Equine Respiratory Viruses Research Articles

Respiratory viruses affecting health and performance in equine athletes.

Some respiratory viruses can affect equine athletes, with acute respiratory clinical signs leading to a reduced ability to perform. The direct association between equine respiratory viruses and athletic performance is unclear in subclinically affected horses. This narrative review summarises the current evidence on respiratory viruses most commonly detected in performing horses, including equine herpesviruses, equine influenza virus, equine rhinitis viruses, equine arteritis virus, and equine adenovirus 1. It covers their virology, clinical manifestations, epidemiology, pathogenesis, diagnosis, and control measures, with a focus on their impact on performance. Molecular diagnostics on nasopharyngeal swabs are the preferred method for detecting equine respiratory viruses nowadays. Studies highlighted in this review reveal a high prevalence of equine herpesviruses -particularly gammaherpesviruses- in the airways of both healthy and diseased horses. In contrast, equine rhinitis A virus, equine arteritis virus, and equine adenovirus 1 are the least common viruses. Transportation contributes to spreading equine infectious diseases across countries and can temporarily weaken the immune system, increasing the risk of respiratory viral infections and reactivation of latent equine herpesviruses. Moreover, respiratory viral infections are frequently observed in young horses starting their training. Although there is limited evidence on the specific impact of equine respiratory viruses on performance, this review emphasises that vaccination and care management are essential strategies for limiting the spread and severity of outbreaks in sport horses.

Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Equine respiratory viruses, airway inflammation and performance in thoroughbred racehorses

Equine asthma is a common cause of poor performance in racehorses but it is unclear if respiratory viruses contribute to its etiology. The objective of the study was to determine if respiratory viruses were associated with clinical signs and bronchoalveolar lavage fluid (BALF) cytology in Thoroughbred racehorses. Equine herpesviruses (EHV-1, 2, 4, 5) and equine rhinitis A and B viruses (ERBV, ERAV) genomes were quantified by qPCR in nasopharyngeal, tracheal, and BALF samples collected after racing. The relationships between virus detection and load and clinical signs, performance, BALF cytology, and environmental exposures were examined with generalized linear mixed models. Ninety-two samples were collected from 31 horses. EHV-1 and ERAV were not found; EHV-4 was detected in only one sample. EHV-2, EHV-5 and ERBV were more likely to be detected in upper airway samples than in BALF (P < 0.0001). Neither respiratory virus detection nor load was associated with clinical signs or performance. Nasopharyngeal detection and load of ERBV and tracheal detection and load of EHV-5 were associated with increased proportions of neutrophils in BALF (P < 0.003). However, nasopharyngeal detection and load of EHV-5 was not (P = 0.11). Nasopharyngeal detection and load of EHV-2 were associated with decreased BALF mast cell proportions. Respirable dust exposures were significantly higher in horses with detection of ERBV when compared to horses with no detectable ERBV (P < 0.001). Our results suggest that ERBV, EHV-2 and EHV-5 are commonly present in upper airways of healthy racehorses; however, the role they play in the etiology of equine asthma remains unclear.

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract.

Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes.

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.

Epidemiology and molecular detection of equine herpesviruses in western Algeria in 2011

An episode of acute equine respiratory infection was reported in western Algeria (Tiaret province) between February and March 2011, affecting a large population of horses. Nasal swabs (n=100) were taken from horses aged between 1 and 27 years, presenting with cough and mucopurulent nasal discharge. The prevalence of equine respiratory virus infections was examined using quantitative polymerase chain reaction (qPCR). One, or more, of four equine respiratory viruses were detected in the nasal swabs of 90 of 100 horses (90%) and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 2 (EHV-2) and equine herpesvirus type 5 (EHV-5) were 2%, 14%, 90% and 75%, respectively. Equine influenza virus and equine arteritis virus were not detected in any samples. Among the 90 infected horses, 70 were co-infected with EHV-2 and EHV-5 and 14 others were co-infected with EHV-4, EHV-2 and EHV-5. The present study shows a positivity rate of 97.3% for EHV-5 in young horses aged <3years; a finding which decreased with age. Viral load of EHV-5 was significantly higher in <3years whereas no effect of age was observed with EHV-2. The study shows that equine herpesviruses 1, 2, 4 and 5 are endemic in horse populations from Algeria as detected for the first time by qPCR.

Surveillance program for Equine Influenza Virus in the United States (2010-2013)

Recent surveillance studies for equine respiratory viruses have shown that equine influenza virus (EIV) remains a prevalent viral respiratory pathogen of horses throughout the USA and Europe. Due to the highly contagious nature and rapid spread of EIV, this virus poses severe financial implications for the horse industry. The objective of this voluntary surveillance study was to gain a better understanding of the prevalence and epidemiology of EIV shed by horses presented to veterinarians with an upper respiratory tract (URT) infection from March 2010 to November 2013. Equine practitioners throughout the United States (239 practices in 38 states) were asked to collect nasal secretions from equine patients with signs of acute URT infection. A questionnaire was used to collect information pertaining to patient demographics and clinical signs. Samples were tested by qPCR for the presence of EIV. Categorical analyses were performed using a Pearson's chi-square test to determine the association between observations (age, breed, gender, use, vaccination and clinical signs) and EIV. Further, categorical observations from EIV positive horses from March 2010 to November 2013 were compared to previous data fromMarch 2008 to February 2010 (60 EIV PCR positive horses). Sequencing analysis of the HA1 gene of EIV was performed on 35 different EIV isolates to determine their sequence homology. A total 2,375 horses were enrolled with 230 (9.7%) index cases testing PCR positive for EIV. EIV positive horses in the 1-5 and 6-10 year age group, as well as Quarter Horses, were over-represented, while stallions/colts were under-represented. Fever, nasal discharge and coughingweremore commonly observed in EIV positive horses than the entire study population. EIV positive cases from the 2010-2013 study period were significantly older and more often vaccinated against EIV compared to EIV positive animals from the 2008-2010 study period. Among the 84/230 EIV (+) horses with a confirmed vaccination status, 46% were vaccinated within the previous 6 months and 31% vaccinated within 12 months. All the sequenced EIV isolates belonged to the clade 1 Florida sublineage. This study provides valuable contemporary information on the frequency of EIV detected by qPCR and underscores the fact that older and previously vaccinated horses were susceptible to EIV. There was evidence for lack of vaccine effectiveness following use of inactivated EIV vaccines currently available, including those containing contemporary strains such as A/equine/Ohio/1/2003.

Voluntary surveillance program for equine influenza virus in the United States from 2010 to 2013.

BackgroundRecent surveillance studies for equine respiratory viruses have shown that equine influenza virus (EIV) continues to be a prevalent respiratory virus of equids throughout the United States and Europe.ObjectivesTo gain a better understanding of the prevalence and epidemiology of EIV shed by horses, mules and donkeys in the United States from March 2010 to November 2013.Animals2,605 equids.MethodsNasal secretions from index cases with acute onset of respiratory disease were tested by qPCR for EIV. Multilevel logistic regression was used to model the association between EIV status and prevalence factors. Furthermore, observations from EIV‐positive study horses were compared to previous data from March 2008 to February 2010.ResultsA total of 230 (9.7%) index cases tested qPCR positive for EIV. A higher‐than‐expected proportion of EIV qPCR‐positive horses occurred in the 1–5, 6–10, and 11–15 age groups when compared to the <1 year of age group. Fever, nasal discharge and coughing were positively associated with EIV‐positive horses. EIV qPCR‐positive study cases were significantly older and more often vaccinated against EIV compared to EIV qPCR‐positive animals from the 2008‐2010 study period.Conclusions and Clinical ImportanceThis study provides valuable and contemporary information on the frequency of EIV detected by qPCR in the United States. The results also underscore that older and previously vaccinated horses were susceptible to EIV.

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.

A survey of respiratory viruses in New Zealand horses

AIMS: To determine which viruses circulate among selected populations of New Zealand horses and whether or not viral infections were associated with development of respiratory disease. METHODS: Nasal swabs were collected from 33 healthy horses and 52 horses with respiratory disease and tested by virus isolation and/or PCR for the presence of equine herpesviruses (EHV) and equine rhinitis viruses. RESULTS: Herpesviruses were the only viruses detected in nasal swab samples. When both the results of nasal swab PCR and virus isolation were considered together, a total of 41/52 (79%) horses with respiratory disease and 2/32 (6%) healthy horses were positive for at least one virus. As such, rates of virus detection were significantly higher (p<0.001) in samples from horses with respiratory disease than from healthy horses. More than half of the virus-positive horses were infected with multiple viruses. Infection with EHV-5 was most common (28 horses), followed by EHV-2 (27 horses), EHV-4 (21 horses) and EHV-1 (3 horses). CONCLUSIONS: Herpesviruses were more commonly detected in nasal swabs from horses with respiratory disease than from healthy horses suggesting their aetiological involvement in the development of clinical signs among sampled horses. Further investigation to elucidate the exact relationships between these viruses and respiratory disease in horses is warranted. CLINICAL RELEVANCE: Equine respiratory disease has been recognised as an important cause of wastage for the equine industry worldwide. It is likely multifactorial, involving complex interactions between different microorganisms, the environment and the host. Ability to control, or minimise, the adverse effects of equine respiratory disease is critically dependent on our understanding of microbial agents involved in these interactions. The results of the present study update our knowledge on the equine respiratory viruses currently circulating among selected populations of horses in New Zealand.

Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses

Background: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. Results: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n=11) or ERBV (n=10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). Conclusion: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.

Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses

BackgroundEquine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections.ResultsThree rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml).ConclusionThe newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.

Development and Evaluation of One-Step TaqMan Real-Time Reverse Transcription-PCR Assays Targeting Nucleoprotein, Matrix, and Hemagglutinin Genes of Equine Influenza Virus

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Equine respiratory viruses in foals in New Zealand

AIMS: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses. METHODS: Foals were recruited to the study in October/November 1995 at the age of 1 month (Group A) or in March/April 1996 at the age of 4–6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date. RESULTS: The only viruses isolated were EHV-2 and EHV-5. EHV-2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2–4 months from January to April (Group A), March to April (Group B) or May to July (Group C). EHV-5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV-1 or EHV-4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV-1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV-1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV. Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres ≥1:10 to EAdV-1were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease. CONCLUSIONS: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

Cultures of equine respiratory epithelial cells and organ explants as tools for the study of equine influenza virus infection

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.

Isolation of equine herpesvirus type 5 in New Zealand

Aim. To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand. Methods. Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes. Results. EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease. Conclusion. EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.

Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus

A recombinant glutathione-S-transferase fusion protein expressing amino acids 55–98 of equine arteritis virus (EAV) GL (rGL55–98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with the EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post-EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rGL55–98 as a diagnostic antigen.

Prevalence of antibodies to equine viruses in the Netherlands

Summary The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested. The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi-1, M. Influenzae A / equi-2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested. The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.

An equine respiratory enterovirus. Some biological and physical properties.

Experiments involving the intranasal inoculation of monkeys, rabbits and guinea pigs with an equine respiratory virus were carried out. The animals were killed at various intervals after inoculation and attempts made to isolate virus in tissue culture from extracts of homogenized tissues and organs. All three species were susceptible, virus reproduction occurring in the respiratory tract and associated lymph glands. Of the three species virus was least readily isolated from the guinea pig tissues. There was a viraemia in monkeys and rabbits, but virus was less readily detected in the blood of guinea pigs. Virus was found in the urine and kidney cultures of some of the animals which had by then developed antibody. A human volunteer intranasally inoculated developed severe pharyngitis and swelling of the pharyngeal lymph glands; viraemia accompanied by fever occurred and lasted 4 days. The virus was unstable at pH 5.0 and below and its stability in distilled water was not increased by the addition of M MgCl2.