Pathogenesis of NGPV and DuCV CO-infection pathogenicity analysis of co-infection with novel goose parvovirus and duck circovirus in Cherry Valley meat ducks

Abstract

The prevalence of short beak and dwarfism syndrome (SBDS) in China is increasing, causing much economic losses to the waterfowl industry. Novel goose parvovirus (NGPV) has been identified as the primary pathogen; however, it is difficult to reproduce SBDS with NGPV infection alone. Duck circovirus (DuCV) orchestrates immunosuppression in ducks, rendering them more prone to other pathogenic infection. Notably, a high co-infection rate between DuCV and NGPV has been observed. Given this context, the present study utilized the Cherry Valley meat duck, the largest breed in China, as the experimental model. In this study, ducks were separately or co-infected with NGPV and DuCV. At 1, 3, 7, 14, 21, 28, and 35 days post-infection (DPI), six ducks were randomly selected to detect relevant indicators and evaluate whether the two viruses have a synergistic pathogenic effect. The results showed that these two viruses were continuously detected in serum and swabs in the co-infection group from 1 DPI onward, and were higher than those in the corresponding single-infection group. The co-infected ducks exhibited symptoms of SBDS. The levels of calcium, phosphorus, and lymphocytes were significantly reduced, whereas the level of total bilirubin was significantly elevated, indicating impaired metabolic and immune functions. Moreover, compared with single-infection groups, ducks in the co-infection group exhibited more severe immunosuppression and tissue damage, along with higher viral loads of these two viruses in many organs. The results showed that co-infection with DuCV and NGPV can promote viral replication, increase pathogenicity, and lead to the occurrence of SBDS. This study will provide a theoretical basis for the prevention and control of SBDS.

Introduction

Since 2015, a new disease named “short beak and dwarfism syndrome (SBDS)” has broken out in duck flocks across multiple regions in northern China (Chen et al., 2015). Although the mortality rate in the affected duck flocks is relatively low (2% to 6%), morbidity rates reach 10% to 30% and even 50% or higher in some regions (Li et al., 2016; Zhao et al., 2019). Infected ducks exhibit beak atrophy, swollen tongues and shorter tibia, along with severe growth retardation, which causes huge economic losses to the poultry industry (Palya et al., 2009). After serial experiments, the pathogen of SBDS was identified as a novel goose parvovirus (NGPV). NGPV belongs to Anseriform dependoparvovirus 1 and is an icosahedrally symmetric, spherical or hexagonal, linear single-stranded DNA virus with a diameter of approximately 20 to 25 nm (Tatár-Kis et al., 2004). The genomic length is approximately 5.0 to 5.1 kb. It has two open reading frames, responsible for encoding two replication proteins and three capsid proteins, and contains two inverted terminal repeats that form hairpin structures (Chen et al., 2015; Deng et al., 2014; Li et al., 2016). NGPV exhibits a high genetic similarity exceeding 92.2% with goose parvovirus (GPV) (Li et al., 2018b; Wan et al., 2019). However, unlike GPV, which only infects geese and Muscovy ducks, NGPV infects multiple duck species such as Cherry Valley meat ducks, Beijing ducks, and Muscovy ducks (François et al., 2016; Li et al., 2018b; Yu et al., 2016). Consequently, the hazards posed by NGPV infection have been receiving increasing attention.

Although NGPV has been shown to be the main causative agent of SBDS, ducks infected with NGPV alone rarely induces the typical symptoms (Ning et al., 2017). Meanwhile, duck circovirus (DuCV) is a contagious immunosuppressive virus that belongs to the genus Circovirus of the family Circoviridae comprising genotypes DuCV-1, DuCV-2, and DuCV-3 (Liao et al., 2022; Wen et al., 2014). The virion is icosahedral and non-enveloped with a diameter of approximately 15 to 16 nm (Hattermann et al., 2003). It is extensively distributed globally and has been detected in multiple duck breeds (Banda et al., 2007; Wan et al., 2011a, 2011b; Zhang et al., 2009). DuCV infection can damage the immune organs, thereby causing long-term immunosuppression (Soike et al., 2004). Consequently, infected ducks exhibit feather disorders and growth retardation (Li et al., 2025). Notably, beak and feather disease in parrots and goose dwarf syndrome caused by other avian circovirus infections also exhibit symptoms of growth retardation and beak abnormalities (McOrist et al., 1984; Soike et al., 1999). These symptoms are similar to those of ducks suffering from SBDS (Li et al., 2018a). Our previous investigation found a high co-infection rate (63.53%) of DuCV and NGPV. Furthermore, the positive rate of DuCV in SBDS-affected ducks (82.35%) was higher than that reported in normal ducks (21.8%) (Cha et al., 2014; Li et al., 2018a). Thus, it is hypothesized that DuCV may be involved in the pathogenic process of SBDS.

However, substantial gaps still remain in understanding the pathogenicity of DuCV and NGPV co-infection. In this study, we used Cherry Valley meat ducks co-infected with DuCV and NGPV to establish an animal model aiming to investigate whether the two viruses have a synergistic pathogenic effect. This research contributes to a deeper understanding of mixed viral infections in ducks, enhances the knowledge of SBDS, and provides a theoretical basis for prevention and control of this disease.

Materials and methods

Virus

The NGPV JS1123 strain (GenBank No. MN415967) was originally isolated from the liver of a 21-day-old Cherry Valley meat duck with SBDS in Shandong province, China, in 2018. The isolated virus was inoculated into the allantoic cavity of 10-day-old duck embryos for culture and the viral load of allantoic fluid was 3.43 × 10² copies/µL by using TaqMan-based fluorescent quantitative real-time PCR (FQ-PCR). Given the limited amplification efficiency of DuCV in duck embryos, the virus was propagated in vivo via inoculation of healthy ducks. The DuCV TAYB03 strain (GenBank No. MN808566) were isolated, identified, and stored employing the methodologies previously established in the laboratory (Li, et al., 2025). The viral load of the suspension was 2.47 × 10⁵ copies/µL. Prior to use, both virus strains were filter-sterilized and screened by polymerase chain reaction (PCR) assays for the absence of other extraneous agents in ducks.

Birds

A total of 168 one-day-old healthy Cherry Valley meat ducklings were purchased from the Jingwei Husbandry Company in Taian City. These ducklings showed no clinical symptoms. Serum and cloacal swabs were collected and tests were conducted according to the previous research methods to confirm the absence of pathogens (including DuCV, NGPV, duck plague virus, duck reovirus, duck hepatitis virus, avian poxvirus, Tembusu virus and avian influenza virus, Riemerella anatipestifer, Pasteurella multocida and Salmonella) (Supplementary Table 1) (Hu et al., 2011; Niu et al., 2016; Peng et al., 2023; Wan et al., 2011a; Wu et al., 2023, 2025a; Xiao et al., 2021; Zhang et al., 2020; Zheng et al., 2015).

Animal experiment

This experiment was approved by the Animal Care and Use Committee of Shandong Agricultural University (approval number, SDAUA-2019-206) and performed by the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China). All ducklings were randomly divided into four groups equally (n = 42): the control group, the DuCV group, the NGPV group and the DuCV + NGPV group. They were housed separately in SPF animal isolators, which were ventilated under positive pressure. No immunizations were administered. Water and food were autoclaved and were provided ad libitum. In the DuCV group, ducklings were challenged orally with 1 mL viral suspension containing 2.47 × 10⁸ copies, whereas those in the NGPV group were challenged in the same manner using 200 μL viral suspension containing 6.86 × 10⁴ copies. Preliminary experiments demonstrated that these dosages would successfully infect ducklings, causing the corresponding clinical symptoms without excessive mortality. In the DuCV + NGPV group, ducklings received the same treatments as those in the two single-infection groups simultaneously. And ducklings in the control group received an equivalent dosage of sterile phosphate-buffered saline (PBS, pH=7.2).

Throughout the experiment, the clinical symptoms of ducks in all groups were monitored, including weight, the length of the beak, and the length of the tibia. At 1, 3, 7, 14, 21, 28, and 35 days post-infection (DPI), six ducks were randomly selected from each group. These ducks were weighed, and swab samples (from the pharynx and cloaca) were collected. Additionally, blood samples were obtained via the jugular vein. Swabs were immediately placed in 1 mL sterile PBS and vortexed thoroughly. They then underwent three freeze-thaw cycles. After centrifugation, the supernatant was collected, filtered through a 0.22 μm filter for sterilization, and stored at −80°C for future experimental applications. Blood samples were centrifuged at 3000 × g for 10 min to obtain serum. The following biochemical parameters were then measured using an automatic biochemical analyzer: A portion of the serum was used to detect biochemical indicators such as calcium (Ca), phosphorus (P), total bilirubin (TBIL) and lymphocyte (LYM), while the remaining portion was stored at −80°C for future experiments. Ducks were subsequently euthanized via intravenous pentobarbital sodium (New Asia Pharmaceutical, Shanghai, China). At necropsy, the heart, liver, lung, spleen, kidney, duodenum, tongue and thymus were collected and weighed, and the immune organ index was calculated. The calculation formula was as follows: Immune organ index (%) = Immune organ weight (g) / Body weight (g) × 100. A portion of each tissue was fixed in a 10% neutral buffered formalin for histopathological examinations. The remaining part of each tissue was homogenized, frozen, thawed, and sterilized for DNA extraction and PCR analysis later.

Histological evaluation

The collected tissues were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Subsequently, paraffin blocks were sectioned at approximately 4 μm by a microtome (Leica RM2255, Germany) and stained with haematoxylin and eosin (H&E). Finally, the tissue sections were visualized with a light microscope (Nikon Eclipse 80i, Japan).

Viral load assays

The total viral DNA from swab, serum, and organ samples was extracted using the phenol-chloroform method. To detect NGPV, a TaqMan-based FQ-PCR assay was conducted following to a previous report (Niu, et al., 2016). The primers and probe were as follows: F: 5′-TCATCAAGAATACACCAGTA-3′, R: 5′-GTATTGAGTTAT-GTAGGAGTTC-3′, Probe: FAM-5′-CCTCCAGTAGAATATGTGAACCAGA-3′-TAMRA. The reaction was performed in a final reaction volume of 20 μL containing 10 μL Premix Ex Taq (TaKaRa, Dalian, China), 0.4 μL of each forward and reverse primer (each at a concentration of 0.2 μM), 2 μL DNA solution, 6.2 μL double distilled H₂O (ddH₂O) and 0.8 μL probe. The thermal profile was set as 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s, using LightCycler96 Real-Time PCR System (Roche, Germany). To target the DuCV, the FQ-PCR primers were designed as described in a previous study (Niu, et al., 2016; Wan et al., 2011a): F, 5′-TGTTATCTTTGGGCGTGG-3′; R, 5′-CATTTCCCGAGTAACCGTC-3′. The reaction was performed in 20 μL reaction mixtures containing 10 μL of kit-supplied SYBR® PCR master mix (TaKaRa, Dalian, China), 0.4 μL concentrations of each forward and reverse primer (each at a concentration of 10 μM) and 2 μL DNA solutions. The final volume was made up to 20 μL with ddH₂O. Amplification was performed using the following thermocycling program: 95°C for 120 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Each sample was tested in triplicate along with positive and negative reference samples in each run.

Statistical Analysis

SPSS 25.0 software (SPSS, USA) was used to analyze the data. Data were expressed as mean ± SEM and a P value less than 0.05 was considered statistically significant. Data were analyzed via multi-factor ANOVA.

Results

Detection of Clinical Indicators

All virus-challenged ducks exhibited systemic symptoms, including ruffled plumage, lethargy, and prolonged recumbency. No ducks died in this experiment. Ducks in the DuCV + NGPV group presented typical symptoms, such as short beaks and swollen tongues. The body weights (Fig. 1A) and tibial lengths (Fig. 1C) of ducks in both the single-infection groups and the co-infection group were significantly lower than those of the control group (p < 0.05). Starting from 14 DPI, the beak lengths (Fig. 1B) of infected ducks in the NGPV group and the DuCV + NGPV group were significantly shorter than those in the control group (p < 0.05). Notably, body weights (Fig. 1A) and beak lengths (Fig. 1B) of ducks in the co-infection group became significantly lower than those in the control group at 14 DPI and 21 DPI, respectively, which were earlier than those observed in the single-infection group (Fig. 1).

Fig. 1.

Clinical indicators of ducks at different DPI. (A) weight, (B) the length of beak, (D) the length of tibia. *, p < 0.05; **, p < 0.01.

Immune organ indexes

Compared with the control group, the immune organ indices of ducks in the DuCV group were significantly reduced (p < 0.05), whereas those in the NGPV and DuCV + NGPV groups showed a significant increase (p < 0.05). In addition, at 3 or 14 DPI, the spleen and thymus indexes of the co-infection group were significantly higher than those in the NGPV group (p < 0.05) (Fig. 2).

Fig. 2.

Changes in immune organ indexes. (A) thymus, (B) spleen. *, p < 0.05; **, p < 0.01.

Histopathology

As shown in Fig. 3A and 3B, compared with the control group, DuCV infection in ducks resulted in hepatocyte degeneration in the liver and cytoplasmic rarefaction in renal tubular epithelial cells. In addition to these lesions, hemorrhage was also observed in the livers and kidneys of ducks in both the NGPV group and the DuCV + NGPV group. The number of parenchymal cells decreased in the spleen of ducks in the DuCV group, and extensive bleeding occurred after NGPV infection (Fig. 3C). The pathological changes appeared to be more severe in the co-infection group. In the thymus, DuCV infection induced lymphocyte apoptosis, while hemorrhage was additionally observed in the NGPV group and the DuCV + NGPV group (Fig. 3D). No obvious pathological changes were detected in the intestine of ducks in the DuCV group; however, NGPV infection and DuCV + NGPV co-infection caused the exfoliation of intestinal epithelial cells, indicating damage to the intestinal mucosa (Fig. 3E).

Fig. 3.

Histopathological changes in (A) liver, (B) kidney, (C) spleen, (D) thymus and (E) intestine in ducks. bar =50 μm.

Serum clinical indexes analyses

In Fig. 4, the results showed that the levels of Ca (Fig. 4A) and P (Fig. 4B) in the serum samples from all virus-infected groups were significantly reduced (p < 0.05), whereas the level of TBIL (Fig. 4C) were significantly increased (p < 0.05). Compared with single DuCV or NGPV infection, co-infection with both viruses resulted in a prolonged duration of abnormal biochemical indicators. Moreover, significant changes (p < 0.05) in the levels of TBIL and Ca were first observed in the DuCV + NGPV group at 3 and 7 DPI, respectively. No significant difference was observed in the LYM (Fig. 4D) between ducks in the NGPV and control groups. In the DuCV + NGPV group, the level of LYM was significantly decreased (p < 0.05) at 28 and 35 DPI, occurring earlier than that in the DuCV group.

Fig. 4.

Changes in the (A) Ca, (B) P, (C) TBIL and (D) LYM in the serum. *, p < 0.05; **, p < 0.01.

Viremia and the patterns of virus excretion

As shown in Fig. 5, throughout the experimental period, DuCV and NGPV were detected individually or simultaneously in both serum and swab samples collected from ducks infected with either or both viruses, whereas samples from ducks in the control group tested negative. In the DuCV + NGPV group, the viral load of NGPV in throat swabs was significantly higher (p < 0.05) than that in the NGPV group from 1 to 3 DPI, and the viral load of DuCV in throat swabs remained significantly higher (p < 0.05) than that in the DuCV group from 14 to 35 DPI (Fig. 5B). The viral loads of DuCV in serum at 7 DPI and in cloacal swabs at 1 DPI of the DuCV + NGPV group were also significantly higher (p < 0.05) than those in the DuCV group (Fig. 5C). The viral loads of both viruses in the co-infection group and the single-infection groups peaked at 3 DPI in serum (Fig. 5A) and at 1 DPI in throat swabs (Fig. 5B). In cloacal swabs, NGPV showed an earlier peak viral load (3 DPI) than DuCV (7 DPI) (Fig. 5C).

Fig. 5.

Quantification of NGPV and DuCV DNA in (A) serum, (B) throat swab and (C) cloacal swab of infected ducks. DuCV and NGPV were consistently detected in infected ducks. At multiple DPIs, the viral loads of NGPV in oral swabs and DuCV in serum, oral swabs and cloacal swabs of ducks in the co-infection group were higher than those in the single-infection groups. *, p < 0.05; **, p < 0.01.

Detection of viral load in tissues

As shown in Fig. 6, after NGPV infection, the presence of NGPV was detected at 1 DPI in all organs of the ducks from both the DuCV + NGPV and NGPV groups, except for the heart, where the detection occurred at 3 DPI. In the DuCV + NGPV group, the viral load of NGPV in the heart reached its peak at 7 DPI versus 14 DPI in the NGPV group (Fig. 6A). In both groups, the viral loads of NGPV in the spleen (Fig. 6C) and intestine (Fig. 6F) reached their respective peaks at 3 and 7 DPI, whereas the viral loads in all other organs peaked at 21 DPI. Overall, the viral loads of NGPV in all organs (excluding the lung) of the ducks in the DuCV + NGPV group were significantly higher (p < 0.05) than those in the NGPV group at multiple time points.

Fig. 6.

Quantification of NGPV DNA in (A) heart, (B) liver, (C) spleen, (D) lung, (E) kidney, (F) intestine, (G) thymus and (H) tongue of infected ducks. NGPV was detected in the heart at 3 DPI, whereas it was detected in all other organs at 1 DPI and persisted throughout the entire experimental period. Except for the liver, the viral loads of NGPV in other organs of ducks in the co-infection group were higher than those in the single-infection group at multiple DPIs. *, p < 0.05; **, p < 0.01.

After DuCV infection, the virus was first detected in the liver of ducks from both the DuCV + NGPV and DuCV groups at 1 DPI (Fig. 7B). Subsequently, it was detected in the heart (Fig. 7A), spleen (Fig. 7C), and kidney (Fig. 7E) at 3 DPI; in the thymus (Fig. 7G) and intestine (Fig. 7F) at 7 DPI; in the tongue at 14 DPI; and finally in the lung (Fig. 7D) at 28 DPI. In the DuCV + NGPV group, the viral loads of DuCV in the spleen and liver reached their peaks at 3 and 7 DPI, respectively. In contrast, in the DuCV group, the peak viral loads in these organs occurred at 21 and 14 DPI. Regarding the other organs in both groups, including the heart, thymus, intestine, kidney/tongue, and lung, the viral loads peaked at 3, 14, 21, 28, and 35 DPI, respectively. Moreover, the viral loads of DuCV in the heart, spleen, and liver of ducks in the DuCV + NGPV group were significantly higher (p < 0.05) than those in the DuCV group at multiple time points (Fig. 7).

Fig. 7.

Quantification of DuCV DNA in (A) heart, (B) liver, (C) spleen, (D) lung, (E) kidney, (F) intestine, (G) thymus and (H) tongue of infected ducks. DuCV was detected in all organs, and the viral loads of DuCV in the heart, liver and spleen of the co-infection group were higher than those in the single-infection group at multiple DPIs. *, p < 0.05; **, p < 0.01.

Discussion

China is a leading country in the global waterfowl breeding industry. However, the incidence of SBDS has caused substantial economic losses. Previous investigations have revealed a relatively high co-infection rate with DuCV and NGPV (Cha et al., 2014; Li et al., 2018a; Yang et al., 2020). In China, Cherry Valley meat ducks are the commercial duck breed with the largest breeding population. Several studies have indicated that DuCV-1 is the dominant prevalent genotype in Cherry Valley meat ducks in China (Li et al., 2018a; Yang et al., 2020; Zhang et al., 2023). To better mimic the actual clinical scenario, we used DuCV-1 and NGPV to infect Cherry Valley meat ducks either individually or in combination, aiming to investigate the pathogenicity of these two viruses.

After NGPV and DuCV infection, both viruses were detected in the serum and swabs of infected ducks at 1 DPI, and detection persisted throughout the experimental period. Persistent viremia provided structural basis for viral invasion of peripheral tissues and immune organs as well as the induction of systemic immunosuppression (Bergthaler et al., 2010). This might account for the detection of both viruses in multiple organs at later stages. In addition, because DuCV and NGPV possess the characteristic of horizontal transmission, the positive detection of viruses in swabs indicates that infected ducks continuously excrete the virus into the external environment, which significantly raises potential risks (Li et al., 2018a; Wang et al., 2022a).

In this experiment, infection of Cherry Valley meat ducks with NGPV or DuCV alone, or in combination, led to lethargy and weight loss. The levels of Ca and P play vital roles in physiological processes such as growth and energy production (Weaver et al., 2016). The levels of these two minerals in the serum of ducks in the DuCV + NGPV group were significantly lower than those in the single-infection group; however, given that our study did not include specific assessments of intestinal absorption, liver function markers, or renal function, this observed finding can only be tentatively associated with the poor growth and development of the ducks, and no definitive causal relationship can be established. Further investigation was required to explore the specific mechanism. The results from this study showed that ducks with only NGPV infection have significantly shorter beak and lower body weight compared to the control group; however, it should be noted that this was derived from statistical analysis of a large sample size. Moreover, only the co-infection group was observed to replicate the distinct symptoms of SBDS clinically. Similarly, in another study on SPF Shaoxing ducks, it was found that co-infection with these two viruses also exacerbates the clinical symptoms (Zhu et al., 2025).

During the necropsy, macroscopic inspection revealed that DuCV infection decreased the size of the thymus. The soluble CD4 (sCD4) and soluble CD8 (sCD8) molecules serve as the major surface markers of helper T lymphocytes and cytotoxic T lymphocytes, respectively, and were considered as indicators for lymphoid system reconstitution (Ho et al., 1994). Lymphocyte Transformation Rate (LTR) refers to an in vitro assay that measures the activity and function of T lymphocytes, thus reflecting the body’s immune ability (Bertram et al., 1997). In a previous study, it was definitively demonstrated that DuCV infection in ducks leads to a decrease in LTR, reduced concentrations of sCD4 and sCD8, as well as suppressed secretion of IL-12 and IFN-γ. These findings, which are well-established in the literature, indicate that DuCV inhibits the proliferation and differentiation of lymphocytes, significantly impairs T cell-mediated immune function, and exerts a great impact on humoral and cellular immunity (Wang et al., 2022b). Based on the significantly reduced indices of immune organs, it can be concluded that DuCV infection markedly impairs the development of these organs. Although we were unable to collect other immune organs such as the bursa of Fabricius due to logistical transportation and sample constraints, the damaging effects of DuCV on immune organs have long been reported in previous studies (Li et al., 2025; Soike et al., 2004). These findings suggested that DuCV infection may induce immunosuppression and potentially render ducks susceptible to secondary infections, though direct evidence of such immunosuppressive effects is not provided in this study. In addition to NGPV, ducks may also be secondarily infected by Riemerella anatipestifer (RA), Escherichia coli (E. coli), Pasteurella multocida, Staphylococcus aureus, and duck hepatitis A virus (DHAV) (Jiang et al., 2008; Soike et al., 2004; Zhang et al., 2009). The level of LYM in serum can reflect the immune function (Tian et al., 2024). In this study, the levels of LYM were significantly reduced in both the DuCV + NGPV group and the DuCV group. Similar to previous study (Wang et al., 2022b), the results of this experiment indicated that DuCV may affect the immune defense function mediated by lymphocytes. Based on our observations, we speculate that the earlier decline in LYM levels in the co-infection group may indicate that co-infection with NGPV exacerbates the immunosuppressive effect. The premature occurrence of immunosuppression will disrupt the host's innate and adaptive immune barriers, thereby creating a favorable microenvironment for the invasion and colonization of secondary pathogens (Wu et al., 2024). However, this study only used serum LYM levels as a macroscopic detection indicator and lacked in-depth research on virus-host protein interactions, thus failing to precisely explain the mechanism of synergistic immunosuppression. It has been definitively demonstrated that the ORF3 protein of duck circovirus (DuCV) can regulate caspase 3 and caspase 9 expression, thereby triggering cell apoptosis via the mitochondrial pathway (Huang et al., 2023). Although research on the mechanisms underlying NGPV-induced apoptosis remained limited, it has been proven that GPV can also induce cell apoptosis via the mitochondrial pathway (Yan et al., 2021). Therefore, it remained to be investigated whether co-infection with NGPV and DuCV can synergistically induce immune cell damage through this pathway.

Similar to another study, NGPV infection in this experiment inflicted damage on multiple organs of ducks (Chen et al., 2016). Co-infection with DuCV exacerbated such pathological damage, accompanied by enhanced viral replication (evidenced by increased viral loads in multiple organs at multiple DPIs) and earlier appearance of viral load peaks, confirming the synergistic pathogenicity between DuCV and NGPV. This synergistic effect may create a favorable environment for viral replication and spread. This potential association might be tentatively attributed to DuCV-mediated immunosuppression (inferred from our observational findings) and the theoretical mechanism—well-established in the literature—by which both avian circoviruses and parvoviruses can induce apoptosis via the mitochondrial pathway (Hong et al., 2018; Huang et al., 2023; Yan et al., 2021). However, this remains speculative and requires further verification.

It has been reported that co-infection of SPF Shaoxing ducks with DuCV and NGPV results in significantly higher viral loads in the liver compared to the single-infection groups (Zhu, et al., 2025). Compared with this study, our research extended the scope of affected organs beyond the liver, confirmed that the promoting effect of co-infection on viral replication can cover multiple organs such as the heart and spleen, and revealed the temporal characteristics of the synergistic effect, thereby further improving the understanding of "DuCV and NGPV co-infection enhancing viral replication". The viral load of NGPV in the tongue of the DuCV + NGPV group was significantly higher than that of the NGPV group since 3DPI. However, the degree of swelling and the changes in length of the tongue were not specifically quantified. In addition, multiple strains were not compared in this study, nor were the mucosal immune indicators of duck tongues detected. Therefore, it remained unclear whether a threshold effect or strain-specific lingual pathological response differences exist. Future studies focusing on these aspects, such as the establishment of in vitro co-infection models and exploration of virus-host protein interactions, will help clarify the underlying mechanisms and confirm the specificity of the observed synergistic effects.

Conclusions

In conclusion, co-infection with NGPV and DuCV in Cherry Valley meat ducks was associated with concurrent enhancement of the replication capacity and pathogenicity of both viruses, accompanied by more severe clinical symptoms and a higher incidence of SBDS. In the future, further in-depth mechanistic studies on the co-infection of these two viruses will facilitate the prevention and control of SBDS.

CRediT authorship contribution statement

Hanqing Li: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Lei Bei: Software, Resources, Investigation, Formal analysis. Hui Yan: Validation, Methodology, Investigation, Formal analysis. Mingrui Zhao: Visualization, Software, Methodology, Investigation. Ruihua Zhang: Validation, Resources, Project administration, Methodology, Investigation. Yu Meng: Software, Resources, Methodology, Data curation. Yanli Zhu: Resources, Project administration, Methodology. Liangmeng Wei: Project administration, Methodology, Formal analysis. Shijin Jiang: Writing – review & editing, Supervision, Resources, Funding acquisition, Data curation, Conceptualization.

Disclosures

The authors declare no conflict of interest.