Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication.

Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.

Equine Influenza Culture Methods.

Equine influenza viruses are cultured in embryonated chicken eggs or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

Mutation W222L at the Receptor Binding Site of Hemagglutinin Could Facilitate Viral Adaption from Equine Influenza A(H3N8) Virus to Dogs.

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., α2,3-linkage or α2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., α2,3-linked and α2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17.

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×106cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9.05 log TCID50/ml for A/Panama/2007/99 influenza H3N2. Porcine strains were also greatly rescued with titres from 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, viral kinetics showed maximal titers reached at 24h post-infection for most of the strains, allowing early harvest time (Time Of Harvest: TOH). The B strains present specific production kinetics with a delay of 24h before reaching the maximal viral particle release. Process optimization on H1N1 2009 human pandemic strain allowed identifying best operating conditions for production (MOI, trypsin concentration, cell density at infection) allowing improving the production level by 2 log. Our results suggest that the DuckCelt®-T17 cell line is a very promising platform for industrial production of influenza viruses and particularly for avian viral strains.

A live-attenuated influenza vaccine for H3N2 canine influenza virus.

Canine influenza is a contagious respiratory disease in dogs caused by two subtypes (H3N2 and H3N8) of canine influenza virus (CIV). Currently, only inactivated influenza vaccines (IIVs) are available for the prevention of CIVs. Historically, live-attenuated influenza vaccines (LAIVs) have been shown to produce better immunogenicity and protection efficacy than IIVs. Here, we have engineered a CIV H3N2 LAIV by using the internal genes of a previously described CIV H3N8 LAIV as a master donor virus (MDV) and the surface HA and NA genes of a circulating CIV H3N2 strain. Our findings show that CIV H3N2 LAIV replicates efficiently at low temperature but its replication is impaired at higher temperatures. The CIV H3N2 LAIV was attenuated in vivo but induced better protection efficacy in mice against challenge with wild-type CIV H3N2 than a commercial CIV H3N2 IIV. This is the first description of a LAIV for the prevention of CIV H3N2 in dogs.

A naturally truncated NS1 protein of influenza A virus impairs its interferon-antagonizing activity and thereby confers attenuation in vitro.

The non-structural protein of influenza A virus (NS1A protein) is a multifunctional protein that antagonizes host antiviral responses and contributes to efficient viral replication during infection. However, most of its functions have been elucidated by generating recombinant viruses expressing mutated NS1 proteins that do not exist in nature. Recently, the novel H3N8 A/Equine/Kyonggi/SA1/2011 (KG11) influenza virus was isolated in Korea from horses showing respiratory disease symptoms. KG11 virus contains a naturally truncated NS gene segment with the truncation in the NS1A coding region, resulting in truncation of the effector domain of the NS1A protein. Using this KG11 virus, we investigated the role of truncated NS1A protein in the virus life cycle and its effect on host immune responses were compared to the A/Equine/Miami/1/1963 H3N8 (MA63) virus, which encodes a full-length NS1A protein. The replication of KG11 virus was attenuated by 2 logs in multiple-cycle growth, and its plaque size was significantly smaller than that of the MA63 virus. To understand the attenuation of KG11 virus, we evaluated the level of activation in Akt and interferon regulatory factor 3 (IRF-3) pathways and measured the induction of downstream genes. Our results showed that the activation of Akt was reduced, whereas phosphorylation of IRF-3 was increased in cells infected with KG11 virus when compared to MA63-virus-infected cells. We also determined that the expression of antiviral and pro-inflammatory genes was significantly increased. Taken together, these results revealed that the KG11 virus expressing the naturally truncated NS1A protein impairs the inhibition of host antiviral responses, thereby resulting in the attenuation of viral replication.

Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence.

UNLABELLED: The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. IMPORTANCE: Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.

Equine influenza culture methods.

Equine influenza viruses are cultured in embryonated hen eggs, or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

Overview of the epidemiology of equine influenza in the Australian outbreak.

This overview of the equine influenza (EI) epidemic as it occurred in two Australian states, New South Wales and Queensland, in 2007 describes the functions and activities of the epidemiology teams that were engaged during the outbreak and also identifies key features of the epidemiology of EI during the outbreak.

Significant features of the epidemiology of equine influenza in New South Wales, Australia, 2007.

Equine influenza (EI) was first diagnosed in the Australian horse population on 24 August 2007 at Centennial Park Equestrian Centre (CPEC) in Sydney, New South Wales (NSW), Australia. By then, the virus had already spread to many properties in NSW and southern Queensland. The outbreak in NSW affected approximately 6000 premises populated by approximately 47,000 horses. Analyses undertaken by the epidemiology section, a distinct unit within the planning section of the State Disease Control Headquarters, included the attack risk on affected properties, the level of under-reporting of affected properties and a risk assessment of the movement of horses out of the Special Restricted Area. We describe the epidemiological features and the lessons learned from the outbreak in NSW.

Analysis of horse movements and events during the 2007 outbreak of equine influenza in New South Wales, Australia.

OBJECTIVE: To analyse horse event and horse movement registration data collected between September 2007 and December 2008 during the outbreak of equine influenza in New South Wales, Australia. RESULTS: A total of 9356 events were registered, involving 504,382 horses and 242,952 riders. Registered horse movements totalled 224,084, involving 349,327 horses (excluding mobs) travelling 34.4 million km with an average of 154 km per journey. The number of recorded events and movements were low while movement controls were most stringent, then increased from December 2007 as restrictions were eased, to peak in April 2008 with up to 290 events and 15,730 movements weekly, after which registrations declined as the disease was eradicated. The main types of events registered were pony clubs (38%), race meetings and trials (17%), competition (13%), and clinics and lessons (11%). CONCLUSIONS: Registration of horse events and movements allowed movement controls to be progressively eased while retaining the ability to trace the movements of large numbers of horses if needed. The number of recorded events, movements and distances travelled confirms the highly mobile nature of the recreational horse industries, helps to explain the rapid and widespread dispersal of the disease before movement restrictions were imposed, and also demonstrates the value of those restrictions as a control measure. The data provide a quantitative snap-shot of horse events and movements, albeit distorted by the prevailing movement restrictions as well as by limitations in the data recording that should be addressed when developing traceability systems for horses in future.

Spatial association and clinical development of equine influenza in horses yarded overnight at an equestrian event at Maitland prior to propagating the 2007 epidemic in Australia.

The interaction and stabling of horses at equine events may have a substantial impact on the spread of a zoonotic disease. This study aimed to investigate the spread of equine influenza (EI) at an equestrian event at the start of the Australian outbreak. Around one-third of the competing horses were stabled overnight at the event and, of these, 70% developed symptoms of EI within 7 days. The index case was never positively identified, but stabling position and disease onset provided clues to its potential identity.

Attack risk on infected properties during the 2007 equine influenza outbreak in New South Wales, Australia.

The aim of this preliminary study was to estimate the proportions of seropositive horses on infected premises (IPs) in order to assess the attack risk of the disease. Logistic regression analyses were conducted to evaluate the differences in attack risks between enterprise sizes and predefined spatial clusters/regions. The average attack risk experienced during the outbreak was 96.88% (median 100%), but it differed according to the size of the enterprise and other geographic and demographic conditions. The highest attack risks were observed in the Dubbo cluster/region and the lowest in the Narrabri-Northern cluster. Properties with fewer horses were generally more likely to have higher attack risks than larger enterprises, though this was not true for all cluster/regions.

Risk of an equine influenza virus reservoir establishing in wild horses in New South Wales during the Australian epidemic.

Australia has the world's largest population of wild equids and equine influenza (EI) was confirmed on several properties in New South Wales (NSW) close to uncontrolled areas of land during the 2007 outbreak. Likelihood and risk management assessments were carried out to determine the risk of EI infection of the wild horse populations. The likelihood of spread to the wild horse population was determined to be extremely low, but the likelihood of spread from an established wild horse reservoir back to domestic horses was considered high. The most effective mechanism of control was determined to be prevention of the spread of EI into the wild horse population through a vaccination buffer zone between EI infection foci and known wild horse populations.

Significant features of the epidemiology of equine influenza in Queensland, Australia, 2007.

An outbreak of equine influenza (EI) caused by influenza A H3N8 subtype virus occurred in the Australian states of Queensland and New South Wales in August 2007. Infection in the Australian horse population was associated with the introduction of infection by horses from overseas. The first case of EI in Queensland was detected on 25 August 2007 at an equestrian sporting event. Infection subsequently spread locally and to other clusters through horse movements prior to the implementation of an official standstill. There were five main clusters of infected properties during this outbreak and several outliers, which were investigated to find the potential mechanism of disease spread. To contain the outbreak, Queensland was divided into infection status zones, with different movement controls applied to each zone. Vaccination was implemented strategically in infected areas and within horse subpopulations. Control and eventual eradication of EI from Queensland was achieved through a combination of quarantine, biosecurity measures, movement control, rapid diagnostic testing and vaccination.

Surveillance and prevention in a non-affected state: Victoria.

The equine influenza outbreak detected in August 2007 in New South Wales and Queensland did not enter Victoria, which was, however, considered at risk because of its sizable border with New South Wales. Accordingly, Victoria implemented a response plan to prevent disease entry and enable early detection of any disease. Horse movement restrictions, surveillance strategies and public awareness formed a large part of this response.

The August 2007 equine influenza response management framework.

This section outlines the most important issues addressed in the management of the response in the two infected states, New South Wales and Queensland. There were differences in the management of the response between the states for logistic, geographic and organisation structural reasons. Issues included the use of control centres, information centres, the problems associated with the lack of trained staff to undertake all the roles, legislative issues, controls of horse movements, the availability of resources for adequate surveillance, the challenges of communication between disparate groups and tracing the movements of both humans and horses.

High-level coordination and strategy in the 2007 equine influenza outbreak response.

The equine influenza (EI) outbreak presented many challenges that required high-level coordination and decision making, as well as the development of new approaches for satisfactory and consistent resolution. This paper outlines the elements of the national coordination arrangements, preparatory arrangements in place prior to the outbreak that facilitated national coordination, and some of the issues faced and resolved in the response.

Industry liaison officers in the infected states of Australia during the 2007 equine influenza outbreak.

We describe the activities of persons who were formally employed as industry liaison officers (ILOs) in the infected states of Australia during the 2007 equine influenza outbreak. The knowledge and communication skills of ILOs allowed them to function very effectively, despite most of them having received no prior training. It is arguable, however, that if ILOs had received prior training, they would have performed more effectively sooner. We suggest that more individuals need ILO training in 'peacetime' and that 'just-in-time' training packages require preparation and trial prior to outbreaks to be effective.