Pathogenesis and Transmission of Genetically Diverse Swine-Origin H3N2 Variant Influenza A Viruses from Multiple Lineages Isolated in the United States, 2011-2016.

While several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, the recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011 to 2016 were evaluated for their replicative ability in both in vitro and in vivo in mammalian models as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacities in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.IMPORTANCE Swine-origin influenza viruses of the H3N2 subtype, with the hemagglutinin (HA) and neuraminidase (NA) derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the United States during the 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the abilities of these viruses to replicate, cause disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlight their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice.

On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.

The influence of seasonality and manufacturer kit lot changes on 17α-hydroxyprogesterone measurements and referral rates of congenital adrenal hyperplasia in newborns.

Newborn screening for congenital adrenal hyperplasia (CAH) is performed by measuring the concentration of 17α-hydroxyprogesterone (17-OHP) in dried blood spots. Unfortunately, the level of 17-OHP varies due to multiple factors, and therefore, the false positive rate for the test is a challenge. We analyzed screening data from 2007 to 2015 to determine the effect of seasonal changes and manufacturer kit lot changes on 17-OHP values and on numbers of infants referred. Data from screening 2.2 million infants over a 9-year period indicates that in the NYS during the colder months, daily mean 17-OHP values are higher, more retests are performed, and more infants are referred even though fewer infants are born. The practice of using fixed cutoffs for referring infants for CAH leads to more false positive results in colder months. In addition, there was an overall 10% increase in the daily mean 17-OHP values from the 2 years before and after a manufacturer kit lot change that occurred in November 2013, suggestive of a functional change in the kit at that time. CONCLUSION: Newborn screening programs should be cognizant of seasonal temperature variations and (un)anticipated manufacturer kit changes because they may affect 17-OHP values and CAH referral rates. What is Known: • Newborn screening for congenital adrenal hyperplasia is generally performed by measuring 17α-hydroxyprogesterone (17-OHP) levels in dried blood spots. • 17-OHP concentrations are affected by gestational age/weight of infant when specimen is collected, specimen collection time after birth, as well as race and sex of infant. What is New: • Seasonal temperature variations and unanticipated manufacturer kit changes affect 17-OHP levels and consequently referral rates in programs that use fixed cutoffs. • Daily mean 17-OHP is generally higher when the ambient temperature is lower.

Screening for cystic fibrosis in New York State: considerations for algorithm improvements.

Newborn screening for cystic fibrosis (CF), a chronic progressive disease affecting mucus viscosity, has been beneficial in both improving life expectancy and the quality of life for individuals with CF. In New York State from 2007 to 2012 screening for CF involved measuring immunoreactive trypsinogen (IRT) levels in dried blood spots from newborns using the IMMUCHEM(™) Blood Spot Trypsin-MW ELISA kit. Any specimen in the top 5% IRT level underwent DNA analysis using the InPlex(®) CF Molecular Test. Of the 1.48 million newborns screened during the 6-year time period, 7631 babies were referred for follow-up. CF was confirmed in 251 cases, and 94 cases were diagnosed with CF transmembrane conductance regulated-related metabolic syndrome or possible CF. Nine reports of false negatives were made to the program. Variation in daily average IRT was observed depending on the season (4-6 ng/ml) and kit lot (<3 ng/ml), supporting the use of a floating cutoff. The screening method had a sensitivity of 96.5%, specificity of 99.6%, positive predictive value of 4.5%, and negative predictive value of 99.5%. CONCLUSION: Considerations for CF screening algorithms should include IRT variations resulting from age at specimen collection, sex, race/ethnicity, season, and manufacturer kit lots. WHAT IS KNOWN: Measuring IRT level in dried blood spots is the first-tier screen for CF. Current algorithms for CF screening lead to substantial false-positive referral rates. WHAT IS NEW: IRT values were affected by age of infant when specimen is collected, race/ethnicity and sex of infant, and changes in seasons and manufacturer kit lots The prevalence of CF in NYS is 1 in 4200 with the highest prevalence in White infants (1 in 2600) and the lowest in Black infants (1 in 15,400).

Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets.

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6-sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.

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.

The "New Deadly Quartet" for cardiovascular disease in the 21st century: obesity, metabolic syndrome, inflammation and climate change: how does statin therapy fit into this equation?

Despite population-based improvements in cardiovascular risk factors, such as blood pressure, cholesterol and smoking, cardiovascular disease still remains the number-one cause of mortality in the United States. In 1989, Kaplan coined the term "Deadly Quartet" to represent a combination of risk factors that included upper body obesity, glucose intolerance, hypertriglyceridemia and hypertension [Kaplan in Arch Int Med 7:1514-1520, 1989]. In 2002, the third report of the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP III) essentially added low HDL-C criteria and renamed this the "metabolic syndrome." [The National Cholesterol Education Program (NCEP) in JAMA 285:2486-2497, 2001] However, often forgotten was that a pro-inflammatory state and pro-thrombotic state were also considered components of the syndrome, albeit the panel did not find enough evidence at the time to recommend routine screening for these risk factors [The National Cholesterol Education Program (NCEP) in JAMA 285:2486-2497, 2001]. Now over a decade later, it may be time to reconsider this deadly quartet by reevaluating the roles of obesity and subclinical inflammation as they relate to the metabolic syndrome. To complete this new quartet, the addition of increased exposure to elevated levels of particulate matter in the atmosphere may help elucidate why this cardiovascular pandemic continues, despite our concerted efforts. In this article, we will summarize the evidence, focusing on how statin therapy may further impact this new version of the "deadly quartet".

Influenza virus aerosol exposure and analytical system for ferrets.

Understanding the transmission ability of newly emerging influenza viruses is central to the development of public health preparedness and prevention strategies. Animals are used to model influenza virus infection and transmission, but the routinely used intranasal inoculation of a liquid virus suspension does not reflect natural infection. We report the development of an inoculation method that delivers an influenza virus aerosol inoculum to ferrets and the characterization of size distribution and viable virus present in aerosols shed from infected ferrets during normal breathing and sneezing. By comparing virus deposition, infectivity, virulence, and transmissibility among animals inoculated intranasally or by aerosols with a human (H3N2) or avian (H5N1) influenza virus, we demonstrate that aerosol inoculations more closely resemble a natural, airborne influenza virus infection and that viable virus is measurable in droplets and droplet nuclei exhaled by infected ferrets. These methods will provide improved risk assessment of emerging influenza viruses that pose a threat to public health.

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets.

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.

Oseltamivir inhibits H7 influenza virus replication in mice inoculated by the ocular route.

The majority of human infections associated with H7 influenza viruses have resulted in ocular and not respiratory disease. While oseltamivir has been prescribed to individuals presenting with conjunctivitis following H7 virus exposure, it is unknown if oseltamivir inhibits virus replication in ocular tissue. We demonstrate that H7 viruses possess sensitivity to neuraminidase inhibitors and that administration of oseltamivir before ocular virus challenge in mice inhibits H7N7 and H7N3 virus replication in ocular and respiratory tissues.

Naturally occurring human monoclonal antibodies neutralize both 1918 and 2009 pandemic influenza A (H1N1) viruses.

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virus in vitro, and one exerted a marked therapeutic effect in vivo.

PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal impact on virulence in animal models.

Unlike previous pandemic viruses, the 2009 H1N1 pandemic influenza virus does not code for the virulence factor PB1-F2. The genome of the 2009 H1N1 virus contains three stop codons preventing PB1-F2 expression; however, PB1-F2 production could occur following genetic mutation or reassortment. Thus, it is of great interest to understand the impact that expression of the PB1-F2 protein might have in the context of the 2009 pandemic influenza virus, A/California/04/2009 (Cal/09). We have addressed this question by generating two Cal/09 viruses with productive PB1-F2 open reading frames containing either an asparagine at position 66 of PB1-F2 (66N) or a serine at position 66 (66S): this N66S change has previously been shown to be associated with increased virulence in mice. We used these viruses to investigate the effect on virulence conferred by expression of the 66N or the 66S PB1-F2 protein in both in vitro and in vivo systems. Our results show enhanced replication of the 66S virus in A549 cells, while studies of BALB/c and DBA/2 mice and ferrets revealed no significant differences in symptoms of infection with wild-type Cal/09 versus the 66N or 66S virus variant. Also, coinfection of mice with Streptococcus pneumoniae and the different viruses (recombinant wild-type [rWT] Cal/09 and the 66N and 66S viruses) did not result in significant differences in mortality. Mice infected with either PB1-F2-expressing virus did demonstrate altered protein levels of proinflammatory cytokines; differences were observed to be greater in infection caused by the 66S virus. In summary, our study demonstrates that PB1-F2 expression by the Cal/09 virus modulates the immune response to infection while having a minimal effect on virus virulence in two mammalian models.

Pathogenesis of pandemic influenza A (H1N1) and triple-reassortant swine influenza A (H1) viruses in mice.

The pandemic H1N1 virus of 2009 (2009 H1N1) continues to cause illness worldwide, primarily in younger age groups. To better understand the pathogenesis of these viruses in mammals, we used a mouse model to evaluate the relative virulence of selected 2009 H1N1 viruses and compared them to a representative human triple-reassortant swine influenza virus that has circulated in pigs in the United States for over a decade preceding the current pandemic. Additional comparisons were made with the reconstructed 1918 virus, a 1976 H1N1 swine influenza virus, and a highly pathogenic H5N1 virus. Mice were inoculated intranasally with each virus and monitored for morbidity, mortality, viral replication, hemostatic parameters, cytokine production, and lung histology. All 2009 H1N1 viruses replicated efficiently in the lungs of mice and possessed a high degree of infectivity but did not cause lethal disease or exhibit extrapulmonary virus spread. Transient weight loss, lymphopenia, and proinflammatory cytokine and chemokine production were present following 2009 H1N1 virus infection, but these levels were generally muted compared with a triple-reassortant swine virus and the 1918 virus. 2009 H1N1 viruses isolated from fatal cases did not demonstrate enhanced virulence in this model compared with isolates from mild human cases. Histologically, infection with the 2009 viruses resulted in lesions in the lung varying from mild to moderate bronchiolitis with occasional necrosis of bronchiolar epithelium and mild to moderate peribronchiolar alveolitis. Taken together, these studies demonstrate that the 2009 H1N1 viruses exhibited mild to moderate virulence in mice compared with highly pathogenic viruses.

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication.

BACKGROUND: Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state. RESULTS: In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice. CONCLUSIONS: Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.

Evaluating the value of intrapartum fetal scalp blood sampling to predict adverse neonatal outcomes: A UK multicentre observational study.

OBJECTIVE: To evaluate the value of fetal scalp blood sampling (FBS) as an adjunct test to cardiotocography, to predict adverse neonatal outcomes. STUDY DESIGN: A multicentre service evaluation observational study in forty-four maternity units in the UK. We collected data retrospectively on pregnant women with singleton pregnancy who received FBS in labour using a standardised data collection tool. The primary outcome was prediction of neonatal acidaemia diagnosed as umbilical cord arterial pH < 7.05, the secondary outcomes were the prediction of Apgar scores<7 at 1st and 5th minutes and admission to the neonatal intensive care unit (NICU). We evaluated the correlation between the last FBS blood gas before birth and the umbilical cord blood and adjusted for time intervals. We constructed 2 × 2 tables to calculate the sensitivity, specificity, positive (PPV) and negative predictive value (NPV) and generated receiver operating curves to report on the Area Under the Curve (AUC). RESULTS: In total, 1422 samples were included in the analysis; pH values showed no correlation (r = 0.001, p = 0.9) in samples obtained within an hour (n = 314), or within half an hour from birth (n = 115) (r=-0.003, p = 0.9). A suboptimal FBS pH value (<7.25) had a poor sensitivity (22%) and PPV (4.9%) to predict neonatal acidaemia with high specificity (87.3%) and NPV (97.4%). Similar performance was noted to predict Apgar scores <7 at 1st (sensitivity 14.5%, specificity 87.5%, PPV 23.4%, NPV 79.6%) and 5th minute (sensitivity 20.3%, specificity 87.4%, PPV 7.6%, NPV 95.6%), and admission to NICU (sensitivity 20.3%, specificity 87.5%, PPV 13.3%, NPV 92.1%). The AUC for FBS pH to predict neonatal acidaemia was 0.59 (95%CI 0.59-0.68, p = 0.3) with similar performance to predict Apgar scores<7 at 1st minute (AUC 0.55, 95%CI 0.51-0.59, p = 0.004), 5th minute (AUC 0.55, 95%CI 0.48-0.62, p = 0.13), and admission to NICU (AUC 0.58, 95%CI 0.52-0.64, p = 0.002). Forty-one neonates had acidaemia (2.8%, 41/1422) at birth. There was no significant correlation in pH values between the FBS and the umbilical cord blood in this subgroup adjusted for sampling time intervals (r = 0.03, p = 0.83). CONCLUSIONS: As an adjunct tool to cardiotocography, FBS offered limited value to predict neonatal acidaemia, low Apgar Scores and admission to NICU.

Pelvic organ prolapse: is there a difference in POPQ exam results based on time of day, morning or afternoon?

OBJECTIVE: The purpose of this study was to determine if there is any difference between AM and PM POPQ exam results. STUDY DESIGN: Prospective, IRB approved study of women presenting with pelvic organ prolapse symptoms. Initial POPQ exams were performed in the morning or afternoon; a second exam was performed within 4 weeks at the opposite time of day. Statistical analysis included Wilcoxon signed rank test, paired t test, Spearman correlation, and Stuart-Maxwell test. RESULTS: The study consisted of 32 subjects. Mean age was 58.8 years, mean BMI 28.6, median parity 2.0, 75% white, 22% black, and 3% Hispanic. The POPQ exam stages were 47% stage II, 50% stage III, and 3.0% stage IV. There was no significant difference for the 9 POPQ exam measures between morning and afternoon exams. There was good agreement between AM and PM POPQ ordinal stages (Stewart Maxwell P = 1.0, kappa 0.76). CONCLUSION: There is no difference between AM and PM POPQ exam results.

Preexisting Immunity, Not Frailty Phenotype, Predicts Influenza Postvaccination Titers among Older Veterans.

Both preexisting immunity to influenza and age have been shown to be correlates of influenza vaccine responses. Frailty, an indicator of functional impairment in older adults, was also shown in one study to predict lower influenza vaccine responses among nonveterans. In the current study, we aimed to determine the associations between frailty, preexisting immunity, and immune responses to influenza vaccine among older veterans. We studied 117 subjects (age range, 62 to 95 years [median age, 81 years]), divided into three cohorts based on the Fried frailty test, i.e., nonfrail (NF) (n = 23 [median age, 68 years]), prefrail (n = 50 [median age, 80 years]), and frail (n = 44 [median age, 82 years]), during the 2010-2011 and 2011-2012 influenza seasons. Subjects received the seasonal trivalent inactivated influenza vaccine, and baseline and postvaccination samples were obtained. Anti-influenza humoral immunity, as measured by hemagglutination inhibition (HI) and microneutralization assays, was measured for influenza B, A(H1N1)pdm09, and A(H3N2) viruses. Postvaccination titers were not different between frail and NF subjects overall in this older subset of veterans. However, preexisting HI titers were strongly correlated with postvaccination titers among all functional status groups. When microneutralization titers were compared, the association between preexisting immunity and vaccine responses varied by frailty status, with the strongest correlation being observed for the NF group. In conclusion, preexisting immunity rather than frailty appeared to predict postvaccination titers in this older veteran cohort.

Enhanced virulence of clade 2.3.2.1 highly pathogenic avian influenza A H5N1 viruses in ferrets.

Sporadic avian to human transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses necessitates the analysis of currently circulating and evolving clades to assess their potential risk. Following the spread and sustained circulation of clade 2 viruses across multiple continents, numerous subclades and genotypes have been described. To better understand the pathogenesis associated with the continued diversification of clade 2A(H5N1) influenza viruses, we investigated the relative virulence of eleven human and poultry isolates collected from 2006 to 2013 by determining their ability to cause disease in the ferret model. Numerous clade 2 viruses, including a clade 2.2 avian isolate, a 2.2.2.1 human isolate, and two 2.2.1 human isolates, were found to be of low virulence in the ferret model, though lethality was detected following infection with one 2.2.1 human isolate. In contrast, three of six clade 2.3.2.1 avian isolates tested led to severe disease and death among infected ferrets. Clade 2.3.2.1b and 2.3.2.1c isolates, but not 2.3.2.1a isolates, were associated with ferret lethality. All A(H5N1) viruses replicated efficiently in the respiratory tract of ferrets regardless of their virulence and lethality. However, lethal isolates were characterized by systemic viral dissemination, including detection in the brain and enhanced histopathology in lung tissues. The finding of disparate virulence phenotypes between clade 2A(H5N1) viruses, notably differences between subclades of 2.3.2.1 viruses, suggests there are distinct molecular determinants present within the established subclades, the identification of which will assist in molecular-based surveillance and public health efforts against A(H5N1) viruses.

Pathogenicity testing of influenza candidate vaccine viruses in the ferret model.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets.

Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses.