# Pathogenesis of Cluster 1 Duck Tembusu Virus in Ducks Reveals the Impact of Viral Genotype on Pathogenicity and Disease Severity

Wikanda Tunterak, Kanana Rungprasert, Suwarak Wannaratana, Nichapat Yurayart, Duangduean Prakairungnamthip, Patchareeporn Ninvilai, Benchaphorn Limcharoen, Teerawut Nedumpun, Rodolphe Hamel, Wijit Banlunara, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Wikanda Tunterak, Kanana Rungprasert, Suwarak Wannaratana, Nichapat Yurayart, Duangduean Prakairungnamthip, et al. Pathogenesis of Cluster 1 Duck Tembusu Virus in Ducks Reveals the Impact of Viral Genotype on Pathogenicity and Disease Severity. Transboundary and emerging diseases, 2023, 2023, pp.1-16. 10.1155/2023/9239953. hal-04314682

HAL Id: hal-04314682

https://hal.umontpellier.fr/hal-04314682v1

Submitted on 17 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Research Article

# Pathogenesis of Cluster 1 Duck Tembusu Virus in Ducks Reveals the Impact of Viral Genotype on Pathogenicity and Disease Severity

Wikanda Tunterak,<fnref n="1" /> Kanana Rungprasert,<fnref n="1" /> Suwarak Wannaratana,<fnref n="2" /> Nichapat Yurayart,<fnref n="3" />
Duangduean Prakairungnamthip,<fnref n="1" /> Patchareeporn Ninvilai,<fnref n="4" /> Benchaphorn Limcharoen,<fnref n="5" />
Teerawut Nedumpun,<fnref n="1" /> Rodolphe Hamel,<fnref n="6" />,<fnref n="7" />,<fnref n="8" /> Wijit Banlunara,<fnref n="9" /> and
Aunyaratana Thontiravong<fnref n="1" />,<fnref n="3" />,<fnref n="10" />,<fnref n="11" />

Correspondence should be addressed to Aunyaratana Thontiravong; aunyaratana.t@chula.ac.th

Received 13 July 2023; Revised 19 September 2023; Accepted 31 October 2023; Published 28 November 2023

Academic Editor: Horacio Bach

Copyright © 2023 Wikanda Tunterak et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. Currently, several clusters of DTMUV, including clusters 1, 2, and 3, have been identified and caused outbreaks in Asia. However, most of the DTMUV pathogenesis evaluation has mainly focused on cluster 2, while limited information is available on the pathogenesis of other DTMUV clusters, particularly cluster 1. In this study, the pathogenesis of a cluster 1 DTMUV was investigated in Cherry Valley ducks and compared to our previously reported cluster 2.1 DTMUV. Our results demonstrated that cluster 1 DTMUV was generally less pathogenic than cluster 2.1 DTMUV in ducks as evidenced by slower body weight loss, lower morbidity and mortality rates, and milder pathological changes. Concordantly, delayed viremia, reduced viral loads in blood and tissues, and shorter shedding period with lower viral loads were also observed in cluster 1 DTMUV inoculated ducks compared with those reported in cluster 2.1 DTMUV. In addition, we also found that cluster 1 DTMUV exhibited significant antigenic difference compared to cluster 2.1 DTMUV. Altogether, our findings suggest distinct pathogenicity and antigenicity between cluster 1 and 2.1 DTMUVs in ducks, highlighting the potential association between DTMUV genotype and pathogenicity/ disease severity. This study enhances our understanding of DTMUV pathogenesis in ducks and provides useful information for the design and development of effective DTMUV vaccines.

<footnote n="1">¹Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote n="2">²Faculty of Veterinary Medicine, Rajamangala University of Technology Tawan-Ok, Chonburi, Thailand</footnote>

<footnote n="3">³Animal Vector-Borne Disease Research Unit, Veterinary Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote n="4">⁴Avian Veterinary Services, CPF (Thailand) Public Company Limited, Bangkok, Thailand</footnote>

<footnote n="5">⁵Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote n="6">⁶MIVEGEC, Univ Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote n="7">⁷Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Nakhon Pathom, Thailand</footnote>

<footnote n="8">⁸Viral Vector Joint Unit and Join Laboratory, Mahidol University, Nakhon Pathom, Thailand</footnote>

<footnote n="9">⁹Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote n="10">¹⁰Center of Excellence for Emerging and Re-emerging Infectious Diseases in Animals (CUEIDAs), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote n="11">¹¹Center of Excellence of Systems Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand</footnote>
# 1. Introduction

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that causes severe neurological disorders and acute egg drop syndrome in ducks and some other avian species, including geese and chickens [1]. Currently, DTMUV is widely spread and becomes one of the most economically important disease of poultry in Asia [2–5]. To establish effective control and prevention strategies, a better understanding of the pathogenesis of this emerging virus is crucial.

DTMUV is classified as a new genotype of Tembusu virus (TMUV), which belongs to the genus *Flavivirus* of the family *Flaviviridae* [6, 7]. Like other flaviviruses, DTMUV genome consists of a positive-sense single-stranded RNA, which encodes three structural proteins (capsid (C), premembrane protein (prM), and envelope (E)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [1]. At present, DTMUV is genetically classified into three distinct clusters, including cluster 1, cluster 2 (2.1 and 2.2), and cluster 3 [2]. All three clusters were found to be circulated in Asia with different geographical distributions [2]. Interestingly, our previous study revealed a marked genetic divergence among these three different clusters of DTMUV [2]. Whether these genetic changes contributed to the antigenicity, pathogenicity, and disease severity, however, remains largely unknown.

Several studies consistently showed that DTMUV generally causes retarded growth and severe neurological syndromes in young ducks and decreased egg production in laying ducks [8–10]. However, the severity of the disease varies greatly from mild to severe clinical symptoms depending on several factors, including the age of duck, the strain of virus, the route of infection, and the virus infectivity titer [10–13]. Besides the duck age, the viral strain/genotype is likely to be a key factor affecting the virulence and pathogenicity of DTMUV [12–14]. However, the pathogenesis evaluation of DTMUV has been mainly restricted to cluster 2. Limited information is available on the pathogenesis of other DTMUV clusters, particularly cluster 1, which was circulated in some Asian countries, including Malaysia and Thailand. Here, the pathogenesis of a cluster 1 DTMUV was investigated in Cherry Valley ducks and compared to our previously reported cluster 2.1 DTMUV [10].

# 2. Materials and Methods

## 2.1. Virus.
DTMUV strain DK/TH/CU-DTMUV2007 (Gen-Bank accession number MF621927) was used for pathogenesis assessment in this study [15]. This virus was originally obtained from ducks infected with DTMUV in Thailand and belongs to the DTMUV cluster 1 [2, 15]. The propagation of DK/TH/CU-DTMUV2007 was carried out in 9-day-old embryonated duck eggs, as described previously [7]. The virus was subsequently collected, clarified through centrifugation, and the 50% embryo lethal dose (ELD50) was determined using the Reed and Muench's [16] method. All viral handling was conducted in a BSL-2 containment facility.

## 2.2. Animal Study.
The pathogenesis assessment of cluster 1 DTMUV was carried out in 4-week-old Cherry Valley ducks, as described previously [10]. This specific age group was chosen for pathogenesis assessment due to its high susceptibility to DTMUV infection [10]. In brief, DTMUV-negative ducks ($n=35$) were inoculated with $10^5$ ELD50/ml of cluster 1 DTMUV (DK/TH/CU-DTMUV2007) through the intranasal (0.5 ml) and intramuscular (0.5 ml) routes. An additional group of 35 ducks was mock inoculated with allantoic fluid from specific pathogen free duck eggs in the same fashion to serve as the noninfected control group. These ducks were acquired from a private research farm operating under high biosecurity standards and were verified to be free from common duck viruses, including DTMUV, by virus-specific RT-PCR/PCR and serological assays [6, 17–20]. To determine the transmission of cluster 1 DTMUV, five naïve ducks were introduced to DTMUV-inoculated group 1 day post-inoculation (dpi) to allow direct contact. Clinical signs and body weights were observed daily throughout the 21-day period. Additionally, oropharyngeal (OP) and cloacal (CL) swabs were taken from each duck on days 1–7, 9, 11, 13, 15, 17, 19, and 21 dpi to examine viral shedding. Five ducks from each group were subjected to blood collection for viremia determination and serum neutralization (SN) testing. Subsequently, these ducks were humanely euthanized at 1, 3, 5, 7, 9, 14, and 21 dpi for gross examination. Various tissue samples, including the brain, spleen, bursa, thymus, heart, pancreas, liver, sciatic nerve, lung, trachea, proventriculus, gizzard, and intestine, were collected. These samples were then either frozen at –80°C for viral load measurement or preserved in 10% neutral buffered formalin for subsequent histopathological and immunohistochemical (IHC) analyses. The animal experiment was conducted in the ABSL-2 containment facility at Chulalongkorn University Laboratory Animal Center (CULAC), and it was carried out with the approval of Chulalongkorn University Animal Care and Use Committee (approval number 2073001).

To compare the pathogenesis of cluster 1 DTMUV with cluster 2 DTMUV, the cluster 2.1 DTMUV pathogenesis data was obtained from our previous study, which was performed in the same way as a cluster 1 DTMUV described in this study [10].

## 2.3. Histopathological and Immunohistochemical Examinations.
To determine the histopathological lesions, tissue samples from the brain, spleen, bursa, thymus, heart, pancreas, and liver were first preserved in 10% neutral buffered formalin solution and then embedded in paraffin. Four-micrometer-thick sections were subsequently cut and subjected to staining with hematoxylin and eosin (H&E) following standard histopathological protocols. The histopathological score of lymphoid organs, brain, and other tissues was determined based on the criteria described previously [10]. Briefly, the sections of lymphoid organs were assessed and assigned scores based on the following criteria: 0 for no lesion, 1 for ≤30% depletion, 2 for 30%–60% depletion, and 3 for ≥60% depletion. Brain sections were evaluated using the following criteria: 0 for no lesion, 1 for mild lesions characterized by mild perivascular cuffing consisting of 2 and 3 layers of mononuclear cells and focal gliosis, 2 for
moderate lesions involving moderate perivascular cuffing com-
prising 4–6 layers of mononuclear cells and multifocal gliosis,
and 3 for severe lesions marked by severe perivascular cuffing
having >6 layers of mononuclear cells and diffuse gliosis. Other
tissue sections were scored based on the criteria of 0 for no
lesion, 1 for mild mononuclear cell infiltration, 2 for moderate
congestion with moderate mononuclear cell infiltration, and
3 for severe congestion and necrosis accompanied by severe
mononuclear cell infiltration.

Additionally, the presence of antigens specific to flavi-
viruses in various tissues, including the brain, spleen, thy-
mus, bursa, heart, pancreas, sciatic nerve, lung, trachea,
proventriculus, gizzard, and intestine, were assessed through
immunohistochemical staining, as described previously [10].
Briefly, the tissue sections were deparaffinized, rehydrated,
and subjected to pretreatment in citrate buffer (pH 6.0) using
a microwave oven. To inhibit endogenous peroxidase activ-
ity, the sections were incubated with a 3% hydrogen peroxide
solution (H₂O₂) at room temperature for 10 min. Further-
more, the sections were blocked with 2% BSA for 30 min at
37°C. Subsequently, the primary antibody, a mouse mono-
clonal antibody specific to the Flavivirus antigen group, clone
D1-4G2-4-15 (EMD Millipore Corporation, CA, USA), was
applied and incubated overnight at 4°C. Following three
washes in PBS, the sections were subjected to incubation
with the secondary antibody using a polymer system
(Dako REALTM EnvisionTM/HRP, Rabbit/Mouse, Dako,
Denmark) at room temperature for 45 min. Subsequently,
the substrate, 3,3'-diamino-benzidine tetrahydrochloride
(DAB), was added. Hematoxylin was used for counterstain-
ing, and the sections were subsequently dehydrated, mounted
with permount medium, and examined under a light micro-
scope. Each test included both positive and negative controls.

2.4. *Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR)*. DTMUV loads in serum, tissue, and swab samples were quantified by DTMUV E-specific RT-qPCR, as previously described [10]. Briefly, viral RNA was extracted from serum, tissue, and swab samples using QIAamp Viral RNA Mini Kit (Qiagen®, Hilden, Germany) and RNeasy Mini Kit (Qiagen®, Hilden, Germany), respectively, according to the manufacturer's instructions. The cDNA was converted from 250 ng of total RNA by random hexamers and the Improm-II reverse transcription system (Promega, Wisconsin, USA), and subsequently served as a template for qPCR using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, TX, USA). To assess DTMUV loads in the samples, a standard curve was established using a recombinant plasmid containing the DTMUV E gene. The absolute quantification of DTMUV in the samples was normalized per 250 ng of total RNA. Both samples and standards were examined in triplicate.

2.5. *Serum Neutralization (SN) Test*. To detect the presence of cluster 1 DTMUV-specific antibodies in serum samples, SN test was conducted using baby hamster kidney (BHK-21) cells as previously described [10, 17]. In brief, triplicate serial twofold dilutions of heat-inactivated sera were incubated for 1 hr at 37°C with 100 TCID₅₀ of cluster 1 DTMUV (DK/TH/

CU-DTMUV2007). After incubation, the virus–serum mixture
was introduced into a 96-well plate containing BHK-21 cells.
These cells were incubated at 37°C, and the development of
cytopathic effects (CPE) was monitored daily for 5 days. The
controls included reference DTMUV seropositive and negative
sera, uninfected BHK-21 cells, and a back titration of used virus.
The titers of cluster 1 DTMUV-specific neutralizing antibodies
were expressed based on the reciprocal of the highest serum
dilution capable of inhibiting CPE.

To evaluate the antigenic relationship between cluster 1 and 2.1 DTMUVs, a cross-neutralization test was performed, as described above using cluster 1 or 2.1 DTMUV and their respective antisera. Cluster 2.1 DTMUV-specific antisera were obtained from our previous study [10]. The antigenic relatedness (R) values were calculated using the Archetti and Horsfall's [21] equation, and the antigenic relationship between these two clusters was assessed based on the R-value [13, 21, 22]. An R-value between 24% and 49% represents a four- to eight-fold difference, and R-value ≤ 24% indicates a greater than eightfold difference [13, 22].

2.6. *Statistical Analysis.* Data were presented as the mean ± standard deviation (SD). A two-tailed, unpaired Student's *t*-test was applied to compare body weights between the DTMUV-inoculated and noninfected control groups, as well as to assess differences in virus shedding, viral loads in serum and tissues, and SN antibody titers between cluster 1 and 2.1 DTMUV-inoculated groups. Gross and histopathological lesion scores differences between cluster 1 and 2.1 DTMUV-inoculated groups were evaluated using a nonparametric Mann–Whitney *U*-test. The statistical analyses were performed with GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA). Any *P*<0.05 was considered to indicate statistical significance.

**3. Results**

3.1. *Clinical Manifestation.* Most ducks inoculated with cluster 1 DTMUV (14/35) displayed depression and loss of appetite as early as 2 dpi (Figures 1(a) and 1(b)). These ducks also exhibited moderate to severe neurological signs, including ataxia, reluctance to walk, and paralysis from 2 to 14 dpi, and continued to show mild neurological signs until the end of observation period (21 dpi) (Figures 1(c) and 1d)). DTMUV-inoculated ducks began to significantly lose weight at 7 dpi and recovered by 21 dpi (Figure 1(e); Table 1). While the morbidity rate of cluster 1 DTMUV was relatively high (40%), none of the inoculated ducks died during the 21-day observation period. Mild to moderate neurological signs were also detected in most contact ducks from 1 to 16 days postcontact (dpc) with 100% (5/5) morbidity; however, no contact ducks died during the observation period. It is noted that these contact ducks were confirmed DTMUV infection by RT-qPCR positive on OP (10¹.⁴–10¹.⁸ copies) and CL (10².⁹–10⁴.⁴ copies) swabs at 4 and 9 dpc. This finding indicated that, like cluster 2.1 DTMUV, cluster 1 DTMUV transmitted efficiently among ducks. Ducks in the noninfected control group remained healthy throughout the 21-day
<figure><img src="image_3.png" /><figcaption>FIGURE 1: Clinical manifestation of 4-week-old ducks inoculated with cluster 1 DTMUV. (a–d) DTMUV-inoculated ducks showed depression, loss of appetite, and severe neurological signs, including ataxia, reluctance to walk, and paralysis (a, c), while ducks in noninfected control group remained healthy throughout the observation period (b, d). (e) Mean body weights of 4-week-old ducks inoculated with cluster 1 DTMUV compared to noninfected control ducks. Each data point represents the mean body weights ± standard deviation (SD) of five ducks. Asterisks (*) indicate statistically significant differences between DTMUV inoculated and noninfected control groups (*P*<0.05, two-tailed Student's unpair *t*-test).</figcaption></figure>

observation period. Compared with a previously reported cluster 2.1 DTMUV [10], cluster 1 DTMUV exhibited slower body weight loss, milder neurological signs, and lower morbidity and mortality rates in 4-week-old ducks (Table 1). Taken together, cluster 1 DTMUV generally induced less severe disease in 4-week-old ducks than cluster 2.1 DTMUV in terms of body weight loss, severity of symptoms, morbidity, and mortality rates.

3.2. *Gross and Histopathological Findings*. The most prominent gross lesions in cluster 1 DTMUV-inoculated ducks were cerebral edema and meningeal congestion and hemorrhages in myocardium and pancreas (Figures 2(a), 2(e), and 2(f)). The major gross lesions of immune organs were spleen enlargement, diffuse petechial hemorrhage and swelling in thymus, and bursa swelling (Figure 2(b)–2(d)). Notably, cluster 1 DTMUV had significantly lower mean gross
<table><caption>TABLE 1: Comparative clinical manifestation of cluster 1 and 2.1 DTMUV infections in 4-week-old ducks.</caption><thead><tr><th>DTMUV cluster</th><th>Neurological signs (no. of duck/severity)</th><th>Morbidity rate (%)</th><th>Mortality rate (%)</th><th>Body weight loss (duration; day)</th></tr></thead><tbody><tr><td>1</td><td>14/++<sup>a</sup></td><td>40</td><td>0</td><td>7–14</td></tr><tr><td>2.1</td><td>18/+++</td><td>51.42</td><td>22.86</td><td>3–21</td></tr></tbody></table>

Note: <sup>a</sup>The severity of neurological signs is shown as + (mild), ++ (moderate), and +++ (severe). Clinical data of cluster 2.1 DTMUV were retrieved from our previous study [10].

<figure><img src="image_4.png" /><figcaption>FIGURE 2: Continued.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIGURE 2: Gross lesions and scores of cluster 1 DTMUV inoculated ducks. (a) Brain: severe congestion and edema. (b) Spleen: swelling and pallor color. (c) Thymus: diffuse petechial hemorrhage and swollen. (d) Bursa: swollen. (e) Heart: petechial hemorrhage in myocardium. (f) Pancreas: multiple petechial hemorrhage and congestion. Bar charts show mean gross lesion scores of cluster 1 DTMUV compared to cluster 2.1 DTMUV. It should be noted that gross lesion scores of cluster 2.1 DTMUV were retrieved from our previous study [10]. Each data point represents the mean gross lesion scores ± standard deviation (SD) of five ducks. Asterisks (*) indicate statistically significant differences between cluster 1 and 2.1 DTMUV infected groups (*P*<0.05), a nonparametric Mann–Whitney *U*-test.</figcaption></figure>

lesion scores on spleen, bursa, and heart than a previously reported cluster 2.1 DTMUV (Figure 2). No gross lesions were observed in tissues from any of the noninfected control ducks. Overall, the severity of the gross lesions in cluster 1 DTMUV inoculated ducks appeared to be lower than those observed for a previously reported cluster 2.1 DTMUV.

The significant histopathological findings in cluster 1 DTMUV inoculated ducks were congestion, perivascular cuffing with mononuclear cells, and necrosis of lymphoid cells in various organs. In the brain, the main histopathological lesion was multifocal gliosis, perivascular cuffing with mononuclear cells, and mild to moderate nonsuppurative encephalitis (Figure 3(a)). In lymphoid tissues, severe lymphoid
<figure><img src="image_6.png" /></figure>

(a)

<figure><img src="image_7.png" /></figure>

(b)

<figure><img src="image_8.png" /></figure>

(c)

<figure><img src="image_9.png" /></figure>

(d)

<figure><img src="image_10.png" /><figcaption>FIGURE 3: Continued.</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>FIGURE 3: Histopathological lesions and scores of cluster 1 DTMUV inoculated ducks. (a) Brain: severe nonsuppurative perivascular cuffing (white arrows), multifocal gliosis (black arrows), and dark neurons (blue arrows). (b) Spleen: severe germinal lymphoid depletion and congestion. (c) Thymus: severe lymphoid depletion. (d) Bursa: moderate lymphoid depletion. (e) Heart: perivascular cuffing and focal mononuclear cells infiltration in myocardium (arrow indicates blood vessel). (f) Pancreas: severe mononuclear cells infiltration observed mostly at the perivascular area in interlobular fibrous tissue. (g) Liver: severe mononuclear cells infiltration at perivascular area of portal triads. Inserts show higher magnification of the selected square-lesions. Bar charts show mean histopathological scores of cluster 1 DTMUV compared to cluster 2.1 DTMUV. It should be noted that histopathological scores of cluster 2.1 DTMUV were retrieved from our previous study [10]. Each data point represents the mean histopathological scores ± standard deviation (SD) of five ducks. Asterisks (*) indicate statistically significant differences between cluster 1 and 2.1 DTMUV infected groups (*P*<0.05), a nonparametric Mann–Whitney *U*-test.</figcaption></figure>

depletion, lymphocytic death, and hemorrhage were observed in
spleen, thymus, and bursa as early as 1 dpi (Figure 3(b)–3(d)). In
addition, nonsuppurative perivascular cuffing by mononuclear
cell infiltration was mostly observed at perivascular area of vari-
ous organs, including heart, pancreas, and liver (Figure 3(e)–
3(g)). Interestingly, ducks inoculated with cluster 1 DTMUV
had significantly lower mean histopathological scores on thymus,

bursa, and heart than cluster 2.1 DTMUV inoculated ducks
(*P* < 0.05) (Figure 3). No histopathological changes were
observed in any of the tested organs from noninfected control
ducks. Corresponding to observations on histopathology,
IHC analysis revealed the presence of DTMUV antigens in
all examined organs of cluster 1 DTMUV inoculated ducks,
with different levels of intensity, indicating systemic infection
(Figure 4). It is interesting to note that positive immunostaining was more intense and widespread in gastrointestinal tissues than in respiratory tissues of cluster 1 DTMUV inoculated ducks, which was consistent with high and prolonged viral shedding in CL swabs compared to OP swabs (Figures 4 and 5). Similar to cluster 2.1 DTMUV [10], DTMUV antigen was mainly identified in monocytes/macrophages in various tissues of cluster 1 DTMUV inoculated ducks (Figure 4). However, DTMUV immunostaining in cluster 1 DTMUV inoculated group appeared to be weaker and fewer than a previously reported cluster 2.1 DTMUV inoculated group [10] (Figure 4). Altogether, histopathology along with clinical findings and gross pathology suggested that cluster 1 DTMUV was virulent in 4-week-old ducks but was less pathogenic than cluster 2.1 DTMUV.

### 3.3. Viremia, Viral Dissemination, and Shedding.
To evaluate the magnitude and kinetic of cluster 1 DTMUV viremia, DTMUV loads in serum samples collected at 1, 3, 5, 7, 9, 14, and 21 dpi were quantified by DTMUV E-specific RT-qPCR. The results demonstrated that viremia in cluster 1 DTMUV inoculated group was first detected at 5 dpi, peaked at 7 dpi, and sustained through 21 dpi (Figure 5(a)). Compared with a previously reported cluster 2.1 DTMUV [10], cluster 1 DTMUV induced delayed and lower levels of viremia (Figure 5(a)), which correlated with reduced disease severity in cluster 1 DTMUV inoculated ducks. No DTMUV RNA was detected in serum samples from any of the noninfected control ducks.

To investigate the tissue dissemination of cluster 1 DTMUV, tissue samples, including brain, spleen, heart, thymus, pancreas, and liver, collected at 1, 3, 5, 7, 9, 14, and 21 dpi were assessed for DTMUV RNA levels. In contrast to delayed serum viremia, DTMUV RNA could be detected in almost all tested tissues of ducks inoculated with cluster 1 DTMUV as early as 1 dpi, suggestive of a rapid systemic spread of cluster 1 DTMUV (Figure 5(b)–5(g)). This observation indicated that the rapid tissue dissemination of cluster 1 DTMUV may occur primarily through cell-associated viremia rather than via cell-free viremia. Although viral loads in all tested organs of cluster 1 DTMUV inoculated ducks reached the maximum levels during 3–7 dpi and mostly sustained through 21 dpi, the levels of viral load in tissues of these ducks were most significantly lower than those observed for cluster 2.1 DTMUV (Figure 5(b)–5(g)). These findings suggested that low viral load levels in visceral organs may be the main cause of decreased disease severity in cluster 1 DTMUV inoculated ducks. It should be noted that, unlike cluster 2.1 DTMUV, the highest viral loads of cluster 1 DTMUV inoculated group were detected in brain (Figure 5(b)–5(g)), indicating that brain might be the major target organ of cluster 1 DTMUV. No DTMUV RNA was observed in any of the noninfected control ducks. Taken together, these findings along with the viremia level suggested that reduced disease severity in cluster 1 DTMUV inoculated ducks might be associated with lower viral load levels both in blood and tissues of cluster 1

DTMUV inoculated ducks compared to cluster 2.1 DTMUV inoculated ducks.

Analysis of cluster 1 DTMUV shedding pattern demonstrated that most ducks inoculated with cluster 1 DTMUV shed the virus in OP swabs as early as 1 dpi; however, the duration of shedding lasted only for 4 days with low-level viral shedding (Figure 5(h)). In contrast to OP shedding, although intermittent viral shedding on CL swabs was observed in some ducks inoculated with cluster 1 DTMUV during the early phase of infection (1–9 dpi), all ducks consistently shed the virus in CL swabs from 13 to 21 dpi, with relatively high shedding level (Figure 5(i)). No DTMUV shedding was found in any of the noninfected control ducks. Overall, cluster 1 DTMUV inoculated ducks generally shed the virus in CL swabs for a longer period and at a higher level than those in OP swabs. This result together with strong and widespread DTMUV immunostaining in gastrointestinal tissues indicated that cluster 1 DTMUV might be spread mainly through the fecal-oral route rather than by the airborne route. Notably, ducks inoculated with cluster 1 DTMUV exhibited significantly lower shedding levels and shorter shedding duration compared to cluster 2.1 DTMUV ($P<0.05$) (Figures 5(h) and 5(i)). Collectively, these findings, in conjunction with the viral load levels in blood and tissues, revealed that cluster 1 DTMUV replicated less efficiently than cluster 2.1 DTMUV, which might lead to the milder pathogenicity of cluster 1 DTMUV.

### 3.4. Neutralizing Antibody Response.
To evaluate the magnitude and kinetic of neutralizing antibody response in ducks inoculated with cluster 1 DTMUV, the titer of cluster 1 DTMUV-specific neutralizing antibodies was assessed by SN test. Following cluster 1 DTMUV inoculation, neutralizing antibodies against cluster 1 DTMUV were first detected at 9 dpi in all ducks, peaked at 14 dpi, and then began to decline but remained detectable at a high level until the end of experiment (Figure 6(a)). Compared with a previously reported cluster 2.1 DTMUV [10], cluster 1 DTMUV induced delayed neutralizing antibody response (Figure 6(a)). However, titers of neutralizing antibodies observed in ducks inoculated with cluster 1 DTMUV were generally comparable to those reported in cluster 2.1 DTMUV (Figure 6(a)). Altogether, these results demonstrated delayed neutralizing antibody response following cluster 1 DTMUV infection in ducks.

To investigate the antigenic relatedness between cluster 1 and 2.1 DTMUVs, a cross-neutralization test was conducted using cluster 1 or 2.1 DTMUVs and their respective antisera. The cross-neutralization results demonstrated partial cross-neutralizations between cluster 1 and 2.1 DTMUVs in some serum samples; however, marked differences in SN titers were observed between homologous and heterologous viruses for all serum samples (Figure 6(b)). Notably, significant antigenic differences were found between cluster 1 and 2.1 DTMUVs ($R$ values < 24%) (Figure 6(b)). Collectively, these results revealed that cluster 1 DTMUV displayed not only virulence difference but also antigenic variation compared to cluster 2.1 DTMUV.
<figure><img src="image_15.png" /><figcaption>FIGURE 4: Continued.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>FIGURE 5: Continued.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>FIGURE 5: Viremia, viral dissemination, and shedding of cluster 1 DTMUV inoculated ducks. Levels of viremia and viral loads in tissues and swabs were evaluated by RT-qPCR. (a) Viremia levels were expressed as log₁₀ DTMUV genome copy number per 50 ng total RNA. Levels of DTMUV RNA in brain (b), spleen (c), heart (d), thymus (e), pancreas (f), liver (g), oropharyngeal (h), and cloacal (i) swabs were expressed as log₁₀ DTMUV genome copy number per 250 ng total RNA. It should be noted that all viral load data of cluster 2.1 DTMUV inoculated group were retrieved from a previous study [10]. Each data point represents the mean ± standard deviation (SD). Asterisks (*) represent statistically significant difference between cluster 1 and 2.1 DTMUV inoculated groups at the indicated time point (*P* < 0.05, two-tailed Student's unpair t-test).</figcaption></figure>
<figure><img src="image_18.png" /></figure>

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Virus cluster</th><th colspan="6">Duck antiserum against</th></tr><tr><th>Cluster 1 (1)</th><th>Cluster 2.1 (1)</th><th>Cluster 1 (2)</th><th>Cluster 2.1 (2)</th><th>Cluster 1 (3)</th><th>Cluster 2.1 (3)</th></tr></thead><tbody><tr><td rowspan="2">SN titer</td><td>Cluster 1</td><td>128</td><td>4</td><td>256</td><td>&lt;2</td><td>128</td><td>4</td></tr><tr><td>Cluster 2.1</td><td>8</td><td>128</td><td>&lt;2</td><td>64</td><td>8</td><td>128</td></tr><tr><td>R-value (%)</td><td></td><td colspan="2">4.42</td><td colspan="2">12.5</td><td colspan="2">4.42</td></tr></tbody></table>

FIGURE 6: Neutralizing antibody response in ducks inoculated with cluster 1 DTMUV (a) and cross-neutralization activity between cluster 1 and 2.1 DTMUVs (b). All serum neutralization (SN) titer data of cluster 2.1 DTMUV inoculated group were retrieved from a previous study [10]. The antigenic relatedness (R) values were calculated using the Archetti and Horsfall's [21] formula and the antigenic relationship between these two clusters was evaluated based on the R values. An R-value between 24% and 49% represents a four- to eight-fold difference, and R-value ≤ 24% indicates a greater than eightfold difference [13, 22]. Asterisks (*) represent statistically significant difference between cluster 1 and 2.1 DTMUV inoculated groups at the indicated time point (P < 0.05, two-tailed Student's unpair t-test).

## 4. Discussion

The viral strain/genotype is believed to be one of the most important factors affecting the virulence and pathogenicity of DTMUV [12–14]. Most of the DTMUV pathogenesis evaluation has mainly focused on cluster 2, while information on the pathogenesis of other DTMUV clusters, particularly cluster 1, is limited. In this study, the pathogenesis of cluster 1 DTMUV was investigated in 4-week-old ducks and compared to our previously reported cluster 2.1 DTMUV [10]. Our results revealed that cluster 1 DTMUV caused less severe clinical disease compared to cluster 2.1 DTMUV. Concordantly, we also demonstrated that cluster 1 DTMUV replicated less efficiently both in blood and tissues and had overall lower pathogenicity in 4-week-old ducks than cluster 2.1 DTMUV. These findings, in conjunction with the previous evidence [12, 13, 23], support the association between DTMUV genotype and disease severity/pathogenicity. It is interesting to note that, besides pathogenicity difference, significant antigenic variation was found between cluster 1 and 2.1 DTMUVs. Collectively, our data suggested distinct pathogenicity and antigenicity between these 2 DTMUV genotypes. To the best of our knowledge, this is the first study reporting the pathogenesis of cluster 1 Thai DTMUV in ducks

and providing the evidence that cluster 1 DTMUV is prone to be less pathogenic than cluster 2.1 DTMUV.

The clinical and pathological analyses demonstrated that cluster 1 DTMUV induced typical clinical features of DTMUV infection in 4-week-old ducks; however, differences in the disease severity and pathogenicity were observed when compared with cluster 2.1 DTMUV [10]. Notably, cluster 1 DTMUV induced milder clinical signs and lesions and had lower morbidity and mortality rates than cluster 2.1 DTMUV in 4-week-old ducks, which clearly indicate that cluster 1 DTMUV was less pathogenic to 4-week-old ducks than cluster 2.1 DTMUV. These observations are in line with the previous studies reporting that cluster 1 Malaysian DTMUV appeared to be less virulent than cluster 2 DTMUV [3, 10, 12]. As reported in cluster 1 Malaysian DTMUV [3] and in the natural infection of cluster 1 DTMUV in Thailand [15], pathological lesions caused by cluster 1 Thai DTMUV were mainly observed in the brain, correlating with severe neurological signs presenting in the inoculated ducks. Apart from brain, cluster 1 Thai DTMUV could also induce pathological changes with positive DTMUV immunostaining in multiple organs of inoculated ducks, indicating systemic infection of cluster 1 DTMUV. Similar to cluster 2.1 DTMUV [10], DTMUV antigen was mainly detected in monocytes/macrophages in various tissues
of cluster 1 DTMUV inoculated ducks, supporting a role of
monocytes/macrophages tropism in DTMUV pathogenicity
[24]. However, consistent with severity of clinical signs and
lesions, only sporadic DTMUV immunostaining was found in
cluster 1 DTMUV inoculated ducks when compared to ducks
inoculated with cluster 2.1 DTMUV [10]. This indicates a
decreased replication ability of cluster 1 DTMUV in tissues
of inoculated ducks, which possibly leads to milder disease
compared with cluster 2.1 DTMUV. Supporting this notion,
several previous studies reported the association between the
degree of pathogenicity and the level of viral replication capac-
ity in vivo [25, 26]. In concordance with a previous observation
of cluster 2.1 DTMUV [10], cluster 1 DTMUV contact ducks
showed higher morbidity than the inoculated ducks. This may
be related to prolonged viral shedding from cloaca of inocu-
lated ducks, which possibly leads to reinfection in contact
ducks. Altogether, clinical and pathological findings suggested
that cluster 1 DTMUV induced milder pathogenicity com-
pared to cluster 2.1 DTMUV.

Like cluster 2 DTMUV [9–11], DTMUV RNA could also
be identified in various organs of ducks inoculated with clus-
ter 1 DTMUV as early as 1 dpi, suggesting the rapid systemic
spread and broad tissue tropism of cluster 1 DTMUV in
ducks. However, cluster 1 DTMUV displayed a markedly
reduced ability to replicate both in tissues and blood com-
pared to cluster 2.1 DTMUV, potentially contributing to the
milder pathogenicity than cluster 2.1 DTMUV. These findings,
in conjunction with the previous observations [12, 25–27],
suggest that the genotype-specific clinical outcomes are likely
linked to viral replication capacity. Interestingly, whereas both
cluster 1 and 2.1 DTMUVs were able to induce rapid systemic
infection, cluster 1 DTMUV caused delayed serum viremia
compared to cluster 2.1 DTMUV. A possible explanation for
this finding is that cluster 1 DTMUV may invade peripheral
organs mainly via cell-associated viremia rather than through
cell-free viremia. Supporting this speculation, a previous study
showed that DTMUV could infect and replicate efficiently
both in monocytes/macrophages and lymphocytes obtained
from duck peripheral blood mononuclear cells [24]. In addi-
tion, our previous study demonstrated that cluster 1 DTMUV
was able to infect and replicate in duck monocytes/macro-
phages (unpublished data). It is interesting to note that the
highest viral loads were detected in brain of ducks inoculated
with cluster 1 DTMUV rather than in spleen as reported in
cluster 2.1 DTMUV [10]. This finding along with strong
DTMUV immunostaining in brain supports the notion that
brain is likely to be the main target organ of cluster 1 DTMUV
in 4-week-old ducks. We also found that cluster 1 DTMUV
persisted in most tissues of infected ducks until the end of
observation period. This might have resulted from delayed
production of neutralizing antibodies in ducks inoculated
with cluster 1 DTMUV, which potentially leads to delayed
viral clearance in the tissues. Supporting this speculation, our
previous studies showed that the induction of high neutraliz-
ing antibody titers early in the course of cluster 2.1 DTMUV
infection was associated with the reduction of viral loads
in various tissues of the infected ducks [7, 10]. However, the

exact mechanism contributing to this event requires further
investigation.

Dynamics of viral shedding following cluster 1 DTMUV
infection were monitored in OP and CL swabs. Our results
revealed a marked difference in shedding pattern between
OP and CL swabs of ducks inoculated with cluster 1
DTMUV. In contrast to cluster 2.1 DTMUV [10], CL shed-
ding was generally higher and longer than OP shedding for
cluster 1 DTMUV, suggesting that CL swabs might be more
appropriate than OP swabs for cluster 1 DTMUV detection.
However, short shedding in OP swabs and intermittent shed-
ding in CL swabs appear to limit the utility of these speci-
mens for detecting cluster 1 DTMUV as compared to tissues,
especially the brain. These findings along with a higher num-
ber of DTMUV antigen-positive cells in gastrointestinal tis-
sues compared to respiratory tissues indicated that cluster 1
DTMUV transmission was more likely via the fecal-oral
route rather than through the airborne route. Concordantly,
high level of DTMUV RNA (10<sup>2.9</sup>–10<sup>4.4</sup> copies) could be
detected mostly in CL swabs from contact ducks showing
typical signs of DTMUV infection, suggesting the potential
transmission of cluster 1 DTMUV mainly through the fecal-
oral route. Supporting this notion, a recent study showed
that, unlike cluster 2 DTMUV [9, 10], cluster 3 DTMUV
could not be transmitted via direct contact, indicating the
potential association between the main route of transmission
and DTMUV genotypes [28]. Interestingly, shorter shedding
duration with lower viral loads could be observed in ducks
inoculated with cluster 1 DTMUV compared with a previ-
ously reported cluster 2.1 DTMUV [10]. This might be
related to a reduced viral replication capacity of this cluster
in ducks compared to cluster 2.1 DTMUV. Collectively, clus-
ter 1 DTMUV exhibited a marked decrease in viral replica-
tion and shedding abilities compared to cluster 2.1 DTMUV,
thus providing a potential explanation as to why current
predominant circulation is mostly attributed to cluster 2.1
DTMUV [2, 23].

Compared with a previously reported cluster 2.1 DTMUV
[10], delayed seroconversion was observed in ducks inocu-
lated with cluster 1 DTMUV. This difference may be attrib-
uted to the distinct viral replication capacity of each cluster in
ducks. It should be noted that although the severity of disease
gradually declined over the course of cluster 1 DTMUV infec-
tion, no obvious correlation was observed between neutraliz-
ing antibody titers and viral loads in blood and tissues, which
contrasted with those observed in cluster 2.1 DTMUV
[10, 29]. This suggested that other immunological mechan-
isms, particularly cellular immune response, may be involved
in the control of cluster 1 DTMUV infection in ducks, which
warrants further investigation. Interestingly, our results dem-
onstrated a marked antigenic difference between cluster 1 and
2.1 DTMUV. This difference may be attributed to amino acid
substitutions in the E protein. Supporting this notion, our
previous study showed that amino acid changes in cluster 1
DTMUV were mainly observed in the E protein, especially in
the domain III (DIII), which is known to be responsible for
the induction of virus-neutralizing antibodies [15]. Consistent
with this finding, a previous study revealed that a significant
antigenic difference was also found between cluster 2 and 3 DTMUVs [13]. Therefore, our data together with this previous finding suggests the potential relationship between neutralizing activity and DTMUV clusters/genotypes. However, whether these antigenic differences affect the cross-protection among DTMUV genotypes should be urgently investigated.

Our results collectively demonstrated that the pathogenicity of cluster 1 DTMUV was lower than those of cluster 2.1 DTMUV as evidenced by lower morbidity and mortality rates, milder pathological changes, and reduced viral loads. The difference in the pathogenicity and disease severity between these two clusters may be partly associated with the variation in inducing host immune responses, particularly cellular immune responses. A previous study showed that highly virulent DTMUV elicited stronger cellular immune response than low virulent DTMUV, whereas both of them induced comparable levels of neutralizing antibodies [14]. Additional studies are required to clarify the role of host immune responses on the pathogenicity and virulence differences between cluster 1 and 2.1 DTMUVs. Furthermore, the pathogenicity variation between cluster 1 and 2.1 DTMUVs could be resulted from functional differences in the viral genome or proteins. Our previous study revealed a marked genetic divergence between cluster 1 and 2.1 DTMUVs, most of which were located in NS5, NS1, and E genes [15]. Several studies have shown that minor changes in the genome of DTMUV, especially the E and NS5 genes, have a large impact on pathogenicity and disease severity [30–34]. However, the molecular determinants responsible for the pathogenicity difference observed between cluster 1 and 2.1 DTMUVs remain unknown and require further investigation.

In conclusion, our data collectively indicated that cluster 1 DTMUV was less pathogenic and virulent than a previously reported cluster 2.1 DTMUV in 4-week-old ducks. Overall, our findings suggest differences in the antigenicity and pathogenicity between DTMUV genotypes in ducks, highlighting the potential association between DTMUV genotype and pathogenicity/disease severity. This study enhances our understanding of the pathogenesis of DTMUV infection in ducks and provides useful information for the design and development of effective DTMUV vaccines.

## Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## Ethical Approval

Animal experiment was approved and conducted in accordance with the ethical guidelines of the Chulalongkorn University Animal Care and Use Committee (approval number 2073001).

## Conflicts of Interest

The authors declare that they have no conflicts of interest.

## Authors' Contributions

All authors have read and approved the manuscript and have contributed significantly to the work. WT, WB, and AT designed the study. WT, KR, SW, NY, DP, PN, BL, TN, WB, and AT conducted the experiment. WT, KR, WB, and AT analyzed the results. WT, RH, WB, and AT wrote and approved the manuscript.

## Acknowledgments

The authors would like to thank the staffs of the Virology Unit, Department of Veterinary Microbiology, Avian Health Research Unit, Department of Veterinary Medicine, Faculty of Veterinary Science, and CULAC, Chulalongkorn University; to Drs. Nataya Charoenvisal, Phornphisut Areeraksakul, and Chaitawat Sirisereewan for assistance with animal experiment; to CPF (Thailand) Public Company Limited for contribution to providing ducks for animal experiment; and also to Chulalongkorn University for its financial support to the Center of Excellence for Emerging and Re-emerging Infectious Diseases in Animals, the Animal Vector-Borne Disease Research Unit, the Animal Virome and Diagnostic Development Research Group, and the Center of Excellence of Systems Microbiology. This work was supported by the Thailand Science Research and Innovation Fund Chulalongkorn University (FOOD66310011), the Second Century Fund (C2F), Chulalongkorn University, and the National Research Council of Thailand (NRCT): R. Thanawongnuwech NRCT Senior scholar 2022 (N42A650553).

## References

[1] R. Hamel, T. Phanitchat, S. Wichit et al., "New insights into the biology of the emerging Tembusu virus," *Pathogens*, vol. 10, no. 8, Article ID 1010, 2021.

[2] P. Ninvilai, W. Tunterak, K. Oraveerakul, A. Amonsin, and A. Thontiravong, "Genetic characterization of duck Tembusu virus in Thailand, 2015-2017: Identification of a novel cluster," *Transboundary and Emerging Diseases*, vol. 66, no. 5, pp. 1982-1992, 2019.

[3] Z. G. Homonnay, E. W. Kovács, K. Bányai et al., "Tembusu-like flavivirus (Perak virus) as the cause of neurological disease outbreaks in young Pekin ducks," *Avian Pathology*, vol. 43, no. 6, pp. 552-560, 2014.

[4] Y.-P. Chen, Y.-H. Shih, F. Lee, and C.-J. Chiou, "The first identification of Tembusu virus in a pekin duck farm in Taiwan," *Journal of Applied Animal Research*, vol. 50, no. 1, pp. 86-92, 2022.

[5] G. Qiu, Y. Cui, Y. Li, Y. Li, and Y. Wang, "The spread of Tembusu virus in China from 2010 to 2019," *Virus Research*, vol. 300, Article ID 198374, 2021.

[6] J. Su, S. Li, X. Hu et al., "Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus," *PLoS ONE*, vol. 6, no. 3, Article ID e18106, 2011.

[7] A. Thontiravong, P. Ninvilai, W. Tunterak et al., "Tembusu-related flavivirus in ducks, Thailand," *Emerging Infectious Diseases*, vol. 21, no. 12, pp. 2164-2167, 2015.

[8] Z. Cao, C. Zhang, Y. Liu et al., "Tembusu virus in ducks, china," *Emerging Infectious Diseases*, vol. 17, no. 10, pp. 1873-1875, 2011.
[9] N. Li, C. Lv, R. Yue et al., "Effect of age on the pathogenesis of duck Tembusu virus in cherry valley ducks," *Frontiers in Microbiology*, vol. 6, Article ID 581, 2015.

[10] P. Ninvilai, B. Limcharoen, W. Tunterak et al., "Pathogenesis of thai duck Tembusu virus in cherry valley ducks: the effect of age on susceptibility to infection," *Veterinary Microbiology*, vol. 243, Article ID 108636, 2020.

[11] C. Lv, R. Li, X. Liu, N. Li, and S. Liu, "Pathogenicity comparison of duck Tembusu virus in different aged Cherry Valley breeding ducks," *BMC Veterinary Research*, vol. 15, no. 1, Article ID 282, 2019.

[12] C. Feng, M. Jin, L. Yang et al., "Pathogenicity of a jinding duck-origin cluster 2.1 isolate of Tembusu virus in 3-week-old pekin ducklings," *Veterinary Microbiology*, vol. 251, Article ID 108870, 2020.

[13] Z. Yu, H. Ren, M. Sun et al., "Tembusu virus infection in laying chickens: evidence for a distinct genetic cluster with significant antigenic variation," *Transboundary and Emerging Diseases*, vol. 69, no. 4, pp. e1130-e1141, 2022.

[14] R. Meng, B. Yang, C. Feng, J. Huang, X. Wang, and D. Zhang, "The difference in CD4(+) T cell immunity between high- and low-virulence Tembusu viruses is mainly related to residues 151 and 304 in the envelope protein," *Frontiers in Immunology*, vol. 13, Article ID 890263, 2022.

[15] P. Ninvilai, N. Nonthabenjawan, B. Limcharoen et al., "The presence of duck Tembusu virus in Thailand since 2007: a retrospective study," *Transboundary and Emerging Diseases*, vol. 65, no. 5, pp. 1208-1216, 2018.

[16] L. J. Reed and H. Muench, "A simple method of estimating fifty per cent endpoints," *American Journal of Epidemiology*, vol. 27, no. 3, pp. 493-497, 1938.

[17] W. Tunterak, D. Prakairungnamthip, P. Ninvilai et al., "Serological evidence of duck Tembusu virus infection in free-grazing ducks, Thailand," *Transboundary and Emerging Diseases*, vol. 65, no. 6, pp. 1943-1950, 2018.

[18] H. Li, S. Liu, and X. Kong, "Characterization of the genes encoding UL24, TK and gH proteins from duck enteritis virus (DEV): a proof for the classification of DEV," *Virus Genes*, vol. 33, no. 2, pp. 221-227, 2006.

[19] H. Liu, Z. Wang, Y. Wu et al., "Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005," *Journal of Virological Methods*, vol. 140, no. 1-2, pp. 206-211, 2007.

[20] D. L. Suarez, A. Das, and E. Ellis, "Review of rapid molecular diagnostic tools for avian influenza virus," *Avian Diseases*, vol. 51, no. s1, pp. 201-208, 2007.

[21] I. Archetti and F. L. Horsfall Jr., "Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum," *Journal of Experimental Medicine*, vol. 92, no. 5, pp. 441-462, 1950.

[22] N. K. Blackburn, D. L. Thompson, and P. G. Jupp, "Antigenic relationship of West Nile strains by titre ratios calculated from cross-neutralization test results," *Epidemiology and Infection*, vol. 99, no. 2, pp. 551-557, 1987.

[23] Y. Zhu, Z. Hu, X. Lv et al., "A novel Tembusu virus isolated from goslings in China form a new subgenotype 2.1.1," *Transbound-ary and Emerging Diseases*, vol. 69, no. 4, pp. 1782-1793, 2022.

[24] Y. Ma, Y. Liang, N. Wang et al., "Avian flavivirus infection of monocytes/macrophages by extensive subversion of host antiviral innate immune responses," *Journal of Virology*, vol. 93, no. 22, 2019.

[25] K. Clyde, J. L. Kyle, and E. Harris, "Recent advances in deciphering viral and host determinants of dengue virus

replication and pathogenesis," *Journal of Virology*, vol. 80, no. 23, pp. 11418-11431, 2006.

[26] C. Sureau and F. Negro, "The hepatitis delta virus: replication and pathogenesis," *Journal of Hepatology*, vol. 64, no. 1, pp. S102-S116, 2016.

[27] C. R. Vicente, K. H. Herbinger, G. Fröschl, C. M. Romano, A. S. A. Cabidelle, and C. C. Junior, "Serotype influences on dengue severity: a cross-sectional study on 485 confirmed dengue cases in Vitória, Brazil," *BMC Infectious Diseases*, vol. 16, Article ID 320, 2016.

[28] D. Yan, X. Li, Z. Wang et al., "The emergence of a disease caused by a mosquito origin Cluster 3.2 Tembusu virus in chickens in China," *Veterinary Microbiology*, vol. 272, Article ID 109500, 2022.

[29] A. Thontiravong, T. Nedumpun, P. Ninvilai et al., "Dynamics of cellular and humoral immune responses following duck Tembusu virus infection in ducks," *Transboundary and Emerging Diseases*, vol. 69, no. 5, pp. e1365-e1373, 2022.

[30] M. Sun, L. Zhang, Y. Cao et al., "Basic amino acid substitution at residue 367 of the envelope protein of Tembusu virus plays a critical role in pathogenesis," *Journal of Virology*, vol. 94, no. 8, 2020.

[31] X. Sun, M. Sun, L. Zhang et al., "Amino acid substitutions in ns5 contribute differentially to Tembusu virus attenuation in ducklings and cell cultures," *Viruses*, vol. 13, no. 5, Article ID 921, 2021.

[32] D. Yan, Y. Shi, H. Wang et al., "A single mutation at position 156 in the envelope protein of Tembusu virus is responsible for virus tissue tropism and transmissibility in ducks," *Journal of Virology*, vol. 92, no. 17, 2018.

[33] D. Yan, B. Wang, Y. Shi et al., "A single mutation at position 120 in the envelope protein attenuates Tembusu virus in ducks," *Viruses*, vol. 14, no. 3, Article ID 447, 2022.

[34] L. Yang, T. Liang, J. Lv et al., "Substantial attenuation of virulence of Tembusu virus strain PS Is determined by an arginine at residue 304 of the envelope protein," *Journal of Virology*, vol. 95, no. 6, 2021.