Equine Influenza Virus Research Articles

Presence of respiratory viruses in equines in Brazil.

Equines are susceptible to respiratory viruses such as influenza and parainfluenza. Respiratory diseases have adversely impacted economies all over the world. This study was intended to determine the presence of influenza and parainfluenza viruses in unvaccinated horses from some regions of the state of São Paulo, Brazil. Blood serum collected from 72 equines of different towns in this state was tested by hemagglutination inhibition test to detect antibodies for both viruses using the corresponding antigens. About 98.6% (71) and 97.2% (70) of the equines responded with antibody protective titers (≥ 80 HIU/25µL) H7N7 and H3N8 subtypes of influenza A viruses, respectively. All horses (72) also responded with protective titers (≥ 80) HIU/25µL against the parainfluenza virus. The difference between mean antibody titers to H7N7 and H3N8 subtypes of influenza A viruses was not statistically significant (p > 0.05). The mean titers for influenza and parainfluenza viruses, on the other hand, showed a statistically significant difference (p < 0.001). These results indicate a better antibody response from equines to parainfluenza 3 virus than to the equine influenza viruses. No statistically significant differences in the responses against H7N7 and H3N8 subtypes of influenza A and parainfluenza 3 viruses were observed according to the gender (female, male) or the age (≤ 2 to 20 years-old) groups. This study provides evidence of the concomitant presence of two subtypes of the equine influenza A (H7N7 and H3N8) viruses and the parainfluenza 3 virus in equines in Brazil. Thus, it is advisable to vaccinate equines against these respiratory viruses.

Complete Genome Sequences of Noncoding Regions of Korean Equine H3N8 Influenza Virus.

We analyzed the complete genome sequence containing the 3′ and 5′ noncoding regions (NCRs) of the Korean H3N8 equine influenza virus (EIV), which will provide a better understanding of the pathogenesis, transmission, and evolution of EIV.

Detection of evolutionarily distinct avian influenza a viruses in antarctica.

ABSTRACTDistinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment.

Continuing evolution of equine influenza virus in Central Asia, 2007-2012.

Equine influenza (EI) continues to be an important respiratory pathogen of horses worldwide. Since 2007 several outbreaks of EI have occurred in Central Asian countries, including Kazakhstan, western Mongolia, India and western China. Phylogenetic analysis showed that two H3N8 equine influenza virus (EIV) isolates from Kazakhstan, A/equine/Almaty/26/2007 and A/equine/South Kazakhstan/236/12, were related to Florida sublineage 2, with high similarity to EIVs circulating in the same period in neighbouring countries. New outbreaks of EI during 2011 and 2012 in Kazakhstan and other Central Asian countries were caused by viruses of the same lineage. Genetic characterization of the viruses showed formation of a small EIV cluster with specific genetic signatures and continued evolution of this lineage in Central Asia between 2007 and 2012. The main genetic changes were observed in hemagglutinin gene without any antigenic drift. Although no vaccination policy was carried out in Kazakhstan, application of Florida clade 2-based vaccines is recommended.

Genetic evolution of equine influenza strains isolated in France from 2005 to 2010.

REASON FOR PERFORMING THIS STUDY: Equine influenza virus (EIV) is considered the most economically important equine respiratory pathogen worldwide. The H3N8 subtype, responsible for all outbreaks of equine influenza globally, evolves perpetually. Mutations in the genome of these viruses have the potential to modify their antigenic properties and recognition by pre-existing antibodies. The aim of this study was to determine the genetic evolution of EIV strains in France and to compare it with the evolution of strains isolated globally. Analysis of the sequence data was performed to investigate any possible links between the outbreaks. Retrospective genetic analysis study of archived material. Analyses were performed on the HA1 domain of haemagglutinin H3 of EIV isolated in a previous study carried out from November 2005 to October 2010. The nucleic acid sequence of 41 strains was analysed and translated. The French viruses were compared with 59 Clade 1 strains and 83 Clade 2 strains. Strains were aligned chronologically and on the basis of their geographical origin. The 16 Clade 1 strains are all derived from the outbreak that started in the Grosbois training yard in 2009. The virus genome appears to have been stable during the outbreak. The 25 Clade 2 strains were isolated over the 5-year period during which several mutations had emerged. Some strains incorporate a sporadic mutation, and others a mutation that may occur several times but does not persist. However, all strains are gradually moving towards definitive mutations. This study demonstrated that EIVs have evolved in France during this period in a similar manner to EIVs globally. The data lend support to the current World Animal Health Organisation recommendation that the vaccines contain a representative of both Clade 1 and Clade 2 of the Florida sublineage.

Virus replication and cytokine production in primary alveolar macrophages and airway epithelial cells induced by emerging canine H3N8 influenza virus (LB510)

Canine H3N8 influenza virus was first recognized as a serious infectious pulmonary disease in racing greyhound dogs in 2004. Disease resulted from direct transmission of commonly circulating equine H3N8 virus to dogs.We hypothesized that transmission of equine influenza virus to dogs was characterized by mutations in one or more genes that allowed the virus to replicate more efficiently in dog airway epithelial cells and/or alveolar macrophages and induce higher levels of proinflammatory cytokines. Primary alveolar macrophages and airway epithelial cells were isolated from dogs and inoculated with emergent and endemic canine H3N8 influenza viruses A/canine/Florida /43/2004 (canine/FL/04), canine/FL/05, canine/PA/09 as well as early and more recent equine viruses equine/KY/91, equine/NY/99 and equine/OH/03.Cytokines responses were measured by multiplex protein assay and/or quantitative PCR. Emergent and endemic canine viruses replicated to higher titers in dog macrophages than did the equine viruses from 1999 and 2003. Only endemic canine viruses from 2005 and 2009 replicated to a higher titer in isolated dog airway epithelial cells than did equine viruses from 1999 and 2005. Induction of TNF‐alpha in macrophages by equine and canine viruses was highly variable among macrophage isolates from different dogs. No significant differences were measured in epithelial cell production of IL‐8 in response to canine or equine H3N8 virus inoculation. Although small differences in productive virus replication were detected for canine influenza virus isolates compared to equine influenza virus isolates in macrophages and epithelial cells, differences in cytokine production were not detected.Grant Funding Source: Supported by NIH AI101625

Flu season

> EMBO reports (ER) In 2012, two publications by Ron Fouchier and Yoshihiro Kawaoka about increasing the transmissibility of highly pathogenic H5N1 avian flu virus created a public debate about flu research and biosecurity. Since Fouchier announced that he would repeat such gain‐of‐function experiments with the H7N9 bird flu virus, he was widely criticised for what some regard as conducting irresponsible high‐risk experiments in the middle of Rotterdam. What are the potential benefits of these experiments, and do you think Fouchier should continue with his gain‐of‐function experiments on bird flu viruses? > Jeffery Taubenberger (JT) There are no simple answers, but it's worth thinking about this historically. People use the term gain‐of‐function as if this were a newly invented concept. But gain‐of‐function is what virologists have done for a hundred years. If you take an isolate of human influenza from a nasal or a throat swab and you grow it in a fertilized chicken egg, it can select for a clone that is able to grow in a chicken egg, which is not a native host. Mutations can rapidly accumulate in the virus, and you have now gained a function. It's the same thing with animal passage, so adapting a human or a chicken influenza virus to a ferret is something that people have done for eighty years. The concern I think comes from several aspects. The H5N1 virus, which has been circulating since 1996, is very pathogenic for chickens and it has caused human infections with a theoretical case fatality rate of approaching 60%. The concern is about gain‐of‐function experiments that could lead to more mammalian transmissibility of these highly pathogenic viruses. We know that a zoonotically‐derived animal influenza virus has to adapt to humans who are generally not susceptible to avian, equine, or canine influenza viruses. If we're ever actually …

Epidemiology and Genetic Characterization of H3N8 Equine Influenza Virus Responsible for Clinical Disease in Algeria in 2011

An outbreak of equine influenza (EI) was reported in Algeria between May and July, 2011. The outbreak started in Tiaret, in west province of Algeria, and spread to the other parts of the country affecting almost 900 horses in many provinces. The population studied was composed of 325 horses from different groups of age. Clinical sign expression was age dependent. Indeed, a morbidity rate of 14.9% was observed in horses under 15months old and a rate of 4.95% in horses over 8years old. Interestingly, the morbidity rate raised sharply to reach 100% in horses aged between 18months and 7years. The virus (H3N8) was detected in nasopharyngeal swabs (n=11) from non-vaccinated horses using a qRT-PCR targeting a portion of the gene encoding the matrix protein (M). The virus isolates were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes and were named from A/equine/Tiaret/1/2011 to A/equine/Tiaret/10/2011. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 2 of the Florida sublineage in the American lineage. Moreover, they are closely related to A/equine/Yokohama/aq13/2010, A/equine/Eyragues/1/2010, A/equine/Bokel/2011 and A/equine/Lichtenfeld/2012. Our data indicate that this strain was also circulating in the European horse population in 2010, 2011 and 2012.

Seroprevalence and Rate of Infection of Equine Influenza Virus (H3N8 and H7N7) and Equine Herpesvirus (1 and 4) in the Horse Population in Israel

Equine influenza and equine rhinopneumonitis are among the Office International des Epizooties or the World Organisation for Animal Health notifiable, contagious respiratory diseases. Although vaccination of horses in Israel against equine influenza virus (EIV) and against equine herpesvirus (EHV) is routinely performed, information regarding the occurrence and the epidemiology of the diseases is lacking. We hereby attempt to determine seroprevalence and rate of infection for EHV-1 and 4 and for EIV in horses distributed throughout Israel and describe demographic and environmental risk factors associated with seroprevalence. Despite the fact that last reported isolation of EIV in Israel occurred in 2007, we found a 26.4% (29/110) (95% confidence interval [CI]: 18.18–34.62) seroprevalence for H3N8, a 16.4% (18/110) (95% CI: 9.49–23.31) for H7N7, and a 6.4% (7/110) (95% CI: 1.83–10.97) rate of seroconversion for H3N8, suggesting current and active circulation of EIV in horses in Israel. Age, housing management type, and type of farm activity were significantly associated with seroprevalence, with activities allowing exposure to new horses positively associated with seroprevalence to EIV and an only pasture housing management negatively associated with seroprevalence. No association was detected between other demographic variables (gender, breed, and color) and environmental factors (climatic regions). Seroprevalence to EHV-1 and 4 were very low (<1%) and very high (>99%), respectively, raising questions regarding the appropriate vaccination guidelines. Our findings of the occurrence of EIV in horses in Israel imply an underdiagnosis of this virus in this country and warrant further investigation as to the strains that circulate in this region and their accordance with the current vaccine strains.

Little Evidence of Avian or Equine Influenza Virus Infection among a Cohort of Mongolian Adults with Animal Exposures, 2010–2011

Avian (AIV) and equine influenza virus (EIV) have been repeatedly shown to circulate among Mongolia’s migrating birds or domestic horses. In 2009, 439 Mongolian adults, many with occupational exposure to animals, were enrolled in a prospective cohort study of zoonotic influenza transmission. Sera were drawn upon enrollment and again at 12 and 24 months. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members confirmed to have acute influenza A infections, permitted respiratory swab collections which were studied with rRT-PCR for influenza A. Serologic assays were performed against equine, avian, and human influenza viruses. Over the 2 yrs of follow-up, 100 ILI investigations in the cohort were conducted. Thirty-six ILI cases (36%) were identified as influenza A infections by rRT-PCR; none yielded evidence for AIV or EIV. Serological examination of 12 mo and 24 mo annual sera revealed 37 participants had detectable antibody titers (≥1∶10) against studied viruses during the course of study follow-up: 21 against A/Equine/Mongolia/01/2008(H3N8); 4 against an avian A/Teal/Hong Kong/w3129(H6N1), 11 against an avian-like A/Hong Kong/1073/1999(H9N2), and 1 against an avian A/Migrating duck/Hong Kong/MPD268/2007(H10N4) virus. However, all such titers were <1∶80 and none were statistically associated with avian or horse exposures. A number of subjects had evidence of seroconversion to zoonotic viruses, but the 4-fold titer changes were again not associated with avian or horse exposures. As elevated antibodies against seasonal influenza viruses were high during the study period, it seems likely that cross-reacting antibodies against seasonal human influenza viruses were a cause of the low-level seroreactivity against AIV or EIV. Despite the presence of AIV and EIV circulating among wild birds and horses in Mongolia, there was little evidence of AIV or EIV infection in this prospective study of Mongolians with animal exposures.

A brief introduction to equine influenza and equine influenza viruses.

Equine influenza virus (EIV) is a common respiratory pathogen of horses and other equids in most parts of the world. EIV are Type A influenza viruses and two subtypes are known: H3N8 and H7N7. Both are believed to have evolved from avian influenza virus ancestors. The H3N8 subtype circulates widely, but the H7N7 subtype is thought to be extinct. The clinical disease in horses, caused by either subtype, is an upper respiratory infection of varying severity depending upon the immune status of the individual animal. It is not normally life-threatening in itself except in very young foals; however it predisposes infected equids to secondary infections capable of producing life-threatening pneumonias. Vaccines are available and widely used in some horse populations, but their effectiveness is limited by antigenic drift and other factors, and vaccinated animals with subclinical infections have been responsible for introduction of EIV into susceptible populations. EIV has spread into canines.

Equine influenza serological methods.

Serologic tests for equine influenza virus (EIV) antibodies are used for many purposes, including retrospective diagnosis, subtyping of virus isolates, antigenic comparison of different virus strains, and measurement of immune responses to EIV vaccines. The hemagglutination-inhibition (HI), single radial hemolysis (SRH), and serum micro-neutralization tests are the most widely used for these purposes and are described here. The presence of inhibitors of hemagglutination in equine serum complicates interpretation of HI assay results, and there are alternative protocols (receptor-destroying enzyme, periodate, trypsin-periodate) for their removal. With the EIV H3N8 strains in particular, equine antibody titers may be magnified by pretreating the HI test antigen with Tween-80 and ether. The SRH assay offers stronger correlations between serum antibody titers and protection from disease. Other tests are sometimes used for specialized purposes such as the neuraminidase-inhibition assay for subtyping, or ELISA for measuring different specific antibody isotypes, and are not described here.

Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance.

RNA extraction from equine samples for equine influenza virus.

The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories, to highlight the advantages and drawbacks of each method, and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab specimens. Furthermore, most of the information presented in this chapter could be directly applicable to isolation and purification of nucleic acids (both DNA and RNA) from other equine clinical samples.

Relevamiento serológico de anticuerpos contra enfermedades virales de interés sanitario en llamas (Lama glama) de la provincia de Jujuy, Argentina

Llama population from Argentina is mainly concentrated in the Andean Puna, Jujuy. Llamas represent an important economic resource for the Andean communities. The aim of this study was to investigate the prevalence of antibodies against viral antigens associated to viral diseases of economic impact (neonatal diarrhea, reproductive and respiratory syndromes). A total of 349 serum samples from adult llamas were analyzed. The obtained antibody prevalence was 100 % for Rotavirus A and 70 % for Bovine parainfluenza virus 3. In contrast, no reactors were detected to Bovine herpesvirus 1, Bovine viral diarrhea virus 1, Human influenza A virus (H1N1) and Equine influenza virus (H3N8). These results confirm the wide circulation of rotavirus and parainfluenza virus in Argentinean llamas and suggest that susceptibility to infection with bovine herpesvirus, pestivirus and influenza A viruses is low. This serologic survey provides novel information regarding the epidemiology of viral diseases affecting llamas from the Argentinean Andean Puna.

Equine influenza diagnosis: sample collection and transport.

In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of virus strains for antigenic characterization and potential inclusion in vaccines. Both nasal swabs and nasopharyngeal swabs are employed in horses. These differ little in their invasiveness, but nasopharyngeal swabs typically yield more virus than nasal swabs and are superior diagnostic specimens. Methods for obtaining nasopharyngeal swab specimens are described.

Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010–2012

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010–2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2.

Comparison of the Virucidal Effects of Disinfectant Agents Against Equine Influenza A Virus

Disinfection is one of the most important biosecurity measures to minimize disease spread during outbreaks of equine influenza. Although many disinfectants are commercially available, information about their effects against equine influenza A virus (EIV) is limited. This report describes an evaluation of the effects of six disinfectants against EIV (∼104.7 egg infectious dose 50/200 μL) under different conditions (reaction time [10 and/or 30 minutes], temperature [4°C–25°C], and the absence and/or presence of uninfected allantoic fluid that served as our best choice of “organic matter” equivalent). Although the efficacy of didecyldimethylammonium chloride decreased with decreasing reaction temperature, the compound showed the highest efficacy of the three quaternary ammonium compounds tested in this study. The effects of sodium dichloroisocyanurate and nonoxynol iodine were not affected by reaction time or temperature, but they were affected by the presence of organic matter. Antec Virkon S containing potassium peroxymonosulfate and sodium chloride consistently inactivated EIV regardless of reaction time, temperature, and the presence of organic matter. These findings will help us to take rational biosecurity measures during outbreaks of equine influenza.

Protein expression changes in cells inoculated with Equine Influenza Virus: antibody microarray analysis

Changes in the level of cellular proteins in cells inoculated with equine influenza virus H7N7 and H3N8 were studied with microarray technique. H3N8 induced pro-apoptotic proteins while H7N7 induced both pro- as well as anti-apoptotic factors. The higher level of some cytoskeleton components and proteins involved in the protein quality control was recorded. Relatively high number of proteins involved in the regulation of transcription was down-regulated. The pattern of changes observed for H7N7 and H3N8 may reflect differences in the biological properties of both serotypes.

The evaluation of a nucleoprotein ELISA for the detection of equine influenza antibodies and the differentiation of infected from vaccinated horses (DIVA)

Antibodies against equine influenza virus (EIV) are traditionally quantified by haemagglutination inhibition (HI) or single radial haemolysis (SRH). To evaluate an ELISA for the detection of antibodies against influenza nucleoprotein in the diagnosis and surveillance of equine influenza (EI). The ELISA was compared with the SRH and HI tests. Serial serum samples from 203 naturally and 14 experimentally infected horses, from 60 weanlings following primary vaccination with five different vaccines (two whole inactivated vaccines, two ISCOM-based subunit vaccines and a recombinant canarypox virus vaccine) and from 44 adult horses following annual booster vaccination with six different vaccines were analysed. Fewer seroconversions were detected in clinical samples by ELISA than by SRH or HI but ELISA was more sensitive than SRH in naïve foals post-experimental infection. The ELISA did not detect the antibody response to vaccination with the recombinant canarypox virus vaccine confirming the usefulness of the combination of this kit and vaccine to differentiate between naturally infected and vaccinated horses, that is, DIVA. No DIVA capacity was evident with the other vaccines. The results suggest that this ELISA is a useful supplementary test for the diagnosis of EI although less sensitive than HI or SRH. It is an appropriate test for EI surveillance in a naïve population and may be combined with the recombinant canarypox virus vaccine but not with other commercially available subunit vaccines, in a DIVA strategy.