Acid Sensitivity of the Influenza Virus Hemagglutinin

Influenza virus hemagglutinin was shown to be acid resistant if precipitates which form during acidification are first removed. Adsorption of virus to precipitates formed during acidification may cause a virus to be described incorrectly as acid sensitive.     

High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Background

The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins.

Methods

Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols (<1 µM, 1–4 µM, and >4 µM aerodynamic diameters) adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay.

Results

Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed.

Conclusion

At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles <4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity >40% will significantly reduce the infectivity of aerosolized virus.     

Progress and Challenge in Computational Identification of Influenza Virus Reassortment

Virologica Sinica - Genomic reassortment is an important evolutionary mechanism for influenza viruses. In this process, the novel viruses acquire new characteristics by the exchange of the intact...     

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.     

Extended Viral Shedding of a Low Pathogenic Avian Influenza Virus by Striped Skunks (Mephitis mephitis)

Background

Striped skunks (Mephitis mephitis) are susceptible to infection with some influenza A viruses. However, the viral shedding capability of this peri-domestic mammal and its potential role in influenza A virus ecology are largely undetermined.

Methodology/Principal Findings

Striped skunks were experimentally infected with a low pathogenic (LP) H4N6 avian influenza virus (AIV) and monitored for 20 days post infection (DPI). All of the skunks exposed to H4N6 AIV shed large quantities of viral RNA, as detected by real-time RT-PCR and confirmed for live virus with virus isolation, from nasal washes and oral swabs (maximum ≤10^6.02 PCR EID₅₀ equivalent/mL and ≤10^5.19 PCR EID₅₀ equivalent/mL, respectively). Some evidence of potential fecal shedding was also noted. Following necropsy on 20 DPI, viral RNA was detected in the nasal turbinates of one individual. All treatment animals yielded evidence of a serological response by 20 DPI.

Conclusions/Significance

These results indicate that striped skunks have the potential to shed large quantities of viral RNA through the oral and nasal routes following exposure to a LP AIV. Considering the peri-domestic nature of these animals, along with the duration of shedding observed in this species, their presence on poultry and waterfowl operations could influence influenza A virus epidemiology. For example, this species could introduce a virus to a naive poultry flock or act as a trafficking mechanism of AIV to and from an infected poultry flock to naive flocks or wild bird populations.     

Evolution of the Influenza A Virus Genome during Development of Oseltamivir Resistance In Vitro

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.     

Calpain 10 Homology Modeling with CYGAK and Increased Lipophilicity Leads to Greater Potency and Efficacy in Cells

Calpain 10 is a ubiquitously expressed mitochondrial and cytosolic Ca(2+)-regulated cysteine protease in which overexpression or knockdown leads to mitochondrial dysfunction and cell death. We previously identified a potent and specific calpain 10 peptide inhibitor (CYGAK), but it was not efficacious in cells. Therefore, we created a homology model using the calpain 10 amino acid sequence and calpain 1 3-D structure and docked CYGAK in the active site. Using this model we modified the inhibitor to improve potency 2-fold (CYGAbuK). To increase cellular efficacy, we created CYGAK-S-phenyl-oleic acid heterodimers. Using renal mitochondrial matrix CYGAK, CYGAK-OC, and CYGAK-ON had IC(50)'s of 70, 90, and 875 nM, respectively. Using isolated whole renal mitochondria CYGAK, CYGAK-OC, and CYGAK-ON had IC(50)'s of 95, 196, and >10,000 nM, respectively. Using renal proximal tubular cells (RPTC) in primary culture, 30 min exposures to CYGAK-OC and CYGAbuK-OC decreased cellular calpain activity approximately 20% at 1 μM, and concentrations up to 100 μM had no additional effect. RPTC treated with 10 μM CYGAK-OC for 24 h induced accumulation of ATP synthase β and NDUFB8, two calpain 10 substrates. In summary, we used molecular modeling to improve the potency of CYGAK, while creating CYGAK-oleic acid heterodimers to improve efficacy in cells. Since calpain 10 has been implicated in type 2 diabetes and renal aging, the use of this inhibitor may contribute to elucidating the role of calpain 10 in these and other diseases.     

Long-Term Shedding of Influenza Virus,Parainfluenza Virus,Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.     

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.     

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

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.     

Immunization of Pigs with a Particle-Mediated DNA Vaccine to Influenza A Virus Protects against Challenge with Homologous Virus

Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.     

Functional and Structural Analysis of Influenza Virus Neuraminidase N3 Offers Further Insight into the Mechanisms of Oseltamivir Resistance

The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.     

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Virus Shedding and Potential for Interspecies Waterborne Transmission of Highly Pathogenic H5N1 Influenza Virus in Sparrows and Chickens

To elucidate the role of sparrows as intermediate hosts of highly pathogenic avian influenza H5N1 viruses, we assessed shedding and interspecies waterborne transmission of A/duck/Laos/25/06 in sparrows and chickens. Inoculated birds shed virus at high titers from the oropharynx and cloaca, and infection was fatal. Waterborne transmission from inoculated sparrows to contact chickens was absent, while 25% of sparrows were infected via waterborne transmission from chickens. The viral shedding and susceptibility to infection we observed in sparrows, coupled with their presence in poultry houses, could facilitate virus spread among poultry and wild birds in the face of an H5N1 influenza virus outbreak.The H5N1 influenza A viruses remain a major global concern because of their rapid evolution, genetic diversity, broad host range, and ongoing circulation in wild and domestic birds. H5N1 influenza viruses have swept through poultry flocks across Asia and have spread westward through Eastern Europe to India and Africa since 2003 (1). Sixty-two countries have reported H5N1 influenza virus in domestic poultry/wild birds during the time period 2003 to 2009 (http://www.oie.int/eng/info_ev/en_AI_factoids_2.htm), and to date, more than 400 human infections have been documented in 16 countries, with a mortality rate of ∼61% (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_05_22/en/index.html). Most human cases of H5N1 influenza have occurred after contact with infected poultry (13).Some of the more recent isolates of H5N1 highly pathogenic avian influenza (HPAI) virus do not cause overt disease in certain species of domestic and wild ducks; however, these viruses are 100% lethal to chickens and gallinaceous poultry. Because of ducks’ ability to “silently” spread H5N1 HPAI virus and their unresolved role as a reservoir, they are the focus of much research (5, 6, 11). In contrast, the possible role of passerine birds has received little attention, despite their widespread interaction with poultry at many sites worldwide (http://www.searo.who.int/LinkFiles/Publication_PHI-prevention-control-AI.pdf). The order Passeriformes includes more than half of all bird species, including sparrows. Since 2001, several outbreaks of H5N1 influenza virus infection have been reported in passerine birds in eastern Asia, often near infected poultry farms (15). Interestingly, the only confirmed presence of asymptomatic infection with HPAI H5N1 in wild birds was in tree sparrows in Henan Province, China. Both tree and house sparrows (Passer montanus and Passer domesticus, respectively) are members of the Old World sparrow family Passeridae, and in fact, the tree sparrow was not recognized as a species separate from that of the house sparrow until 1713 (http://www.arkive.org/tree-sparrow/passer-montanus/info.html?displayMode=factsheet). The four avian influenza virus isolates obtained from these asymptomatic infections were of the A/Goose/Guangdong/1/96 lineage and were highly pathogenic to experimentally infected chickens (4, 8).Under experimental conditions, passerine species have shown varied susceptibility to HPAI H5N1 viruses. Among sparrows, starlings, and pigeons inoculated with HPAI H5N1 virus isolates, only sparrows experienced lethal infection, and transmission to contact birds was extremely rare (2). Similarly, in sparrows and starlings inoculated with the H5N1 HPAI A/chicken/Hong Kong/220/97 virus, clinical signs were observed only in sparrows, and no deaths occurred (9).To assess the duration and routes of virus shedding and the waterborne virus transmission of HPAI H5N1 virus between sparrows and chickens, we inoculated groups of birds with A/duck/Laos/25/06, which had caused extremely high morbidity and mortality in domestic ducks (7) and was highly pathogenic to chickens, geese, and quail (J.-K. Kim and R. G. Webster, unpublished data). The virus was obtained from our collaborators in Lao People''s Democratic Republic and was grown in the allantoic cavities of 10-day-old embryonated chicken eggs (eggs) for 36 to 48 h at 35°C. The allantoic fluid was harvested, titrated (50% egg infective dose [EID₅₀] per milliliter), and stored at −80°C. All experiments were approved by the U.S. Department of Agriculture and the U.S. Centers for Disease Control and Prevention and were performed in biosafety level 3+ facilities at St. Jude Children''s Research Hospital. Wild house sparrows (Passer domesticus) were captured locally (Memphis, TN), and specific-pathogen-free outbred White Leghorn chickens (Gallus domesticus) were purchased from Charles River Laboratories (North Franklin, CT). All animal experiments were approved by the St. Jude Animal Care and Use Committee and complied with the policies of the National Institutes of Health and the Animal Welfare Act.Before inoculation, oropharyngeal and cloacal swabs were collected from sparrows, and baseline blood samples were collected from chickens to exclude preexisting H5N1 influenza virus infection. Eight sparrows were inoculated intranasally with 10⁶ EID₅₀ of virus in a volume of 100 μl, and five chickens were inoculated with 10² EID₅₀ of virus in a volume of 1 ml (0.5 ml intranasally, 0.5 ml intratracheally, and 1 drop per eye). All birds in each experimental group were housed in a single cage. Inoculated sparrows were provided with 1 liter of water in a shallow stainless steel pan at the bottom of the cage, and chickens were given 3 liters of water in a trough inside the cage. Twenty-four hours after inoculation, 1 liter of water was removed from the inoculated chickens’ cage and placed undiluted in a cage housing 8 contact sparrows; similarly, 1 liter of water was taken from the inoculated sparrows’ cage, mixed with 2 liters of fresh water, and placed in a cage housing 5 contact chickens. Clinical disease signs, including depression, huddling at the cage bottom, and ruffled feathers, were monitored through daily observation, and oropharyngeal and cloacal swabs obtained from all birds were collected daily for 14 days. Swab samples were titrated in eggs and expressed as log₁₀ EID₅₀/ml (10). The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.Blood samples were taken from all surviving contact birds on day 14 of the study. Sera were treated with a receptor-destroying enzyme (Denka Seiken, Campbell, CA), as instructed by the manufacturer, and heat inactivated at 56°C for 30 min. Hemagglutination inhibition (HI; using 0.5% packed chicken red blood cells) titers were determined as the reciprocal of the highest serum dilution that inhibited 4 hemagglutinating units of virus. HI titers of ≥10 were considered suggestive of recent influenza virus infection.Inoculation with A/duck/Laos/25/06 was lethal to all birds (Table ​(Table1).1). While chickens succumbed to infection within 2 days postinoculation (p.i.), the mean time until death for sparrows was 4.1 days; mortality occurred rapidly (overnight) without prior observation of clinical signs. Expected clinical signs, should they have occurred, included moderate to severe depression, huddling at the cage bottom, and ruffled feathers (9). All inoculated birds shed virus from the oropharynx and, to a lesser extent, from the cloaca (Fig. 1A and B). The mean virus titers of inoculated chickens and sparrows were comparable on day 1 p.i.; however, on day 2 p.i., the mean oropharyngeal and cloacal viral titers of chickens were approximately 2 and 2.5 times greater, respectively, than those of sparrows (Fig. 1A and B). The virus titer in water used by inoculated sparrows was 10^0.75 EID₅₀/ml at 1 day p.i. and peaked at 10^1.75 EID₅₀/ml on days 2 and 4 p.i. (Fig. ​(Fig.1C).1C). No virus was detected in water from the inoculated chickens’ cage.Open in a separate windowFIG. 1.Mean oropharyngeal and cloacal virus titers in sparrows (A) and chickens (B) inoculated with a lethal dose of A/duck/Laos/25/06 (H5N1) virus. (C) Virus titers in the drinking water of inoculated sparrows. Sparrows were inoculated with 10⁶ EID₅₀/ml of virus, and chickens were inoculated with 10² EID₅₀/ml of virus. The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.

TABLE 1.

Transmission rates, mortality rates, and mean peak titers of A/duck/Laos/25/06 (H5N1) virus in inoculated and contact birds

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.