Cross-protection against influenza viruses by chimeric M2e-H3 stalk protein or multi-subtype neuraminidase plus M2e virus-like particle vaccine in ferrets.

Current influenza vaccine is not effective in providing cross-protection against variants. We evaluated the immunogenicity and efficacy of multi-subtype neuraminidase (NA) and M2 ectodomain virus-like particle (m-cNA-M2e VLP) and chimeric M2e-H3 stalk protein vaccines (M2e-H3 stalk) in ferrets. Our results showed that ferrets with recombinant m-cNA-M2e VLP or M2e-H3 stalk vaccination induced multi-vaccine antigen specific IgG antibodies (M2e, H3 stalk, NA), NA inhibition, antibody-secreting cells, and IFN-γ secreting cell responses. Ferrets immunized with either m-cNA-M2e VLP or M2e-H3 stalk vaccine were protected from H1N1 and H3N2 influenza viruses by lowering viral titers in nasal washes, trachea, and lungs after challenge. Vaccinated ferret antisera conferred broad humoral immunity in naïve mice. Our findings provide evidence that immunity to M2e and HA-stalk or M2e plus multi-subtype NA proteins induces cross-protection in ferrets.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Comparative Analysis of Gut Microbiomes in Laboratory Chinchillas, Ferrets, and Marmots: Implications for Pathogen Infection Research.

Gut microbes play a vital role in the health and disease of animals, especially in relation to pathogen infections. Chinchillas, ferrets, and marmots are commonly used as important laboratory animals for infectious disease research. Here, we studied the bacterial and fungal microbiota and discovered that chinchillas had higher alpha diversity and a higher abundance of bacteria compared to marmots and ferrets by using the metabarcoding of 16S rRNA genes and ITS2, coupled with co-occurrence network analysis. The dominant microbes varied significantly among the three animal species, particularly in the gut mycobiota. In the ferrets, the feces were dominated by yeast such as Rhodotorula and Kurtzmaniella, while in the chinchillas, we found Teunomyces and Penicillium dominating, and Acaulium, Piromyces, and Kernia in the marmots. Nevertheless, the dominant bacterial genera shared some similarities, such as Clostridium and Pseudomonas across the three animal species. However, there were significant differences observed, such as Vagococcus and Ignatzschineria in the ferrets, Acinetobacter and Bacteroides in the chinchillas, and Bacteroides and Cellvibrio in the marmots. Additionally, our differential analysis revealed significant differences in classification levels among the three different animal species, as well as variations in feeding habitats that resulted in distinct contributions from the host microbiome. Therefore, our data are valuable for monitoring and evaluating the impacts of the microbiome, as well as considering potential applications.

Mycobacteriosis in a Pet Ferret (Mustela putorius furo) Caused by Mycobacterium xenopi: A Case Report on Neglected Risk of Zoonotic Transmission.

Ferrets are highly susceptible to a wide range of mycobacteria, mainly M. bovis, M. avium, and M. triplex. Therefore, ferrets pose a risk of transmission of mycobacteriosis, especially zoonotically relevant tuberculosis. The aim of this study was to describe the findings of M. xenopi mycobacteriosis in a pet ferret and emphasize its zoonotic potential. A pet ferret had a history of weight loss, apathy, hyporexia, and hair loss. Abdominal ultrasound revealed splenomegaly with two solid masses and cystic lesions of the liver. Fine-needle aspiration cytology revealed numerous acid-fast bacilli in epithelioid cells, thus leading to the suspicion of mycobacterial infection. Because of its poor general condition, the ferret was euthanized. Necropsy examination revealed generalized granulomatous lymphadenitis, pneumonia, myocarditis, splenitis, and hepatitis. Histologically, in all organs, there were multifocal to coalescing areas of inflammatory infiltration composed of epithelioid macrophages, a low number of lymphocytes, and plasma cells, without necrosis nor multinucleated giant cells. Ziehl-Neelsen staining detected the presence of numerous (multibacillary) acid-fast bacteria, which were PCR-typed as M. xenopi. This is the first study showing the antimicrobial susceptibility testing of M. xenopi in veterinary medicine, describing the resistance to doxycycline. Overall, our results could facilitate further diagnosis and provide guidelines for the treatment protocols for such infections.

Treatment of Three Ferrets Diagnosed with Ferret Systemic Coronaviral Disease Using the Nucleoside Analogue GS-441524.

Ferret Systemic Coronaviral Disease (FSCD) is a systemic disease caused by ferret systemic coronavirus, which is considered lethal in most of the ferrets that are affected by it. To our knowledge, no treatment has been shown to be effective against FSCD in vivo, and most of the ferrets are euthanized or die after the development of clinical disease. GS-441524 has been shown to be effective in successfully treating cats with Feline Infectious Peritonitis (FIP), a disease that shares similarities with FSCD. However, to our knowledge, treatment with GS-441524 has not been reported for the treatment of FSCD in ferrets. Here, we describe three cases of ferrets diagnosed with FSCD successfully cured utilizing oral GS-441524. FSCD may be effectively treated following similar protocols utilized for feline infectious peritonitis in cats.

Polymeric nanoparticle-based mRNA vaccine is protective against influenza virus infection in ferrets.

New therapies and vaccines based on nucleic acids combined with an efficient nanoparticle delivery vehicle have a broad applicability for different disease indications. An alternative delivery technology for the successfully applied lipid nanoparticles in mRNA SARS-CoV-2 vaccines are nanoparticles composed of biodegradable poly(amido)amine-based polymers with mRNA payload. To show that these polymeric nanoparticles can efficiently deliver influenza hemagglutinin mRNA to target tissues and elicit protective immune responses, a relevant ferret influenza challenge model was used. In this model, our nanoparticle-based vaccine elicited strong humoral and cellular immune responses in the absence of local and systemic reactogenicity. Upon virus challenge, vaccinated animals exhibited reduced clinical signs and virus load relative to unvaccinated control animals. Based on these findings, further investigation of the polymeric nanoparticles in the context of prophylactic vaccination is warranted. Future studies will focus on optimizing the payload, the nanoparticle stability, the efficacy in the context of pre-existing immunity, and the applicability of the technology to prevent other infectious diseases.

Pharmacokinetics of hydrorphone hydrochloride after intravenous and subcutaneous administration in ferrets (Mustela putorius furo).

OBJECTIVE: To determine the pharmacokinetic profile of hydromorphone 0.2 mg kg-1 administered by the intravenous (IV) and subcutaneous (SC) route in ferrets. STUDY DESIGN: Randomized, crossover study. ANIMALS: A group of eight adult ferrets weighting (mean ± standard deviation) 1.02 ± 0.22 kg. METHODS: Hydromorphone hydrochloride 0.2 mg kg-1 was administered IV or SC with a washout period of 7 days. Blood samples were collected from a jugular catheter before administration of hydromorphone and at 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 360, 480 and 720 minutes after hydromorphone administration. Plasma hydromorphone concentrations were determined by liquid chromatography/tandem mass spectrometry. Data were analyzed using a non-linear mixed effects model. RESULTS: The hydromorphone effective half-life was (t1/2) 45 min-1. Systemic clearance (Cls) and the volume of distribution (Vdss) following IV administration were 84.8 mL kg-1 min-1 and 5.59 L kg-1, respectively. The maximum observed plasma concentration was 59.53 ± 14.02 ng mL-1 within 10 minutes following SC administration. The SC bioavailability was 102.0%. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of IV and SC hydromorphone (0.2 mg kg-1) was characterized by a high clearance, short terminal half-life and large volume of distribution. Hydromorphone plasma concentrations remained greater than 2 ng mL-1 for 2 hours in most ferrets, a threshold reported to provide antinociceptive effects in other species. Hydromorphone was well absorbed following SC injection, providing an alternative administration route for clinical use in ferrets.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

IMPORTANCE: Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

Twenty-five metagenome-assembled genomes recovered from the gut microbiome of the domestic ferret, Mustela putorius.

With the advent of metagenomics has come an increased appreciation for the gut microbiome's role in overall health of mammalian organisms. Even so, studies characterizing taxonomic and functional diversity of the ferret gut microbiome remain limited. Here, we present 25 metagenome-assembled genomes recovered from the gut microbiome of domestic ferrets.

Subcutaneous administration of hydromorphone (0.2 mg/kg) provides antinociception in ferrets (Mustela putorius furo).

OBJECTIVE: To evaluate antinociceptive efficacy of SC administration of hydromorphone hydrochloride and buprenorphine hydrochloride in ferrets (Mustela putorius furo). ANIMALS: 14 healthy adult ferrets (6 neutered males, 8 spayed females). METHODS: In a randomized, blind, controlled, complete crossover design, all 14 ferrets received a single, SC injection of hydromorphone low dose (0.1 mg/kg), hydromorphone high dose (0.2 mg/kg), buprenorphine low dose (0.02 mg/kg), buprenorphine high dose (0.04 mg/kg), or saline solution (0.2 mL/kg). Sedation and forelimb withdrawal latency from a noxious thermal stimulation were evaluated, and behavior was recorded for a total of 8 hours postinjection. RESULTS: Compared to saline, administration of hydromorphone at 0.2 mg/kg resulted in an estimated increase of withdrawal latencies of 7.4 seconds (95% CI, 3.2 to 11.6) at 60 minutes, of 6.6 seconds (2.4 to 10.8) at 90 minutes, of 6.0 seconds (1.8 to 10.2) at 120 minutes, of 7.0 seconds (2.9 to 11.1) at 180 minutes, and of 4.5 seconds (0.5 to 8.6) at 240 minutes. These differences were statistically significant. Hydromorphone administered at a lower dose and buprenorphine at either dose did not increase withdrawal latencies compared to saline. Based on the sedation score used in this study, signs of sedation increased over time in a similar fashion with all treatments, including saline. Erratic dysphoric-like behaviors occurred in all groups except for saline. CLINICAL RELEVANCE: SC administration of hydromorphone at a dose of 0.2 mg/kg provided antinociception from 1 to 4 hours postinjection. Further validation of sedation scores in ferrets is warranted.

The Mucin-Like Domain of the Ebola Glycoprotein Does Not Impact Virulence or Pathogenicity in Ferrets.

BACKGROUND: Ebola virus (EBOV) is considered among the most dangerous viruses with case fatality rates approaching 90% depending on the outbreak. While several viral proteins (VPs) including VP24, VP35, and the soluble glycoprotein are understood to contribute to virulence, less is known of the contribution of the highly variable mucin-like domain (MLD) of EBOV. Early studies have defined a potential role in immune evasion of the MLD by providing a glycan shield to critical glycoprotein residues tied to viral entry. Nonetheless, little is known as to what direct role the MLD plays in acute EBOV disease (EVD). METHODS: We generated an infectious EBOV clone that lacks the MLD and assessed its virulence in ferrets compared with wild-type (WT) virus. RESULTS: No differences in growth kinetics were observed in vitro, nor were there any differences in time to death, viremia, or clinical picture in ferrets infected with recombinant EBOV (rEBOV)-WT or rEBOV-Δmucin. CONCLUSIONS: The EBOV MLD does not play a critical role in acute pathogenesis of EVD in ferrets.

Infection of ferrets with wild type-based recombinant canine distemper virus overwhelms the immune system and causes fatal systemic disease.

Canine distemper virus (CDV) causes systemic infection resulting in severe and often fatal disease in a large spectrum of animal host species. The virus is closely related to measles virus and targets myeloid, lymphoid, and epithelial cells, but CDV is more virulent and the infection spreads more rapidly within the infected host. Here, we aimed to study the pathogenesis of wild-type CDV infection by experimentally inoculating ferrets with recombinant CDV (rCDV) based on an isolate directly obtained from a naturally infected raccoon. The recombinant virus was engineered to express a fluorescent reporter protein, facilitating assessment of viral tropism and virulence. In ferrets, this wild type-based rCDV infected myeloid, lymphoid, and epithelial cells, and the infection resulted in systemic dissemination to multiple tissues and organs, especially those of the lymphatic system. High infection percentages in immune cells resulted in depletion of these cells both from circulation and from lymphoid tissues. The majority of CDV-infected ferrets reached their humane endpoints within 20 d and had to be euthanized. In that period, the virus also reached the central nervous system in several ferrets, but we did not observe the development of neurological complications during the study period of 23 d. Two out of 14 ferrets survived CDV infection and developed neutralizing antibodies. We show for the first time the pathogenesis of a non-adapted wild type-based rCDV in ferrets. IMPORTANCE Infection of ferrets with recombinant canine distemper virus (rCDV) expressing a fluorescent reporter protein has been used as proxy to understand measles pathogenesis and immune suppression in humans. CDV and measles virus use the same cellular receptors, but CDV is more virulent, and infection is often associated with neurological complications. rCDV strains in current use have complicated passage histories, which may have affected their pathogenesis. Here, we studied the pathogenesis of the first wild type-based rCDV in ferrets. We used macroscopic fluorescence to identify infected cells and tissues; multicolor flow cytometry to determine viral tropism in immune cells; and histopathology and immunohistochemistry to characterize infected cells and lesions in tissues. We conclude that CDV often overwhelmed the immune system, resulting in viral dissemination to multiple tissues in the absence of a detectable neutralizing antibody response. This virus is a promising tool to study the pathogenesis of morbillivirus infections.

Upregulation of multiple toll-like receptors in ferret brain after blast exposure: Potential targets for treatment.

Although blast-induced traumatic brain injury (bTBI) has been designated as the signature injury of recent combat operations, its precise pathological mechanism(s) has not been identified thus far. Prior preclinical studies on bTBI demonstrated acute neuroinflammatory cascades which are known to be contributing to neurodegeneration. Danger-associated chemical patterns are released from the injured cells, which activate non-specific pattern recognition receptors, such as toll-like receptors (TLRs) leading to increased expression of inflammatory genes and release of cytokines. Upregulation of specific TLRs in the brain has been described as a mechanism of injury in diverse brain injury models unrelated to blast exposure. However, the expression profile of various TLRs in bTBI has not been investigated thus far. Hence, we have evaluated the expression of transcripts for TLR1-TLR10 in the brain of a gyrencephalic animal model of bTBI. We exposed ferrets to tightly coupled repeated blasts and determined the differential expression of TLRs (TLR1-10) by quantitative RT-PCR in multiple brain regions at 4 hr, 24 hr, 7 days and 28 days post-blast injury. The results obtained indicate that multiple TLRs are upregulated in the brain at 4 hr, 24 hr, 7 days and 28 days post-blast. Specifically, upregulation of TLR2, TLR4 and TLR9 was noted in different brain regions, suggesting that multiple TLRs might play a role in the pathophysiology of bTBI and that drugs that can inhibit multiple TLRs might have enhanced efficacy to attenuate brain damage and thereby improve bTBI outcome. Taken together, these results suggest that several TLRs are upregulated in the brain after bTBI and participate in the inflammatory response and thereby provide new insights into the disease pathogenesis. Therefore, inhibition of multiple TLRs, including TLR2, 4 and 9, simultaneously might be a potential therapeutic strategy for the treatment of bTBI.

Enhanced fitness of SARS-CoV-2 B.1.617.2 Delta variant in ferrets.

Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.2) variant displaced the prototype Washington/1/2020 (WA/1) strain. While WA/1 virus exhibited a moderately increased proportion compared to that in the inoculum following co-infection in human respiratory cells, Delta variant possessed a substantial in vivo fitness advantage as this virus becoming predominant in both inoculated and contact animals. This work identifies critical traits of the Delta variant that likely played a role in it becoming a dominant variant and highlights the necessities of employing multiple model systems to assess the fitness of newly emerged SARS-CoV-2 variants.

[Morphology of the esophagus of ferrets and expression profile of molecular markers].

OBJECTIVE: To examine the morphological characteristics and the expression profile of molecular markers of ferret esophagus and assess the feasibility of using ferrets as animal models for studying human esophageal diseases. METHODS: Frozen sections and paraffin- embedded specimens of the esophageal tissues were obtained from adult ferrets (aged 6 to 8 months) and ferrets aged 1 day, 3 days, 5 days, 1 week and 2 weeks. HE staining and periodic acid-Schiff (PAS) staining were used for morphological analysis of the esophageal submucosal glands (SMGs) of adult ferrets, and the expressions of MUC5B and MUC5AC were tested using Mucin staining; The expressions of cytokeratins (CK4, CK5, CK7, CK8, CK14, CK17, CK18, CK19, and CK20) in adult ferret esophagus were examined using HE staining and immunofluorescence assay. The expressions of LEF1 in the esophageal epithelium and SMGs were detected with immunofluorescence assay. RESULTS: In adult ferrets, the esophageal SMGs were connective tissues below the muscularis mucosa of the esophagus with secretory functions. Cytokeratins were expressed differentially in different esophageal cells: CK4, CK8 and CK20 were expressed mainly in the mucous cells, ductal cells and epithelial cells, respectively, while the mucous cells expressed the largest variety of cytokeratins. Mucin staining showed positive MUC5B and MUC5AC expression in the cytoplasm and lumen of adult ferret esophageal glands. Lectin from DBA, ECL, GSLI, GSL Ⅱ, SBA, Tacalin bioylated, ULEX, WGA, GSL Ⅰ and GSL Ⅱ were expressed on ductal cell membrane, and ECL, PNA and WGA were detected on epithelial cell membrane. Lectin with ConA, PHA-E and PHA-L were expressed on serous cell membrane. Immunofluorescence assay showed that LEF1 in the developing glands were visible from 3 days to 1 week of age and then disappeared as the glands matured. The intensity of LEF1 expression in the esophageal glands differed significantly between ferrets aged 1 to 7 days and those aged two weeks. CONCLUSION: Ferrets and human share similar esophageal tissue structures and some common molecular markers, suggesting the possibility of using ferrets as animal models of human esophageal diseases.

Problem-Oriented Approach in Exotic Companion Mammals.

Dermatologic disorders are some of the most common conditions affecting exotic companion mammals. This article provides a clinical approach of the conditions presenting with alopecia, pruritus, scaling/crusting, erosion/ulceration, and nodules in order to select and interpret the appropriate diagnostic tests to achieve a diagnosis for a successful treatment.

Zoonotic Dermatoses of Exotic Companion Mammals.

Integumentary disorders caused by zoonotic agents are very common in exotic companion mammals. This article provides an understanding of the main zoonotic dermatoses including parasitic, fungal, bacterial, and viral diseases to provide the most updated information on their epidemiology, diagnosis, reported clinical signs, and therapies.

Enhanced mucosal immune responses and reduced viral load in the respiratory tract of ferrets to intranasal lipid nanoparticle-based SARS-CoV-2 proteins and mRNA vaccines.

BACKGROUND: Unlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies. RESULTS: We developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1ß and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses. CONCLUSIONS: Intranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.

Ferrets (Mustela furo) Are Aware of Their Dimensions.

Self-awareness is a complex phenomenon expressed as the ability of an individual to separate "self-entity" from "other entity". One of its earliest evolutionary components is body size awareness, namely, the ability to consider the boundaries of one's own body as factors influencing interaction with surrounding objects. For ferrets, Mustela furo, the task requiring the penetration of various holes is ecologically relevant. We designed an experimental study in which the ferrets were supposed to select one opening out of three to get the bait. The first experiment was aimed at studying whether ferrets would prefer the holes basing on the hole size. In the second experiment, we tested the ferrets' ability to select a single passable hole on the first try while the impassable ones were larger in area. Results from the first experiment show that when choosing from the three passable openings, the animals preferred the shortest path to the bait and ignored the size of the holes. In the second experiment, all tested ferrets preferred to penetrate the passable opening on the first attempt, even though the areas of the two impenetrable ones were larger. We argue that these data indicate that ferrets are aware of their own body size.

Multi-Influenza HA Subtype Protection of Ferrets Vaccinated with an N1 COBRA-Based Neuraminidase.

The influenza neuraminidase (NA) is a promising target for next-generation vaccines. Protection induced by vaccination with the computationally optimized broadly reactive NA antigen (N1-I COBRA NA) was characterized in both influenza serologically naive and pre-immune ferret models following H1N1 (A/California/07/2009, CA/09) or H5N1 (A/Vietnam/1203/2004, Viet/04) influenza challenges. The N1-I COBRA NA vaccine elicited antibodies with neutralizing ELLA activity against both seasonal and pandemic H1N1 influenza, as well as the H5N1 influenza virus. In both models, N1-I COBRA NA-vaccinated ferrets that were challenged with CA/09 virus had similar morbidity (weight loss and clinical symptoms) as ferrets vaccinated with the CA/09 HA control vaccine. There were significantly reduced viral titers compared to the mock-vaccinated control animals. Ferrets vaccinated with N1-I COBRA NA or Viet/04 NA vaccines were protected against the H5N1 virus infection with minimal clinical symptoms and negligible weight loss. In contrast, ferrets vaccinated with the CA/09 NA vaccine lost ~10% of their original body weight with 25% mortality. Vaccination with either HA or NA vaccines did not inhibit contact transmission of CA/09 virus to naïve cage mates. Overall, the N1-I COBRA vaccine elicited protective immune responses against both H1N1 and H5N1 infections and partially mitigated disease in contact-transmission receiving ferrets. These results indicate that the N1-I COBRA NA performed similarly to the CA/09 HA and NA positive controls. Therefore, the N1-I COBRA NA alone induces protection against viruses from both H5N1 and H1N1 subtypes, indicating its value as a vaccine component in broadly protective influenza vaccines.