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.     

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.     

Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.     

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.     

Environmental Conditions Affect Exhalation of H3N2 Seasonal and Variant Influenza Viruses and Respiratory Droplet Transmission in Ferrets

The seasonality of influenza virus infections in temperate climates and the role of environmental conditions like temperature and humidity in the transmission of influenza virus through the air are not well understood. Using ferrets housed at four different environmental conditions, we evaluated the respiratory droplet transmission of two influenza viruses (a seasonal H3N2 virus and an H3N2 variant virus, the etiologic virus of a swine to human summertime infection) and concurrently characterized the aerosol shedding profiles of infected animals. Comparisons were made among the different temperature and humidity conditions and between the two viruses to determine if the H3N2 variant virus exhibited enhanced capabilities that may have contributed to the infections occurring in the summer. We report here that although increased levels of H3N2 variant virus were found in ferret nasal wash and exhaled aerosol samples compared to the seasonal H3N2 virus, enhanced respiratory droplet transmission was not observed under any of the environmental settings. However, overall environmental conditions were shown to modulate the frequency of influenza virus transmission through the air. Transmission occurred most frequently at 23°C/30%RH, while the levels of infectious virus in aerosols exhaled by infected ferrets agree with these results. Improving our understanding of how environmental conditions affect influenza virus infectivity and transmission may reveal ways to better protect the public against influenza virus infections.     

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。     

Decoding the distribution of glycan receptors for human-adapted influenza A viruses in ferret respiratory tract

Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface. It is generally known that upper respiratory tract of humans and ferrets predominantly express α2→6 sialylated glycan receptors. However much less is known about the fine structure of these glycan receptors and their distribution in different regions of the ferret respiratory tract. In this study, we characterize distribution of glycan receptors going beyond terminal sialic acid linkage in the cranial and caudal regions of the ferret trachea (upper respiratory tract) and lung hilar region (lower respiratory tract) by multiplexing use of various plant lectins and human-adapted HAs to stain these tissue sections. Our findings show that the sialylated glycan receptors recognized by human-adapted HAs are predominantly distributed in submucosal gland of lung hilar region as a part of O-linked glycans. Our study has implications in understanding influenza A viral pathogenesis in ferrets and also in employing ferrets as animal models for developing therapeutic strategies against influenza.     

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.     

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.     

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。     

Metagenomic Analysis of the Ferret Fecal Viral Flora

Ferrets are widely used as a small animal model for a number of viral infections, including influenza A virus and SARS coronavirus. To further analyze the microbiological status of ferrets, their fecal viral flora was studied using a metagenomics approach. Novel viruses from the families Picorna-, Papilloma-, and Anelloviridae as well as known viruses from the families Astro-, Corona-, Parvo-, and Hepeviridae were identified in different ferret cohorts. Ferret kobu- and hepatitis E virus were mainly present in human household ferrets, whereas coronaviruses were found both in household as well as farm ferrets. Our studies illuminate the viral diversity found in ferrets and provide tools to prescreen for newly identified viruses that potentially could influence disease outcome of experimental virus infections in ferrets.     

Low pathogenic avian influenza isolates from wild birds replicate and transmit via contact in ferrets without prior adaptation

Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential.     

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.     

Differential Pathological and Immune Responses in Newly Weaned Ferrets Are Associated with a Mild Clinical Outcome of Pandemic 2009 H1N1 Infection

Young children are typically considered a high-risk group for disease associated with influenza virus infection. Interestingly, recent clinical reports suggested that young children were the smallest group of cases with severe pandemic 2009 H1N1 (H1N1pdm) influenza virus infection. Here we established a newly weaned ferret model for the investigation of H1N1pdm infection in young age groups compared to adults. We found that young ferrets had a significantly milder fever and less weight loss than adult ferrets, which paralleled the mild clinical symptoms in the younger humans. Although there was no significant difference in viral clearance, disease severity was associated with pulmonary pathology, where newly weaned ferrets had an earlier pathology improvement. We examined the immune responses associated with protection of the young age group during H1N1pdm infection. We found that interferon and regulatory interleukin-10 responses were more robust in the lungs of young ferrets. In contrast, myeloperoxidase and major histocompatibility complex responses were persistently higher in the adult lungs; as well, the numbers of inflammation-prone granulocytes were highly elevated in the adult peripheral blood. Importantly, we observed that H1N1pdm infection triggered formation of lung structures that resembled inducible bronchus-associated lymphoid tissues (iBALTs) in young ferrets which were associated with high levels of homeostatic chemokines CCL19 and CXCL13, but these were not seen in the adult ferrets with severe disease. These results may be extrapolated to a model of the mild disease seen in human children. Furthermore, these mechanistic analyses provide significant new insight into the developing immune system and effective strategies for intervention and vaccination against respiratory viruses.     

Designing Anti-Influenza Aptamers: Novel Quantitative Structure Activity Relationship Approach Gives Insights into Aptamer – Virus Interaction

This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA) that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus’ infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer’s binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship) tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers’ binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R² = 0.702 for the training set, and R² = 0.66 for the independent test set. Moreover, in the test set the model’s sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer’s binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction with the influenza virus are discussed.     

Neurovirulence of H5N1 Infection in Ferrets Is Mediated by Multifocal Replication in Distinct Permissive Neuronal Cell Regions

Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence.     

Cloned Defective Interfering Influenza Virus Protects Ferrets from Pandemic 2009 Influenza A Virus and Allows Protective Immunity to Be Established

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.     

Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [¹⁸F]-2-deoxy-2-fluoro-D-glucose ([¹⁸F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [¹⁸F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([¹⁸F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [¹⁸F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.     

Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.