First human case of avian influenza A (H10N3) in Southwest China

Abstract

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. Despite this, only two cases of human infection with the H10N3 strain have been documented. Here, we present the initial instance of human infection with avian influenza virus H10N3 in Yunnan Province, Southwest China. The patient, a previously healthy 51-year-old male, presented with recurrent fever peaking at 39℃, accompanied by symptoms such as cough, expectoration, chest tightness, and shortness of breath. Diagnosis revealed severe pneumonia, type I respiratory failure, and infection with avian influenza virus H10N3. Additionally, the patient experienced complications from Candida albicans and Staphylococcus epidermidis infections. Following treatment with appropriate antiviral drugs and antibiotics, the patient's condition improved. Molecular analysis of the viral strain identified four mutations potentially hazardous to human health. This underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus.

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First human case of avian inuenza A (H10N3) in
Southwest China
Guiming Liu
The Third People's Hospital of Kunming City
Jingyi Dai
The Third People's Hospital of Kunming City
Jun Zhao
Hubei University of Medicine
Jiawei Xia
The Third People's Hospital of Kunming City
Pei Zhang
The Third People's Hospital of Kunming City
Yadi Ding
The Third People's Hospital of Kunming City
Qiujing Li
The Third People's Hospital of Kunming City
Min Hou
Kunming City Center for Disease Control and Prevention
Xianhui Xiong
Kunming City Center for Disease Control and Prevention
Qianqi Jian
Kunming City Center for Disease Control and Prevention
Yanyan Liu
Kunming City Center for Disease Control and Prevention
Article
Keywords:
Posted Date: April 4th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4181286/v1

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License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Additional Declarations: There is NO Competing Interest.

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Abstract
In recent years, the avian inuenza virus has emerged as a signicant threat to both human and public
health. Despite this, only two cases of human infection with the H10N3 strain have been documented.
Here, we present the initial instance of human infection with avian inuenza virus H10N3 in Yunnan
Province, Southwest China. The patient, a previously healthy 51-year-old male, presented with recurrent
fever peaking at 39℃, accompanied by symptoms such as cough, expectoration, chest tightness, and
shortness of breath. Diagnosis revealed severe pneumonia, type I respiratory failure, and infection with
avian inuenza virus H10N3. Additionally, the patient experienced complications from Candida albicans
and Staphylococcus epidermidis infections. Following treatment with appropriate antiviral drugs and
antibiotics, the patient's condition improved. Molecular analysis of the viral strain identied four
mutations potentially hazardous to human health. This underscores the importance of continuous and
vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian inuenza virus.
Introduction
Inuenza A virus belongs to the family Orthomycoviridae. Inuenza A viruses can infect a variety of birds,
humans, and animals such as pigs, horses, seals and whales1. It is an important pathogen that poses
risks to both human and animal health. Currently, only two subtypes of inuenza A viruses, H1N1 and
H3N2, are known to circulate in humans1. However, humans can also be infected by other subtypes,
collectively referred to as avian inuenza viruses (AIV).
In recent years, human infection with AIV has been frequently reported around the world, particularly
involving subtypes H5, H7, and other2–4. Among these, human infections with H10 avian inuenza virus
have been reported globally, including subtypes H10N7, H10N8, and H10N3 5,6. The H10N3 subtype of
avian inuenza virus has been circulating among waterfowl and poultry in East and South Asia for
decades, with rare instances of human infection7. The rst recorded human cases of Avian-Origin
Inuenza A (H10N3) virus occurred in Jiangsu, China, in April 20218, followed by a second case reported
in Zhejiang in June 20229. Importantly, no instances of human-to-human transmission were detected in
either case.
Results
A previously healthy 51-year-old male experienced recurrent fever for a week, reaching a maximum
temperature of 39°C, along with symptoms of cough, expectoration, chest tightness, and shortness of
breath. Despite seeking medical attention at the local community health service center, his symptoms did
not signicantly improve. Consequently, he was transferred to the Department of Respiratory and Critical
Care Medicine at a hospital in Yunnan Province, Southwest China, on March 6, 2024. The patient had a
history of raising various birds, including chickens, ducks, geese, pigeons, peacocks, and ostriches.
Notably, more than 20 chickens and geese died in the week preceding the onset of his illness, and he had

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a history of slaughtering these birds. There was no reported contact with individuals exhibiting respiratory
symptoms within the preceding month.
Upon admission (7 days after the onset of illness), the patient presented with a temperature of 39℃, a
pulse rate of 110 beats per minute, a respiratory rate of 28 breaths per minute, oxygen saturation of 78%,
and blood pressure measuring 105/70 mmHg. Laboratory tests revealed a low white blood cell count,
elevated neutrophil percentage, decreased platelet count, and elevated levels of infectious markers.
Additionally, the nucleic acid test for inuenza A virus was positive (Table1). Chest computed
tomography revealed multiple patchy and increased density shadows in both lungs, characterized by
unclear boundaries and uneven density (Fig.1). The initial diagnosis upon admission included severe
pneumonia, type I respiratory failure, and inuenza attributed to inuenza A virus.
The patient was administered oseltamivir (150mg, twice daily) and methylprednisolone (80mg, once
daily) for treatment. Subsequent sputum culture results revealed infection with Candida albicans and
Staphylococcus epidermidis, prompting the administration of appropriate antibiotics. Following this,
Samples were sent for mNGS detection on March 8th, processed on March 12th, with the detection of
positive inuenza A virus on March 13th. Conrmation of the H10N3 subtype was achieved through
sequence analysis and alignment on March 14th, and samples were subsequently sent to the CDC
(Centers for Disease Control and Prevention). Conrmation of the H10N3 subtype through PCR was
obtained on March 15th. Following this, the CDC conducted nanopore sequencing (Nanopore, GridION
X5) on the samples and obtained the whole genome information of the samples (GenBank accession
number SUB14344866, PP555666-555673). The patient's fever subsided on March 17th (18 days after
illness onset), and on March 19th (20 days after illness onset), the nucleic acid test for inuenza A virus
returned negative results for the rst time. Subsequent test results on March 21st (22 days after illness
onset) indicated normalization of the patient's white blood cell count, along with a decrease or return to
normal levels of infection markers. However, the patient exhibited prolonged prothrombin time. Chest
computed tomography scans showed a reduction in lesions compared to previous scans (Fig.1).
Through online analysis using BLASTN software on the GISAID website, it was determined that all eight
gene segments of the H10N3 virus strain in our case originated from Eurasian avian inuenza viruses.
The phylogenetic tree indicated that the H10N3 strain from this patient belonged to the same group as
the rst patient in Jiangsu and the H10N3 strains found in poultry across various provinces in China
(Fig.2). Specically, the H10N3 strain from this patient showed a closer genetic relationship to a chicken
(GISAID#EPIISL15737164) from Jiangsu Province. Molecular characterization revealed a mutation at the
226th amino acid residue in the receptor binding site of the HA protein, where the amino acid changed
from Q to L. This mutation makes the virus more adept at binding to human α-2,6-sialic acid receptors,
signicantly increasing the likelihood of human infection10. The mutation D701N in the PB2 protein has
been shown to enhance the replication activity of avian inuenza RNA polymerase within the human
body. This mutation also increases the adaptability and pathogenicity of the virus to the human host,
potentially serving as a crucial factor in avian inuenza viruses crossing the host species barrier11. The
presence of the S409N mutation in the PA protein suggests the potential for infectivity in humans and

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may contribute to increased pathogenicity of this particular virus strain12. The S31N mutation in the M2
protein has been associated with resistance to adamantanes, a class of antiviral drugs13.
Discussion
The patient in this case not only exhibited infection with the H10N3 subtype of inuenza A virus but also
presented with a mixed infection involving bacteria and fungi, making the condition complex. It's worth
noting that severe pneumonia patients infected with avian inuenza often experience concurrent or
secondary bacterial and fungal infections. Therefore, it is recommended to conduct repeated sputum
culture, respiratory tract aspirate culture, or metagenomic Next Generation Sequencing (mNGS) detection
in clinical settings to identify the types of bacteria or fungi present, as well as their susceptibility or
resistance patterns. This approach enables clinicians to make informed decisions regarding antibiotic
selection and guide appropriate clinical treatment strategies.
The clinical manifestations of avian inuenza virus infection vary depending on the virus subtypes
involved. For instance, infection with H5N1 and H7N9 subtypes can lead to severe pneumonia and related
complications in patients. Conversely, certain subtypes such as H7 and H9 may only induce conjunctivitis
or mild respiratory symptoms. It's important for healthcare providers to be aware of these differences in
clinical presentation when diagnosing and managing cases of avian inuenza virus infection1. As of now,
only two cases of human infection with the H10N3 subtype have been reported. The symptoms observed
in the patient infected with H10N3 in this case closely resemble those documented in the two previously
known cases of H10N3 infection. Notably, all cases resulted in severe pneumonia in the affected
patients8,9. In light of our ndings, the identication of HA-Q226L, PB2-D701N, PA-S409N, and M2-S31N
mutations in the protein of the Yunnan H10N3 virus strain underscores the potential for increased harm
posed by H10N3 in humans. Therefore, it is imperative to closely monitor the dynamics of this subtype.
The case of human infection with H10N3 avian inuenza virus highlighted in this study involved close
contact with live birds, particularly through the handling and slaughtering of dead birds. This contact
ultimately led to the patient contracting avian inuenza and experiencing severe illness. This underscores
the importance of paying special attention to instances of unexpected bird deaths and promptly reporting
such cases. Moreover, it emphasizes the necessity of establishing a comprehensive avian inuenza
surveillance system, not only within Yunnan but also globally, to continuously and vigilantly monitor the
H10N3 virus strain and its potential impact on human health.
Methods
Data collection
On March 6, 2024 the patient went to Kunming Third People's Hospital for treatment due to continuous
fever for many days, and was diagnosed with severe pneumonia, type I respiratory failure and infection

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by avian inuenza virus. After the diagnosis of avian inuenza virus infection, the patient investigated by
using questionnaires, including demographic information, poultry contact history, basic diseases, etc.
Genomic analysis and genome assembly
Multiple amplication products were obtained by using inuenza A virus genotyping gene targeted
amplication kit (BaiyiTech, Hangzhou). The amplied products were puried using ampure XP beads
nucleic acid magnetic bead Purication Kit (Beckman, USA) and the library was constructed. The library
was constructed by ligation method with the kit sqk-nbd114.24 (Nanopore, UK). After the library was
constructed, it was added to the o-min 114 sequencing chip (Nanopore, UK), and high-throughput
sequencing was performed on the gridion X5 third-generation sequencer. All experimental steps were
carried out in strict accordance with the relevant kit instructions and nanopore third-generation high-
throughput sequencing requirements.
Phylogenetic analysis
The nucleotide sequences obtained were analyzed in the Genbank and GISAID databases using the
BLASTn tool to initially determine the virus subtypes. Similar HA nucleotide sequences were downloaded
for phylogenetic analysis. The nucleotide and amino acid sequences were aligned using MAFFT (v7.310),
and the phylogenetic tree was constructed based on the neighbor-joining method using MEGA-X.
Declarations
Acknowledgements
The authors thank the study subject and collaborating clinicians for their participation and contribution to
the work. This research was supported by Kunming Science and Technology Bureau (2023-1-NS-007),
Kunming Health Commission, Kunming infectious disease precise diagnosis and treatment center 2023-
SW(JI)-28. This research was also supported by “The Project of Health Science and Technology Talents
Ten Hundred Thousand’ in Kunming” 2021-SW(DAITOU)-06.
Author contributions
The author contributions are as follows. J.Y.D., J.Z., J.W.X., Y.Y.L. and G.M.L. conceived, designed and
supervised the study. J.W.X. treated the patient. P.Z., Y.D.D. and Q.J.L. gathered data and interviewed
patients. M.H., X.H.X., Q.Q.J. and Y.Y.L did the laboratory tests, P.Z., Y.D.D. and Q.J.L. performed the data
analyses and explored the mutation site. J.Y.D., J.Z., J.W.X., Y.Y.L. and G.M.L. wrote the drafts of the
manuscript and interpreted the ndings. All authors read the manuscript, provided feedback, and
approved the nal version.
Ethics declaration
The patient and his family members signed consent forms approving the investigation, sample collection
and its publication. The procedures were in accordance with the Helsinki declaration of 1975, as revised

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in 1983. According to local regulations in China, institutional review board approval is not required for
case reports, but only the written consent of the patient. The Third People’s Hospital of Kunming City
ethics committee reviewed this work and determined that institutional review board approval was not
required.
Competing interests
The authors declare that there is no conict of interest.
Data availability statement
The sequence data generated in this study have been deposited in the NCBI GenBank database under
accession number SUB14344866, PP555666- PP555673.
Peer review information
Nature Communications
thanks the anonymous reviewer(s) for their contribution
to the peer review of this work.
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Table
Table 1
Laboratory Test Results
Day 7 Day 22 Normal range
White Blood Cell (×109cells/L) 2.12 8.58 3.50–9.50
Neutrophil (×109cells/L) 1.80 7.19 1.80–6.30
Neutrophil percentage (%) 84.90 83.90 40.00–75.00
Lymphocyte (×109cells/L) 0.26 0.74 1.10–3.20
Lymphocyte percentage (%) 12.30 8.60 20.00–50.00
Blood platelet (×109cells/L) 79 223 125–350
Prothrombin time (s) 15.9 16.6 14.0–16.0
Hypersensitive C-reactive protein (mg/L) 249.41 21.93 0.00–6.00
Lnterleukin-6 (pg/mL) 78.99 8.26 0.00–7.00
Procalcitonin (ng/mL) 14.040 0.248 < 0.500
pO2 (mmHg) 32.00 68.40 80.00-100.00
pCO2 (mmHg) 32.00 52.10 35.00–45.00
Nucleic acid testing for inuenza A virus Positive Negative Negative
Figures

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Figure 1
Computed tomography of lung. (A and B). Results on March 6, 2024 showed that multiple patchy and
patchy increased density shadows were seen in both lungs, with unclear boundary and uneven density. (C
and D). Results on March 23, 2024 showed a reduction in lesions compared to previous scans.

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Figure 2
Phylogenetic tree of 24 H10N3 strains from China. The Phylogenetic tree was downloaded from the
GISAID database (https://gisaid.org) using the neighbor-joining method in MEGA X. The diamond
indicates the H10N3 strain in this study, and the octagon indicates the H10N3 strain from the rst case in
Jiangsu.