminaMiSeq System [28]. Phylogenetic analysis showed that our Moroccan H9N2 viruses isolated from chickens were in the same cluster as the other Moroccan viruses detected in 2016, and grouped into G1 lineage. They were compared with relevant virus sequences available in GenBank.
Based on HA and NA phylogenetic trees, our Moroccan viruses were closely related to viruses previously isolated in the Emirates (2015), Morocco (2016), Burkina Faso (2017), and Algeria (2017), with bootstrap values of 100 and 60 for HA and NA, respectively (Figure 1). Regarding the internal genes, they grouped with the Moroccan viruses of 2016–2017, Algerian viruses of 2017, and Ghana viruses of 2017–2018 (Figure A1).
The sequence analyses of the seven Moroccan isolates showed several substitutions in both HA and NA sequences when compared to 2016 strain SF1 (Table A2 and Table A3).
All seven Moroccan isolates had the RSSR*GLF motif at the HA cleavage site, which is a characteristic and signature of the low pathogenic H9N2 viruses.
Potential HA glycosylation sites were identical to 2016 Moroccan viruses (29 NSTE, 82 NPSC, 105 NGTC, 141 NVTY, 298 NSTM, 492 NGTY, H3 numbering throughout), except for position site (297 NISK→NVSK) for four out of seven samples.
Our viruses did not present HA Receptor Binding Site (RBS) sequence associated with greater affinity for 6′-sialylacetyllactosamine (6SLN) [33] (Table 3), nor, when compared to Moroccan 2016 viruses, new critical amino acids defined as supporting mammalian replication [34,35,36].
Among mutations associated to resistance to antiviral molecules, no changes from SF1 strain were identified for NA H274Y substitution [37] or M2 S31N mutation [38] (Table 3).

4. Discussion

Our analysis included 151 field samples from 108 poultry farms suspected of being infected by the LPAI H9N2 virus and presenting mainly respiratory signs as well as poor zootechnical performances (decrease in production, decrease in feed consumption and mortalities), as reported previously [39].
The results revealed that 58% of the samples were positive for LPAIV H9N2. However, we cannot extrapolate these results on the epidemiological profile of the LPAI H9N2 virus in Morocco since our sampling was not representative enough of the national territory and only 8 regions out of 12 (Fes-Meknes, Rabat-Sale-Kenitra, Casablanca-Settat, Draa-Tafilalet, Béni Mellal-Khenifra, Souss-Massa, Marrakech-Safi and the Oriental) were sampled.
The spread of LPAI H9N2 virus in Morocco can be explained mainly by the movement of farmers, workers, and feed suppliers without compliance with biosecurity rules, to which can be added the transport of live chickens [13,15,26]. It should be noted that the application of sanitary biosecurity measures in broiler farms has been shown to be insufficient to prevent the entry of the virus [26]. In addition, mutations associated with resistance to antiviral molecules are still present in our LPAI H9N2 strains. The M2 S31N mutation is known to increase resistance to antiviral molecules, especially amantadine and rimantadine [38]. Likewise, other studies have shown that the absence of the H274Y substitution in the NA protein can confer to the virus a sensitivity to neuraminidase inhibitors such as oseltamivir [37].
The positivity rate of AI H9N2 positive farms was estimated to be 50% in unvaccinated chicken farms (32 positive farms out of 64 chicken-unvaccinated farms tested), while it reached 70% in vaccinated chicken farms (31 positive farms out of 44 chicken-vaccinated farms tested). A recent study evaluating the efficacy of four different commercial vaccines on H9N2 LPAIV SF1 strain has shown that they conferred a very limited protection against the infection [40]. Park and collaborators indicated that vaccination against H9N2 virus coupled with continuous infection of vaccinated flocks is an advantage for mutant viruses selection [41], whereas other studies report that vaccination decreases viral pressure in the field by reducing the level and duration of viral shedding [42]. Other explanations include the quality of the vaccine (either that it was not of the same strain as the virus currently circulating, or that it had a low antigen concentration [43,44]). We were not able to gather sufficient information relative to the vaccines used and their composition to be able to conclude on this point.
In addition, the vaccination rate against H9N2 was low (41%), which can be explained by the high cost of vaccination or by the fact that some farmers consider the vaccine is ineffective.
For unvaccinated specimens that tested negative, despite respiratory signs, other respiratory diseases, including BI or NDV, might be the cause of the observed clinical signs.
In this study, we demonstrated a relationship between our viruses, isolated in 2018–2019, and those isolated in Morocco in 2016, which all belong to the G1 lineage [26]. In order to evaluate the evolution of the Moroccan H9N2 virus over time (i.e., after its first introduction into Morocco), phylogenetic and genetic analyses were carried out.
On the HA and NA phylogeny, the 2018–2019 Moroccan viruses were close to those from Emirates (2015), Morocco (2016), Burkina Faso (2017), and Algeria (2017). As for internal genes, they were grouped in the same cluster as the Moroccan viruses of 2016–2017, Algerian viruses of 2017, and Ghana viruses of 2017–2018. This similarity can be explained by the common border between Morocco and Algeria, and by the history of commercial exchanges within western Africa countries. The evolution of the influenza virus directly depends on its genomic properties, which leads us to follow and verify the presence of possible mutations over time (especially on the HA and NA genes, which are the main proteins targeted by antibodies). Moroccan viruses harbor several mutations in HA and NA. Some have already been characterized, such as HA Q226L, which is known to enhance binding to mammalian-like receptors [45]. For other mutations, further studies are necessary in order to determine whether they could affect the virulence of the virus in poultry, or increase transmissibility to human. Potential glycosylation sites were identified in our Moroccan strains. As compared to Moroccan strains isolated in 2016, there was one amino acid change within a glycosylation site (297 NISK→NVSK, H3 numbering throughout) in four of the seven sequenced isolates. Changes in glycosylation sites may affect the host range and virulence of influenza viruses [46], though we do not know if it is the case here.

5. Conclusions

The low pathogenic avian influenza virus H9N2 is endemic within the country despite vaccination. Biosecurity issues in farm management, combined with high mutation potential are likely to cause dynamic changes in LPAI H9N2 strains. This prompts us to propose appropriate surveillance and adaptation of vaccines to circulating strains in order to better understand and fight public health risks.

Author Contributions

F.-Z.S. participated in the design of the study, drafted the manuscript, isolated the viruses and realized the genetic and the sequence alignment phylogenetic studies. S.F. participated in the design of the study, helped in isolation of the viruses and corrected the manuscript. A.R. and R.W. carried out the sequencing. C.C. helped analyzing the data and corrected the manuscript. M.D. corrected the manuscript. C.D.T., N.-S.T., and M.E.H. helped in the draft of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by PHC TOUBKAL 19/76, grant number 41439WM and by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (CEIRS HHSN272201400006C).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank partners and veterinarians for their help in this project. This project was financially supported by Ministry of Europe and Foreign Affairs, Ministry of Higher Education, Research and Innovation and the French Institute of Rabat (PHC TOUBKAL 2019 French-Morocco bilateral program) Grant Number: 41439WM. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Viruses 14 00529 g0a1a 550Viruses 14 00529 g0a1b 550Viruses 14 00529 g0a1c 550
Figure A1. Phylogenetic trees of Moroccan PB2 (A), PB1 (B), PA (C), NP (D), M (E), and NS (F) segments. The nucleotide sequences of Moroccan H9N2 viruses characterized in this study (black squares) were compared with relevant virus sequences available in GenBank and GISAID databases, reference viruses, and relevant sequences from neighboring areas.
Figure A1. Phylogenetic trees of Moroccan PB2 (A), PB1 (B), PA (C), NP (D), M (E), and NS (F) segments. The nucleotide sequences of Moroccan H9N2 viruses characterized in this study (black squares) were compared with relevant virus sequences available in GenBank and GISAID databases, reference viruses, and relevant sequences from neighboring areas.
Viruses 14 00529 g0a1aViruses 14 00529 g0a1bViruses 14 00529 g0a1c
Table
Table A1. List and status of samples used in this study.
Table A1. List and status of samples used in this study.
SampleFarmSampling DateLocation of FarmAge of BirdsH9N2 Vaccination StatusH9N2 RT-qPCRCt ValueFarms Status
AS1F128/06/2018Rabat27DV25P
AS2F216/07/2018Rabat46DV-N
AS3 46D-
AS4 46D-
AS5F308/08/2018Oriental45DNV-N
AS6 45D-
AS7F408/08/2018Oriental36DNV-N
AS8F517/07/2018Casablanca28DV-N
AS9F617/09/2018Temara43DV-N
AS11F719/10/2018Meknes32DNV-N
AS12B1 *F823/10/2018Kenitra23DNV20P
AS12B223/10/201823D33
AS13 *+F918/10/2018El hajeb32DNV17P
AS14 *+F1024/10/2018Meknes30DNV18P
AS15F1121/10/2018Meknes37DNV-N
AS16 *F1217/10/2018Midelt40DNV18P
AS17F1311/12/2018Fes38DNV26P
AS18F1411/12/2018Fes35DNV26P
AS19F1511/12/2018Fes41DNV-N
AS20F1623/11/2018Fes32DV25P
AS21F1712/11/2018Salé36DV-N
AS22F1807/12/2019Meknes34DNV22P
AS2307/12/201934D26
AS2407/12/201934D22
AS26F1919/02/2019Benslimane30DNV-N
AS27F2020/02/2019Rabat24DNV-N
AAS28F2104/11/2018Ait brahim (Fes)32DV24P
AS29 *+F2216/11/2018Fes36DV16P
AS30F2326/01/2019Hajeb20DV24P
AS31F2411/02/2019Sefrou30–36DV17P
AS32 *+F2512/02/2019Ain chegag37DV19P
AS33 *F2613/02/2019Zerarda tahla40DNV16P
AS34F2725/01/2019Khemisset33DNV-N
AS35F2804/03/2019Meknes32D -N
AS36F2913/02/2019Fes34D 14P
AS37F3013/02/2019Fes37DV-N
AS38F3101/02/2019Meknes32DNV-N
AS39F3212/02/2019Hadeb34DNV-N
AS40F3312/02/2019Salé42DNV12P
AS41F3425/02/2019Khemisset44DV-N
AS42F3525/02/2019Meknes35DNV-N
AS43 *F3607/02/2019Casablanca29DV16P
AS44F3707/02/2019Casablanca32DV16P
AS45 *F3810/02/2019Rabat45DV17P
AS4610/02/201945D14
AS47F3911/02/2019Tiflet28DNV-N
AS54F4020/02/2019Rabat32DV24P
AS55F4120/02/2019Oriental37DNV-N
AS56 *F4220/02/2019Oriental38DNV26P
AS57F4321/02/2019Taza29DNV26P
AS58F4421/02/2019Tahla36DNV-P
AS5921/02/201936D26
AS60F4521/02/2019Tahla39DNV-N
AS61 *F4624/02/2019Meknes44DNV21P
AS62F4724/02/2019Elhajeb37DNV23P
AS6324/02/201937D12
AS64F4824/02/2019Elhajeb35DNV26P
AS65F4925/02/2019Meknes41DNV-N
AS66F5025/02/2019Meknes35DNV-N
AS67F5125/02/2019Meknes32DNV-N
AS68 *F5226/02/2019Khenifra38DNV14P
AS69F5325/02/2019Khenifra28DNV25P
AS70F5428/02/2019Salé23DNV22P
AS71 *+F5501/04/2019Sidi slimane14DNV22P
AS72F5611/12/2017Khenifra33DNV-N
AS73F5720/02/2019Khenifra36DNV14P
AS74F5811/04/2019Sidi slimane37DNV-N
AS75F5906/03/2019Salé29DNV-N
AS76 *+F6023/02/2019Meknes38DNV20P
AS77 *+F6117/01/2019Meknes29DNV16P
AS78F6201/04/2019Tiznit25DNV-P
AS7901/04/201925DNV30
AS80F6301/04/2019Tiznit32DNV30P
AS8101/04/201932DNV28
AS82F6401/04/2019Tiznit32DNV-N
AS8301/04/201937DNV-
AS84F6501/04/2019Tiznit37DNV-N
AS85 *F6601/04/2019Tiznit28DNV16P
AS86F6701/04/2019Tiznit37DNV-N
AS8701/04/201934DNV-
AS88F6801/04/2019Tiznit34DNV-N
AS89F6901/04/2019Tiznit15DNV26P
AS90F7031/05/2019Rabat32DNV-N
AS9131/05/201932D-
AS9231/05/201932D-
AS9331/05/201932D-
AS9431/05/201932D-
AS9531/05/201932D-
AS9631/05/201932D-
AS9731/05/201932D-
AS9831/05/201932D-
BS1F7109/04/2019Fes28DNV-N
BS2F7209/04/2019Fes36DNV-N
BS3F7316/09/2019Ait moussa35DNV-N
BS416/09/201935D-
BS5F7408/10/2019Ait moussa36DV-N
BS609/10/201936D-
BS7F7523/10/2019Marrakech18DNV-N
BS8F7624/10/2019Haouz29DNV-N
BS9F7730/10/2019Marrakech34DV-N
BS10F7831/10/2019Rhamna13DNV-N
BS11F7905/11/2019Marrakech12DNV-N
BS12F8023/11/2019Casablanca30DNV29P
BS13F8123/11/2019Casablanca21DNV28P
BS14F8223/11/2019Casablanca24DNV33P
BS15F8323/11/2019Casablanca24DNV31P
BS18F8425/11/2019Tiznit34DV-N
BS1925/11/201934D-
BS2025/11/201934D-
BS21F8505/12/2019Tiznit29DV-N
BS2205/12/201929D-
BS2305/12/201929D-
BS47F8614/11/2019Casablanca28DV26P
BS48F8714/11/2019Casablanca28DV26P
BS49F8814/11/2019Casablanca28DV27P
BS50F8924/10/2019Mohammedia28DV-N
BS57F9005/03/2020Casablanca34DV29P
BS5805/03/202034D-
BS59F9105/03/2020Casablanca30DV-P
BS6005/03/202030D33
BS61F9202/07/2020Tiznit42DV-N
BS62F9302/07/2020Tiznit34DV30P
BS63F9402/07/2020Tiznit30DV30P
BS64F9502/07/2020Tiznit22DV30P
BS65F9602/07/2020Tiznit30DV29P
BS66F9702/07/2020Tiznit35DV32P
BS67F9802/07/2020Tiznit29DV27P
BS68F9902/07/2020Tiznit21DV29P
BS69F10002/07/2020Tiznit13DV27P
BS70F10102/07/2020Tiznit31DV28P
BS71F10202/07/2020Tiznit44DV30P
BS72F10302/07/2020Tiznit36DV28P
BS73F10402/07/2020Tiznit24DV29P
BS76F10502/07/2020Tiznit42DV30P
BS7702/07/202042D28
BS7802/07/202042D29
BS7902/07/202042D27
BS8002/07/202042D30
BS8102/07/202042D32
BS8202/07/202042D39
BS8302/07/202042D39
BS8402/07/202042D36
BS85F10609/09/2020Casablanca33DNV31P
BS8609/09/202033D30
BS87F10709/09/2020Casablanca27DNV30P
BS8809/09/202027D30
BS8909/09/202027D29
BS9009/09/202027D30
BS91 *F10810/09/2020Rabat29DV24P
BS9210/09/202029D29
BS9310/09/202029D34
F: Farm; D: day; V: vaccinated; NV: Non-Vaccinated; P: positive; N: negative; Ct: Cycle Threshold; *: isolated sample; +: sequenced sample.
Table
Table A2. HA mutations as compared to 2016 Moroccan strain SF1 (H3 numbering).
Table A2. HA mutations as compared to 2016 Moroccan strain SF1 (H3 numbering).
137188190222226298325364375397402408493
SF1TDALLIHMVDENT
AS13TDTLLIHIVDENT
AS14TDALLIQMVDENI
AS29TDVLLIHMVNENT
AS32TDALLVHMVDDNT
AS71TDALLVHMIDEST
AS76TNALQVHMVDENT
AS77TDALQVHMVDENT
Table
Table A3. NA mutations as compared to 2016 Moroccan strain SF1 (N2 numbering).
Table A3. NA mutations as compared to 2016 Moroccan strain SF1 (N2 numbering).
8163142465056575860658088101116127254261290329332368385390400416
SF1ITTYSAIIIRITSSVSIKVNSKTSNI
AS13ITTYSAIIIRITSSVSIRVDSKNASI
AS14ITTYSTTTTRIPLSVNVKVNSTTSNM
AS29ITTHPAIIIKITSSVSIRVNSKTASI
AS32IITYSAIIIRTTSSISIKVNSKTSSI
AS71MITYSAIIIRTTSAVSIKANFKTSSI
AS76MITYSAIIIRTTSAVSIKVNFKTSSI
AS77IIMYSAIIIRTTSSISIKVNSKTSSI

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Viruses 14 00529 g001 550
Figure 1. Phylogenetic trees of Moroccan HA (a) and NA (b) gene segments. The nucleotide sequences of Moroccan H9N2 viruses (black squares) characterized in this study were compared with relevant virus sequences available in GenBank and GISAID databases, reference viruses, and relevant sequences from neighboring areas.
Figure 1. Phylogenetic trees of Moroccan HA (a) and NA (b) gene segments. The nucleotide sequences of Moroccan H9N2 viruses (black squares) characterized in this study were compared with relevant virus sequences available in GenBank and GISAID databases, reference viruses, and relevant sequences from neighboring areas.
Viruses 14 00529 g001
Table
Table 1. Accession numbers of segments sequences of studied Moroccan H9N2 viruses.
Table 1. Accession numbers of segments sequences of studied Moroccan H9N2 viruses.
SegmentStrain
AS13AS14AS29AS32AS71AS76AS77
PB2MW165151MW165079MW165121MW165089MW165136MW165110MW165106
PB1MW165154MW165125MW165122MW165088MW165142MW165113MW165101
PAMW165158MW165082MW165117MW165085MW165139MW165116MW165103
HAMW165152MW165084MW165120MW165090MW165137MW165111MW165105
NPMW165157MW165083MW165124MW165086MW165140MW165109MW165108
NAMW165155MW165078MW165119MW165092MW165135MW165115MW165104
NSMW165156MW165080MW165123MW165091MW165141MW165114MW165102
MMW165153MW165081MW165118MW165087MW165138MW165112MW165107
AS13, A/chicken/Morocco/AS13/2018; AS14, A/chicken/Morocco/AS14/2018; AS29, A/chicken/Morocco/AS29/2018; AS32, A/chicken/Morocco/AS32/2019; AS71, A/chicken/Morocco/AS71/2019; AS76, A/chicken/Morocco/AS76/2019; AS77, A/chicken/Morocco/AS77/2019.
Table
Table 2. Farms positivity rate.
Table 2. Farms positivity rate.
Number of FarmsPositive FarmsPositivity Rate
Fes-Meknes342059%
Rabat-Sale-Kenitra18844%
Casablanca-Settat161381%
Draa-Tafilalet11100%
BeniMellal-Khenifra4375%
Souss-Massa261765%
Oriental4125%
Marrakech-Safi500%
Morocco (Total)1086358%
Vaccinated44 (41%)3170%
Unvaccinated64 (59%)3250%
Table
Table 3. Residues associated to 6′-sialylacetyllactosamine-affinity and to drug resistance.
Table 3. Residues associated to 6′-sialylacetyllactosamine-affinity and to drug resistance.
HA * RBS NAM2
190Q226L227274S31N
SF1 **VLIQN
AS13TLIQN
AS14ALIQN
AS29VLIQN
AS32ALIQN
AS71ALIQN
AS76AQIQN
AS77AQIQN
HA, hemagglutinin; RBS, receptor-binding site. * H3 numbering; ** GenBank accession number: SCA48100.
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Sikht, F.-Z.; Ducatez, M.; Touzani, C.D.; Rubrum, A.; Webby, R.; El Houadfi, M.; Tligui, N.-S.; Camus, C.; Fellahi, S. Avian Influenza a H9N2 Viruses in Morocco, 2018–2019. Viruses 2022, 14, 529. https://doi.org/10.3390/v14030529

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Sikht F-Z, Ducatez M, Touzani CD, Rubrum A, Webby R, El Houadfi M, Tligui N-S, Camus C, Fellahi S. Avian Influenza a H9N2 Viruses in Morocco, 2018–2019. Viruses. 2022; 14(3):529. https://doi.org/10.3390/v14030529

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Sikht, Fatima-Zohra, Mariette Ducatez, Charifa Drissi Touzani, Adam Rubrum, Richard Webby, Mohammed El Houadfi, Nour-Said Tligui, Christelle Camus, and Siham Fellahi. 2022. "Avian Influenza a H9N2 Viruses in Morocco, 2018–2019" Viruses 14, no. 3: 529. https://doi.org/10.3390/v14030529

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