Experimental infection of pigs and ferrets with "pre-pandemic," human-adapted, and swine-adapted variants of the H1N1pdm09 influenza A virus reveals significant differences in viral dynamics and pathological manifestations.

Influenza A viruses are RNA viruses that cause epidemics in humans and are enzootic in the pig population globally. In 2009, pig-to-human transmission of a reassortant H1N1 virus (H1N1pdm09) caused the first influenza pandemic of the 21st century. This study investigated the infection dynamics, pathogenesis, and lesions in pigs and ferrets inoculated with natural isolates of swine-adapted, human-adapted, and "pre-pandemic" H1N1pdm09 viruses. Additionally, the direct-contact and aerosol transmission properties of the three H1N1pdm09 isolates were assessed in ferrets. In pigs, inoculated ferrets, and ferrets infected by direct contact with inoculated ferrets, the pre-pandemic H1N1pdm09 virus induced an intermediary viral load, caused the most severe lesions, and had the highest clinical impact. The swine-adapted H1N1pdm09 virus induced the highest viral load, caused intermediary lesions, and had the least clinical impact in pigs. The human-adapted H1N1pdm09 virus induced the highest viral load, caused the mildest lesions, and had the least clinical impact in ferrets infected by direct contact. The discrepancy between viral load and clinical impact presumably reflects the importance of viral host adaptation. Interestingly, the swine-adapted H1N1pdm09 virus was transmitted by aerosols to two-thirds of the ferrets. Further work is needed to assess the risk of human-to-human aerosol transmission of swine-adapted H1N1pdm09 viruses.

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus.

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.

Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America.

Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly followed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically and phenotypically diverse, with many causing severe disease with dramatic neurologic involvement in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous system warrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants.

Swine H1N1 Influenza Virus Variants with Enhanced Polymerase Activity and HA Stability Promote Airborne Transmission in Ferrets.

Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor hemagglutinin (HA) stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCE Diverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage as a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both α-2,6- and α-2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

HA stabilization promotes replication and transmission of swine H1N1 gamma influenza viruses in ferrets.

Pandemic influenza A viruses can emerge from swine, an intermediate host that supports adaptation of human-preferred receptor-binding specificity by the hemagglutinin (HA) surface antigen. Other HA traits necessary for pandemic potential are poorly understood. For swine influenza viruses isolated in 2009-2016, gamma-clade viruses had less stable HA proteins (activation pH 5.5-5.9) than pandemic clade (pH 5.0-5.5). Gamma-clade viruses replicated to higher levels in mammalian cells than pandemic clade. In ferrets, a model for human adaptation, a relatively stable HA protein (pH 5.5-5.6) was necessary for efficient replication and airborne transmission. The overall airborne transmission frequency in ferrets for four isolates tested was 42%, and isolate G15 airborne transmitted 100% after selection of a variant with a stabilized HA. The results suggest swine influenza viruses containing both a stabilized HA and alpha-2,6 receptor binding in tandem pose greater pandemic risk. Increasing evidence supports adding HA stability to pre-pandemic risk assessment algorithms.

A(H9N2) influenza viruses associated with chicken mortality in outbreaks in Algeria 2017.

In late 2017, increased mortality was detected in chicken farms in Algeria undergoing A(H9N2) influenza outbreaks. Analysis of viruses isolated from affected farms showed that they were monophyletic, were of the G1 hemagglutinin (HA) lineage, and were antigenically and genetically similar to viruses detected contemporaneously in other countries in Northern Africa and the Middle East. The virus was able to spread via contact transmission between ferrets but did not cause disease in intravenously inoculated chickens.

Highly pathogenic avian influenza H5N1 clade 2.3.2.1 and clade 2.3.4 viruses do not induce a clade-specific phenotype in mallard ducks.

Among the diverse clades of highly pathogenic avian influenza (HPAI) H5N1 viruses of the goose/Guangdong lineage, only a few have been able to spread across continents: clade 2.2 viruses spread from China to Europe and into Africa in 2005-2006, clade 2.3.2.1 viruses spread from China to Eastern Europe in 2009-2010 and clade 2.3.4.4 viruses of the H5Nx subtype spread from China to Europe and North America in 2014/2015. While the poultry trade and wild-bird migration have been implicated in the spread of HPAI H5N1 viruses, it has been proposed that robust virus-shedding by wild ducks in the absence of overt clinical signs may have contributed to the wider dissemination of the clade 2.2, 2.3.2.1 and 2.3.4.4 viruses. Here we determined the phenotype of two divergent viruses from clade 2.3.2.1, a clade that spread widely, and two divergent viruses from clade 2.3.4, a clade that was constrained to Southeast Asia, in young (ducklings) and adult (juvenile) mallard ducks. We found that the virus-shedding magnitude and duration, transmission pattern and pathogenicity of the viruses in young and adult mallard ducks were largely independent of the virus clade. A clade-specific pattern could only be detected in terms of cumulative virus shedding, which was higher with clade 2.3.2.1 than with clade 2.3.4 viruses in juvenile mallards, but not in ducklings. The ability of clade 2.3.2.1c A/common buzzard/Bulgaria/38 WB/2010-like viruses to spread cross-continentally may, therefore, have been strain-specific or independent of phenotype in wild ducks.

The immune correlates of protection for an avian influenza H5N1 vaccine in the ferret model using oil-in-water adjuvants.

Because of the pathogenicity and low incidence of avian influenza virus infections in humans, the immune correlates of protection for avian influenza vaccines cannot be determined from clinical studies. Here, we used the ferret model to address this for an avian influenza H5N1 vaccine. Using oil-in-water adjuvants, we generated groups of ferrets with undetectable (geometric mean titer [GMT] < 10), low (GMT = 28.3), or high (GMT > 761.1) hemagglutination-inhibition (HAI) titers to the A/Viet Nam/1203/2004 (H5N1) virus. Ferrets were then challenged with the wild-type virus and disease severity and immunologic parameters were studied. The severity of infection and symptom profile were inversely associated with pre-challenge HAI titers in a dose-dependent manner. A vaccinated ferret with no detectable HAI-antibodies but high flu-specific IgG-antibody titers mounted rapid functional antibodies after infection and experienced milder disease compared to other ferrets in the group. Compared to naïve ferrets, all vaccinated ferrets showed improved cellular immunity in the lungs and peripheral blood. High number of IFNγ+ CD8- T cells in the airways was associated with early viral clearance. Thus, while neutralizing antibodies are the best correlate of protection, non-neutralizing antibodies can also be protective. This should be taken into consideration in future avian influenza vaccine trials.

Pandemic Seasonal H1N1 Reassortants Recovered from Patient Material Display a Phenotype Similar to That of the Seasonal Parent.

UNLABELLED: We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients. IMPORTANCE: Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.

Correlation Between the Interval of Influenza Virus Infectivity and Results of Diagnostic Assays in a Ferret Model.

BACKGROUND: The relationship between influenza virus infectivity and virus shedding, based on different diagnostic methods, has not been defined. METHODS: Three donor ferrets infected with 2009 pandemic influenza A(H1N1) underwent daily quantitative culture, antigen-detection testing, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Eight contacts were sequentially cohoused with each of the donors for 24 hours during days 3-10 after inoculation. RESULTS: Transmission was observed until day 5 after inoculation, corresponding to high culture titers and positive results of antigen-detection tests. Real-time RT-PCR showed no relation to the cessation of transmission. CONCLUSIONS: Antigen-detection testing and virus culture but not real-time RT-PCR identified the end of the infectious period.

Influenza Virus Surveillance in Coordinated Swine Production Systems, United States.

To clarify the epidemiology of influenza A viruses in coordinated swine production systems to which no animals from outside the system are introduced, we conducted virologic surveillance during September 2012-September 2013. Animal age, geographic location, and farm type were found to affect the prevalence of these viruses.

Impact of Adjuvants on the Immunogenicity and Efficacy of Split-Virion H7N9 Vaccine in Ferrets.

BACKGROUND: An effective vaccine is urgently needed against the H7N9 avian influenza virus. We evaluated the immunogenicity and protective efficacy of a split-virion H7N9 vaccine with or without the oil-in-water adjuvants in ferrets. METHODS: Ferrets were vaccinated with 2 doses of unadjuvanted, MF59 or AS03-adjuvanted A/Shanghai/2/2013 (H7N9) vaccine, and the induction of antibodies to hemagglutinin (HA) or neuraminidase proteins was evaluated. Ferrets were then challenged with wild-type H7N9 virus to assess the vaccine's protective efficacy. The vaccine composition and integrity was also evaluated in vitro. RESULTS: Adjuvanted vaccines stimulated robust serum antibody titers against HA and neuraminidase compared with the unadjuvanted vaccines. Although there was a difference in adjuvanticity between AS03 and MF59 at a lower dose (3.75 µg of HA), both adjuvants induced comparable antibody responses after 2 doses of 15 µg. On challenge, ferrets that received adjuvanted vaccines showed lower viral burden than the control or unadjuvanted vaccine group. In vitro examinations revealed that the vaccine contained visible split-virus particles and retained the native conformation of HA recognizable by polyclonal and monoclonal antibodies. CONCLUSIONS: The adjuvanted H7N9 vaccines demonstrated superior immunogenicity and protective efficacy against H7N9 infection in ferrets and hold potential as a vaccination regimen.

A single dose of whole inactivated H7N9 influenza vaccine confers protection from severe disease but not infection in ferrets.

The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50 µg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50 µg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.

Long-term vaccine-induced heterologous protection against H5N1 influenza viruses in the ferret model.

BACKGROUND: Highly pathogenic H5N1 influenza viruses reemerged in humans in 2003 and have caused fatal human infections in Asia and Africa as well as ongoing outbreaks in poultry. These viruses have evolved substantially and are now so antigenically varied that a single vaccine antigen may not protect against all circulating strains. Nevertheless, studies have shown that substantial cross-reactivity can be achieved with H5N1 vaccines. These studies have not, however, addressed the issue of duration of such cross-reactive protection. OBJECTIVES: To directly address this using the ferret model, we used two recommended World Health Organization H5N1 vaccine seed strains - A/Vietnam/1203/04 (clade 1) and A/duck/Hunan/795/02 (clade 2.1) - seven single, double, or triple mutant viruses based on A/Vietnam/1203/04, and the ancestral viruses A and D, selected from sequences at nodes of the hemagglutinin and neuraminidase gene phylogenies to represent antigenically diverse progeny H5N1 subclades as vaccine antigens. RESULTS: All inactivated whole-virus vaccines provided full protection against morbidity and mortality in ferrets challenged with the highly pathogenic H5N1 strain A/Vietnam/1203/04 5 months and 1 year after immunization. CONCLUSION: If an H5N1 pandemic was to arise, and with the hypothesis that one can extrapolate the results from three doses of a whole-virion vaccine in ferrets to the available split vaccines for use in humans, the population could be efficiently immunized with currently available H5N1 vaccines, while the homologous vaccine is under production.

Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States.

As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.

Evaluation of ABT-751 against childhood cancer models in vivo.

OBJECTIVE: ABT-751 is a novel antimitotic agent that binds tubulin at the colchicine binding site. ABT-751 is undergoing Phase I trials in children, but has not been evaluated against a range of pediatric tumor models in vivo. MATERIALS AND METHODS: ABT-751 was evaluated against 27 subcutaneously implanted xenograft models of childhood cancer including neuroblastoma [4], osteosarcoma [4], Ewing sarcoma [2] rhabdomyosarcoma [8], medulloblastoma [1] and eight kidney cancer lines (six Wilms tumors, two rhabdoid). ABT-751 was administered at 100 mg/kg P.O. on a schedule of 5 days on, 5 days off, 5 days on, repeating the cycle at 21 days. Tumor diameters were measured at 7 day intervals for a period of 12 weeks. Three measures of antitumor activity were used: (1) clinical response criteria [e.g., partial response (PR), complete response (CR), etc.]; (2) treated to control (T/C) tumor volume at day 21; and (3) a time to event measure based on the median event free survival (EFS) of treated and control lines. RESULTS: ABT-751 induced regression in 4 of 25 models (16%) including models of neuroblastoma that are refractory to vincristine and paclitaxel. Other regressions occurred in rhabdomyosarcoma and Wilms tumor models. ABT-751 significantly increased event free survival (EFS > 2.0) in eight models (33%) in addition to those with objective responses. CONCLUSIONS: ABT-751 demonstrated intermediate activity against this tumor panel. Neuroblastoma models appear somewhat more sensitive to this agent, with objective regressions also in rhabdomyosarcoma and Wilms tumor. ABT-751 was also active in several tumor lines intrinsically refractory to vincristine or paclitaxel.