DISPATCHES

Highly Pathogenic Avian

Virus in Wild Birds, Chile

Naomi Ariyama,1 1

The Study
- In early December 2022, increased wild bird deaths
were detected across the north coast of Chile (Figure
genome sequences of strains from wild birds. Phyloge- 1). Wild birds, mainly pelicans, were found dead or
netic analysis suggests multiple viral introductions into
- Veterinary Services of the Agricultural and Livestock
Service of Chile had collected 1,368 samples for HPAI
virus detection and epidemiologic investigation:
H A(H5N1) viruses grouped within hemaggluti-
nin (HA) gene clade 2.3.4.4b are spreading globally
1,080 from domestic birds and 288 from wild birds
(Appendix Table 1, [Link]
article/29/9/[Link]). We performed a
and causing high mortality among domestic and wild
total of 578 real-time qualitative reverse transcrip-
birds (1). In addition, the viruses have spilled over to
tion PCR (qRT-PCR) reactions to detect active avian
several nonavian species, including humans (2). To
-
contain HPAI outbreaks, poultry exposed to or in-
munodiffusion (AGID) tests to detect previous AIV
fected with HPAI viruses have been culled, resulting
exposure (Appendix Table 2).
in disposal of 131 million domestic birds globally in
We initially performed qRT-PCR by using Vet-
2022 (3). Therefore, HPAI viruses pose a threat not
MAX-Gold AIV Detection Kit (Applied Biosystems/
only to public health because of zoonotic potential
but also to food security.
com), targeting the AIV matrix gene. Then, we tested
In late 2021, HPAI H5N1 virus clade 2.3.4.4b,
which had spread predominantly in Europe, Asia,
to US Department of Agriculture (USDA) National
and Africa, was detected in wild birds in North
Veterinary Services Laboratories standard protocols
America and, shortly after, in domestic poultry (3–5).
(nos. 1732.02, 1767.01, and 1768.01). We tested 13 tis-
In October 2022, this virus reached South America
sue samples, 2 (15%) of which were positive; 248 tra-
cheal swab samples, 43 (17%) of which were positive;
also reported in Peru, Ecuador, and Venezuela (2).
314 cloacal swab samples, 47 (15%) of which were pos-
We describe detection of this virus clade in wild birds
itive; and 3 oral swab samples, 1 (33%) of which was
in Chile.
positive. Among all samples tested by qRT-PCR, 93
(16%) were H5 AIV–positive with cycle threshold (Ct)
values <40. Among positive samples, 18 were from
the Arica y Parinacota region, 18 were from Tarapa-
ca, 53 were from Antofagasta, and 4 were from Ata-
cama (Figure 1). No domestic poultry samples were
AIV-positive, but among wild bird species, H5 AIV
was detected most frequently among Peruvian peli-
cans (Pelecanus thagus) (n = 50, 54%), turkey vultures
1

1842 Emerging Infectious Diseases • [Link]/eid • Vol. 29, No. 9, September 2023
(Cathartes aura) (n = 12, 13%), and Peruvian boobies sequencing by using the Native Barcoding Kit 96 and
(Sula variegata) (n = 10, 11%) (Appendix Table 1). MinION platform (Oxford Nanopore Technologies,
We evaluated 754 serum samples by using the of- [Link] according to the manu-

and matrix protein, according to USDA protocols FASTQ ([Link] accord-

([Link] ing to the average quality (Phred score >7) and length
info_services/downloads/Avian_AGID_SOP.pdf) <2,600 bp by using NanoFit (8). We assembled ge-
(6). We found no positive serum samples (Appendix nomes according to reference by using the nanopore
Table 2). ARTIC pipeline version 1.2.3 ([Link]
We selected 11 H5 AIV–positive samples from
the initial outbreak according to Ct values (Ct <27) and to account for the primer sets (Appendix). We
and location; 9 samples were from pelicans and 2 Falco_rusticolus/EdoMex/
from gulls, representing 3 administrative regions of CPA-19638–22/2022(H5N1) (GenBank accession nos.
Chile. We obtained whole-genome AIV sequences by OP691321–28) as the reference. We used the National

PCR (7), then performed next-generation nanopore Sequence Annotation Tool (9) to check and annotate

Figure 1. Distribution of
samples collected and tested

color of circles indicate sample

Emerging Infectious Diseases • [Link]/eid • Vol. 29, No. 9, September 2023 1843
 Figure 2. Images of Peruvian
Pelecanus thagus)
collected and sampled for highly

Dead pelican found on land near

near shoreline.

assembled genomes and conducted H5 clade classi- 2022, and the Chile–Peru sequences had closely re-
lated ancestors among strains from North America
tool (10). We conducted a BLAST search (https:// (Appendix Figure 1). A sequence from Ecuador
[Link]/[Link]) to choose the refer- grouped in a paraphyletic branch with different se-
ence from preliminary assembled contigs constructed quences from North America and had a time to most
- recent common ancestor estimated at August 27,
ing Canu (11). We obtained sequences with a mean 2022 (95% highest posterior density July 10–Septem-
coverage depth of 33,381 for 10 samples; 9 of 10 ge- ber 21). The sequences from Venezuela had a lon-
nomes were complete (Appendix Table 3). ger branch in the phylogeny reconstruction. Those
- sequences were more closely related to strains col-
type clade 2.3.4.4b. We inferred Bayesian evolution- lected earlier in the year from North America and
ary analysis sampling trees for HA and neuramini- had a time to most recent common ancestor esti-
dase (NA), and maximum-likelihood trees for internal mated at February 2, 2022 (95% highest posterior
segments (Appendix). A/Peru/LIM-003/2022 and density January 15–February 23). The NA, matrix,
A/Peru/LAM-002/2022 (GISAID accession nos. EPI_ and polymerase acidic sequences from Venezuela
ISL_16249730 and EPI_ISL_16249681) were the most also grouped outside the Chile–Peru subcluster in
closely related HA sequences found in the GISAID the maximum-likelihood phylogenies (Appendix
database (12) (Appendix Figure 1). We observed Figures 2, 5, 7). The phylogenetic clustering with dif-
similar results from phylogenies for NA and internal ferent sequences from North America suggests that
genes (Appendix Figures 2–8). Sequences from Peru viruses from Venezuela might have resulted from
corresponded to isolates collected in November 2022 separate introductions into South America. How-
ever, because of the low availability of HPAI H5N1
virus sequences from Central and South America,
HPAI virus sequences available from Ecuador and conclusions on the origin of the cluster in Chile
Mexico grouped closely to the Chile–Peru subcluster are limited.
(Appendix Figures 3–8). For HA, the Chile–Peru sub- Previous studies suggest that the HPAI H5N1 vi-
cluster showed a nonsynonymous mutation, T392A rus clade 2.3.4.4b was introduced into North Amer-
(L131Q), previously associated with antigenic vari- ica multiple times across the East Asia–Australasia/
ability in H5N1 virus strains (13). We found other
synonymous and nonsynonymous mutations in NA 4,14,15
(L269M and S339P) and internal genes (Appendix Ta- reach the southernmost tip of South America, repre-
ble 4), but those mutations have not been associated senting a high-risk route for HPAIV dissemination
with phenotypic changes. across the continent.
The phylogenetic tree for the HA segment
showed that the sequences from Chile and Peru were Conclusions
closely related to a recent ancestor from North Amer-
ica that was detected during October–November HPAI H5N1 viruses have disseminated as far as the

1844 Emerging Infectious Diseases • [Link]/eid • Vol. 29, No. 9, September 2023
Maule region (35 south latitude) of Chile; no poul- 12]. [Link]

4. Bevins SN, Shriner SA, Cumbee JC Jr, Dilione KE,

Livestock Service of Chile implemented a contingen- Douglass KE, Ellis JW, et al. Intercontinental movement
cy plan to perform extensive surveillance and rein-
force biosecurity measurements to avoid introduction [Link] virus to the United States, 2021. Emerg Infect Dis.
of HPAI virus into domestic poultry. The impact of 2022;28:1006–11. [Link]
HPAI H5N1 virus in the country, and the potential
for introduction of the virus from Chile to Antarctica, [cited 2022 Dec 27]. [Link]
remain to be fully elucidated. newsroom/stakeholder-info/sa_by_date/sa-2022/hpai-sc

This article was preprinted at [Link] antibody in mammalian and avian sera by immunodiffusion.
Bull World Health Organ. 1970;42:779–85.
2023.04.07.535949.
7. Mena I, Nelson MI, Quezada-Monroy F, Dutta J,
Cortes-Fernández R, Lara-Puente JH, et al. Origins of the
Acknowledgments
We thank the Agricultural and Livestock Service (SAG) 2016;5:e16777. PubMed [Link]
personnel for their support and contributions, especially 8. De Coster W, D’Hert S, Schultz DT, Cruts M,
in sample collection. We are grateful to Belen Aguero and Van Broeckhoven C. NanoPack: visualizing and processing
long-read sequencing data. Bioinformatics. 2018;34:2666–9.
Felipe Berrios for their help in sample processing. We are [Link]
grateful to the GISAID EpiFlu Database, laboratories, and 9. Bao Y, Bolotov P, Dernovoy D, Kiryutin B, Tatusova T.

especially to the Servicio Nacional de Sanidad Agraria del annotation. Nucleic Acids Res. 2007;35(Web Server
issue):W280–4. [Link]
Perú–SENASA, Peru, source of the closest strains. 10. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C,
Davis JJ, et al. Introducing the Bacterial and Viral
This study was funded by Fondecyt 1211517 (to V.N.) and
Bioinformatics Resource Center (BV-BRC): a resource
combining PATRIC, IRD and ViPR. Nucleic Acids Res.
Transmission (contract no. 75N93021C00014, awarded 2023;51(D1):D678–89. [Link]
to R.A.M. and V.N.) and Emory CEIRR (contract no. gkac1003
11. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH,
75N93021C00017 awarded to C.P.R, V.N., and R.A.M.),
Phillippy AM. Canu: scalable and accurate long-read
both part of the National Institute of Allergy and assembly via adaptive k-mer weighting and repeat
separation. Genome Res. 2017;27:722–36. [Link]
Research and Surveillance. 10.1101/gr.215087.116
12. Khare S, Gurry C, Freitas L, Schultz MB, Bach G, Diallo A,
et al. GISAID’s role in pandemic response. China CDC Wkly.
About the Author 2021;3:1049–51. [Link]
13. Li J, Gu M, Liu K, Gao R, Sun W, Liu D, et al. Amino acid
Dr. Ariyama is a veterinarian and PhD candidate at the substitutions in antigenic region B of hemagglutinin play a
University of Chile, Santiago, Chile. Her primary focus critical role in the antigenic drift of subclade [Link] highly
is the study of emerging viral zoonotic pathogens.
2020;67:263–75. [Link]
Dr. Pardo-Roa is an assistant professor at the Molecular 14. Prosser DJ, Chen J, Ahlstrom CA, Reeves AB, Poulson RL,
Sullivan JD, et al. Maintenance and dissemination of

A viruses and next generation sequencing technologies. Flyway of North America. PLoS Pathog. 2022;18:e1010605.
[Link]
15. Alkie TN, Lopes S, Hisanaga T, Xu W, Suderman M,
Koziuk J, et al. A threat from both sides: Multiple
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