Emerging Highly Pathogenic Avian Influenza (H5N8) Virus in Podiceps nigricollis in Northwest China in 2021

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Introduction
Avian infuenza virus (AIV) is a member of the infuenza A virus of the Orthomyxoviridae family and can cause avian disease, a public health threat.In recent years, AIVs, particularly the highly pathogenic AIV (HPAIV) A (H5N8), have caused numerous outbreaks worldwide in both poultry and wild birds, especially in autumn and winter.Migratory birds such as waterfowl are natural hosts of AIV.Te longdistance spread of HPAIV is closely associated with wild bird migration, particularly H5N8 and H5N1 [1,2].
H5N8 HPAIV of branch 2.3.4 was frst detected in Chinese poultry in 2010 [3], although it did not cause a largescale outbreak.In 2014, multiple H5N8 HPAI outbreaks were reported in South Korea, China, Japan, Europe, and North America, triggering the frst intercontinental epidemic [4,5].By 2016, H5N8 HPAIV spread to Europe, the Middle East, Africa, and other countries and regions, leading to a second global epidemic [6].More importantly, several H5Nx AIVs have a zoonotic potential and cross the species barrier from birds to humans, confrmed by the frst H5N8 HPAIV infection in humans in December 2020.H5N8 HPAIV has become one of the important biosecurity issues threatening the world; thus, the zoonotic potential of AIVs warrants continuous, vigilant monitoring to avert further spread and disastrous pandemics.

Materials and Methods
From May 27 to June 10, 2021, dead or seriously sick birds were found in the Hongjiannao Nature Reserve, Shenmu County, Yulin City, Shaanxi, Northwest China.14 migratory birds were collected, including 13 black-necked grebes (Podiceps nigricollis) and one great crested grebe (Podiceps cristatus) in the Hongjiannao Nature Reserve (latitude: 39.071876 °N, longitude: 109.929433°E) from June 2 to 6, 2021.Some organs of the 14 dead birds were dissected and collected by the Shaanxi Institute of Zoology (Northwest Institute of Endangered Zoological Species).Tese samples were then sent to our laboratory.
Te fnal amplifcation reaction was performed with the doublestrand cDNA template and specifc primer (5′-GCCGGA-GCTCTGCAGAATTC-3′).GridION library preparation was performed according to the manufacturer's instructions for barcoding cDNA/DNA and native DNA (SQK-LSK109 and EXP-NBD104).Oxford Nanopore Technologies Min-KNOW software was used to collect raw sequence data.GridION sequencing was run for up to 26 h.Te Guppy base-calling tool was used to trim adaptor and barcode sequences for demultiplexing ONT reads from FAST5 fles.Te fltered reads were assembled and blasted with the avian infuenza virus (AIV) database to search for the bestmatched reference sequences.Subsequently, the fltered reads were mapped to the reference sequences using Min-imap2 and converted with SAM tools [8,9].
Meanwhile, we downloaded the AIV sequences from the GenBank and GISAID databases to create a local AIV database (updated December 31, 2021).Ten, using each sequence of the eight H5N8 viruses as a query, Blastn was performed with default parameters against the local AIV database.Subsequently, the frst 100 gene sequences in the output were collected.Tus, eight datasets corresponding to the eight AIV sequences were obtained.Subsequently, the optimal model and the maximum likelihood phylogenetic trees were constructed by Mega-X.After selecting the optimal model calculated before, the phylogenetic tree was constructed using the maximum likelihood method, and the bootstrap value was set to 1000.
In addition, the lung, liver, heart, trachea, and kidneys were collected from dead migratory birds.Tese tissues were fxed in 4% formaldehyde (neutral bufered) and embedded in parafn.Hematoxylin-eosin staining was performed on sections of these tissues to observe the histopathological changes.

Results and Discussion
On May 27, 2021, 4,249 dead wildfowl, primarily Podiceps nigricollis, were found in the Hongjiannao Nature Reserve, Shenmu, Shaanxi.Tese infected birds had visible clinical features such as weakness and dyspnea.Oropharyngeal swabs and organs from birds were collected for pathogen identifcation.Before the laboratory confrmation of the pathogen, the Ministry of Agriculture and Rural Afairs implemented prompt and strict control measures, including    Transboundary and Emerging Diseases the harmless treatment of all dead and infected wildfowl, demarcating epidemic areas, strictly controlling vehicles and people entering and leaving the area, and disinfecting the lake area contaminating with the virus and reduce the risk of human infection.Total viral RNA was extracted from organ samples and assessed for nucleic acid concentration and purity to confrm the pathogen.Full-length H5N8 virus genome sequences of three strains were obtained using direct RNA sequencing (i.e., Oxford Nanopore Technologies sequencing).All eight segment sequences of the three strains were blasted against the NCBI database, and the highest homology with the eight segments was the strain found in China and Korea in 2021 (Table 1).Te ML-tree of hemagglutinin (HA) is shown in Figure 1, while the other fragment sequences are shown in Figure 2. Te ML-tree of the hemagglutinin (HA) sequence showed that the three H5N8 strains had the highest sequence homology with H5N8 strains isolated from poultry and wild birds in Korea (i.e., 99.5%; Table 1), all of which belonged to the 2.3.4.4b clade.In October 2020, the H5N8 virus was detected in swans in Inner Mongolia, China [10].
Meanwhile, similar viruses were reported in Shandong, China, in late November 2020 [11].According to the reports, birds can pass through China through three bird migratory routes: the East Asian-Australasian fyway, the Central Asian fyway, and the Black Sea-Mediterranean fyway.Several migratory fyways in Eurasia have overlapping breeding areas.However, the most common bird migration pattern is fying north to breed in spring and summer and returning to warmer regions in autumn [12].Swans spend the winter in Southern China and Southeast Asia and breed in Mongolia or East Asia (such as South Korea and Japan) along the East Asian-Australasian fyway.Migratory birds such as cardinals spend the winter in South Asia (i.e., India, Nepal, Bangladesh, and Myanmar) and breed along the Central Asian fyway in Qinghai or Mongolia [13].Shaanxi (including Yulin City) is a key stopover site during the migration of wild birds in China, and the Hongjiannao Nature Reserve is an ideal habitat for migratory birds, especially waterfowl.Tus, H5N8 HPAIV detected in P. nigricollis was likely to be transmitted during the migration and reproduction of migratory birds.Combining the phylogenetic analysis of the virus with the migration pattern of wild birds, these birds likely spent the winter in Southern China in the fall and winter of 2020 and then bred along the East Asian-Australasian fyway to spread the virus to Northwest China.
To further explore the pathogenicity of the H5N8 virus in migratory birds, tissues of three dead migratory birds were stained (Figure 3).Histopathological analysis showed infammatory cell infltration and necrosis in these organs.In detail, severe bleeding in the lungs and numerous erythrocytes in the respiratory lumen, accompanied by necrotic foci, were found surrounded by infammatory cell infltration.Infammatory cells near the portal area, and severe steatosis were observed in the liver.Te mucosal layer of the trachea was hypertrophic, with infammatory cell infltration in the stroma.Blood vessels of the cerebral parenchyma were congested to diferent degrees.Te renal interstitium was congested with obvious infammatory cell infltration.However, no obvious pathological changes were observed in the heart.According to the histopathological analysis, the pathological changes in the lungs, liver, trachea, brain, and kidney were highly pathogenic, consistent with related HPAIV reports.However, whether severe liver steatosis is associated with H5N8 needs further research.Interestingly, we found that the sequence obtained in this study was highly homologous with the sequence (A/grebe/Shaanxi/SD001/ 2021 (A/H5N8), A/grebe/Ningxia/SD001/2021 (A/H5N8)) uploaded by Professor Cui of Harbin Veterinary Research Institute in the GISAID database, indicating that it is feasible and efective to combine the fltering extraction method with the Oxford Nanopore technology.
In conclusion, H5N8 HPAIV was detected during the spring migration of P. nigricollis in Shaanxi, China, and phylogenetic analysis illustrated the pathways of the introduction of the H5N8 virus to Central China.Tis fnding warrants the need to strengthen the monitoring of various migratory waterfowl at the main stopover sites and breeding habitats to prevent the development and spread of HPAIV.

Figure 3 :
Figure 3: Pathological changes in the lungs, liver, heart, trachea, brain, and kidney of dead migratory birds infected with H5N8.(a) Pathological changes in the lung.Infammatory cell infltration (green arrow), focal necrosis (black arrow), and erythrocyte (blue arrow).(b) Pathological changes in the liver, infammatory cells (blue arrow) near the portal area, and severe steatosis.(c) No obvious pathological changes in the heart.(d) Pathological changes in the trachea, hypertrophy of tracheal mucosa, and infltration of infammatory cells in stroma (black arrows).(e) Pathological changes in the brain and diferent degrees of cerebral parenchymal vascular congestion (black arrows).(f ) Pathological changes in the kidney.Interstitial hemorrhage (black arrows) was obvious.

Table 1 :
Highest nucleotide identity of the eight H5N8 virus sequences with the sequences from the NCBI and GISAID.