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Abstract: In horses, natural infection confers long lasting protective immunity characterised by mucosal IgA and humoral IgGa and IgGb responses. In order to investigate the potential of locally administered vaccine to induce a protective IgA response, responses generated by vaccination with an immunostimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F) containing A/eq/Newmarket/77 (H7N7), A/eq/Borlänge/91 (H3N8) and A/eq/Kentucky/98 (H3N8) using a systemic prime/mucosal boost strategy were studied. Seven ponies in the vaccine group received EQUIP F vaccine intranasally 6 weeks after an initial intramuscular immunisation. Following intranasal boosting a transient increase in virus-specific IgA was detected in nasal wash secretions. Aerosol challenge with the A/eq/Newmarket/1/93 reference strain 4 weeks after the intranasal booster resulted in clinical signs of infection and viral shedding in seven of seven influenza-naive control animals whereas the seven vaccinated ponies had statistically significantly reduced clinical signs and duration of virus excretion. Furthermore, following this challenge, significantly enhanced levels of virus-specific IgA were detected in the nasal washes from vaccinated ponies compared with the unvaccinated control animals. These data indicate that the intranasal administration of EQUIP F vaccine primes the mucosal system for an enhanced IgA response following exposure to live influenza virus. [Copyright &y& Elsevier]

Abstract: A microcarrier-based process was used to produce equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93) in Madin Darby Canine kidney (MDCK) cells. The virus was purified in a sequence of downstream processing steps comprising of depth filtration, inactivation, ultrafiltration (UF) and gel filtration. In the ultrafiltration step, the hemagglutinin (HA) was recovered to 100%. A high increase of neuraminidase (NA) activity indicated the removal of some inhibitory compounds during this step. At the same time, the level of contaminating proteins and DNA was reduced by more than 88%. In the subsequent size exclusion chromatography (Sepharose CL 2B), the recovery of HA and NA in the “virus peak” was 37.8 and 59.8%, respectively compared to the concentrated feed material. Inconsistencies in the overall mass balance for HA and NA (70.0 and 69.2%) during gel filtration indicated non-specific interactions of the inactivated virus to the gel matrix which is supported by a HA recovery of about 50% in shake flask experiments performed as a control. Overall 35.8% of HA and 291.6% of NA were recovered. More than 95.7% of the host cell proteins and 98.7% of the host cell DNA were removed during downstream processing. [Copyright &y& Elsevier]

Equine influenza is a common disease of the horse, causing significant morbidity worldwide. Here we describe the establishment of a plasmid-based reverse genetics system for equine influenza virus. Utilizing this system, we generated three mutant viruses encoding carboxy-terminally truncated NS1 proteins. We have previously shown that a recombinant human influenza virus lacking the NS1 gene (delNSl) could only replicate in interferon (IFN)-incompetent systems, suggesting that the NS1 protein is responsible for IFN antagonist activity. Contrary to previous findings with human influenza virus, we found that in the case of equine influenza virus, the length of the NS1 protein did not correlate with the level of attenuation of that virus. With equine influenza virus, the mutant virus with the shortest NS1 protein turned out to be the least attenuated. We speculate that the basis for attenuation of the equine NS1 mutant viruses generated is related to their level of NS1 protein expression. Our findings show that the recombinant mutant viruses are impaired in their ability to inhibit IFN production in vitro and they do not replicate as efficiently as the parental recombinant strain in embryonated hen eggs, in MDCK cells, or in vivo in a mouse model. Therefore, these attenuated mutant NS1 viruses may have potential as candidates for a live equine influenza vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Reported here are the results of antigenic and genetic characterisation of equine influenza strains causing local outbreaks reported to the Equine Diagnostic Centre in Berlin, Germany. In 2000, equine influenza virus was detected in a nasal swab from a non-vaccinated horse using a rapid diagnostic kit, but was not successfully isolated. Partial direct sequencing of the haemagglutinin (HA1) gene, indicated that the virus was a European lineage H3N8 subtype strain representative of strains isolated in several European countries during 2000. In 2002, two equine influenza viruses were isolated from nasal swabs both taken from unvaccinated horses with acute respiratory symptoms housed at the same stables. Antigenic characterisation using a panel of ferret antisera suggested that these isolates also belonged to the European lineage of H3N8 viruses. Analysis of deduced HA1 amino acid sequences confirmed that the HA1 of both isolates were identical and belonged to the European lineage. However, from phylogenetic analysis, both strains appeared to be more closely related to viruses isolated between 1989 and 1995 than to viruses isolated more recently in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. The importance of expanding current equine influenza surveillance efforts is emphasised. [Copyright &y& Elsevier]

Abstract: Protective responses generated by vaccination with an immuno-stimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F), containing a new ‘American lineage’ H3N8 virus, were studied. Seven ponies in the vaccine group received two intramuscular injections of EQUIP F given 6 weeks apart. Aerosol challenge with an A/eq/Newmarket/1/93 reference strain 4 weeks after booster vaccination resulted in clinical signs of infection and viral shedding in 7 influenza-naive control animals whereas the vaccinated ponies were significantly protected from both clinical signs and virus excretion. Influenza virus-specific IgG responses in serum following immunisation with the ISCOM vaccine were predominantly of the IgGa and IgGb sub-isotypes, a pattern similar to that generated by equine influenza virus infection. However, in contrast to the response following infection, virus-specific antibody responses in nasal washes following immunisation were characterised by the presence of IgG but not IgA.These results demonstrated that an ISCOM-based vaccine containing A/eq/Kentucky/98 provides strong protective immunity against challenge with an ‘American lineage’ H3N8 reference virus. [Copyright &y& Elsevier]

Two lineages of antigenically distinct equine influenza A H3N8 subtype viruses, American and European, co-circulate. Experiments were conducted in ponies to investigate the protection induced by vaccines containing virus from one lineage against challenge infection with homologous or heterologous virus. Regression analysis showed that vaccinated ponies with average pre-challenge single radial haemolysis (SRH) antibody levels (i.e. 45–190 mm2) had a higher probability of becoming infected if they were vaccinated with virus heterologous to the challenge strain than if they were vaccinated with homologous virus. Field studies in Thoroughbred racehorses also showed that SRH antibody levels of ≥150 mm2 induced by vaccines containing a European lineage strain are protective against infection with a virus from the same lineage, but that the same or higher antibody levels may not be protective against an American lineage virus. In conclusion, vaccines should contain virus strains representative of both H3N8 subtype lineages to maximise protection against infection. [Copyright &y& Elsevier]

We develop semiparametric methods for matched case-control studies using regression splines. Three methods are developed: 1) an approximate cross-validation scheme to estimate the smoothing parameter inherent in regression splines, as well as 2) Monte Carlo expectation maximization (MCEM) and 3) Bayesian methods to fit the regression spline model. We compare the approximate cross-validation approach, MCEM, and Bayesian approaches using simulation, showing that they appear approximately equally efficient; the approximate cross-validation method is computationally the most convenient. An example from equine epidemiology that motivated the work is used to demonstrate our approaches. [ABSTRACT FROM AUTHOR]

A stochastic model of equine influenza (EI) is constructed to assess the risk of an outbreak in a Thoroughbred population at a typical flat race training yard. The model is parameterised using data from equine challenge experiments conducted by the Animal Health Trust (relating to the latent and infectious period of animals) and also published data on previous epidemics (to estimate the transmission rate for equine influenza). Using 89 ponies, an empirical relationship between pre-challenge antibody and the probability of becoming infectious is established using logistic regression. Changes in antibody level over time are quantified using published and unpublished studies comprising 618 ponies and horses. A plausible Thoroughbred population is examined over the course of a year and the model is used to assess the risk of an outbreak of EI in the yard under the current minimum vaccination policy in the UK. The model is adapted to consider an alternative vaccination programme where the frequency of vaccination in older horses (2-year-olds and upwards) is increased. Model results show that this practical alternative would offer a significant increase in protection. Spread of infection between yards is also considered to ascertain the risk of secondary outbreaks. [Copyright &y& Elsevier]

An outbreak of equine influenza H3N8 in a riding school is described retrospectively with emphasis on diagnosis and putative vaccine failure. In March 1995 an outbreak of equine influenza occurred among 11 horses in a riding school, where most horses had received basic primary immunizations and several booster vaccinations against influenza. Six of the 11 diseased horses had received their last booster vaccination within 5 months of the outbreak. Nevertheless, the influenza infection spread rapidly and clinical manifestations were prominent with frequent, harsh, dry coughing often accompanied by high fever.Nasal swabs were taken from 11 diseased horses. Influenza A virus of the equine H3N8 (equi-2) subtype was isolated from five nasal swab extracts. Stored nasal swab extracts were also retrospectively investigated in two different enzyme immunoassays designed to detect the type-specific conserved nucleoprotein of influenza A viruses, and in a single-tube reverse transcription-PCR (RT-PCR) using a set of primers based on highly conserved regions of the matrix gene of influenza A viruses. Five nasal swab extracts were found positive in a DAS-ELISA and seven in the Directigen® Flu A (DFA) assay, respectively. Two nasal swab extracts from which virus was isolated did not give a positive result in the DAS-ELISA, and one of these also did not give a positive result in the DFA assay. Nine nasal swab extracts were found positive by RT-PCR. Moreover, all virus isolation and/or ELISA positive nasal swab extracts were confirmed by RT-PCR. Three nasal swab extracts were negative by virus isolation, PCR and ELISA. A significant rise in HI titre against influenza A/eq/Miami/63 (H3N8) virus was detected in seven of the nine paired sera available.In acute phase serum samples from 10 horses, SRH antibody levels varied widely. However, some horses with high, or at least putatively clinically protective SRH antibody levels, showed clinical signs and infection was confirmed. Antigenic analysis of two isolates showed that A/eq/Holland/1/95 (H3N8) and A/eq/Holland/2/95 (H3N8) cluster with the UK isolate Osgodsby/92, the Swedish isolate Borla¨nge/91 and some other European isolates, with H/2/95 identical in reactivity to Borla¨nge/91 and H/1/95 more similar in reactivity to Osgodsby/92 than H/2/95. Nucleotide and deduced amino-acid sequences showed large differences of both isolates as compared with Miami/63, Fontainebleau/79 and Kentucky/81, the influenza A H3N8 subtype strains incorporated in the vaccines used in this riding school. The role of antigenic drift in vaccine breakdown is discussed in the light of evidence for vaccine breakdown in the UK in 1989, Sweden in 1991 and in the USA since 1991. [Copyright &y& Elsevier]

Summary. Equine nasal turbinate epithelial cells and tracheal rafts were maintained with sustained viability in culture. Both types of culture supported productive replication of equine influenza virus (equine-2, subtype H3N8) and cell death occurred through apoptosis following viral infection. Thus, primary respiratory epithelial cell and organ cultures of equine origin may be valuable as alternatives to the intact animal for studying the virus-host interaction of equine respiratory viruses including influenza. [ABSTRACT FROM AUTHOR]

Field outbreaks of influenza that occurred in vaccinated Thoroughbred racehorses in Newmarket in 1995 and 1996 were investigated by nucleoprotein ELISA and serology. Investigations showed that serum levels of vaccine-induced single radial haemolysis (SRH) antibody correlated closely with protective immunity against equine influenza and were consistent with observations made in previous experimental studies using nebulised aerosol challenge. In the second part of this study, antibody levels stimulated by vaccination were investigated to examine probable protection in high risk groups, such as yearlings and horses in training. Results for yearlings correlated closely with experimentally derived antibody profiles described for several equine influenza vaccines. The horses in training had levels of antibody immediately prior to revaccination, which were higher than those measured in the yearlings. In conclusion, SRH antibody, used in the investigation of outbreaks and surveillance of post vaccination responses, was shown to correlate with and validate experimental vaccination and challenge models currently used in ponies in the licensing of modern vaccines. There may be benefit from serological monitoring of horses following vaccination through identification of susceptible periods to infection and demonstration of poor vaccine responders. This would allow appropriate and timely amendment of vaccination strategies to maximise protective immunity against influenza. [ABSTRACT FROM AUTHOR]

Simple Summary: The objective of the present study was to determine the molecular frequency of three recently identified parvoviruses (equine parvovirus hepatitis, equine parvovirus CSF and equine copivirus) in blood and respiratory secretions of 667 equids with acute onset of fever and respiratory signs and 87 clinically healthy horses. One hundred and seventeen sick horses tested qPCR-positive for at least one of the three parvoviruses. Ten clinically healthy horses tested qPCR-positive for one of the equine parvoviruses. The frequency of detection of the three equine parvoviruses was similar between sick and clinically healthy horses, suggesting that these newly characterized viruses do not appear to contribute to the clinical picture of equids with respiratory disease. In order to prove the clinical relevance of any of these newly identified equine parvoviruses, experimental challenge studies using pure, clonal inocula will be required. Three newly identified equine parvoviruses (equine parvovirus hepatitis (EqPV-H), equine parvovirus CSF (EqPV-CSF) and equine copivirus (Eqcopivirus)) have recently been discovered in horses with respiratory signs. However, the clinical impact of these three equine parvoviruses has yet to be determined. Nasal fluid samples and blood from 667 equids with acute onset of fever and respiratory signs submitted to a diagnostic laboratory were analyzed for the presence of common equine respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, S. equi subspecies equi) as well as EqPV-H, EqPV-CSF and Eqcopivirus by qPCR. An additional 87 clinically healthy horses served as controls. One hundred and seventeen sick horses tested qPCR-positive for at least one of the three parvoviruses. Co-infections with common respiratory pathogens and parvoviruses were seen in 39 sick equids. All 87 clinically healthy horses tested qPCR-negative for all tested common respiratory pathogens and 10 healthy horses tested qPCR-positive for one of the equine parvoviruses. When the frequency of detection for EqPV-H, EqPV-CSF and Eqcopivirus of equids with respiratory signs was compared to that of clinically healthy horses, the difference was not statistically significant (p > 0.05), suggesting that the three recently identified equine parvoviruses do not contribute to the clinical picture of equids with respiratory disease. [ABSTRACT FROM AUTHOR]

• Viral respiratory diseases are common in horses. • Quantitative PCR has become the desired testing platform for equine respiratory viruses. • Molecular results are valuable in assessing infective status and epidemiological risk. • Understanding testing procedures and limitations aids interpretation of results. Equine respiratory viruses remain a leading cause of equine morbidity and mortality, with the resurgence of certain infections, an increasing population of elderly, more susceptible horses, the growth of international equine commerce, and an expansion in geographic distribution of pathogens. The focus of rapid diagnosis of infectious diseases has also shifted recently, with the appearance and increasing importance of nucleic acid amplification-based techniques, primarily polymerase chain reaction (PCR), at the expense of traditional methods such as clinical microbiology. While PCR is fast, reliable, cost-effective, and more sensitive than conventional detection methods, careful interpretation of diagnostic test results is required, taking into account the clinical status of the patient, sample type, assay used and biological relevance of the detected viruses. The interpretation of common equine respiratory viruses such as influenza virus (EIV), alpha herpesviruses (EHV-1, EHV-4), arteritis virus (EAV) and rhinoviruses (ERAV, ERBV) is straight forward as causality can generally be established. However, the testing of less-characterized viruses, such as the gamma herpesviruses (EHV-2, EHV-5), may be confusing, considering their well-established host relationship and frequent detection in both diseased and healthy horses. For selected viruses, absolute quantitation (EHV-1 and EHV-4) and genotyping (EIV and EHV-1) has allowed additional information to be gained regarding viral state and virulence, respectively. This information is relevant when managing outbreaks so that adequate biosecurity measures can be instituted and medical interventions can be considered. The goal of this review is to help the equine practitioner navigate through the rapidly expanding field of molecular diagnostics for respiratory viruses and facilitate the interpretation of results. [ABSTRACT FROM AUTHOR]

Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world's horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI. [ABSTRACT FROM AUTHOR]

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy. [ABSTRACT FROM AUTHOR]

To prevent and control H3N8 subtype equine influenza, we prepared virus-like particles (VLPs) comprising the HA, NA and M1 proteins of H3N8 equine influenza virus (EIV) through the insect cell-baculovirus expression system. The results of Western blot and hemagglutination analyses demonstrated that the constructed VLPs comprising HA, NA and M1 proteins have good hemagglutination activity. Immunoelectron microscope revealed that the VLPs share similar morphology and structure with natural virus particles. The hyperimmune serum from horses immunized with the VLPs were injected into mice by means of artificial passive immunization and then challenge, or challenge following by injecting hyperimmune serum. The results showed that the equine hyperimmune serum has good preventive and therapeutic efficacy against the infection of H3N8 EIV. The study provides a technical foundation for the development of H3N8 EIV VLP vaccine. • H3N8 EIV VLP vaccine was successfully constructed. • H3N8 EIV VLPs share similar morphology and structure with natural virus particles. • Hyperimmune serum has good preventive and therapeutic efficacy against the infection of H3N8 EIV. [ABSTRACT FROM AUTHOR]