Isolation and characterisation of equine influenza viruses (H3N8) from Europe and North America from 2008 to 2009

Like other influenza A viruses, equine influenza virus undergoes antigenic drift. It is therefore essential that surveillance is carried out to ensure that recommended strains for inclusion in vaccines are kept up to date. Here we report antigenic and genetic characterisation carried out on equine influenza virus strains isolated in North America and Europe over a 2-year period from 2008 to 2009. Nasopharyngeal swabs were taken from equines showing acute clinical signs and submitted to diagnostic laboratories for testing and virus isolation in eggs. The sequence of the HA1 portion of the viral haemagglutinin was determined for each strain. Where possible, sequence was determined directly from swab material as well as from virus isolated in eggs. In Europe, 20 viruses were isolated from 15 sporadic outbreaks and 5 viruses were isolated from North America. All of the European and North American viruses were characterised as members of the Florida sublineage, with similarity to A/eq/Lincolnshire/1/07 (clade 1) or A/eq/Richmond/1/07 (clade 2). Antigenic characterisation by haemagglutination inhibition assay indicated that the two clades could be readily distinguished and there were also at least seven amino acid differences between them. The selection of vaccine strains for 2010 by the expert surveillance panel have taken these differences into account and it is now recommended that representatives of both Florida clade 1 and clade 2 are included in vaccines.     

Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population.     

The molecular epidemiology of equine influenza in Ireland from 2007-2010 and its international significance

Reasons for performing the study: Antigenic and genetic drift of equine influenza (EI) virus is monitored annually by the Expert Surveillance Panel (ESP), which make recommendations on the need to update vaccines. Surveillance programmes are essential for this process to operate effectively and to decrease the risk of disease spread through the international movement of subclinically infected vaccinated horses. Not only is surveillance necessary to inform vaccine companies which strains are in circulation, but it serves as an early warning system for horse owners, trainers and veterinary clinicians, facilitating the implementation of appropriate prophylactic and control measures. Objective: To summarise the genetic analysis of EI viruses detected in Ireland from June 2007 to January 2010. Methods: The HA1 gene of 18 viruses was sequenced and phylogenetic analysis undertaken. Results: All viruses belonged to the Florida sublineage of the American lineage. Clade 2 viruses predominated up to 2009. The viruses identified on 4 premises in 2007 displayed 100% nucleotide identity to A/eq/Richmond/1/07, the current clade 2 prototype. The first clade 1 virus was identified in November 2009 and, thereafter, clade 1 viruses were responsible for all the outbreaks identified. The Irish clade 1 viruses differ from the clade 1 virus responsible for the EI outbreaks in Japan and Australia in 2007. No virus of the Eurasian lineage was isolated during this surveillance period. Conclusions: In 2010 the ESP recommended that the vaccines should not include a H7N7 virus or a H3N8 virus of the Eurasian lineage but that they should contain both a clade 1 and clade 2 virus of the Florida sublineage. The surveillance data presented here support these recommendations and indicate that they are epidemiologically relevant. Potential relevance: These data also serve as a scientific basis for investigating the source of epizootics and outbreaks both nationally and internationally.     

Detection of clade 2 equine influenza virus in an adult horse recently imported to the USA

A 4‐year‐old Warmblood mare presented to the William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California at Davis with bilateral mucoid nasal discharge and pyrexia. The mare had recently been imported from Germany, arriving at a quarantine holding facility 72 h prior to presentation. Based on clinical presentation and culture results of tracheal fluid, the mare was diagnosed with bacterial bronchopneumonia secondary to equine influenza. The equine influenza virus (EIV) identified in the imported mare displayed 99.1% nucleotide homology of the HA1 gene to the prototype Florida sublineage clade 2 isolate A/equine/Richmond/1/2007 (H3N8). This case illustrates the risk of introducing a clade 2 EIV in North America.     

Antibody responses induced by Japanese whole inactivated vaccines against equine influenza virus (H3N8) belonging to Florida sublineage clade2

In 2010, the World Organisation for Animal Health recommended the inclusion of a Florida sublineage clade2 strain of equine influenza virus (H3N8), which is represented by A/equine/Richmond/1/07 (Richmond07), in equine influenza vaccines. Here, we evaluate the antigenic differences between Japanese vaccine strains and Richmond07 by performing hemagglutination inhibition (HI) assays. Ferret antiserum raised to A/equine/La Plata/93 (La Plata93), which is a Japanese vaccine strain, reacted with Richmond07 at a similar titer to La Plata93. Moreover, two hundred racehorses exhibited similar geometric mean HI antibody titers against La Plata93 and Richmond07 (73.1 and 80.8, respectively). Therefore, we can expect the antibody induced by the current Japanese vaccines to provide some protection against Richmond07-like viruses.     

Description of the outbreak of equine influenza (H3N8) in the United Kingdom in 2003, during which recently vaccinated horses in Newmarket developed respiratory disease

Between March and May 2003, equine influenza virus infection was confirmed as the cause of clinical respiratory disease among both vaccinated and unvaccinated horses of different breeds and types in at least 12 locations in the UK. In the largest outbreak, 21 thoroughbred training yards in Newmarket, with more than 1300 racehorses, were affected, with the horses showing signs of coughing and nasal discharge during a period of nine weeks. Many of the infected horses had been vaccinated during the previous three months with a vaccine that contained representatives from both the European (A/eq/Newmarket/2/93) and American (A/eq/Newmarket/1/93) H3NN8 influenza virus lineages. Antigenic and genetic characterisation of the viruses from Newmarket and elsewhere indicated that they were all closely related to representatives of a sublineage of American viruses, for example, Kentucky/5/02, the first time that this sublineage had been isolated in the uk. In the recently vaccinated racehorses in Newmarket the single radial haemolysis antibody levels in acute sera appeared to be adequate, and there did not appear to be significant antigenic differences between the infecting virus and A/eq/Newmarket/1/93, the representative of the American lineage virus present in the most widely used vaccine, to explain the vaccine failure. However, there was evidence for significantly fewer infections among two-year-old horses than older animals, despite their having similar high levels of antibody, consistent with a qualitative rather than a quantitative difference in the immunity conveyed by the vaccination.     

Isolation and characterisation of an H3N8 equine influenza virus in Australia, 2007

Before 2007, equine influenza had never been diagnosed in Australia. On 22 August 2007, infection was confirmed in horses at Eastern Creek Animal Quarantine Station near Sydney. The virus subsequently isolated (A/equine/Sydney/2888-8/2007) was confirmed by sequence analysis of the haemagglutinin (HA) gene as an H3 virus of the variant American Florida lineage that is now referred to as Clade 1. The HA sequence of the virus was identical to that of a virus isolated from a contemporaneous outbreak in Japan and showed high homology to viruses circulating in North America.     

Genetic analysis of the matrix and non-structural genes of equine influenza virus (H3N8) from epizootic of 2008-2009 in India

India faced an epizootic of equine influenza in 2008-2009. The isolated viruses were typed as H3N8 and grouped with the clade 2 viruses of Florida sublineage on the basis of haemagglutinin (HA) gene sequence analysis. This report describes the genetic analysis and selection pressure of matrix (M) and non-structural 1 (NS1) genes of the Indian isolates. All isolates shared 98.41% and 99.54% homology with other clade 2 viruses of Asian origin for M1 and M2 amino acid (aa) sequences, respectively. There were 3 and 4 unique aa residue changes respectively in M1 and M2 proteins in all Asian isolates. Phylogenetic analysis revealed clustering of Indian and Chinese isolates in a separate group designated here as Asian clade for M gene. Indian and Chinese isolates shared homology ranging from 98.17% to 99.08% at aa level. The M and NS1 genes were under negative selection pressure with estimated magnitude of pressure (ω) 0.054, 0.581 and 0.30 for M1, M2 and NS1, respectively.     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

Genetic Analyses of an H3N8 Influenza Virus Isolate, Causative Strain of the Outbreak of Equine Influenza at the Kanazawa Racecourse in Japan in 2007

In August 2007, an outbreak of equine influenza occurred among vaccinated racehorses with Japanese commercial equine influenza vaccine at Kanazawa Racecourse in Ishikawa prefecture in Japan. Apparent symptoms were pyrexia (38.2-41.0 degrees C) and nasal discharge with or without coughing, although approximately half of the infected horses were subclinical. All horses had been shot with a vaccine that contained two inactivated H3N8 influenza virus strains [A/equine/La Plata/93 (La Plata/93) of American lineage and A/equine/Avesta/93 (Avesta/93) of European lineage] and an H7N7 strain (A/equine/Newmarket/1/77). Influenza virus, A/equine/Kanazawa/1/2007 (H3N8) (Kanazawa/07), was isolated from one of the nasal swab samples of diseased horses. Phylogenetic analysis indicated that Kanazawa/07 was classified into the American sublineage Florida. In addition, four amino acid substitutions were found in the antigenic sites B and E in the HA1 subunit protein of Kanazawa/07 in comparison with that of La Plata/93. Hemagglutination-inhibition (HI) test using 16 serum samples from recovering horses revealed that 1.4- to 8-fold difference in titers between Kanazawa/07 and either of the vaccine strains. The present findings suggest that Japanese commercial inactivated vaccine contributed to reducing the morbidity rate and manifestation of the clinical signs of horses infected with Kanazawa/07 that may be antigenically different from the vaccine strains.     

云南地区马流感病毒检测及其HA基因序列分析

马流感能引起马属动物的高度接触性呼吸道传染病。该病毒已知仅存在H7N7(马流感病毒1型)、H3N8(马流感病毒2型)两个亚型,目前仅H3N8亚型毒株在全球范围内引起马流感流行和暴发。采用A型及H3亚型特异性RT-PCR技术,检测云南疑似马流感病例鼻腔棉拭子样品;HA基因PCR扩增产物经纯化后,克隆至pMD18-T载体进行测序,并与已知代表性毒株对应序列进行比对及系统发育分析。研究发现云南马流感病毒与已知代表毒株血凝素基因核苷酸及氨基酸序列同源性分别介于91.2%~99.2%和89.5%~98.4%,属于美洲谱系(American lineage)佛罗里达亚谱系(Florida sub-lineage)毒株。     

Protection, systemic IFNgamma, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host

In the horse, conventional inactivated or subunit vaccines against equine influenza virus (EIV) induce a short-lived antibody-based immunity to infection. Alternative strategies of vaccination have been subsequently developed to mimic the long-term protection induced by natural infection with the virus. One of these approaches is the use of immune-stimulating complex (ISCOM)-based vaccines. ISCOM vaccines induce a strong antibody response and protection against influenza in horses, humans, and a mouse model. Cell-mediated immunity (CMI) has been demonstrated in humans and mice after ISCOM vaccination, but rarely investigated in the horse. The aim of this study was to evaluate EIV-specific immune responses after intra-muscular vaccination with an ISCOM-EIV vaccine (EQUIP F) containing both equine influenza H7N7 (A/eq/Newmarket/77) and H3N8 (A/eq/Borl?nge/91 and A/eq/Kentucky/98) strains. The antibody response was measured by single radial haemolysis (SRH) assay using different H3N8 EIV strains. Stimulation of type-1 immunity was evaluated with a recently developed method that measures EIV-specific IFNgamma synthesis by peripheral blood lymphocytes (PBL). The protective efficacy of this ISCOM-based vaccine against challenge infection with a recent equine influenza (H3N8; A/eq/South Africa/4/03) strain was also evaluated. Vaccinated ponies developed elevated levels of EIV-specific SRH antibody and increased percentage of EIV-specific IFNgamma(+) PBL, whereas these responses were only detected after challenge infection in unvaccinated control ponies. Vaccinates showed minimal signs of disease and did not shed virus when challenged shortly after the second immunisation. In conclusion, evidence of type-1 immunity induced by an ISCOM-based vaccine is described for the first time in horses.     

福建省鸭禽流感病毒分离株（A/Duck/Fujian/FQ107/2007〔H9N2〕）全基因测序及遗传进化分析

为了解一株可引起产蛋鸭产蛋异常的H9N2亚型禽流感病毒A/Duck/Fujian/FQ107/2007（H9N2）（以下简称Dk/FQ107/07）分离株的分子特性及其遗传进化地位,运用RT-PCR方法对其基因组进行扩增,克隆至pMD18-T载体后测序。结果显示,Dk/FQ107/07病毒株的血凝素（haemagglutini,HA）蛋白裂解位点的氨基酸组成为-PARSSR↓GLF-,其静脉接种指数（intravenous pathogenicity index,IVPI）为0.04,符合低致病性禽流感病毒特征;Dk/FQ107/07株HA基因与A/chicken/Shantou/5269/2005（H9N2）同源性最高,为98.9%,和我国首次哺乳动物流感病毒分离株A/Swine/HongKong/9/98（H9N2）有较近的遗传进化关系,三者均属于经典的H9N2/Y280群系;神经氨酸酶（neuraminidase,NA）基因与中国大陆首次分离株A/chicken/Beijing/1/1994（H9N2）相比,在63、64、65位点上缺失3个氨基酸（T、E、I）;核蛋白（nucleoprotein,NP）基因与高致病性鸭源禽流感分离株A/Duck/Fujian/1734/05（H5N1）和A/Duck/Fujian/9713/2005（H5N1）在同一遗传进化分支上,而从聚合酶（polymerase PA,PA）基因的遗传进化分析发现其基因属于H9N2/Y439群系。由此可见,Dk/FQ107/07可能是由不同禽流感病毒基因亚群间发生自然重排的产物。     

马流感病毒双抗体夹心ELISA检测方法的建立

为建立一种快速、有效的检测马流感病毒(Equine influenza virus,EIV)的方法,以EIV中国分离株A/马/新疆/07(H3N8)制备的多克隆抗体为捕获抗体,原核表达的核蛋白(NP)制备的单克隆抗体为检测抗体,在国内首次建立了检测EIV的双抗体夹心ELISA方法.用该检测方法分别检测EIV、马动脉炎病毒、马疱疹病毒1型、马疱疹病毒4型和马乙型脑炎病毒阳性样品.结果表明,该ELISA方法具有良好的特异性;与常规检测EIV的血凝试验相比,其敏感性是后者的2.5～10倍;同时与H7N7亚型EIV有交叉反应.攻毒试验结果表明该方法可有效检测鼻腔分泌物中的EIV.该方法的建立为EIV的检测及早期防控提供了有效工具.     

湖北株H3N8亚型马流感病毒HA基因的序列测定及其HA蛋白遗传特性分析

2008年从湖北省分离到1株H3N8亚型马流感病毒A/equine/Hubei/6/08。以2002年美国KENTUKY株为模板设计HA基因测序引物,进行RT-PCR,然后测定该分离株的HA基因核苷酸序列。经NCBI上Blast同源性比较发现,与A/equine/Newmarket/5/2003(H3N8)同源性较高为98.7%。HA蛋白遗传进化分析表明该毒株隶属于H3N8亚型马流感病毒中的美洲系福罗里达亚系。该株与OIE现在推荐的疫苗候选株A/equine/Kentuck-y/5/2002(H3N8)HA1蛋白氨基酸序列比对发现有3处氨基酸替换位点;与OIE以往推荐的疫苗候选株A/e-quine/Kentucky/1/1994(H3N8)比对发现有11处氨基酸替换位点。研究结果表明该分离株可作为中国研制马流感疫苗的候选株。     

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.     

Efficacy of a canarypox-vectored recombinant vaccine expressing the hemagglutinin gene of equine influenza H3N8 virus in the protection of ponies from viral challenge

OBJECTIVE: To determine onset and duration of immunity provided by a 2- or 3-dose series of a new canarypox-vectored recombinant vaccine for equine influenza virus (rCP-EIV vaccine) expressing the hemagglutinin genes of influenza H3N8 virus strains A/eq/Kentucky/94 and A/eq/Newmarket/2/93 in ponies. ANIMALS: Forty-nine 1- to 3-year-old male Welsh Mountain Ponies that were seronegative for equine influenza virus. PROCEDURES: Vaccinated and control ponies were challenged with aerosolized influenza virus A/eq/Sussex/89 (H3N8), representative of the Eurasian lineage of circulating influenza viruses. In trial 1, control ponies and ponies that received rCP-EIV vaccine were challenged 2 weeks after completion of the 2-dose primary vaccination program. In trial 2, ponies were challenged 5 months after 2 doses of rCP-EIV vaccine or 1 year after the first boosting dose of rCP-EIV vaccine, administered 5 months after completion of the primary vaccination program. After challenge, ponies were observed daily for clinical signs of influenza and nasal swab specimens were taken to monitor virus excretion. RESULTS: The challenge reliably produced severe clinical signs consistent with influenza infection in the control ponies, and virus was shed for up to 7 days. The vaccination protocol provided clinical and virologic protection to vaccinates at 2 weeks and 5 months after completion of the primary vaccination program and at 12 months after the first booster. CONCLUSION AND CLINICAL RELEVANCE: The rCP-EIV vaccine provided protection of ponies to viral challenge. Of particular importance was the protection at 5 months after the second dose, indicating that this vaccine closes an immunity gap between the second and third vaccination.     

H3N8亚型马流感病毒间接ELISA抗体检测方法建立及应用

为建立马流感血清学ELISA诊断方法,本研究以马流感病毒中国分离株A/马/新疆/07(H3N8)通过SPF鸡胚培养和增殖,收取含病毒尿囊液经蔗糖密度梯度离心纯化后作为ELISA包被抗原,首次在我国建立了检测H3N8亚型马流感抗体的间接ELISA诊断方法。试验的最佳反应条件为:最佳抗原稀释度7μg/mL,封闭液5%脱脂乳,血清稀释度1∶100,二抗稀释度1∶10000,稀释液PBS(pH7.4),血清反应时间1.5h,二抗反应时间1h。通过本方法对555份临床样品进行检测并与血凝抑制(HI)试验检测结果比较,证明本方法特异、敏感,具有良好的稳定性和可重复性,适于马流感的流行病学调查和监测工作。     

中国类禽型H1N1亚型猪流感病毒的发现和遗传分析

采用禽流感病毒通用引物,对2006年发现的1株H1N1亚型的类禽型猪流感病毒的全基因组进行了测序,并进行了遗传学分析。序列分析表明它的8个片段与欧洲的类禽型猪流感病毒A/swine/Ile et Vilaine/1455/99(H1N1)病毒和A/swine/Cotes d'Armor/1488/99(H1N1)病毒的相应基因具有高度的同源性,同源性可达97%~99%,表明类禽型猪流感病毒已在中国出现。其血凝素基因的190E→D和225G→E的突变使得其结合NeuAc-a2,6Gal受体的能力高于NeuAca2,3Gal受体。欧洲的类禽型猪流感病毒可以直接感染人,并且可导致人的肺炎和死亡。中国类禽型猪流感病毒的发现及其的NeuAca2,6Gal受体结合特性使其成为一个潜在可感染人的病毒。     

Cardiac troponin I concentrations in ponies challenged with equine influenza virus

Background: Myocarditis is thought to occur secondary to equine influenza virus (EIV) infections in horses, but there is a lack of published evidence. Hypothesis/Objectives: We proposed that EIV challenge infection in ponies would cause myocardial damage, detectable by increases in plasma cardiac troponin I (cTnI) concentrations. Animals: Twenty‐nine influenza‐naïve yearling ponies: 23 were part of an influenza vaccine study (11 unvaccinated and 12 vaccinated), and were challenged with 10⁸ EID₅₀ EIV A/eq/Kentucky/91 6 months after vaccination. Six age‐matched healthy and unvaccinated ponies concurrently housed in a separate facility not exposed to influenza served as controls. Methods: Heparinized blood was collected before and over 28 days after infection and cTnI determined. Repeated measures analysis of variance, chi‐square, or clustered regression analyses were used to identify relationships between each group and cTnI. Results: All EIV‐infected ponies developed clinical signs and viral shedding, with the unvaccinated group displaying severe signs. One vaccinated pony and 2 unvaccinated ponies had cTnI greater than the reference range at 1 time point. At all other times, cTnI was <0.05 ng/mL. All control ponies had normal cTnI. There were no significant associations between cTnI and either clinical signs or experimental groups. When separated into abnormal versus normal cTnI, there were no significant differences among groups. Conclusions and Clinical Importance: This study demonstrated no evidence of severe myocardial necrosis secondary to EIV challenge with 10⁸ EID₅₀ EIV A/eq/Kentucky/91 in these sedentary ponies, but transient increases in cTnI suggest that mild myocardial damage may occur.