Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission

bs_bs_banner Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission DJ Middleton,* S Riddell, R Klein, R Arkinstall, J Haining, L Frazer, C Mottley, R Evans, D Johnson and J Pallister Objective Characterisation of experimental Hendra virus (HeV) infection in dogs and assessment of associated transmission risk. Methods Beagle dogs were exposed oronasally to Hendra virus/australia/horse/2008/redlands or to blood collected from HeVinfected ferrets. Ferrets were exposed to oral fluids collected from dogs after canine exposure to HeV. Observations made and samples tested post-exposure were used to assess the clinical course and replication sites of HeV in dogs, the infectivity for ferrets of canine oral fluids and features of HeV infection in dogs following contact with infective blood. Results Dogs were reliably infected with HeV and were generally asymptomatic. HeV was re-isolated from the oral cavity and virus clearance was associated with development of virus neutralising antibody. Major sites of HeV replication in dogs were the tonsils, lower respiratory tract and associated lymph nodes. Virus replication was documented in canine kidney and spleen, confirming a viraemic phase for canine HeV infection and suggesting that urine may be a source of infectious virus. Infection was transmitted to ferrets via canine oral secretions, with copy numbers for the HeV N gene in canine oral swabs comparable to those reported for nasal swabs of experimentally infected horses. Conclusion HeV is not highly pathogenic for dogs, but their oral secretions pose a potential transmission risk to people. The timewindow for transmission risk is circumscribed and corresponds to the period of acute infection before establishment of an adaptive immune response. The likelihood of central nervous system involvement in canine HeV infection is unclear, as is any longterm consequence. Keywords disease transmission; dogs; ferrets; Hendra virus; infections Abbreviations CNS, central nervous system; Ct, cycle threshold; dpi, days post-infection; HeV, Hendra virus; VNT, virus neutralisation test Aust Vet J 2017;95:10 18 doi: 10.1111/avj.12552 Hendra virus (HeV) is a zoonotic paramyxovirus that emerged in 1994 in the Brisbane (Australia) suburb of Hendra. 1 It was the first member to be characterised within a new viral genus Henipavirus in the order Mononegavirales and family Paramyxoviridae, wherein it forms a distinct clade with Nipah virus and Cedar virus. 2,3 HeV infection causes a fulminating systemic vasculitis with high mortality rate in diverse animal species, *Corresponding author. CSIRO Australian Animal Health Laboratory, PB24 Geelong, Victoria, 3220, Australia; Deborah.middleton@csiro.au naturally in the horse and humans and experimentally in the ferret and cat; some animals such as rats and chickens appear to be resistant to HeV infection. In an early laboratory study of two dogs exposed to HeV, one developed neutralising antibody following asymptomatic virus replication and the other showed no evidence of infection. 4 More recently, antibody to HeV has been found in asymptomatic dogs on two properties that were experiencing HeV outbreaks in horses. 5,6 In at least one of those cases, the infected dog may have had exposure to the blood of an infected horse. 6 The close relationship between dogs and people has raised a question regarding the potential of dogs to act as a source of transmission of HeV to people. A particular concern is that HeV infection may be clinically silent in the canine species and so there may be no indication of when appropriate infection control procedures should be instituted. The aim of this work was to further characterise experimental HeV infection in dogs and to form a preliminary assessment of the potential for dogs to transmit HeV infection to susceptible humans or other animals. The work was conducted as four studies with temporally overlapping components. These comprised (i) an observational study to evaluate the clinical course of HeV infection in dogs and to record the duration and routes of viral shedding, (ii) a time-course study to determine the sites of virus replication and to provide canine-derived inoculum for the following study, (iii) an assessment of infectivity for ferrets of an inoculum comprising canine oral fluids and (iv) a preliminary characterisation of HeV infection in dogs following their exposure to infective blood. Materials and methods Animals, accommodation, handling and biosafety Each stage of the work was approved by the CSIRO AAHL Animal Ethics Committee (Animal Ethics Committee approval no. 1502) and conducted according to the Australian code for the care and use of animals for scientific purposes, 8th edition. We used 16 purpose-bred male and female Beagle dogs, designated 1 16 and aged 5 8 months, in the studies. All dogs had received a single immunisation against Bordetella bronchiseptica; they had not been given canine distemper virus vaccine, even though serum containing antidistemper virus antibodies does not cross-neutralise HeV (P Selleck, unpubl. data). Dogs were extensively handled during the critical human socialisation period to ensure they interacted positively with staff, including ready acceptance of food treats, exposed to novel environments to ensure their resilience when transferred to the Biosafety Level 4 (BSL4) accommodation and trained to accept a collar, lead and muzzle. These preparations ensured that the dogs could be managed safely under the BSL4 containment conditions required for work with infectious HeV. All dogs were housed at least 10

in pairs for the duration of the study. They were fed with a balanced proprietary canine kibble, offered water ad lib and provided with soft sleeping platforms as well as a changing array of enrichment items, including slow-release food treats. We also used 8 male and female ferrets, designated 1 8 and approximately 12 months old, in these studies. Ferrets were sourced commercially and had not been given canine distemper virus vaccine. Ferrets were housed in cages in pairs, given a dry ration appropriate to the species and also dietary treats, offered water ad lib and provided with soft, dark sleeping quarters and a varying array of toys. Temperature-sensing microchips (Lifechip, Destron Fearing, TX, USA) were injected under the skin on the lateral thorax of all study animals. For exposure to HeV, and collection of biological samples during the observational study, dogs were sedated with SC combination butorphanol (1 mg/kg; Dolorex, MSD Animal Health) and medetomidine (0.01 mg/kg; Domitor, Zoetis) which was reversed with atipamezole (Antisedan, Zoetis) at the same volume of medetomidine. For euthanasia, sedation as described was augmented with IM ketamine (5 mg/kg; Ketamil, Ilium) and then intracardiac barbiturate (150 mg/kg; Lethabarb, Virbac Animal Health) was administered. For exposure to potentially infective inoculum and collection of biological samples in the ferrets, anaesthesia was induced with IM combination of ketamine (5 mg/kg; Ketamil ) and medetomidine (0.05 mg/kg; Domitor ), which was reversed with atipamezole (Antisedan ) at half the medetomidine dose. For euthanasia of the ferrets, anaesthesia was augmented by intracardiac barbiturate (150 mg/kg; Lethabarb ). The animal rooms were maintained at 22 C with 15 air changes/h; humidity ranged from 40% to 60%. While in the BSL4 animal room, staff wore fully encapsulated suits with an external air supply. Animal infection Dogs. Under BSL4 containment conditions, 14 of the 16 dogs were exposed to the third passage in standard Vero cells of an equine isolate of HeV (Hendra virus/australia/horse/2008/redlands [GenBank accession no. HM044317]) via mouth and nose drops (2.5 ml per site), a route selected on the basis that it was a plausible natural route of exposure. The virus dose used was 2 10 6 TCID 50 (median tissue culture infective dose) because this amount of virus reliably induces disease in other susceptible species such as horses, cats and ferrets and leads to death in those species if intervention by euthanasia is not carried out. Under BSL4 containment conditions, 2 of the 16 dogs were exposed to the third passage in standard Vero cells of an equine isolate of HeV (Hendra virus/australia/horse/2008/redlands [GenBank accession no. HM044317]), which had undergone a further single in vivo passage in dogs, followed by a single in vivo passage in ferrets. This inoculum was prepared as follows. Blood was collected into EDTA and plain blood tubes from ferrets 3 and 4 (exposed to day 4 canine oral wash) when they were euthanased for HeV-associated disease. Stored aliquots of EDTA blood were then combined with serum and clot from the plain blood tubes to maximise the volume of inoculum. Although virus re-isolation was not attempted using the ferrets blood samples, HeV was re-isolated from the spleen of both animals. Serum cycle threshold (Ct) values (defined as the number of cycles required for the fluorescent signal to cross the threshold i.e. to exceed the background level) for the HeV N gene were 33.89 (ferret 3) and 36.46 (ferret 4), and the ferret sera had relative estimated TCID 50 titres of 1.5 10 4 /ml and 4.3 10 3 /ml based on comparison with a standard curve. The pooled ferret blood was administered to the two dogs via mouth and nose drops (2.5 ml per site). Residual blood inoculum was mixed with dry kibble and later consumed by the dogs on their recovery from sedation. Ferrets. Two ferrets were moved into the BSL4 facility and housed in clean cages on each of days 2, 4, 6 and 8 after exposure of dogs to Hendra virus/australia/horse/2008/redlands. Ferrets moved on day 8 were housed in a BSL4 facility separate to any of the dogs because there was insufficient ferret caging for them in the first facility. Each ferret was exposed to 1 ml of pooled canine oral wash that had been collected earlier on the same day (see later). The oral wash material was administered to the ferrets by the oronasal route, as used for our standard ferret challenge in henipavirus studies. 7 Where oral wash inoculum was retained after ferret exposure, an aliquot was tested by PCR for viral genome and by virus isolation. Sample collection and analysis For the observational study, six dogs were monitored twice daily for 3 4 weeks after exposure to HeV. Nasal, oral, urethral and rectal swabs and blood were collected prior to HeV exposure and on days 2, 4, 6, 8, 10, 12, 14, 17, 20 and 27 post-infection (dpi). In two dogs, additional samples of oral secretions were regularly collected from the back of the throat by means of surgical gauze swabs that had been soaked in saline to minimise mucosal trauma. All clinical samples were tested for HeV genetic material (N gene) and for infectious virus as previously described. 7 Dogs were euthanased electively at 20 27 dpi. For the time-course study, eight dogs were monitored twice daily for up to 8 dpi. Two dogs were euthanased on each of days 2, 4, 6 and 8. On the day of euthanasia, dogs were first anaesthetised for collection of clinical samples as described earlier, including an oral wash. Oral washes were obtained by rinsing the oral cavity of each dog with 3 ml phosphate-buffered saline, focussing the lavage on the back of the throat. Approximately 1 ml of the wash was retrieved from each dog; the wash material from the two dogs was pooled in sterile sample tubes and stored on wet ice for blind passage to naive ferrets later on the same day. For the ferret infectivity study, eight ferrets were monitored once or twice daily prior to either elective euthanasia on day 8 or 21 postexposure to canine oral fluids or at a predetermined humane endpoint for HeV-associated disease. Clinical samples, including nasal washes, oral and rectal swabs, and blood were collected immediately prior to euthanasia. For the study using infective ferret blood inoculum, the two dogs were monitored twice daily prior to elective euthanasia at 6 dpi. This day was selected on the basis of information about HeV infection dynamics, which had been provided by the earlier time-course study. Postmortem examinations were carried out on each animal. Diverse tissues were collected, tested for viral genome by PCR and virus 11

isolation if viral genome was detected, or examined by histology and immunohistochemistry. 7 Seroconversion was assessed by conventional virus neutralisation test (VNT) 7 and/or by Luminex (blocking and binding) assays. 8 Results Observational study Clinical findings and virological analysis of shedding samples. Dogs 1, 2, 3, 5, 7 and 10 were used in this study. One dog remained asymptomatic. Where clinical signs were observed in the other dogs, they were subtle and comprised lack of usual enthusiasm for food treats, mild reduction in intake of dry food and mild conjunctivitis or scleral injection; signs lasted up to 48 h and all dogs were normal by 9 dpi. Tonsillar hyperplasia was recorded in dog 7 at 2 and 6 dpi, and in dog 10 at 8 dpi. Body temperature was assessed daily from the microchip and every 2 days by rectal thermometer; temperatures remained within the normal range for dogs (<39.3 C), apart from readings of 39.5 C in dogs 1 and 2 at 6 dpi and 39.6 C in dog 1 at 10 dpi. HeV was re-isolated from oral or throat swabs from four of the six dogs between 2 and 4 dpi; virus was not re-isolated from nasal swabs, urethral swabs, rectal swabs or blood at any sampling time (data collated in Table 1). HeV genetic material was not recovered from any clinical sample after 10 dpi (data collated in Table 1 and Figure 1). Postmortem findings: gross pathology, immunohistopathology and virology. No significant abnormalities were noted at postmortem examination of dogs 1, 2 and 5, and no significant histological lesions were detected in adrenal glands, bladder, brain (including olfactory pole), gonads, heart, kidneys, large intestine, liver, lungs, lymph nodes (superficial cervical, bronchial, inguinal, lumbar, mandibular, retropharyngeal), meninges, pharynx, prostate or uterus, salivary glands, small intestine, spinal cord, spleen, trachea or trigeminal ganglion. There was follicular hyperplasia of the tonsillar lymphoid tissue and several focal areas of infiltration of tonsillar epithelium by neutrophils. In dog 3, significant abnormality was confined to a small focus of chronic fibrosing bronchopneumonia in the right lung. In dog 7, there was focal erosive tonsillitis associated with superficial bacterial colonies. There was also very mild chronic interstitial nephritis, and subacute focal bronchiolitis and bronchopneumonia in the left diaphragmatic lung lobe. Dog 10 had tonsillar hyperplasia and hyperaemia. Mild focal subacute bronchiolitis was also identified in its left apical lung lobe, as well as a scanty perivascular cuff in the cerebellar white matter. HeV antigen was not identified in any tissue examined from any dog. Samples assessed for viral genome from each dog included adrenal gland, bladder, brain (including olfactory pole), cerebrospinal fluid, heart, kidney, large intestine, liver, lung, lymph nodes (superficial cervical, inguinal, lumbar, mandibular, retropharyngeal, tonsil), meninges, nerve, pharynx, prostate or uterus, small intestine, spinal cord, spleen, gonad, turbinate and trigeminal ganglion. Generally low levels of HeV genome were identified at 20 or 27 dpi and were confined to lymphoid tissues; the data are summarised in Figure 2. HeV was not re-isolated from any sample that was positive by PCR test. Re-isolation of virus was not attempted on postmortem specimens that were negative by PCR. Table 1. Observational study: HeV re-isolation and HeV genome recovery from clinical samples Dog no./sex Day 2 Day 4 Day 6 Day 8 Day 10 1/M Oral swab / /+ /+ / / Throat swab NT NT NT NT NT Rectal swab / / / /+ / Blood / / / /+ /+ 2/M Oral swab / /+ / / / Throat swab NT NT NT NT NT Rectal swab / / / / / Blood / /+ / / /+ 3/M Oral swab +/+ +/+ /+ /+ / Throat swab +/+ /+ /+ /+ /+ Rectal swab / / / / / Blood / / /+ / / 5/M Oral swab / +/+ /+ / / Throat swab /+ /+ /+ /+ / Nasal swab / /+ / / / Rectal swab / / / /+ / Blood / / / / / 7/F Oral swab +/+ /+ / / / Throat swab /+ /+ / / / Rectal swab / / / / / Blood / / / / / 10/M Oral swab / +/+ / / / Throat swab /+ +/+ / / / Rectal swab / / / / / Blood / / / / / +/+, positive for virus/viral genome; /, negative for virus/viral genome. HeV, Hendra virus; NT, not tested. Serology. Dogs 1 and 2 were tested by Luminex (blocking and binding) assays and were positive for antibody to HeV at 14 dpi (data not shown). All dogs were repeatedly tested by conventional VNT and had developed serum neutralising antibody to HeV by 14 dpi (Table 2). Time-course study (days post-infection) Dogs 4, 6, 8, 9, 11 and 12 were used in this study. Day 2. Dogs 11 and 12 were clinically well until euthanasia on day 2. Rectal temperatures at euthanasia were 40.1 Cand39.4 C, respectively, consistent with fever. Abnormalities detected on postmortem examination of both dogs were confined to the oral cavity, lower respiratory tract and associated lymph nodes. There was mild tonsillar 12

Figure 1. Observational study: quantitative reverse transcription-pcr detection of Hendra virus N gene in canine clinical samples. Figure 2. Observational study: quantitative reverse transcription-pcr detection of Hendra virus N gene in canine tissue samples (median + interquartile range). BLN, bronchial lymph node; RPLN, retropharyngeal lymph node; SMLN, submandibular lymph node. Table 2. Observational study: HeV neutralising antibody titres in serum days post-infection (two replicates) Dog no./sex Day 0 Day 14 Day 17 Day 20 Day 27 1/M <8 64, 64 NS 128, 128 2/M <8 32, 32 NS 256, 512 3/M <8 128, 128 256, 256 512, 512 512, 1024 5/M <8 8, 16 16, 32 32, 32 64, 64 7/F <8 64, 128 128, 256 256, 256 10/M <8 32, 32 32, 32 128, 128, euthanased 20 days dpi. HeV, Hendra virus; NS, no sample. enlargement accompanied by small haemorrhages, enlargement of the bronchial lymph nodes in dog 12 and early consolidation of the left cardiac lung lobe in both animals. Histological examination revealed mild acute ulcerative tonsillitis with epithelial syncytia. Mild to moderately severe acute focal bronchioloalveolitis was identified in the left apical, left diaphragmatic, right apical and right cardiac lung lobes, together with mild focal subacute pneumonia in the right diaphragmatic lung lobe. Hyperplasia of the retropharyngeal lymph node was noted. HeV antigen was detected in tonsillar lesions and isolated tonsillar epithelial cells, in isolated cells within the bronchial lymph node and in the acute pulmonary lesions, including epithelial cells, bronchiolar muscle cells and intraluminal debris. No histological lesions or HeV antigen were noted in other tissue examined, including brain. HeV and HeV genome were not recovered from oral, throat, nasal, urethral or rectal swabs, or from blood of either dog euthanased on day 2 (HeV genome data for clinical samples from all dogs collated in Figure 3a). Virus was grown from lung samples of both animals and viral genome was found in lung, bronchial lymph node and spleen (HeV genome data for tissue samples from all dogs summarised in Figure 3b). Day 4. Dogs 8 and 9 were clinically well until euthanasia on day 4. Rectal temperatures at euthanasia were 37.7 C and 38.8 C, respectively. Abnormalities detected on postmortem examination were confined to the oral cavity, lower respiratory tract and associated lymph nodes. There was hyperplasia and hyperaemia of the tonsils in dog 9 (Figure 4), congestion of the bronchial lymph nodes and localised early haemorrhagic consolidation within, variously, the right apical, left cardiac and left or right diaphragmatic lung lobes. Histological and immunohistological changes in grossly abnormal tissues were similar to those described for dogs at 2 dpi, but HeV antigen was also identified in tonsillar lymphoid tissue, pulmonary vascular endothelium and perivascular cells, and in endothelial cells, subcapsular sinuses and parafollicular areas of the bronchial lymph node. No histological lesions or HeV antigen were noted in other tissues examined, including brain. HeV was re-isolated from oral and throat swabs of dog 8, tonsillar and lung tissue of both animals, soft palate and pharynx (dog 9) and retropharyngeal and bronchial lymph nodes (dog 8). HeV genome was recovered from oral and throat swabs in both animals and from the blood of dog 8. Tissues that were positive for HeV RNA were tonsil, soft palate, pharynx and lung in both dogs, and retropharyngeal and bronchial lymph nodes in dog 8. Day 6. Dogs 4 and 6 were clinically well until euthanasia on day 6. Rectal temperatures at euthanasia were 38.7 C and 38.8 C, respectively. Abnormalities detected on postmortem examination were confined to the oral cavity, lower respiratory tract and associated lymph nodes. There was marked bilateral tonsillar hyperplasia with superficial reddening and focal erosion of tonsillar tissue. There was hyperplasia of tracheobronchial lymph nodes (Figure 5: black arrow), hyperplasia with haemorrhage and congestion of the mediastinal lymph nodes and marked hyperplasia (6 3 2 cm) of the retropharyngeal lymph nodes, which also had focal areas of haemorrhage and congestion visible from the capsular surface. There was 13

A B Figure 3. (a) Time-course study: quantitative reverse transcription-pcr detection of Hendra virus N gene in canine clinical samples (median + range). (b) Time-course study: quantitative reverse transcription-pcr detection of Hendra virus N gene in canine tissue samples (median + range). LN, lymph node. Figure 5. Hyperplasia of tracheobronchial lymph nodes (black arrow) and pulmonary consolidation (white arrow) in dog 4 at 6 days post-infection. Figure 4. Tonsillar hyperplasia and hyperaemia in dog 9 at 4 days post-infection. red purple consolidation of the left cardiac lung lobe in dog 4 (Figure 5: white arrow) and purple consolidation of the dorsal hilar region of the right diaphragmatic lung lobe (dog 6). Histological examination revealed acute ulcerative tonsillitis (Figure 6a), associated with the presence of HeV antigen in residual tonsillar epithelium accompanied by smaller antigen deposits in the underlying lymphoid tissues (Figure 6b). In the retropharyngeal and bronchial lymph nodes there was focal necrotising lymphadenitis with syncytial cell formation; HeV antigen deposition was associated with areas affected by lymphadenitis. In grossly abnormal areas of lung, there was necrotising bronchoalveolitis, but vasculitis with endothelial syncytia was rarely identified. Positive immunostaining was noted in bronchiolar epithelium and luminal debris and also in vascular endothelium. No histological lesions or HeV antigen were noted in other tissues examined, including brain. HeV was not recovered from oral, throat, nasal, urethral or rectal swabs, or from the blood of either dog euthanased on day 6; however, viral genome was detected in the nasal and oral swabs of both dogs and in the throat swab and blood of dog 4. HeV was re-isolated from tonsillar and lung tissue of both animals, and from the lung and retropharyngeal and bronchial lymph nodes of dog 4. Tissues that were positive for HeV RNA were tonsil, soft palate, lung, spleen and the retropharyngeal and bronchial lymph nodes in each dog, the pharynx and periaortic lymph nodes of dog 6 and the kidney, liver and intestine of dog 4. Day 8. Dogs 13 and 14 were clinically well until euthanasia on day 8. Rectal temperatures at euthanasia were 38.3 Cand39.1 C, respectively. Abnormalities detected on postmortem examination were 14

Figure 6. (A) Ulcerative tonsillitis in dog 4 at 6 dpi (H&E). (B) Ulcerative tonsillitis in dog 4 at 6 dpi (IPX. anti-nipah N polyclonal antibody). confined to the lower respiratory tract and comprised consolidation, variously, of the caudal aspects of the right apical and right cardiac lung lobes or the ventral aspects of the left apical and left cardiac lung lobes. On histological examination there was mild to severe subacute bronchointerstitial pneumonia with focal necrosis and desquamation of bronchiolar epithelium, plus vasculitis and fibrinous alveolar exudate, affecting both left- and right-sided lung lobes. Mild acute erosive tonsillitis, reactive hyperplasia and focal lymphadenitis of the retropharyngeal lymph node, mild interstitial nephritis and focal glomerular necrosis (dog 14) were also identified. Very mild focal nonsuppurative meningitis was noted in the brainstem and forebrain (dog 13), as well as occasional small perivascular cuffs in the brain of dog 14; these observations have uncertain significance and may have been incidental findings. Small amounts of HeV antigen were found in the pulmonary lesions, including the walls of blood vessels, as well as within lesional areas of the retropharyngeal lymph node, tonsil (including tonsillar exudate) and renal glomeruli and interstitial tissue. No histological lesions or immunostaining were noted in other tissues examined and viral antigen was not identified in brain. HeV was not recovered from oral, throat, nasal, urethral or rectal swabs, or from blood of either dog euthanased on day 6 but virus was isolated from the lung of dog 14. Viral genome was detected in the lung and bronchial lymph nodes of both dogs and in throat swab, soft palate, pharynx, spleen and retropharyngeal lymph node of dog 13, and in kidney, periaortic lymph nodes and liver of dog 14. Serology. As expected from the short duration of this study, no dog in the time-course study developed serum neutralising antibody to HeV by the day of euthanasia; all titres by VNT were <8. Infectivity of canine oral fluids for ferrets Day 2 canine oral fluid inoculum. HeV genome was not detected in the oral wash from either of the donor dogs. We already know that exposure dose does not influence the incubation period of HeV in ferrets, 7 so for logistical reasons we elected to euthanase these two ferrets on day 8 post-exposure to canine oral fluids, when acute systemic HeV infection would have been detectable had it occurred. 7,9,10 Both ferrets remained clinically well until electively euthanased and no significant gross or histological lesions were identified in the target organs for HeV infection, including lung, spleen, kidney and brain. Viral antigen was not detected in any tissue examined. Viral genome was not recovered from any tissue sampled; attempts at virus reisolation were therefore not undertaken. There was no detectable serum antibody to HeV by VNT. It was concluded that the ferrets had not been infected by oronasal exposure to canine oral fluids. Day 4 canine oral fluid inoculum. HeV genome was detected in the oral washes from both donor dogs (dog 8: Ct 31.64; dog 9: Ct 34.33). One of the two ferrets was febrile (41.5 C) and showed reduced play activity on day 8 post-exposure to canine oral secretions, and the other showed reduced play activity on day 7 and hindlimb ataxia by day 8. Both ferrets were euthanased. Immunohistological lesions consistent with HeV infection were confirmed in ferret lung, spleen and kidney. HeV antigen was also found in meningeal and brain parenchymal blood vessels, gonads and bronchial lymph nodes. HeV genome was detected in numerous tissues; virus was re-isolated from several of these, including lung, spleen, retropharyngeal lymph nodes and gonads of both animals. There was no detectable serum antibody to HeV by VNT. It was concluded that each ferret had been successfully infected with HeV. The incubation period was consistent with infection of ferrets having occurred after exposure to the canine oral fluids. 7,9,10 15

Day 6 canine oral fluid inoculum. There was insufficient canine oral wash remaining after ferret exposure for testing by PCR or virus isolation, but HeV genome was detected in the oral swabs from both dogs and in the throat swab from dog 4. One ferret was febrile (40.4 C) on day 6 post-exposure to the oral fluid inoculum, showed reduced play activity on day 7 and was euthanased. The other ferret had HeV genome in blood on day 9 (Ct 32.69), consistent with virus replication, was febrile (41.1 C) and showed reduced play activity by day 11; it was euthanased. Both animals had necrotising lymphadenitis, necrotising bronchopneumonia and splenic and glomerular necrosis consistent with acute HeV infection. HeV antigen and genome was detected in numerous tissues and virus was re-isolated from lung, spleen and kidney. There was no detectable serum antibody to HeV by VNT. It was concluded that each ferret had been successfully infected with HeV. The incubation period was consistent with infection of ferrets having occurred after exposure to the canine oral fluids. 7,9,10 Day 8 canine oral fluid inoculum. A low level of viral genome (Ct 38.66) was detected in the pooled oral wash from both dogs, but virus was not re-isolated from the fluid. Both ferrets remained clinically well and, on this occasion, were able to be electively euthanased 21 days post-exposure to the inoculum. No significant histological lesions were identified in target organs for HeV infection, including lung, spleen, kidney and brain. Viral antigen was not detected in any tissue examined. Viral genome was not recovered from any tissue sampled; virus re-isolation was therefore not undertaken. There was no detectable antibody to HeV by VNT. It was concluded that the ferrets had not been infected by oronasal exposure to canine oral fluids. HeV in dogs following exposure to infective ferret blood Clinical observations. Dogs 15 and 16 were used in this study and remained clinically well until they were electively euthanased 6 dpi. Temperatures prior to euthanasia were 37.5 C and 38.8 C, respectively. Postmortem findings: gross pathology, immunohistopathology and virology. In dog 15, there were focal areas of consolidation in the left apical, cardiac and diaphragmatic lung lobes. In grossly abnormal areas of lung there was moderately severe subacute bronchointerstitial pneumonia. Peracute multifocal necrotising tonsillitis was also identified, as well as increased numbers of lymphocytes in the subcapsular sinuses and perinodal tissue, acute haemorrhage and syncytial cells in the subcortical regions of the bronchial lymph node. Small necrotic foci associated with a neutrophilic infiltrate were noted in the spleen. Low levels of HeV antigen were seen in pneumonic areas of lung, including the walls of blood vessels; HeV antigen was also found in bronchial lymph nodes, the alveolar wall of histologically normal lung, tonsillar lymphoid but not epithelial tissue, occasional germinal centres in the spleen and in some renal glomeruli. In dog 16, abnormalities detected at postmortem examination comprised tonsillar hyperplasia and a solitary 1 cm area of consolidation in the left diaphragmatic lung lobe. In the grossly abnormal area of lung there was mild focal bronchointerstitial pneumonia; only mild focal alveolitis was identified in other lung lobes. Focal necrosis of Figure 7. Hendra virus antigen in a renal glomerulus of dog 16 at 6 dpi (ferret blood inoculum) (IPX. anti-nipah N polyclonal antibody). Scale bar = 50 μm. dpi, days post-infection. tonsillar epithelium was also noted. HeV antigen was identified in some of the pulmonary lesions, including vascular endothelium, parafollicular cells of the bronchial lymph nodes, periarteriolar lymphoid sheaths of the spleen, occasional renal glomeruli (Figure 7) and scattered pericortical cells (possibly in afferent lymphatics) of the periaortic lymph nodes. Histological lesions or immunostaining were not noted in other tissue examined, including no viral antigen in the brain of either dog. HeV was not re-isolated from clinical samples, but HeV genome was detected in the tonsil and periaortic lymph nodes of dog 15 and in the lungs, bronchial lymph nodes, spleen and kidneys of both animals (Figure 8). HeV was re-isolated from the spleen of dog 15. Figure 8. Quantitative reverse transcription-pcr detection of Hendra virus N gene in canine tissue samples at 6 days post-exposure to infective ferret blood (median + interquartile range). BLN, bronchial lymph node. 16

Serology. As expected from the short time between exposure to inoculum and euthanasia, neither dog in the ancillary transmission study had developed serum neutralising antibody to HeV by the day of euthanasia; all titres by VNT were < 8. Discussion In the current studies, dogs were reliably infected with HeV following exposure to either a tissue culture isolate from the spleen of a naturally infected horse or to blood acquired from ferrets at the peak of their experimentally induced disease. Although some dogs developed subtle, non-specific and short-lived signs of infection, others remained clinically well throughout the period of virus replication; virus clearance was temporally associated with the development of virus neutralising antibody. These observations are consistent with the two canine field cases, in which neutralising antibodies against HeV were found in reportedly asymptomatic dogs that were tested during investigation of HeV events in horses 5,6 and from which infectious virus was not recovered. HeV was repeatedly but not always recovered from the oral cavity of experimentally infected dogs between 2 and 4 days after virus exposure, but not from other mucosal sites or from blood at any time. As expected, because of the higher test sensitivity, HeV genome was identified more consistently than live virus and in all animals. Gene copy numbers were highest in oral swab samples, although positive test results were also obtained from a nasal swab, as well as from rectal swabs and blood samples later in the course of infection. Based on current knowledge, we suggest that oral swabs are the preferred diagnostic sample for confirmation of acute HeV infection in the live dog. All samples were negative for HeV genome after 10 dpi and all dogs had virus neutralising antibody by 14 dpi. Canine oral secretions collected during acute infection (on days 4 and 6 post-exposure to HeV) were capable of direct transmission of virus to ferrets, which then died from HeV-associated disease. HeV infection did not develop in ferrets exposed to canine oral fluids that were collected 2 and 8 dpi in the time-course study, but HeV was re-isolated from canine oral swabs as early as 2 dpi in the observational study. Importantly, we also noted that gene copy numbers for the HeV N gene recovered from canine oral swab samples were comparable to those recovered from the nasal swabs of experimentally infected horses 11,12 that had not been vaccinated against HeV. As infection of people by HeV is epidemiologically associated with contact with the body fluids of acutely infected horses, our data overall suggested that oral secretions from HeV-infected dogs pose a potential transmission risk to people and that the timeframe for transmission risk is circumscribed and corresponds to the period of acute infection prior to establishment of an adaptive immune response. HeV replication was judged to have occurred in tissues where viral antigen was identified by immunohistochemistry and/or from which virus was re-isolated together with consistent histopathology. Identification of the HeV genome alone was not regarded as sufficient evidence that a particular tissue was a site of virus replication, as a positive result may merely have reflected sequestration of virus or genomic fragments in, for example, phagocytes or in cellular debris within lymph or blood. Accordingly, the major sites of HeV replication in dogs were the tonsillar epithelium and underlying lymphoid follicles, lower respiratory tract including pulmonary vascular endothelium, bronchiolar epithelium and muscle cells, and the subcapsular sinuses and parafollicular areas of associated lymph nodes. On that basis, exudates from the inflamed tonsillar tissues and/or lower respiratory tract airway debris are plausible sources of infectious virus present in oral fluids. Virus replication was also documented in the canine kidney and spleen from 6 dpi, confirming that there is a viraemic phase for HeV infection in the dog, involving virus replication in leucocytes or lymphocyte-mediated transfection. 13 Although in this study virus was not recovered from urine samples, the demonstration of HeV replication within the canine kidney suggests that the urine of infected dogs should be also regarded as a potential source of infectious virus. HeV and the closely related paramyxovirus, Nipah virus, exhibit broad species tropism and demonstrate an infection capacity that spans six mammalian Orders. 14 The viruses enter host cells via attachment to evolutionarily conserved cellular receptors comprising ephrin-b2 or ephrin-b3 proteins: 15 these receptors are regulators of axonal pathfinding, migration of neuron precursors and vasculogenesis and are expressed in endothelial and smooth muscle cells in the arterial walls, as well as in the lungs and central nervous system (CNS). 16,17 However, although the range of host species is extensive, there is great variability between species in the clinical effect of infection following either natural or experimental exposure to HeV. For example, humans, non-human primates, horses, cats, ferrets, golden hamsters and guinea pigs are susceptible to acute infection by HeV, leading to systemic vasculitis, encephalitis and pneumonia with significant likelihood of death. 7,18 20 In contrast, HeV has not been associated with any disease in the natural reservoir species, Australian pteropid bats. 21 Aged mice, on the other hand, reliably develop a mild infection phenotype comprising transient and self-limiting infection of the upper and lower respiratory tract accompanied by minor inflammatory response, without viraemia or progression to generalised disease and in the absence of a robust neutralising adaptive immune response. 22 Although encephalitis has been observed in mice, it was most likely established via anterograde entry of virus into the CNS through olfactory sensory neurons with subsequent transneuronal spread, as opposed to the viraemia and haematogenous dissemination of virus that appear to be important in other species (reviewed by Weingartl et al. 23 ). The tissue tropism of HeV in the present experimental dogs was generally similar to that recorded for other species, but the inflammatory lesions induced by the virus in the dogs were considered to be more cellular, less haemorrhagic and less necrotising by comparison with those found in either ferrets or cats under similar exposure conditions, and associated with fewer syncytial cells and smaller amounts of viral antigen. 7,24 An important differentiating feature, and one that likely translated to the good clinical outcome in the experimental dogs, was that virus replication was largely confined to the oropharynx, respiratory tract and associated lymph nodes, without disseminated vasculitis or significant pathology in the spleen and kidneys. On the spectrum of HeV pathogenicity for mammals, dogs appear to be intermediate between the mouse and either ferrets, cats, horses or people. The basis of such species differences in the 17

pathogenicity of HeV and Nipah virus is not understood, although the functionally similar binding of the attachment glycoprotein to ephrin-b2 and ephrin-b3 in humans, horses, pigs, cats, dogs, bats (Pteropus alecto and P. vampyrus) and mice suggests it is mediated by post-entry steps. 25 Our experimental observations of HeV infection in dogs generally align with the description of a canine field case of HeV infection that also underwent detailed postmortem examination. 6 However, one point of divergence was the presence of non-suppurative meningoencephalitis, including cerebral vasculitis, in the field case, lesions that were clearly attributable to HeV. In contrast, there was no evidence in any of our experimental dogs of virus, genome or lesions in the brain that could be reliably attributed to HeV infection, even in dogs directly exposed to infective ferret blood rather than to a tissue culture isolate of HeV. Moreover, all experimental dogs were exposed to HeV via the nasal as well as the oral route, providing ample opportunity for CNS infection via olfactory sensory neurons. 22 In the horse, experimental HeV infection using the same challenge virus faithfully reflects the tissue tropism encountered in the field disease. 11,12 So it may be that CNS involvement in canine HeV infection is in fact rare and its observation in the reported field case was a chance event. But in the dog, in which there is clearly a difference in the downstream response to HeV infection compared with the horse, it may also be that even minor adaptation of the virus to cell culture influences its pathogenicity for canine CNS. Bearing in mind that CNS complications are an important and currently uncontrolled feature of human infection with HeV, clarifying the molecular basis of HeV neuropathogenesis has a broader biological relevance. To guide future studies, it may be helpful to expose additional dogs to infective secretions or blood rather than to tissue culture isolates of HeV, with a view to studying them for longer into the course of acute infection (after 6 dpi) than was possible to evaluate here. This may provide a more accurate assessment of the likelihood of CNS infection developing in dogs that have undergone field exposure to HeV, as well as the potential for any longer term effects. In summary, and consistent with the incidental diagnosis of two field cases of HeV infection in dogs, we found that HeV is not highly pathogenic for dogs. Dogs that are acutely infected with HeV may not come to the attention of a veterinarian and in some cases may go unmarked even by the most observant of owners. Acutely infected dogs pose a potential transmission risk via infective oral fluids and it is also possible that HeV may be shed within urine. Viral shedding occurs over several days only and resolution of acute infection is associated with the detection of virus neutralising antibody in serum. Although it does occur, the overall likelihood of CNS infection by HeV in dogs is unclear, as is any long-term consequence. Acknowledgments M Fisher, B Clayton, J Dups, A Burroughs, E Croser, J Payne and J Harper for their assistance with animal husbandry, sample collection and tissue processing. These studies were supported by the National Hendra Virus Research Program and funded by the Commonwealth of Australia, and the New South Wales and Queensland State Governments. This work was performed at an NCRIS funded facility. References 1. Murray K, Selleck P, Hooper P et al. A morbillivirus that caused fatal disease in horses and humans. Science 1995;268:94 97. 2. Chua KB, Goh KJ, Wong KT et al. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999;354:1257 1259. 3. Marsh GA, de Jong C, Barr JA et al. 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