Experimental Infection of Rats with Influenza A Viruses: Implications for Murine Rodents in Influenza A Virus Ecology

1. Introduction

In recent decades, many influenza A viruses (IAVs) of zoonotic origin have emerged and caused subsequent epidemics or even pandemics in humans, such as the swine-origin 2009 H1N1 pandemic, and the avian-origin H7N9, H10N8, H9N2 and highly pathogenic avian influenza (HPAI) H5Nx outbreaks [1,2,3]. These events have repeatedly highlighted the ecological importance of animals in the genesis and transmission of IAVs.

IAVs have a very wide range of hosts and interspecies spillover is not uncommon in nature. Historically, IAVs have been established in birds, bats, horses, pigs, dogs and hu-mans [4,5]. Sporadic infections in other ungulates and carnivores (e.g., camels, dairy cows, whales, cats, minks, etc.) have also been documented [4,5,6,7,8,9]. While wild waterfowl are thought to serve as a natural reservoir for IAVs, domestic animals, either birds or mammals, may act as intermediate hosts, facilitating the adaptation and emergence of novel variants capable of infecting humans [4,5]. It is therefore crucial to monitor IAVs in different animal species and to better understand the ecology of influenza viruses.

Rodents, including commensal rats, can carry many different zoonotic pathogens but are not generally considered to be natural hosts of IAVs [4,5,10,11], although the presence of IAV nucleic acids or antibodies against viruses such as HPAI H5N1 and H5N6 have been reported [12,13,14,15,16,17]. On the other hand, although laboratory mice (Mus musculus) and rats (Rattus norvegicus) are widely used in biomedical research, and mice are popularly used in influenza research, rats were not generally considered a susceptible host for IAVs. There are limited reports on influenza infection in Rattus species, mostly using adapted viral strains [18,19,20], infant rat models [18,21,22,23], or other non-generalizable conditions [24,25].

Here we propose to experimentally infect rats with different subtypes of IAVs isolated from the field. In particular, the study will focus on prevalent IAV lineages with zoonotic risk and aims to provide direct evidence of the susceptibility of rats to IAVs without prior adaptation. As common synanthropes worldwide, rats are ubiquitous in both rural and urban areas with dense populations [26,27]. They not only share habitats with wild and free-ranging animals, but also have free access to human settlements and live-stock houses. Understanding the role of rats in the ecology of IAVs will aid risk assessment and disease control and prevention.

2. Materials and Methods

2.1. Ethical Statement

All animal experiments were approved and conducted in accordance with institutional guidelines. The design and conduct of the research were endorsed by the Medical Animal Care and Welfare Committee of Shantou University Medical College (Reference No. SUMC2013-111 and SUMC2016-034), and all experiments involving infectious agents were carried out in Biosafety Level 3 (BSL-3) facilities. The research also complied with the Guangdong Provincial Regulations on the Administration of Laboratory Animals and strictly followed the Operational Guidelines for Ethics Committees that Review Biomedical Research issued by the Ministry of Health of China.

2.2. Viruses

Viruses of prevalent and recently emerging subtypes in China were selected for this study. These viruses were initially isolated in 9-day-old embryonated chicken eggs. For avian strains, viral stocks were prepared by propagation in 9-day-old embryonated chicken eggs, while for human isolates, stocks were prepared in Madin-Darby canine kidney (MDCK) cells [28]. All stocks were aliquoted, titrated and frozen at -80°C until use. The representative strains used in this study are summarized in Table 1.

2.3. Animal Experiments

Female specific-pathogen-free (SPF) Sprague-Dawley (SD) rats, 8 to 9 weeks old, were purchased from Beijing Vital River Laboratory Animals Ltd. Co. (Beijing, China) and randomly divided into groups. Prior to inoculation and necropsy, animals were anesthetized by intramuscular injection of sodium pentobarbital (0.2 mg/g). Animals that lost more than 30% of their initial body weight were humanely euthanized and their respiratory tissues and blood were collected where possible.

To test the infectivity of different subtypes of IAVs in SD rats, two groups of three rats and one group of eight rats were inoculated with 100 μl of PBS containing 10², 10⁴ and 10⁶ 50% tissue culture infectious dose (TCID₅₀) of each virus strain, respectively. Eight rats were sham-infected with 100 μl PBS as a control. Five rats in the 10⁶ TCID₅₀ dose group were monitored in comparison to the control group (n=5) and weighed throughout the 14-day experimental period, while the remaining rats were necropsied on day 4 post-inoculation (dpi) to collect respiratory tract tissues for virus replication analysis. At 4 dpi, three rats in each virus dose group were euthanized for airway tissue collection. Each tissue was cut into two pieces. One part was placed in cryo-vials containing 1 ml of PBS, homogenized and stored at -80℃ for titration on MDCK cells. The remainder was fixed in 10% neutral buffered formalin for histopathological examination. Meanwhile, blood was also collected for serum preparation.

2.4. Hemagglutination Inhibition Test

Seroconversion was tested at 4 or 21 dpi before the euthanasia of the animals. Rat serum was treated with receptor-destroying enzyme II (RDE II; Denka Seiken, Tokyo, Japan) overnight at 37 °C to inactivate non-specific inhibitors, followed by heat-inactivation at 56 °C for 30 min to remove non-specific agglutinins by adsorption with freshly prepared turkey red blood cells (TRBCs). The sera obtained were tested by hemagglutination inhibition (HAI) assay using the IAVs listed in Table 1, according to the standard protocol of the World Health Organization (WHO) [28]. Briefly, four hemagglutination units per 25 μl of the IAVs listed in Table 1 were added to an equal volume of two-fold serially diluted sera in U-bottom 96-well microtiter plates and incubated for 45 min to 1 h. Finally, 50 μl of 0.5% TRBCs were added to the plates and incubated for 30 min at room temperature. HAI titers were defined as the reciprocal of the highest serum dilution required for complete hemagglutination inhibition.

2.5. Immunohistochemical and Histopathological Studies

Fresh nasal, tracheal and lung tissues were fixed in 10% (vol/vol) neutral buffered formalin for 48 h at room temperature. After fixation, the tissues were dehydrated in grad-ed ethanol solutions and xylene and embedded in paraffin. Finally, 3 μm tissue sections were obtained using a Leica slicer. Serial sections were subjected to standard immuno-histochemical (IHC) staining for viral nucleoprotein (NP) antigen detection and pathological examination by hematoxylin and eosin (H&E) staining as previously described [29,30].

2.6. Lectin Staining Assay

To determine the expression of sialic acid (SA) receptors in the airways of rats, we used biotinylated conjugated lectins from Maackia amurensis agglutinin I and II (MAA I and MAA II, specific for SAα2,3Galβ1,4GalNAc and SAα2,3Galβ1,3GalNAc, respectively; Vector Laboratories, Burlingame, California, USA) and Sambucus nigra agglutinin (SNA; Vector Laboratories, Burlingame, California, USA) to label α2,3-linked and α2,6-linked SA residues, respectively [31,32,33]. Tissues from non-inoculated SPF SD rats were processed using the same methods as described for immunohistochemical and histopathological studies. After blocking, sections were incubated with MAA (1:1500 dilution) or SNA (1:2000 dilution) for 1 h at room temperature. To ensure reliability, we also analyzed the distribution of IAV receptors in the respiratory tract of female 8- to 9-week-old C57BL/6 mice (Beijing Vital River Laboratory Animals Ltd. Co., Beijing, China).

2.7. Statistical Analysis

GraphPad Prism 10.1.0 (GraphPad Software) was used for calculations of area under the curve (AUC) and statistical analyses. Data are presented as mean ± standard deviation unless otherwise stated. Student’s t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used to compare treatment groups. Statistical significance was set at P < 0.05.

3. Results

3.1. Asymptomatic Infection with IAVs in SD Rats

Clinical signs such as ruffled hair, inactivity, reduced food and water intake and other influenza-like illness were not observed in any of the rats. The body weight of the control rats increased steadily and there were no deaths in any of the inoculated groups (Figure 1). Following inoculation with different virus subtypes, rats in each group experienced no weight loss or a slight weight loss of less than 5% within the first day or two. They then gained weight from baseline over the observation period. However, some HPAI H5 strains (GY5096, SZ2400 and HZ4258) caused a brief weight loss at 8 or 9 dpi, immediately after a 5- or 6-day period of weight gain (Figure 1C,D,F). In contrast, rats in the other groups gained body weight at a stable rate over the 14-day period. Nevertheless, compared to the mock-inoculated control rats, none of the virus-inoculated groups exhibited significant changes in body weight during the infection (Figure 1L, P > 0.05). These results demonstrate that infection with either endemic or emerging IAVs, including HPAI H5 and HPAI H7 viruses, does not cause overt morbidity in the SD rat model, despite the subtle impairment in weight gain.

3.2. Replication and Distribution of Different IAVs in the Rat Respiratory Tract

To examine virus replication in the rat respiratory tract, groups of three rats were administered different doses of each virus (10², 10⁴ and 10⁶ TCID₅₀) and necropsied at 4 dpi to determine the presence and titer of virus. At the 10² TCID₅₀ dose, limited replication of CA7, the pandemic 2009 H1N1 (pdmH1N1) prototype virus, was detected in the nasal turbinate and trachea (Figure 2C). However, nasal viral replication increased to 10⁴ TCID₅₀/ml and 10⁵ TCID₅₀/ml in the groups challenged with 10⁴ TCID₅₀ and 10⁶ TCID₅₀ doses of CA7, respectively (Figure 2A,B). Among the HPAI H5 viruses, only VN1203 (H5N1 clade 1.0) was detected in the respiratory tract after inoculation with the low 10² TCID₅₀ dose, and viral replication titers were low (< 10² TCID₅₀/ml, Figure 2C). Even at the 10⁴ TCID₅₀ dose, only VN1203 and SP378 were detected in the airways (Figure 2B). These two viruses replicated at moderate levels, with the highest titers observed in the nasal turbinates of the rats. All HPAI H5 viruses were detected after inoculation with the 10⁶ TCID₅₀ dose (Figure 2A), but in general, the nasal titers of the H5 groups were lower compared to other subtypes. For H7N9, AH1 replicated to over 10⁴ TCID₅₀/ml in the nasal turbinate of the 10² TCID₅₀ group, which was approximately two logs higher than SH2 and SP440, and was also detected in the lungs (Figure 2C). At other doses, nasal titers for SH2 and SP440 augmented remarkably with the increased inoculation doses, reaching a level closer to that of AH1 (Figure 2A,B). We observed that WZ598 (H9N2) and JX346 (H10N8) displayed comparable replicative capacity to SH2 or SP440 (H7N9) when inoculated at the same dose, and their nasal titers also elevated with increasing inoculation dose (Figure 2A–C). In summary, for most of the viruses tested, titers were much higher in the nasal turbinate than in the trachea and lung, indicating a preference for replication of these viruses in the upper respiratory tract of rats.

Blood samples were collected for serological testing when the rats were euthanized. We performed the HAI assay and observed successful seroconversion at 14 dpi after virus challenge in this rat model (Figure 3, Table S1), but we were unable to detect antibodies in the sera of rats euthanized at 4 dpi. Although the rats did produce antibodies against HPAI H5 and HPAI H7N9 viruses by 14 dpi, the titers were significantly lower than in groups infected with other viruses we tested. In the HPAI groups, all five rats inoculated with VN1203 (H5N1 clade 1.0) and SP440 (H7N9) became seropositive and produced antibodies with titers ranging from 40 to 320, which were lower than those observed for the low pathogenic pdmH1N1, H7N9, H9N2 and H10N8 (all above 320 in each individual). For the other HPAI H5 virus groups (i.e., clade 2.3.4.4 virus groups), some animals remained seronegative and antibodies in seropositive rats were at low or even marginal levels, with titers as low as 10 to 40.

Overall, our findings demonstrated that the viruses tested in our study were ready to infect rats without prior adaptation and replicated productively in this species, but their antibody production levels varied from strain to strain.

3.3. Pneumonia and Histopathological Changes Induced by IAVs in SD Rats

Pneumonia was not observed in the control group (Figure 3D). Although the gross view of the excised lungs appeared normal and no visible lesions were found in the inoculated rats, H&E staining revealed obvious lung injury and comparable pathological changes among the different virus groups at the same dose (Figure 3B,E–O), but distinct between the different doses (Figure 3A–C). The virus-infected rat lungs exhibited typical histopathological changes compared to the mock controls. Alveolitis, widened alveolar septum, inflammatory infiltration, and erythrocyte extravasation were commonly observed across all three doses (Figure 3). At the 10² TCID₅₀ dose, rat pneumonia was relatively mild, and intact alveolar cells could still be observed, but there was obvious widening of the alveolar septum (Figure 3A). In the other two higher dose groups, alveolar cells were destroyed, and there was marked exudation due to inflammation (Figure 3B,C,E–O). Alveolar fusion and markedly thickened alveolar walls, necrosis, and loss of bronchial epithelial cells were also observed (Figure 3). Rats inoculated with 10⁶ TCID₅₀ IAVs displayed the most severe pneumonia, with pulmonary consolidation occurring in most parts of the lung, characterized by complete filling of the alveolar space with exudates and inflammatory factors instead of alveolar air (Figure 3C). To provide a comprehensive picture of the IAV-induced pneumonia in rats, here we shown the representative pathological changes in the lungs of the 10⁴ TCID₅₀ groups for all viruses (Figure 3E–O). Collectively, all IAVs tested here were able to elicit an inflammatory response and lung injury in SD rats, indicating a productive infection with pathogenicity caused by these viruses.

To confirm productive infection and the presence of IAVs in the respiratory tract of SD rats, we performed IHC staining to detect viral NP antigen in the nasal turbinate and lung tissues. Staining for IAV NP revealed significant differences between viruses. In the nasal turbinate, substantial NP antigen from pdmH1N1 was observed in rats inoculated with CA7 at the dose of 10⁶ TCID₅₀ (Figure 4A). Although we had challenged groups of SD rats with different virus doses, only a few NP proteins from the HZ4258 group were detected in one rat in the 10⁶ group among the five HPAI H5 virus strains (Figure 4B, green arrows). Conversely, NP antigens of all H7N9 strains were detected in the nasal turbinate after inoculation (Figure 4C–J). AH1 antigens were detected in the nasal turbinate of the 10² and 10⁴ TCID₅₀ groups (Figure 4C,D), but not in the 10⁶ TCID₅₀ group. For the other two H7N9 viruses, SH2 and SP440, viral antigens were present in all three groups, as shown in Figure 4E–G and Figure 4H–J, respectively. In the H9N2 and H10N8 groups, viral NP from WZ598 and JX346 were detected in nasal tissues from the 10⁶ TCID₅₀ group (Figure 4K) and the 10⁴ TCID₅₀ group (Figure 4L), respectively. On the other hand, our results revealed negative staining for viral NP in the lungs of most groups (Figure 4M). In fact, only NP antigens from WZ598 (H9N2) and AH1 (H7N9) were recorded in a single rat of each group (Figure 4O,N). In the H9N2-positive lung section, bronchiolar epithelial cells were focally stained positive for WZ598 in one rat from the 10⁴ TCID₅₀ group (Figure 4O). For H7N9, stained antigens were only observed in the lung of one rat inoculated with 10² TCID₅₀ of AH1 (Figure 4N), although each H7N9 strain was detected in the lung tissues as indicated previously by virus titration (Figure 2B). Of the NP-positive nasal and lung sections, we found that viral NP was mainly distributed in the ciliated mucosal epithelial cells.

Taken together, our immunohistochemical staining results revealed a predominant distribution of viral NPs in the rat nasal cavity, consistent with the replication pattern of higher viral titers in the nasal turbinate compared to the lungs. These findings suggest that the IAVs tested tend to replicate preferentially in the upper respiratory tracts of SD rats, providing further evidence of viral replication and pathogenesis.

3.4. Expression of Sialic Acid-Linked Receptors in the Respiratory Tract of SD Rats

The attachment and entry of IAVs into host cells are dependent on the presence of specific SA-linked receptors. To investigate the expression of these receptors in the airways of rodents, we first examined the presence of α2,3- and α2,6-linked SA receptors in the airways of mice, using positive staining for both MAAs and SNA (Figure S1). In contrast to the weak MAA II staining observed in mice (Figure S1B, S1E, S1H), SD rats exhibited significantly stronger expression of α2,3-linked SA throughout the upper and lower respiratory tracts (Figure 6B,E,H). This indicates a notable difference in the presence of SAα2,3Galβ1,3GalNAc in the airways between these two rodent species, as MAA II is specific for the detection of this “traditional” avian influenza receptor [31].

In the nasal turbinate of SD rats (Figure 6A–C), both MAAs and SNA bound to ciliated epithelial cells, indicating the presence of α2,3- and α2,6-linked SA receptors, respectively. However, MAAs (Figure 6A,B) exhibited much denser staining in these nasal epithelial cells compared to SNA (Figure 6C). Additionally, cells in the basal and connective tissues were exclusively stained by MAAs but not by SNA, suggesting the presence of α2,3-linked SA receptors and the absence of α2,6-linked SA receptors in these areas. In the lower respiratory tract, staining for both MAA isoforms was observed in the trachea of rats (Figure 6D,E) and mice (Figure S1D,E). However, we found less SNA-positive staining in the epithelial cells of the rat trachea (Figure 6F) compared to MAAs, and there was minimal staining noted in mice (Figure S1F). In the lungs of SD rats (Figure 6G–I), α2,6-linked SA receptors were more abundant in the bronchi and bronchioles, as indicated by denser SNA staining compared to MAAs. In the alveoli, the staining patterns of MAA I and SNA were similar due to their diffuse distribution, although MAA II staining was significantly reduced. These results demonstrate that α2,3-linked SA receptors are predominantly expressed in the nasal turbinate, trachea and alveoli, whereas α2,6-linked SA receptors are more prevalent in the lungs of the SD rat model. This differential expression may provide insight into the mechanisms underlying the variable replication and transmission of IAVs in this model.

4. Discussion

The role of rats and other rodents in the influenza ecology is understudied and controversial. The present study provides compelling experimental evidence that SD rats, a representative model of the Rattus species, are susceptible to productive infection by diverse subtypes of contemporary IAVs that pose a major threat to both public health and agriculture, including avian HPAI H5Nx (clades 1.0 and 2.3.4.4a, b, e, g, either human or avian isolates), H7N9 (HPAI and LPAI), H9N2, H10N8, and the mammalian-adapted pandemic 2009 H1N1 viruses (Table 1). Notably, these infections occurred without prior viral adaptation, challenging the conventional assumptions that rats are refractory to IAV infection. Our findings, together with other previous reports [11,12,13,14,15,16,17,34,35,36,37], underscores the need to re-evaluate rodents as potential reservoirs, mechanical vectors, or bridging hosts in the zoonotic transmission of IAVs. The absence of overt clinical signs, despite robust viral replication, seroconversion and histopathological evidence of lung injury (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5), positions rats as cryptic carriers capable of sustaining IAV infections undetected in natural settings.

A striking feature of IAV-infected SD rats is the dissociation between their subclinical manifestations and significant virological and immunological findings. While mice and ferrets develop observable disease or mortality following experimental IAV challenge [29,30], rats show only transient weight fluctuations (Figure 1) and no influenza-like symptoms, even when infected with HPAI H5 or H7N9 strains. This asymptomatic phenotype is similar to that observed in wild waterfowl, which are natural reservoirs of IAV [4,5]. The absence of disease presentation and the induction of seroconversion in most rats suggests that rats may use effective immune mechanisms to mitigate systemic spread of the virus while permitting local replication in the upper respiratory tract [this study and 19, 20, 34]. This balance may facilitate viral persistence without compromising host survival, allowing rats to act as stealth vectors in ecosystems where they interact with domestic animals, wildlife and humans.

The receptor distribution landscape has been previously reported in BABL/c and C57BL/6J mice and in mouse tracheal epithelial cells (mTEDC) [32,33]. To our knowledge, here we provide the first report on the distribution of α2,3- and α2,6-linked SA in the respiratory tracts of SD rats (Figure 6). The preferential replication of IAVs in the nasal turbinate, as evidenced by higher viral titers and NP antigen localization (Figure 2 and Figure 5), highlights a distinct tissue tropism in rats. This pattern correlates with the predominance of α2,3-linked SA receptors in the upper respiratory tract (Figure 6), which are preferentially recognized by avian-adapted IAVs. However, the concurrent detection of both α2,3- and α2,6-linked SA receptors in the rat lung suggests dual receptor expression that could permit infection of the lower respiratory tract, as observed with H7N9, H9N2, H10N8 and some H5 strains, such as the human isolates VN1203 and SP378 (Figure 2). The discordance between SA receptor distribution (α2,6-SA dominance in the lung with MAA I-positive staining of α2,3-SA) and relatively low pulmonary viral titers (especially for the mammalian-adapted strains like pdmH1N1) implies that additional host factors, such as mucin barriers, antiviral interferon responses, protease availability, and other unknown mechanisms may restrict systemic dissemination. This receptor-replication mismatch warrants further investigation and caution when extrapolating murine data to other rodents, particularly given the contrasting SA expression profiles in mice, which exhibit much weaker α2,3-SA expression compared to rats.

Although viable virus was detected for all virus strains, our results shown significant differences in replication efficiency in rats between IAV strains. This disparity may reflect subtype-, clade- or strain-specific adaptations. Notably, all strains with G57 H9N2-like internal genes (i.e., SZ378, AH1, SH2, SP440, WZ598 and JX346) [38,39,40,41,42,43] replicated robustly in the upper respiratory tract of rats. This may indicate a growth advantage conferred by this specific internal gene cassette. Of note, the efficient replication of H9N2, an enzootic subtype in Eurasia with a broad host range and pandemic potential, and its derivatives suggests that rats may contribute to the evolution of the related viruses. The predominance of the G57 H9N2 genotype in China [38], which is associated with human infections and the emergence of novel avian strains (such as H7N9/H7N7 [39,40], H10N8/H10N6 [41,42], H5N6 [43] and H3N8 [44]), further underlies this risk.

Despite the absence of clinical disease, all IAVs induced significant pulmonary pathology characterized by alveolitis, inflammatory infiltration, and epithelial necrosis (Figure 4). The dose-dependent severity of lung injury implies the potential for subclinical infections to cause covert tissue damage that could predispose rats to secondary infections or alter their respiratory dynamics in natural settings. Of particular concern is the capacity of clade 2.3.4.4 HPAI H5 viruses to replicate in rats without adaptation, albeit with variable viral titers. Although these strains exhibited relatively low nasal titers compared to H7N9 or H9N2 viruses, their detection in the respiratory tract raises questions about the risk of rat-mediated viral persistence or reassortment in regions where HPAI circulates enzootically. Although we have not tested the currently devastating H5N1 clade 2.3.4.4b strains circulating in the Americas, the Animal and Plant Health Inspection Service recently detected this HPAI virus in several black rats (Rattus rattus) in Riverside, California during February 2025, alongside numerous prior reports of infections in deer mice (Peromyscus maniculatus) and house mice (Mus musculus) across the United States [45]. These findings collectively underscore the ever-expanding host spectrum of HPAI H5N1 and emphasize the underappreciated role of murine rodents in influenza ecology, highlighting the critical need to investigate their capacity to sustain viral transmission in natural settings.

The ubiquity of rats in human-dominated ecosystems amplifies their potential role as intermediaries in IAV transmission cycles. As synanthropes, rats have free access to poultry and livestock farms, wet markets, wildlife, and urban garbage dumps, creating opportunities for cross-species virus exchange. Their preferential replication in the upper respiratory and shedding of virus in nasal secretions could facilitate environmental contamination, enabling mechanical transmission to susceptible hosts. Animals that prey on rats or scavenge carcasses, including but not limited to cats, dogs and other carnivores, may also be infected. Although IAV transmission by rats has not been confirmed, animal infections with IAVs through exposure to contaminated environments via virus-laden fomites and consumption of contaminated food and water have been recorded in cats, chickens, mice and SD rats naturally or experimentally [7,35,45,46,47,48,49].

It is also worth noting that the coexistence of α2,3- and α2,6-SA receptors raises the possibility that rats may act as “mixing vessels” for reassortment, particularly when co-infected with avian and human IAVs. While no natural reassortants have been reported in rats, the detection of IAV nucleic acids in wild Rattus norvegicus populations (11.04% in Boston, Massachusetts and 3.47% in southern China) [14,15] suggests ongoing spillover events that merit closer surveillance. Furthermore, the asymptomatic nature of infection complicates surveillance efforts, necessitating active molecular or serological screening in wild rat populations to assess their contribution to IAV maintenance.

This study focused on a laboratory rat model under controlled conditions that may not fully replicate the physiological or immunological state of wild rats. Virus replication outside the respiratory tract was not explored. Natural routes of exposure (e.g., oral, aerosol, fomite, etc.) and co-infections with other pathogens could alter infection outcomes. Additionally, the lack of transmission experiments leaves unresolved whether rats can horizontally transmit IAVs from rat to rat or to the other mammals—a critical question for evaluating their role in viral ecology. While receptor binding specificity has been inferred from previous studies, direct characterization of viral hemagglutinin affinity for rat SA receptors is also needed to clarify tropism mechanisms. Future studies should prioritize in vivo transmission assays, comparative receptor profiling in various wild rodents, and investigations into the molecular determinants of viral fitness in rat respiratory tissues. Additional strains could be included in the experiments to further expands our understanding of IAV infections in this animal, and systematic screening of wild rat populations for IAV RNA and antibodies, particularly in regions with enzootic H5, H7, H9 or H10 viruses, is also warranted to assess the role of rats in influenza A virus transmission and evolution.

5. Conclusions

This study redefines Rattus norvegicus as a permissive host for multiple IAV subtypes prevalent in birds or humans, and highlights its ability to sustain subclinical infections with potential ecological consequences. The convergence of broad viral susceptibility, synanthropic behavior, and dual SA receptor expression in the respiratory tracts positions rats as underrecognized players in influenza ecology. While their role as “mixing vessels” remains speculative, the risk of environmental virus amplification and spillover to domestic animals or humans cannot be dismissed. Strengthening surveillance in rodent populations and integrating rats into One Health frameworks will be essential for mitigating zoonotic threats in an era of escalating avian influenza activity.