Circulation of highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b in highly diverse wild bird species from Peru

doi:10.21203/rs.3.rs-2814674/v1

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Circulation of highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b in highly diverse wild bird species from Peru

https://doi.org/10.21203/rs.3.rs-2814674/v1

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published 13 Feb, 2024

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Highly pathogenic avian Influenza virus (HPAIV) has emerged in unprecedented records in recent years. Despite the large reports of cases in Asia, Europe, and North America, little is known about its circulation in South America. Here, we describe the isolation, and whole genome characterization of HPAIV obtained from sampling 26 wild bird species in Peru, representing one of the largest studies in our region. Out of 147 samples analyzed, 20 were positive for detection of avian influenza virus using a qRT-PCR-based assay. Following inoculation into embryonated chicken eggs, fourteen viral isolates were obtained from which six isolates were selected for genome characterization, based on their host relevance. Our results identified the presence of HPAIV H5N1 subtype. Phylogenetic analysis revealed that these isolates correspond to the clade 2.3.4.4b, sharing a common ancestor with North American isolates and forming a novel subclade along with isolates from Chile. Altogether, changes at the amino acid levels compared to their closest relatives indicates the virus is evolving locally, highlighting the need for constant genomic surveillance. This data evidence the chances for spillover events increases as the virus spreads into large populations of immunologically naïve avian species and adding conditions for cross species transmission.

Biological sciences/Microbiology/Virology/Viral reservoirs

Biological sciences/Microbiology/Microbial genetics/Viral genetics

Biological sciences/Microbiology/Virology/Influenza virus

Influenza viruses represent a major threat for animal and public health worldwide since the viruses have started to evolve rapidly in recent years. The underlying mechanisms for evolution in Influenza viruses rely on both viral genetics and their animal hosts factors. In terms of viral genetics, its genome comprises of single-stranded, eight negative-sense viral RNAs segments (Webster et al., 1992). Viral genome encodes for eleven proteins including some accessory proteins (Hutchinson & Yamauchi, 2018). These eight segments are named and presented from the largest to shortest, as follows: PB2, PB1, PA, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), non-structural (NS) (Bouvier & Palese, 2008; Krammer et al., 2018). These proteins have individual function or act as protein complex during viral replication. Thus, viral genome segmentation is the basis for reassortments, and recombination as evolutionary mechanisms commonly found in influenza viruses. Furthermore, influenza viruses are known to infect a wide host range(Bourret, 2018; Olsen et al., 2006) which is the basis for spillover events and viral adaptation to novel species (Webster et al., 1992). Altogether, influenza viruses are of the most rapidly evolving pathogens of major concern for animal and public health (Medina & García-Sastre, 2011).

Hemagglutinin (HA) and Neuraminidase (NA) are the most important viral proteins playing fundamental roles in viral replication (Air, 2012; Wilks et al., 2012). HA is a relevant protein for viral entry into target cells, therefore it is the main target for neutralizing antibodies and vaccine development. On the other hand, NA is a tetrameric protein with enzymatic activity that allow viral release from infected cells following replication (Gamblin & Skehel, 2010). In addition to their relevance in viral replication, these proteins are used for viral classification purposes. Hence, there are sixteen (16) HA and nine (9) NA described to date, with most of them being identified in avian species such as wild birds belonging to the Anseriformes and Charadriiformes orders (Slemons et al., 1973; Venkatesh et al., 2018). In addition, there is a description of two HA and NA subtypes of influenza-like viruses found in bats from Peru (Ma et al., 2015; Tong et al., 2012).

Furthermore, avian influenza is classified based on the clinical severity of infections in chickens (Alexander & Brown, 2009). Thus, most avian influenza viruses harboured by wild birds correspond to low pathogenicity avian influenza viruses (LPAIVs) causing asymptomatic or mild infections in poultry (Krammer et al., 2018). On the other hand, some H5 and H7 subtypes are known to produce a severe infection leading to massive death when domestic birds are infected and, specially H5 subtypes, causing death in their wild bird hosts. Thus, the co-circulation of Low- and High-pathogenicity AIVs in wild bird species is a contributing factor for the emergence of novel Influenza viruses with distinctive features.

Highly pathogenic avian influenza viruses (HPAIVs) have caught the attention from scientists due to its concern in animal and public health (Fuller et al., 2010). Since the emergence of the HPAIV H5N1 GS/Gd lineage in China in 1996, the virus has evolved into ten clades and multiple subclades (Lee et al., 2017). More recently, one of these genetic clades, such as the 2.3.4.4b clade, represents the main influenza virus type causing the current outbreak around the world (Lee et al., 2017; Lewis et al., 2021; Mosaad et al., 2023; Ndumu et al., 2018). Despite this virus characterization in multiple geographical areas, little is known about the circulation of the HPAIV H5N1 in South America.

Since 2006, we have implemented a local system for monitoring avian influenza viruses in wild birds in Peru. This has led to the detection, isolation, and characterization of multiple AIV isolates that corresponded to those LPAIVs (Castro-Sanguinetti et al., 2022; Ghersi et al., 2009; Segovia et al., 2013). Nevertheless, the detection of HPAIV H5 was reported on November 14th in 2022, following an outbreak of high mortality in pelicans of Paita, Piura (North coast of Peru) by the national authorities. Since the arrival of this HPAIV, more than 55,000 wild birds have died due to these HPAIV H5 infections and 46 outbreaks in poultry farms have been controlled. More recently, spillover events from avian into marine wildlife have been reported by the National Service of Natural Protected areas (SERNANP). Thus, our objective was the identification, isolation, and whole genome characterization of this HPAIV detected in Peru in cases suggestive of avian influenza infections in wild birds from Peru. Our study represents the first complete genetic description of this virus in South America in a large group of local wild bird species.

2.1 Avian influenza virus is detected in a large group of wild bird species with clinical presentation of highly pathogenic avian influenza (HPAIV) infection.

Samples analysed were obtained from oropharyngeal, cloacal and fecal swabs from animals as part of our active surveillance for influenza viruses or those showing clinical sings of HPAIV infection in a collaboration with SERFOR. In total, we evaluated 154 samples that corresponded to 29 wild bird species ranging from migratory birds, waterfowl and some local birds that are part of the natural ecosystem in the pacific coast of Peru. A large proportion of the samples analyzed corresponded to Franklin’s gulls (Leucophaeus pipixcan), Peruvian pelicans (Pelecanus thagus), Sanderlings (Calidris alba), American oystercatchers (Haematopus palliates), mallard ducks (Anas platyrhynchos) among others, which appeared to be within those most common affected by this outbreak. A list of the species and number of samples evaluated is presented in Table 1. Among those animals who presented clinical signs, we observed depression, lethargy, moderate and severe respiratory distress, and neurological disorders. In exceptional cases, some animals were remitted to our laboratory for sample collection. Here, some findings revealed lung edema and general congestion. We did not find hemorrhagic lesions in intestinal tract lymphoid aggregates which has been described to be frequent in cases of HPAIV. Following sample collection, PCR showed that 20 out of 147 samples (13.6%) resulted positive for avian influenza. Cts values of these samples ranged from 16 to 30 cycles out of the 45 PCR cycles protocol. These results evidenced that avian influenza virus was circulating in these populations and were the probable cause of the clinical presentation in these animals. Positive samples were processed for inoculation into embryonated chicken eggs for viral isolation.

Table 1

Sample distribution among wild bird species evaluated for HPAIV infection.
Number	Scientific name	Common name	Spanish name	Number of samples
1	Leucophaeus pipixcan	Franklin’s gull	Gaviota de Franklin	46
2	Pelecanus thagus	Peruvian pelican	Pelícano Peruano	12
3	Calidris alba	Sanderling	Playero arenero	13
4	Haematopus palliatus	American Oystercatcher	Ostrero Americano	8
5	Anas platyrhynchos	Mallard	Pato silvestre	11
6	Larus belcheri	Belcher’s gull	Gaviota peruana	4
7	Parabuteo unicinctus	Harris’s hawk	Gavilán acanelado	7
8	Zenaida meloda	West Peruvian dove	Paloma cuculí	7
9	Larus dominicanus	Kelp gull	Gaviota dominicana	6
10	Zenaida auriculata	Eared dove	Tórtola orejuda	5
11	Phalacrocorax brasilianus	Neotropical cormorant	Cormorán neotropical	4
12	Phalacrocorax bougainvillii	Guanay cormorant	Cormorán guanay	3
13	Cathartes aura	Turkey vulture	Gallinazo cabeza roja	4
14	Nyctanassa violacea	Yellow-crowned night heron	Guaco manglero	3
15	Spheniscus humboldti	Humboldt penguin	Pingüino de Humboldt	1
16	Falco sparverius	American kestrel	Cernícalo Americano	2
17	Sula variegata	Peruvian booby	Piquero Peruano	1
18	Geranoaetus melanoleucus	Black-chested buzzard-eagle	Águila mora	2
19	Columba livia	Domestic pigeon	Paloma castilla	1
20	Ara macao	Scarlet macaw	Guacamayo escarlata	1
21	Amazona amazonica	Orange-winged parrot	Amazona alinaranja	1
22	Amazona ochrocephala	Yellow-crowned parrot	Loro real amazónico	1
23	Ramphastos cuvieri	Cuvier’s Toucan	Tucán de Cuvier	1
24	Pteroglossus castanotis	Chesnut-eared aracari	Tucaneta parda	1
25	Tyto alba	Western barn owl	Lechuza de campanario	1
26	Falco peregrinus	Peregrine falcon	Halcón peregrino	1
				147

2.2. Avian influenza virus was successfully isolated in embryonated eggs from wild bird species.

Embryonated chicken eggs have shown to be the ideal support for avian influenza virus isolation (Spackman E., 2020). Here, we inoculated the 20 qPCR-positive samples in eggs selected previously. Out of those inoculated, 14 samples had evidence of viral replication through embryo death and hemagglutination assay. These samples were obtained from Peruvian pelican (4/6), Belcher’s gull (2/2), Harri’s hawk (1/3), American kestrel (1/2), Guanay cormorant (1/1), Humboldt penguin (1/1), Franklin’s gull (2/2), Peregrine falcon (1/1) and Western barn owl (1/1). We did not obtain the viral isolate from the Chesnut-eared aracari sample that was positive by PCR. These findings were detected in a single round of inoculation. A summary of the results is presented in Table 2. Following isolation, positive samples were selected based on species source and geographical location for whole genome characterization.

Table 2

Positive samples isolated and genome sequenced in the current study.
Species	Positives PCR/total samples	Positive location	Positive collection date	Isolation	Hemagglutination test	Genome sequencing
Franklin’s gull	2/46	Lurín, Lima	November 24th, 2022	+	+	N.S.*
Peruvian pelican	6/12	Puerto Viejo wetlands, Cañete	November 23rd, 2022	+	+	Complete
		Chorrillos, Lima	November 24th, 2022	+	+	Complete
		Punta Hermosa, Lima	November 24th, 2022	+	+	N.S.
Belcher’s gull	2/4	Puerto Viejo wetlands, Cañete	November 24th, 2022	+	+	Complete
Belcher’s gull	2/4	Lima, downtown (Cercado)	December 5th, 2022	+	+	Incomplete
Harris hawk	3/7	Chorrillos, Lima	December 12nd, 2022	+	+	N.S.
Guanay cormorant	1/3	Villa el Salvador, Lima	December 12nd, 2022	+	+	N.S.
Humboldt penguin	1/1	Chorrillos, Lima	December 20th, 2022	+	+	N.S.
American kestrel	2/2	Carabayllo, Lima	December 7th, 2022	+	+	Complete
Western barn owl	1/1	Comas, Lima	December 20th, 2022	+	+	Incomplete
Peregrin falcon	1/1	Callao, Callao	December 22th, 2022	+	+	N.S.
Chesnut-eared aracari	1/1	Villa el Salvador, Lima	December 14th, 2022	-	-	-
*Not sequenced

2.3. Avian influenza virus H5N1 was detected in wild birds with severe disease.

Genetic analysis allows the subtyping of influenza viruses based on the types of HA and NA proteins. Moreover, HA amino acid sequences show differential patterns of cleavage sites to differentiate highly and low pathogenic AIVs (Steinhauer, 1999). Here, we selected 06 isolates for subtyping and genomic reconstruction. Genetic analysis at the nucleotide and amino acid levels identified that our isolates corresponded to the H5N1 subtypes. Furthermore, cleavage sites at the HA regions identified sequences corresponding to those highly pathogenic avian influenza viruses. Hence, we found the multi basic motif (PLREKRRKR↓GLF) in all highly pathogenic avian influenza viruses. A geographical map showing the sample origin distribution is shown in Fig. 1.

2.4 Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b was identified in the outbreak in wild birds in Peru.

Phylogenetic reconstruction allows determining the closest genetic relatives among species including viruses. Thus, we took advantage of a Bayesian model to estimate the most recent common ancestor and define the phylogenetic relationship of the viruses identified here compared with those described in America, Asia and Europe. Based on H5 phylogenetic reconstruction, our results showed that our isolates corresponded to highly pathogenic avian influenza clade 2.3.4.4b, sharing a common ancestor with isolates obtained from snow goose in North Dakota and Kansas, in the US, 2022. Furthermore, tree topology suggested that this virus is forming a subclade along with those isolated from Chile, within the 2.3.4.4b clade, and appears to accumulate changes in this protein. Phylogenetic trees of the HA and NA proteins are shown in Figs. 2 and 3.

2.5 Genetic analysis of HA shows unique changes in the HA of HPAIV H5N1 from wild birds in Peru.

Following phylogenetic reconstruction, we evaluated whether the HPAIV circulating in Peru is presenting some relevant changes compared to those members of the 2.3.4.4b. Hence, we identified some changes that were absent in those observed in most isolates from the US, while others were present in those closely related to our Peruvian isolates. Genetic distances in HA ranged from 99.08 to 99.5% in the samples sequenced. A summary of the genetic distances among all the genome segments with their closest relatives is presented in Table 3. Some novel changes in the HA protein were located into the 152 and 171 amino acid positions. Samples 1, 2 and 6 show the P152S change compared to their closest relatives, while samples 3 and 5 showed the D171N change. These changes suggest that our isolates along with those closely related from Chile share a common ancestor with a North American isolate, while depicting a subclade within the 2.3.4.4b phylogroup. Further studies are required to assess whether these changes are relevant for HA function or vaccine evasion. The most common amino acid changes observed in the viral genomes is shown in Table 4.

Table 3

Genetic diversity of HPAIV H5N1 compared to their closest relatives described.
Sample	Gene	Genetic identity (%)	Closest relative (Accession number)
Sample_1	PB2	99.4	OP377589
	PB1	99.54	OP377588
	PA	93.52	OP377587
	HA	99.29	OP221285
	NP	99.19	OP377585
	NA	99.85	OQ352550
	M	99.24	OP470767
	NS	99.01	OP377586
Sample_2	PB2	99.5	OP377589
	PB1	99.08	OP377588
	PA	99.63	OP377587
	HA	99.35	OP221285
	NP	99.19	0P377585
	NA	99.86	OQ352550
	M	99.24	OP470767
	NS	99.3	OP377586
Sample_3	PB2	99.5	OP377589
	PB1	99.54	OP377588
	PA	99.67	OP377587
	HA	99.35	OP221285
	NP	99.19	OP377585
	NA	99.72	OQ352550
	M	98.35	OP470767
	NS	97.41	OP377586
Sample_5	PB2	99	OP377589
	PB1	99.45	OP377588
	PA	99.58	OP377587
	HA	99.5	OP221285
	NP	99.12	OP377585
	NA	99.77	OQ352550
	M	99.46	OP470767
	NS	99.01	OP377586
Sample_6	PB2	99.6	OP377589
	PB1	99.5	OP377588
	PA	99.58	OP377587
	HA	99.35	OP221285
	NP	99.05	OP377585
	NA	99.64	OQ352550
	M	99.46	OP470767
	NS	99.16	OP377586
Sample_7	PB2	99.5	OP377589
	PB1	99.54	OP377588
	PA	N.D.	N.D.
	HA	99.08	OP221285
	NP	96.76	OP377585
	NA	99.86	OQ352550
	M	N.D.	N.D.
	NS	N.D.	N.D.

Table 4

Summary of the most common amino acid changes detected in the H5N1 influenza A viruses isolated in Peru compared to the reference genome.
Gene		Amino acid changes
HA		K3N, R69K, N88R, K98R, S100N, D110S, F111L, L120M, T124I, S139P, D142E,A143T,S145L,S149A,P152S, H154Q, R156A, S157P, S171D,R178I,Q185R,V190I,P197S,D199N, A201E, K205N, Q208K,V226A,E228K,P233S,K234Q,S239R, E243D, N252D,N256H,A279T,E284G,L285V,N289H,M298V, R326K, T336S,Q338L,R341K,K345-,R346-,Q429K,K499R, N515K, M527V, V539A,V549M
NA		I8T,V17I,I20V,H44Y,A46P,T76A,K78Q,A81T, V99I,H100Y,H155Y,T188I,M258I,L269M,E287D,T289M, G336S,V338M, S339P,P340S,N366S,N366S,G382E,A395E, I418M,S434N,D460G
PA		R57Q,T61M,I63V,T85A,T129I,C212R,K228N,R361K, K399E,P400S,M449V, L504I,R536K,R544E,E553A,P585L, F586L,T608S,R716K
NP		M136L,F230L,A353V,S451A
PB1		K134N,G177E,I182T,R215K,E264D,L378M,G399D,K429R, E460Q,T478S,C490F,D536N,P558T,P598L,G610C,G754R
PB2		N334S,I463V,L464M,A471T,V478I,Q508R,S579P,S590G, I616V,P628Q,R699K,M727G, F741S
M	M1	N85S,N87T,K101R,T140A,F144L,M165I,A200V,K230R, N232D,M248L
M	M2	E8G,K12N,V22I,I45V,R55G
NSP	NS1	I6V, Y14F, I18V, L21R, L22F, S23A, M24D, R25D, D26E, M27L, C28G, D33L, A42S, D53G, L54I, R55E, V56T, M59R, E60A, K63Q, D67R, K70E, S71E, T73
NSP	NS2	I6V, T7S, Q14M, E22G, V26E, R37S, S48A, M49V, N60S,A63G, T64K, N67E, E68Q

Migratory and resident wild birds are considered the natural reservoirs for avian influenza viruses. These avian species are responsible for viral spread in large geographical areas due to their migratory routes. Thus, these species play a major role in the emergence of novel highly pathogenic avian influenza viruses since these viruses also undergo constant reassortment and recombination events. Currently, the emergence of HPAIV affecting multiple species of wild and domestic birds has been described in Asia, Europe, and America, becoming of major concern for animal and public health. Despite this, little is known about the viral circulation and genetic characterization in South America.

In Peru, the national service for animal health (SENASA) reported the first detection of a HPAIV infection in November 2022. This case was reported in a coastal city of the Northern Peru, following the occurrence of Peruvian pelicans found death in the Paita beach in Piura. This event preceded multiple reports of sudden death in wild birds during the late of 2022 and the beginning of 2023. Our initial findings suggested that a viral infection was responsible for causing severe disease in the animals evaluated. These observations were based on a limited number of cases that correlated with clinical presentation despite some differences to those typically described for severe influenza infections among avian species. Thus, based on the classic presentation of HPAIV infections, hemorrhagic intestinal lesions are commonly observed in birds. In contrast, these findings were absent in the animals evaluated in our institution. Hence, although this topic needs to be further addressed through a different approach, the occurrence of some distinctive patterns of disease progression are described in the current outbreak. Thus, additional studies are required to define whether this might be due to either viral or host factors. Nevertheless, we cannot make definitive conclusions about clinical presentations in these species based on our current data.

Even though the epidemiology of cases presented as severe disease suggested the HPAIVs circulation in a highly diverse group of wild bird species, viral presence had to be confirmed using a commercially available test that detects avian influenza viruses. Our results allowed the identification of avian influenza viruses with a highly variable viral load based on the Ct values observed. These results were the first confirmation of the involvement of avian influenza virus in these cases. Even though Ct values can be used as approximation method to assess the viral load for other viral infections (Icochea et al., 2023), we did not find evidence of correlation in terms of severity and viral levels based on Cts values (data not shown). Although the clinical presentation and the PCR results indicate this virus is responsible for the severe cases, further genetic characterization was required to confirm the presence of a highly pathogenic avian influenza virus.

Genetic characterization of these viruses detected the presence of HPAIV H5N1 clade 2.3.4.4b. Thus, this virus is affecting a broad range of species based on our data. We speculate that these species play a differential role into the viral spread since some of them appeared to be more affected than others. Moreover, movement dynamics of these species might add contributing factors in viral dissemination into local areas. Thus, our findings suggest that the viral spread might be driven by multiple wild bird species in our region while remains unclear whether there is a differential contribution in viral spread by these species. Despite these differences in the host source, our isolates showed a high degree of genetic conservation. Our isolates belonging to a monophyletic group share a common ancestor with isolates from Chile. However, some genetic changes have been detected compared to those isolates. This reveals that the virus is undergoing evolution locally. In this regard, along with the circulation of LPAIV, these factors confer the genetic basis for the emergence of novel reassortments with these HPAIV as it has been shown in other regions (Lewis et al., 2021). Hence, genetic surveillance needs to be reinforced in the upcoming months to closely track the viral evolution in our region. Phylogenetic analysis of the most important proteins in Influenza viruses showed that these isolates have common ancestors from H5 and N1 segments in the US, within the Eurasian group. Evolutive analysis shows that these viruses have been imported into the US from Europe and Asia and are co-circulating with other viral lineages in the Americas. It is well known, the H5N1 viruses circulating are reassortants of viruses with different origins(Shi et al., 2023) and these events might continue in South America.

Fluctuations of avian influenza virus circulation is assessed by studies over multiple years in a period (Brown & Stallknecht, 2008). Thus, avian influenza viruses in Peru have been tracked in wild birds since 2006 when we deployed a local year-period based viral surveillance that allowed the detection and characterization of only LPAIVs prior to the current outbreak. For instance, during the 2019 and 2020 period, we identified that these LPAI viruses arose from common ancestors of viruses isolated in North America (Castro-Sanguinetti et al., 2022). These findings indicated that migratory routes of wild birds play a major role into the viral entry in the South American region and are the potential route for those highly pathogenic avian influenza viruses as these birds arrives to this region in the summer period in the south hemisphere (Hill et al., 2016; Olsen et al., 2006). As expected, our current results confirmed this since these HPAIVs shared a common ancestor with those in the US.

In terms of positivity rate, we found interesting differences between the evaluation in the 2019–2020 period and the current outbreak. Within March 2019 and March 2020, we collected 421 samples that allowed the isolation of five LPAIV (Castro-Sanguinetti et al., 2022). In contrast, our current study shows that out of 147 samples evaluated, we isolated 14 avian influenza viruses, with six of them corresponding to HPAIV while the sequencing procedure for the remaining six is still pending. Although these numbers offer a limited perspective in terms of frequency or prevalence, they do suggest higher avian influenza virus circulation during the current period which increases the chances for the emergence of novel viral forms.

Furthermore, although this current outbreak represents the first introduction of the virus in the Peruvian ecosystem, we speculate that multiple introductions might occur in the upcoming migration periods. Further studies are required to define other multiple introductions of the virus and whether there are alternative routes of viral entry (Li et al., 2018). These conditions are the fundamental basis for the emergence of viruses with novel characteristics that might infect other species including mammals. Although no human-to-human transmission has been reported, national authorities have reported the occurrence of infections in marine mammals in Peru. Even though this is not under the scope of this paper, we emphasize that genetic surveillance of these spillover events must be monitored closely since the viral adaptation to these species might reveal the capacity for infection in humans and the human-to-human transmission.

Peru is one of the most biodiverse countries around the world. Thus, the entry and spread of HPAIVs into immunologically naïve wildlife populations is rapidly diminishing the local species, with major impact in those in an endangered situation. Our study shows that multiple local avian species are affected by this virus, and some of those studied here especially Peruvian pelicans (Pelecanus thagus), and Guanay cormorants (Phalacrocorax bougainvillii) and others have been placed into an even more endangered condition. We call the authorities to pay close attention to these species of ecosystem importance. In addition, to its intrinsic importance for the local ecosystem, they do play a major economic role. The guano produced by these species is commercialized as a natural fertilizer of great value. Thus, we urge the national and regional authorities to take actions in a coordinated manner to avoid the impact on these species.

Eventually, our study represents the first report of isolation and genome characterization of HPAIV H5N1clade 2.3.4.4b from the largest wild bird species pool affected of severe disease in South America. Furthermore, this report shows evidence that despite the close genetic relationship of our isolates with North American strains, there are some nucleotide and amino acid changes detected in the virus circulating in Peru, indicating the potential for spillover events into other species.

3.1. Samples

One hundred and fifty-four samples encompassing oropharyngeal, cloacal, and fecal swabs were collected and submitted to the Laboratory of Avian Pathology at the Universidad Nacional Mayor de San Marcos in Peru, between November 2022 and January 2023. These samples included those taken as part of our active surveillance of avian influenza in Peru, along with those obtained as a research collaboration with the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR). All samples were taken in a universal transport media (VTM) tube and transported to the lab during the same collection day at 4° C and processed immediately. In brief, all swabs were resuspended in 500 µl PBS with antibiotics, filtered through 0.22-micron syringe filters to remove bacterial contamination and centrifuged for clarification (8000 x g for 10 min). Supernatants were saved for further molecular test by a specific PCR-based assay for detection of Influenza A viruses and isolation into embryonated chicken eggs. In some instances, samples were pooled into 4–5 samples according to geographical location, species, date, etc. Some live birds samples obtained from the National Forest and wildlife Service (SERFOR), presented neurological and respiratory clinical signs suggestive of influenza infections. A summary of the samples submitted is presented in Table 1 and video recordings of representative species affected by the infection is presented in the supplementary information (Sup. 8 and 9). Protocols and methods were carried out in accordance and approved by the Ethics and animal welfare committee (CEBA) by the Faculty of Veterinary Medicine in the Universidad Nacional Mayor de San Marcos (CEBA202133).

3.2. Viral detection

One hundred and forty (140) µL of samples processed were used for RNA extraction. For this purpose, we used the QIAamp Viral RNA Mini Kit (Qiagen, US), according to manufacturer’s instructions. For viral gene detection, we used the RealPCR Influenza A RNA mix for avian influenza A virus detection following the manufacturer’s instructions (IDEXX, US). The protocol, based on 45 amplification cycles, allow the identification of avian Influenza A viruses. The positive status was used as a primary criteria of sample selection for further isolation and sequencing procedures.

3.3. Viral isolation

Viral isolation was performed by inoculation of 0.2 mL of samples into the allantoid cavity of 9-11-days-old embryonated chicken eggs in five replicates (Spackman E., 2020). Following inoculation, eggs were incubated at 37.5°C for 4 days and checked daily to assess embryo survival. A round of inoculation was performed to increase the viral load for improving the metagenomic sequencing of all eight genomic segments. Following inoculation, an hemagglutination test was used as screening method to evaluate the presence of an hemagglutinating agent. In brief, allantoid fluid (25 uL) was serially diluted with two-fold volume of PBS (pH 7,4). This mix was incubated into an equal proportion of 1% chicken red blood cells (RBCs). After 20 minutes, the bottom formation or red blood cells agglutination was recorded. Following clarification (20000 x g for 10 minutes), positive samples were selected based on wild bird species host and viral load for genome sequencing.

3.4. Next generation sequencing

Positive samples for PCR against Influenza A virus and hemagglutination test following inoculation into embryonated eggs, were selected for genomic characterization using a metagenomic approach. First-strand cDNA was performed using the SuperScript™ III First-Strand Synthesis kit (Invitrogen™, US) with the FR20RV primer (5’- GCCGGAGCTCTGCAGATATC-3’) prior to library preparation. Library preparation was performed using Nextera XT method and sequenced on a MiSeq platform using paired 150 base read chemistry (Cambridge Technologies, Worthington, MN, USA).

3.5. Genome assembly and annotation

Raw data (fastq files) was evaluated based on Phred score using FastQC v. 0.11.9 software (Simon Andrews, 2010), while adapters were removed using Trimmomatic version 0.32 (Bolger et al., 2014). The genome was assembled using SPAdes Genome Assembler version 3.13.1 (Bankevich et al., 2012) and annotated using Prokka genome annotation version 1.14.6 (Seemann, 2014), along with the use of the Bacterial and Viral Bioinformatics Research Center, version 3.28.22 (Olson et al., 2023). Gene annotation was performed using the tools of the Influenza virus genome database to address all eight genome segments. Where low coverage detected the sequences were deposited as partial CD and submitted to Genbank under the accession numbers: OQ747758-OQ747763 (HA), OQ747765-OQ747770 (NA), OQ747863-OQ747868 (PB2), OQ747878-OQ747883 (PB1), OQ747873-OQ747877 (PA), OQ747895-OQ747899 (MP), OQ747884-OQ747888 (NS), OQ747889-OQ747894 (NP).

3.6. Avian Influenza subtyping and phylogenetic analysis

Following genome assembly, we focused on the HA and NA subtyping. For this purpose, we collected the coding region of HA and NA genes from AIV viruses submitted to the National Center for bioinformatics information (NCBI), Influenza virus genome database (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=genomeset). The alignment was performed in MEGA X (Kumar et al., 2018), with posterior manual editing and files were saved as nexus files and used for phylogenetic reconstruction. We used the Markov Chain Monte Carlo (MCMC) algorithm in Mr. Bayes to estimate a Bayesian inference for phylogenetic reconstruction (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). A general time reverse (GTR) substitution model with gamma distribution was used. Markov Chain Monte Carlo chains were run for 2 million iterations and sampled every 100 steps to allow all parameters converge with effective sample size values greater than 200, and with a burn-in of 0.25 to discard the 25% of samples at the beginning of the analysis. The consensus tree was summarized and further visualized using Figtree (Rambaut A., 2018). For the other six genome segments, sequences were retrieved from the NCBI database and selecting sequences collected from 2018 to date, for phylogenetic reconstruction, including sequences from America, Asia and Europe. A schematic representation of our methodology for detection, isolation and genome characterization is presented in Fig. 4.

Acknowledgements

The authors would like to express their gratitude to the professionals from the National Service for Animal Health (SENASA) and Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) especially to Ms. DVM Wendy Rojas and Mr. Renato Colán. We acknowledge the financial support from the Asociación Peruana de Avicultura (APA) and the Universidad Nacional Mayor de San Marcos.

Author contributions

G.C-S., E.I., J.M-B., conceptualized the research plan. J.M-B., G.C-S., E.I., R.G-V., designed and developed the methodology. A. A-C., A.C-L., G.C-S., R.G-V. J.J., W.S. performed field investigation (sample collection and wild bird identification). G.C-S., E.I., R.G-V., performed and supervised the laboratory assays. J.M-B., G.C-S., E.I., R.G-V., composed the original draft of the manuscript. All authors contributed to the manuscript revision and approved the submitted version.

Data availability statement

Nucleotide sequences produced in the current study have been submitted to GenBank. Here are the accession numbers for each gene segment:

HA: OQ747758, OQ747759, OQ747760, OQ747761, OQ747762, OQ747763

NA: OQ747765, OQ747766, OQ747767, OQ747768, OQ747769, OQ747770

PB2: OQ747863, OQ747864, OQ747865, OQ747866, OQ747867, OQ747868

PB1: OQ747878, OQ747879, OQ747880, OQ747881, OQ747882, OQ747883

NP: OQ747889, OQ747890, OQ747891, OQ747892, OQ747893, OQ747894

PA: OQ747873, OQ747874, OQ747875, OQ747876, OQ747877

MP: OQ747895, OQ747896, OQ747897, OQ747898, OQ747899

NS: OQ747884, OQ747885, OQ747886, OQ747887, OQ74788

Additional data is available upon request.

Competing interests

The authors declare no competing interests.

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Circulation of highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b in highly diverse wild bird species from Peru

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Abstract

Figures

INTRODUCTION

RESULTS

2.2. Avian influenza virus was successfully isolated in embryonated eggs from wild bird species.

2.3. Avian influenza virus H5N1 was detected in wild birds with severe disease.

DISCUSSION

MATERIALS AND METHODS

3.1. Samples

3.2. Viral detection

3.3. Viral isolation

3.4. Next generation sequencing

3.5. Genome assembly and annotation

3.6. Avian Influenza subtyping and phylogenetic analysis

Declarations

References

Additional Declarations

Supplementary Files

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