The ferret as a model organism to study influenza A virus infection

Influenza is a human pathogen that continues to pose a public health threat. The use of small mammalian models has become indispensable for understanding the virulence of influenza viruses. Among numerous species used in the laboratory setting, only the ferret model is equally well suited for studying both the pathogenicity and transmissibility of human and avian influenza viruses. Here, we compare the advantages and limitations of the mouse, ferret and guinea pig models for research with influenza A viruses, emphasizing the multifarious uses of the ferret in the assessment of influenza viruses with pandemic potential. Research performed in the ferret model has provided information, support and guidance for the public health response to influenza viruses in humans. We highlight the recent and emerging uses of this species in influenza virus research that are advancing our understanding of virus-host interactions.     

Oseltamivir Population Pharmacokinetics in the Ferret: Model Application for Pharmacokinetic/Pharmacodynamic Study Design

The ferret is a suitable small animal model for preclinical evaluation of efficacy of antiviral drugs against various influenza strains, including highly pathogenic H5N1 viruses. Rigorous pharmacokinetics/pharmacodynamics (PK/PD) assessment of ferret data has not been conducted, perhaps due to insufficient information on oseltamivir PK. Here, based on PK data from several studies on both uninfected and influenza-infected groups (i.e., with influenza A viruses of H5N1 and H3N2 subtypes and an influenza B virus) and several types of anesthesia we developed a population PK model for the active compound oseltamivir carboxylate (OC) in the ferret. The ferret OC population PK model incorporated delayed first-order input, two-compartment distribution, and first-order elimination to successfully describe OC PK. Influenza infection did not affect model parameters, but anesthesia did. The conclusion that OC PK was not influenced by influenza infection must be viewed with caution because the influenza infections in the studies included here resulted in mild clinical symptoms in terms of temperature, body weight, and activity scores. Monte Carlo simulations were used to determine that administration of a 5.08 mg/kg dose of oseltamivir phosphate to ferret every 12 h for 5 days results in the same median OC area under the plasma concentration-time curve 0–12 h (i.e., 3220 mg h/mL) as that observed in humans during steady state at the approved dose of 75 mg twice daily for 5 days. Modeling indicated that PK variability for OC in the ferret model is high, and can be affected by anesthesia. Therefore, for proper interpretation of PK/PD data, sparse PK sampling to allow the OC PK determination in individual animals is important. Another consideration in appropriate design of PK/PD studies is achieving an influenza infection with pronounced clinical symptoms and efficient virus replication, which will allow adequate evaluation of drug effects.     

Inhibition of ciliary activity in organ cultures of ferret trachea in reference to genetic and biological characters in influenza virus strains

As reported previously, attenuated stable inhibitor-resistant influenza viruses can be screened by a 50% ciliary activity inhibition test in ferret tracheal organ cultures. This test was further applied to a 5 attenuated cold-adapted influenza strains and to 11 strains with known a percentage of RNA-RNA hybridization with the parental A/PR/8/34 (HON1) virus strain. Again, with one exception, attenuated strains could be clearly differentiated from virulent ones. It was concluded that virulence of influenza strains for man can be detected using this test regardless of the techniques used to prepare attenuated variants. A preliminary screening of attenuated candidates for live influenza vaccines can be achieved with confidence on ferret tracheal organ cultures.     

A Novel Video Tracking Method to Evaluate the Effect of Influenza Infection and Antiviral Treatment on Ferret Activity

Ferrets are the preferred animal model to assess influenza virus infection, virulence and transmission as they display similar clinical symptoms and pathogenesis to those of humans. Measures of disease severity in the ferret include weight loss, temperature rise, sneezing, viral shedding and reduced activity. To date, the only available method for activity measurement has been the assignment of an arbitrary score by a ‘blind’ observer based on pre-defined responsiveness scale. This manual scoring method is subjective and can be prone to bias. In this study, we described a novel video-tracking methodology for determining activity changes in a ferret model of influenza infection. This method eliminates the various limitations of manual scoring, which include the need for a sole ‘blind’ observer and the requirement to recognise the ‘normal’ activity of ferrets in order to assign relative activity scores. In ferrets infected with an A(H1N1)pdm09 virus, video-tracking was more sensitive than manual scoring in detecting ferret activity changes. Using this video-tracking method, oseltamivir treatment was found to ameliorate the effect of influenza infection on activity in ferret. Oseltamivir treatment of animals was associated with an improvement in clinical symptoms, including reduced inflammatory responses in the upper respiratory tract, lower body weight loss and a smaller rise in body temperature, despite there being no significant reduction in viral shedding. In summary, this novel video-tracking is an easy-to-use, objective and sensitive methodology for measuring ferret activity.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.     

Comparison of the Levels of Infectious Virus in Respirable Aerosols Exhaled by Ferrets Infected with Influenza Viruses Exhibiting Diverse Transmissibility Phenotypes

Influenza viruses pose a major public health burden to communities around the world by causing respiratory infections that can be highly contagious and spread rapidly through the population. Despite extensive research on influenza viruses, the modes of transmission occurring most often among humans are not entirely clear. Contributing to this knowledge gap is the lack of an understanding of the levels of infectious virus present in respirable aerosols exhaled from infected hosts. Here, we used the ferret model to evaluate aerosol shedding patterns and measure the amount of infectious virus present in exhaled respirable aerosols. By comparing these parameters among a panel of human and avian influenza viruses exhibiting diverse respiratory droplet transmission efficiencies, we are able to report that ferrets infected by highly transmissible influenza viruses exhale a greater number of aerosol particles and more infectious virus within respirable aerosols than ferrets infected by influenza viruses that do not readily transmit. Our findings improve our understanding of the ferret transmission model and provide support for the potential for influenza virus aerosol transmission.     

Clinical Profiles Associated with Influenza Disease in the Ferret Model

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.     

Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

雪貂感染流感病毒H3N2动物模型的建立

目的建立甲型流感病毒H3N2感染的雪貂动物模型。方法按实验要求筛选出流感抗体反应阴性的雪貂,经兽用氯胺酮轻度麻醉后进行滴鼻感染H3N2流感病毒株A/Brisbane/10/07,设立两个稀释度106和107 TCID50,每个稀释度接种3只雪貂,感染后第5天安乐处死。感染前采集鼻甲骨活检,感染后1～5 d鼻甲骨活检检测病毒载量,每天记录雪貂一般临床变化。处死时取雪貂肺、肝、脾、小肠、脑组织作病毒滴度检测,肺组织做病理检查。结果 106和107TCID50的H3N2病毒分别感染雪貂,没有雪貂死亡。雪貂感染后都出现一过性的体温升高,体重的下降,流涕、打喷嚏等症状。在鼻甲骨活检物中可测到病毒载量,肠组织可分离到病毒。肺组织以轻度性间质性肺炎为主要病理变化。结论雪貂感染H3N2病毒株A/Brisbane/10/07后,临床表现、病毒学、分子生物学、病理学方面的检测都可以证实雪貂感染H3N2病毒动物模型已建立,其中106 TCID50病毒滴度的是一个建立感染动物模型比较合适的剂量。     

Arginine deficiency, hyperammonemia and Reye's syndrome in ferrets

Young male ferrets developed hyperammonemia and encephalopathy shortly after eating a diet lacking in arginine. The dietary supplementation of arginine or intraperitoneal injection of ornithine prevented hyperammonemia and shortened the duration of encephalopathy. Therefore, young ferrets were assumed to be unable to meet their ornithine needs from sources other than arginine. Adult ferrets did not develop hyperammonemia and encephalopathy after eating arginine-free diet. Because young ferrets are also susceptible to human influenza infections, they were further tested as animal model of Reye's syndrome. Reye's syndrome is a serious childhood disorder that develops following influenza infections and is characterized in part by an encephalopathy, hyperammonemia and elevated serum transaminases. In young ferrets, concurrent administration of aspirin with human influenza inoculation and an arginine-free diet produced symptoms similar to those seen in humans with Reye's syndrome. The ferret model appears to be useful for studying the roles of various etiologic agents and their interactions in producing Reye's syndrome-like disorders. The ammonia metabolism in ferrets is reviewed and the ferret model for Reye's syndrome and its applications for the better understanding of this disorder in humans are discussed.     

Blood collection from the cranial vena cava of the ferret

The domestic ferret, though not as common a laboratory animal as the rat or mouse, serves as a model in critical research areas, including influenza biology and vaccine development. Studies involving ferrets necessitate knowledge of proper blood collection methods, such as cranial vena cava puncture.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

H7N9禽流感病毒与其他流感病毒对雪貂致病性与传播力的比较

目的针对2013年3月中国爆发的人感染H7N9禽流感病毒,在雪貂体内进行致病性及传播力的研究,并与甲型H1N1流感病毒、H5N1禽流感病毒进行比较。方法对新发H7N9毒株、甲型H1N1流感病毒、H5N1禽流感病毒感染雪貂后的临床症状、体征,呼吸道排毒情况,组织病理学变化等进行评价和比较,并对H7N9毒株在雪貂群体中的传播力进行研究。结果雪貂模型的临床症状、死亡率、病毒传播以及组织病理学分析显示:H7N9病毒的致病性低于H5N1,与2009年起源于北美的甲型H1N1流感病毒相当。新发H7N9禽流感病毒可以在雪貂的呼吸道、心脏、肝脏以及嗅球进行复制。值得注意的是H7N9禽流感可以通过飞沫在雪貂间进行低水平的传播,并且在传播过程中,病毒基因组内有多个位点的氨基酸发生了替换。结论 H7N9禽流感病毒对雪貂的致病性较H5N1禽流感病毒低,与甲型H1N1流感病毒对雪貂的致病性相当,H7N9禽流感病毒可在雪貂间进行传播。     

In Vivo Selection of H1N2 Influenza Virus Reassortants in the Ferret Model

Although the ferret model has been extensively used to study pathogenesis and transmission of influenza viruses, little has been done to determine whether ferrets are a good surrogate animal model to study influenza virus reassortment. It has been previously shown that the pandemic 2009 H1N1 (H1N1pdm) virus was able to transmit efficiently in ferrets. In coinfection studies with either seasonal H1N1 or H3N2 strains (H1N1s or H3N2s, respectively), the H1N1pdm virus was able to outcompete these strains and become the dominant transmissible virus. However, lack of reassortment could have been the result of differences in the cell or tissue tropism of these viruses in the ferret. To address this issue, we performed coinfection studies with recombinant influenza viruses carrying the surface genes of a seasonal H3N2 strain in the background of an H1N1pdm strain and vice versa. After serial passages in ferrets, a dominant H1N2 virus population was obtained with a constellation of gene segments, most of which, except for the neuraminidase (NA) and PB1 segments, were from the H1N1pdm strain. Our studies suggest that ferrets recapitulate influenza virus reassortment events. The H1N2 virus generated through this process resembles similar viruses that are emerging in nature, particularly in pigs.     

A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines

This paper describes two methods of analysis using monoclonal antibodies and RNA hybridization to characterize variation in the haemagglutinins of seven high-yielding influenza virus reassortants used for inactivated vaccine production. The results show that variants' were selected in producing these genetic reassortants. The haemagglutinins of two reassortants showed both antigenic and structural differences from their wild-type (wt) parents as detected by the two methods of analysis. These variants were more closely related to other subtype strains which had previously been differentiated from the wt parent by post-infection ferret sera and which also had amino acid sequence differences in antigenically significant sites on the HA 1 polypeptide chain of the haemagglutinin molecule. The haemagglutinins of four of the seven reassortants showed antigenic differences but no apparent structural differences from their wt parents. The haemagglutinin of only on reassortant was antigenically and structurally identical to its wt parent. The variants could not be reliably distinguished with hyperimmune rabbit serum or immune ferret serum to the wt parent virus. It is therefore important to use more discriminatory tests to identify influenza strains correctly. It is also essential for vaccine strains to be as completely characterized as possible. It is considered desirable that both methods of analysis be used to characterize influenza virus reassortant strains.     

Inefficient transmission of H5N1 influenza viruses in a ferret contact model

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two na?ve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other na?ve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.     

The ferret as a model for inner ear research

Viral infections have long been suspected to be causative agents in a number of inner ear dysfunctions. With few exceptions, the virus has not been demonstrated as the direct agent leading to hearing loss and/or vertigo. Selective inner ear changes have been observed recently in sensory and nonsensory epithelial cells in the ferret model for Reye's syndrome after intranasal inoculation with influenza B combined with aspirin administration and the creation of an arginine deficiency. Such findings suggest that these agents act synergistically on the inner ear, particularly on cells that are metabolically active, and that the ferret may now be a useful model to examine the role of certain upper respiratory tract viruses implicated in inner ear disorders, singly and in combination with other agents that may cause metabolic alterations.     

Issues to consider for preparing ferrets as research subjects in the laboratory

The domestic or European ferret (Mustela putorius furo) has been domesticated for thousands of years. Ferrets have been used for hunting and fur production, as pets, and as models in biomedical research. Despite the relatively small numbers used in the laboratory, ferrets have some unique applications including study of human influenza and severe acute respiratory syndrome (SARS)-associated corona virus. They have served as models for peptic ulcer disease, carotenoid metabolism, cystic fibrosis, and drug emesis screening, among others. Most research ferrets are males, due to estrus-related health problems in females. They may be housed conventionally and are easy to care for when their biology and behavior are understood. Due to the small number of ferret suppliers, animals are often shipped long distances, requiring air transport and intermediate handlers. It is important to minimize shipment stress, especially with weanling and pregnant animals. Additional expertise is required for success with pregnant and whelping ferrets and for rearing of neonates. The animals have specific dietary requirements, and proper nutrition is key. Successful housing requires knowledge of ferret behaviors including social behavior, eating habits, a general inquisitive nature, and a species-typical need to burrow and hide. Regular handling is necessary to maintain well-being. A ferret health care program consists of physical examination, immunization, clinical pathology, and a working knowledge of common ferret diseases. Various research methodologies have been described, from basic procedures such as blood collection to major invasive survival surgery. Ferrets have a distinct niche in biomedical research and are hardy animals that thrive well in the laboratory.     

Cloned ferrets produced by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.     

Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system

Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus‐expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated “HA‐Bac‐K”) was compared with those of a commercially available baculovirus‐expressed HA (designated “HA‐Bac‐C”) and an Escherichia coli‐expressed HA (designated “HA‐E. Coli‐K”). HA‐Bac‐K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.