Transmission routes for Nipah Virus from Malaysia and Bangladesh.
Subject: Disease transmission
Infection
RNA
Authors: Clayton, Bronwyn A.
Middleton, Deborah
Bergfeld, Jemma
Haining, Jessica
Arkinstall, Rachel
Wang, Linfa
Marsh, Glenn A.
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Accession Number: 313345646
Full Text: Nipah virus (NiV), a bat-borne paramyxovirus, has caused outbreaks of human disease with high mortality rates in Malaysia, Singapore, India, and Bangladesh. Two divergent NiV strains (NiV-Malaysia and NiV-Bangladesh) share 91.8% nt sequence identity (1).

NiV-Malaysia emerged in 1998 during an outbreak of infectious respiratory and neurologic disease in commercially farmed pigs, presumably after virus spillover from Malaysian flying foxes (2). Pigs were the source of infection for farm and abattoir workers, resulting in a widespread outbreak of severe febrile encephalitic disease among humans (3-5); >250 cases were reported in Malaysia and Singapore, and the case-fatality rate approached 40% (2,5,6). No cases of human-to-human transmission were reported during the outbreak (7,8). However, rare instances of human-to-human transmission have been suggested by asymptomatic seroconversion against NiV-Malaysia in a health care worker, which was recognized after the outbreak (9), and by a recently reported case of late-onset NiV encephalitis attributed to transmission from infected family members (10).

NiV-Bangladesh emerged in 2001 in Bangladesh (11,12), and subsequent outbreaks of disease have occurred almost annually (12-20). Since 2001, >200 cases in humans have been identified in Bangladesh; the overall case-fatality rate is >70% (21). In contrast to the rare instances of human-to-human transmission of NiV-Malaysia, human-to-human transmission of NiV-Bangladesh is a major pathway for human infection (13).

The different transmission characteristics of NiV-Malaysia and NiV-Bangladesh might be attributable to differences in infectivity and pathogenicity of virus strains and in tissue tropism, reflected by higher incidence of respiratory disease in NiV-Bangladesh-infected patients (14,21). We assessed the role that tissue tropism and shedding characteristics of NiV-Malaysia and NiV-Bangladesh might play in clinical outcomes and increasing transmission risk. For this purpose, we used a mammalian infection model, the ferret, in which NiV causes fulminating systemic disease, with fever and neurologic and/or respiratory signs, similar to those in humans (15). Here we describe a ferret model for NiV-Bangladesh infection and our comparison of the characteristics of infections caused by NiV-Malaysia and NiV-Bangladesh in the ferret.

Materials and Methods

Animal Infection, Handling, and Housing

A total of 15 male ferrets, 12-18 months of age, were oronasally exposed to 5,000 50% tissue culture infective doses of low-passage isolates of Nipah virus from humans. Animals were randomly assigned to receive NiV-Bangladesh (n = 8, ferrets B1-B8) or NiV-Malaysia (n = 7, ferrets M9-M15). The 2 groups were housed under separate biosafety level 4 conditions as described (17). The specific NiV-Bangladesh isolate was Nipah Bangladesh/human/2004/Rajbari, R1, which came from the oropharynx of 1 of 12 patients infected during an outbreak of NiV encephalitis in Rajbari district, Bangladesh (1,16). The patient was a 10-year-old boy with neurologic disease and respiratory involvement characterized by coughing, wheezing, and difficulty breathing (S. Luby, pers. comm.). The specific NiV-Malaysia isolate was Nipah virus/Malaysia/human/99, which came from the cerebrospinal fluid of a patient with encephalitis. We selected a challenge dose that was expected to infect all exposed animals with NiV-Malaysia (15) and NiV-Bangladesh (D. Middleton, unpub. data). Ferrets were anesthetized as described before viral challenge and for subsequent sample collection (17). Procedures involving live animals were approved by the Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health Laboratory, Animal Ethics Committee.

Animal Monitoring and Sampling

After receiving the challenge dose, animals were assessed daily for clinical signs of disease. Every 48 hours, nasal wash samples, oral and rectal swab samples, and blood (axillary vein) were collected and temperature and weight were recorded. Sampling days were staggered so that sampling of ferrets B1-B4 and M9-M11 started on 1 day postinfection (dpi) and sampling of ferrets B5-B8 and M12-M15 started on 2 dpi. Environmental samples of urine and feces were obtained daily from cage floors.

Ferrets were euthanized at predetermined humane end points as described (15). Clinical samples and various tissues were collected immediately before euthanasia or during postmortem examination.

Sample Collection, Processing, and Analysis

Nasal wash, swab, urine, whole blood (EDTA treated), tissue, and fecal samples were collected and processed in the same manner as tissue samples and then used for virus isolation, RNA extraction, and TaqMan reverse transcription PCR (RT-PCR; selective for the NiV N gene) as described (17-19). Samples with a mean NiV N gene cycle threshold value [less than or equal to] 39.1 were defined as positive for NiV RNA. For tissue samples, NiV N gene values were normalized to host cell 18S rRNA by multiplex RT-PCR as described (22) but by using probe (5' -VIC-TGCTGGCACCAGACTTGCCCTC-TAMRA-3'). Tissues were also processed for histopathologic and immunohistochemical examination with rabbit a-NiV N protein antiserum (20).

Statistical Analysis

To compare trends in virus shedding over time, we analyzed transformed RT-PCR data from nasal, oral, and rectal swab samples by using a residual maximum-likelihood (REML) model in GenStat statistical software, version 3 (VSN International, Hemel Hempstead, UK). Data were collapsed into 48-hour periods, thereby generating 4 time points for comparison: dpi 1-2, 3-4, 5-6, and 7-8. We omitted dpi 9-10 from analysis because few animals in either group survived this long. To analyze the trend of shedding over time for each virus, we fitted the interaction of virus and days to the model.

On the basis of REML analysis outcomes, we also estimated the amount of NiV-Bangladesh and NiV-Malaysia shed by individual animals over the course of infection by calculating the area under the curve (AUC) for viral RNA (by using the trapezoidal rule) for nasal wash and oral and rectal swab samples. Blood samples were similarly assessed. Estimates were transformed to the [log.sub.10] scale, and mean AUCs for NiV-Bangladesh (n = 8) and NiV-Malaysia (n = 7) samples were compared by using an independent-samples t test.

At the time of euthanasia, we compared levels of viral RNA in nasal, oral, rectal, urine, and blood samples between the 2 groups by using independent-sample t tests of transformed data. Also at the time of euthanasia, we similarly assessed levels of viral RNA in tissue. Analysis by t test did not assume equal differences. All tests used were 2-sided, and p<0.05 was defined as statistically significant.

Results

Clinical Features of Infection

Clinical signs were those of lower respiratory tract and neurologic system infection. Clinical signs were similar for all 8 ferrets challenged with NiV-Bangladesh and for 6 of 7 challenged with NiV-Malaysia (Table 1). In 1 ferret challenged with NiV-Malaysia (ferret M11), localized bacterial lymphadenitis (confirmed by histopathologic examination) developed, and the ferret was euthanized on humane grounds at 5 dpi, at which time no clinical signs of NiV infection had been observed. The first sign of disease was pyrexia (rectal temperature [greater than or equal to] 40[degrees]) for most animals; disease progressed rapidly to its humane end point within 72 hours of pyrexia onset.

Hemorrhage was another clinical sign, but it differed between the 2 groups. At the terminal stage of disease, NiV-Malaysia-infected animals experienced cutaneous petechial hemorrhage, accompanied by bleeding from oral, nasal, and rectal mucosa; whereas, only 1 NiV-Bangladesh-infected animal experienced bleeding (of the oral mucosa at the time of euthanasia).

Virus Loads

Viral RNA and virus isolation results are presented in Tables 2 and 3. Viral RNA was recovered from clinical samples from all animals with clinical disease, and virus was isolated from some samples. For ferret M11, viral RNA was detected in clinical samples as early as 3 dpi and in blood, indicating a productive infection after experimental challenge; data for ferret M11 were therefore included in analysis of shedding. RNA was detected in nasal, oral, or rectal samples from similar numbers of animals exposed to NiV-Malaysia or NiV-Bangladesh (by linear mixed-model analysis; data not shown). Mean levels of viral RNA in clinical samples increased throughout the course of infection and were highest at 7-8 dpi, the time of onset of severe clinical disease for most animals (Figure 1). In 4 NiV-Bangladesh-infected animals and all NiV-Malaysia-infected animals, viral RNA was detected in nasal secretions at least 24 hours before it was detected in blood and/or before onset of pyrexia.

Levels of viral RNA were significantly higher in oral secretions from NiV-Bangladesh-infected than from NiV-Malaysia-infected animals; predicted mean viral RNA levels were at least 10-fold higher in the NiV-Bangladesh-infected group at 5-6 and 7-8 dpi (Figure 1; REML analysis; p = 0.038 for virus by days), and mean AUC was >30-fold higher (Figure 2; p = 0.001; [t.sub.12] = 4.3). However, the rate of virus isolation from oral swab samples did not differ significantly between infection groups (Z test of 2 proportions; data not shown). NiV-Bangladesh was isolated from oral swab samples from ferrets B5 (4 dpi and at euthanasia), B6 (6 dpi), and B7 (4 and 6 dpi). Oral swabs from ferrets infected with NiV-Malaysia yielded isolates in ferret M9 at 3 dpi and in ferrets M10, M13, M14, and M15 at euthanasia (Table 3).

Nasal shedding of viral RNA for both viruses over time was higher than oral and rectal shedding (Figure 1). However, the shedding trend over time was similar between the 2 groups, and no significant difference was found between the mean AUCs (data not shown).

Rectal shedding of viral RNA was observed for most animals with clinical disease in both groups. Predicted means for rectal shedding were higher for the NiV-Malaysia-infected group at 5-6 and 7-8 dpi; for the NiV-Bangladesh-infected group, detection of viral RNA in rectal swab samples was delayed until 7-8 dpi (Figure 1; REML analysis p = 0.006 for virus by days). However, the total amount of viral RNA shed in rectal swab samples over the course of infection did not differ significantly between the 2 viruses (by AUC analysis; data not shown).

Viral RNA was generally detected in blood from 5 dpi on (Tables 2, 3). AUC analysis did not demonstrate a difference between the groups in total viral RNA in blood over the course of infection (data not shown).

Urine collection was rarely achieved by manual bladder expression during the course of infection. However, at the time of euthanasia, small volumes of urine were collected from all animals in the NiV-Bangladesh-infected group and from 5 of 6 animals in the NiV-Malaysia-infected group. The rate of detection of viral RNA and isolation of virus in urine was similar for each group (data not shown).

Environmental urine and fecal samples from both groups were NiV positive by RT-PCR and by virus isolation over the course of infection from as early as 3 dpi for 1 cage in the NiV-Malaysia-infected group (Table 4). The rate of detection and isolation of NiV from environmental samples was highest at 7 dpi, coinciding with onset of severe clinical disease in most animals.

[FIGURE 1 OMITTED]

Histopathologic and Immunohistochemical Findings NiV-Bangladesh

The main histopathologic findings for ferrets from both groups are summarized in Table 5. In ferrets exposed to NiV-Bangladesh, multisystemic inflammatory lesions developed, most consistently affecting the upper and lower respiratory tract, lymphoid tissue, kidneys, and liver. Lesions comprised mild to severe acute necrotizing rhinitis affecting olfactory and respiratory epithelium, focal necrotizing bronchoalveolitis, and marked lymphadenitis (most notably involving the submandibular and retropharyngeal lymph nodes, caudal cervical lymph nodes, and associated peritracheal and periesophageal lymph vessels). Tonsillitis and nasopharyngitis were also noted. In some animals, lymph node lesions were confined to subcapsular and cortical regions; in others, the entire nodal architecture was effaced. There was also glomerular necrosis with hyaline tubular casts; focal proximal renal tubular necrosis and interstitial nephritis; focal adrenal, splenic, and hepatic necrosis; and mild esophagitis and tracheitis. Vasculitis was detected in nasal submucosae, lungs, lymph nodes, spleen, and testes; syncytial cells of epithelial (bronchiole, renal tubule), endothelial (lymph node, testis), and unknown derivation (spleen, lymph node) were also identified. Viral antigen was found in tissues from each animal, including tonsillar (Figure 3, panel A) and nasopharyngeal (Figure 3, panel B) epithelium; vascular endothelium; syncytia; foci of inflammation in lung, bronchial, and bronchiolar epithelium; necrotic areas within lymphoid tissues and adrenal glands; necrotic glomeruli and renal tubular cells; necrotic hepatic acinar tissue; and the esophageal exudate from 1 animal. Within the nasal cavity, viral antigen was identified not only in respiratory and olfactory epithelium but also adjacent to submucosal nerve fibers in 2 animals (Figure 4). Mild testicular degeneration was observed in all animals and was attributed to fever.

Although encephalitis was not detected in any animal, viral antigen was found in endothelial cells within brain parenchyma of 7 and within meninges of 4 of these. Antigen was occasionally detected in neurons and glial cells adjacent to affected capillaries (Figure 5), consistent with hematogenous spread.

NiV-Malaysia

In ferrets exposed to NiV-Malaysia, multisystemic inflammatory disease

developed as described (15), which was generally similar to that observed in ferrets exposed to NiV-Bangladesh. Mild myocarditis was found in 1 animal and cholecystitis was found in another. Unlike findings in NiV-Bangladesh-infected ferrets, focal hepatic necrosis was found in only 1 NiV-Malaysia-infected ferret, a difference that was statistically significant (p = 0.001, Fisher exact test) but of uncertain pathogenic relevance. Mild nonsuppurative meningitis was found in 2 of 7 animals, and vasculitis in the choroid plexus was found in 1 of these. NiV antigen was identified in meningeal endothelial cells of 5 ferrets; in 3 of these 5 ferrets, it was found in the choroid plexus (Figure 6, panel A), ependyma (Figure 6, panel B), parenchymal vascular endothelium, and adjacent neurons and glia, consistent with hematogenous spread.

[FIGURE 2 OMITTED]

Virus in Clinical Samples and Tissues at Euthanasia

Because ferret M11 did not fulfill the defined criteria for reaching humane end point attributable to NiV infection, euthanasia data for this animal were omitted from statistical analysis. Viral RNA levels in oral swab, nasal wash, and rectal swab samples and urine collected at euthanasia were comparable between ferrets infected with NiV-Bangladesh or NiV-Malaysia (by t test; data not shown). All animals with clinical disease had detectable viral RNA in blood at euthanasia. Mean viral load in blood at euthanasia was 10-fold higher in the NiV-Malaysia-infected group (p = 0.008; [t.sub.12] = 3.2; difference between means: [10.sup.11] [95% CI 100.3-101.8]). Viral RNA was detected in virtually all tissues examined from all animals with clinical disease, and levels were generally higher in tissues from the NiV-Malaysia-infected group; this difference was significant for tissue from the olfactory pole of the brain, the nasal turbinates, pharynx, retropharyngeal lymph nodes, spleen, and bladder (online Technical Appendix, wwwnc.cdc.gov/ EID/pdfs/12-0875-Techapp.pdf) and was attributed to the higher RNA levels in blood in this group at euthanasia. Although rectal shedding was detected in most animals over the course of clinical disease, virus reisolation from the jejunoileum was not successful. Virus was reisolated from rectal tissues from 1 of 6 and from 3 of 5 animals positive for viral RNA in the NiV-Bangladesh and NiV-Malaysia infection groups, respectively.

Discussion

We compared the characteristics of viral shedding and tissue tropism between NiV-Malaysia and NiV-Bangladesh in the ferret model to examine whether these characteristics might contribute to observed differences in the clinical outcome and transmission of disease during outbreaks among humans. We found that viral shedding by nasal, oral, rectal, and urinary routes occurred in ferrets infected with both strains, as has been reported for outbreaks among humans (23), and we found that levels of viral genome over time were significantly higher in oral secretions from ferrets infected with NiV-Bangladesh than with NiV-Malaysia.

Although this finding was not reflected by a higher rate of virus isolation in that group, an observation that was attributed at least in part to the relative insensitivity of virus isolation assay compared with RT-PCR (24), increasing viral RNA over time was consistent with active virus replication in the oropharynx of these animals. Accordingly, this finding in oral secretions suggests that strain differences in replication at sites relevant to transmission might occur.

[FIGURE 3 OMITTED]

Although, to our knowledge, data on viral shedding in humans over the course of infection have not been reported, epidemiologic studies identified exposure to infectious saliva or respiratory secretions from patients as a major risk factor for human-to-human transmission of NiV-Bangladesh (13,25,26). Lower respiratory tract involvement and associated signs, including coughing, are more commonly reported for humans infected with NiV-Bangladesh than with NiV-Malaysia and have been suggested as a contributing factor in the higher likelihood of transmission from patients so affected (21). We did not observe differences between the 2 strains in the form of viral antigen distribution, lesion distribution and severity, or levels of viral RNA in the oropharynx or lower respiratory tract at the time of advanced clinical disease that would offer an immediate explanation for the increased oral shedding of NiV-Bangladesh. It might be that a higher level of oral shedding of NiV-Bangladesh reflects additional, more extensive, or more efficient viral replication in the oropharynx or lower respiratory tract earlier in the infection process that is later masked by fulminant NiV infection. In addition, our criteria for euthanasia might not have reflected a consistent biological time point in the infection process with each strain. More in vivo studies of viral infection of the oropharynx and lower respiratory tract, particularly soon after exposure, are warranted to explore these points further. Differences in infection and replication efficiency between virus strains might also be elucidated by in vitro comparisons of NiV-Bangladesh and NiV-Malaysia replication kinetics in respiratory cell lines.

It is noteworthy that with both NiV strains, shedding was observed in nasal wash and oral swab samples before the onset of pyrexia, as has been reported for hamsters infected with NiV-Malaysia (27). This finding suggests risk for transmission during the incubation period and before hematogenous virus spread.

Isolation of virus and detection of viral RNA from rectal swab samples from animals in both infection groups supports the potential for oral-fecal NiV transmission with a comparatively higher risk for NiV-Malaysia during terminal disease. Because rectal shedding typically occurred later in the course of infection and was not associated with viral localization in the gastrointestinal tract at euthanasia, it was attributed to effusion of blood-borne virus from compromised gastrointestinal tract vasculature. The higher mean levels of viral RNA in blood at euthanasia in the group infected with NiV-Malaysia are of uncertain pathogenic significance, but it is noteworthy that increased severity of hemorrhagic diathesis was observed in this group. Thrombocytopenia and gastrointestinal bleeding have been reported for some humans with advanced NiV-Malaysia infection (28-30), and a hemorrhagic syndrome has been observed in green monkeys (24) and ferrets (18) after infection with NiV-Malaysia. In our study, animals with hemorrhagic disease tended to be those that had reached their end points for euthanasia late (9 dpi in the NiV-Bangladesh-infected group and [greater than or equal to] 8 dpi for 4 of the 6 animals in the NiV-Malaysia-infected group), and they might have had more prolonged endothelial infection.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

We were unable to assess shedding in urine over time, but virus was reisolated from urine collected at euthanasia from ferrets infected with NiV-Bangladesh and NiV-Malaysia. This finding is consistent with findings for NiV-Malaysia-infected human patients (23). Virus reisolation from urine and feces collected from cages containing infected animals in both groups also suggests that environmental contamination might pose an infection risk.

Viral antigen was observed in neurons and glia and in meningeal and parenchymal vascular endothelium of ani mals in both groups. This finding is consistent with the dual mechanism proposed for the pathogenesis of NiV neurologic disease in humans, namely, direct cellular dysfunction resulting from neuronal infection and vasculitis-associated ischemic injury to the brain (2,31-33).

Our observation that oropharyngeal shedding occurred at higher levels in NiV-Bangladesh-infected ferrets suggests a mechanism for the higher risk for human-to-human transmission that is observed for this NiV strain in the field, although the mechanism for enhanced shedding of NiV-Bangladesh has not yet been elucidated. However, it is recognized that heterogeneity of NiV-Bangladesh isolates (1,34) is more substantial than has been observed for NiV-Malaysia isolates. It would be of value to compare the shedding characteristics of the NiV-Bangladesh isolate studied here (from an outbreak in which human-to-human transmission was not observed) (16) with characteristics of isolates obtained during outbreaks with differing epidemiologic features. Transmission of NiV-Malaysia has been recently described in the hamster model (35), but the application of this observation to enhancing infection control with human NiV-Bangladesh is unclear, and natural transmission of NiV-Malaysia among humans occurs at low frequency (9,10). Our observations for ferrets support the view that although transmission of NiV-Malaysia between humans is possible, an increased propensity for oral shedding of NiV-Bangladesh (of pharyngeal or lower respiratory tract origin) within the context of social environmental factors in play during outbreaks of human disease leads to a higher incidence of human-to-human transmission of NiV-Bangladesh. Whether increased oral shedding of NiV-Bangladesh is predictive for increased transmissibility under controlled conditions in an animal model remains to be seen. In addition to time-course studies, in vivo studies that simulate various levels of interaction between infected and in-contact animals are warranted. On the basis of the virus shedding reported here for the ferret model, we propose that the ferret is a suitable human surrogate for further investigation of NiV transmission.

[FIGURE 6 OMITTED]

Acknowledgments

We thank Jean Payne, John Bingham, and Jenni Harper for assistance with histopathologic and immunohistochemical analyses; John White and Tyrone McDonald for RNA extraction; Adam Foord for helping conduct Taqman RT-PCRs; Elena Virtue for assistance with processing of animal samples; and Sandy Clarke and the Statistical Consulting Centre, University of Melbourne, for advice and help with data analysis.

Dr Clayton is a postgraduate student at the Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation, Livestock Industries, enrolled in a PhD program through the University of Melbourne. Her research interests include viral pathogenesis and emerging viral diseases.

Author affiliations: Commonwealth Scientific and Industrial Research Organisation Livestock Industries, Geelong, Victoria, Australia (B.A. Clayton, D. Middleton, J. Bergfeld, J. Haining, R. Arkinstall, L. Wang, G.A. Marsh); University of Melbourne, Parkville, Victoria, Australia (B.A. Clayton); and Duke-National University of Singapore Graduate Medical School, Singapore (L. Wang)

DOI: 10.3201/eid1812.120875

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(33.) Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, et al. Nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol. 2002;161:2153-67. http:// dx.doi.org/10.1016/S0002-9440(10)64493-8

(34.) Lo MK, Lowe L, Hummel KB, Sazzad HM, Gurley ES, Hossain MJ, et al. Characterization of Nipah virus from outbreaks in Bangladesh, 2008-2010. Emerg Infect Dis. 2012;18:248-55. http://dx.doi. org/10.3201/eid1802.111492

(35.) de Wit E, Bushmaker T, Scott D, Feldmann H, Munster VJ. Nipah virus transmission in a hamster model. PLoS Negl Trop Dis. 2011;5:e1432. http://dx.doi.org/10.1371/journal.pntd.0001432

Address for correspondence: Glenn A. Marsh, CSIRO Livestock Industries, Australian Animal Health Laboratory, 5 Portarlington Rd East, Geelong, Victoria 3219, Australia; email: glenn.marsh@csiro.au
Table 1. Clinical disease in ferrets after experimental infection
with NiV from Bangladesh or Malaysia *

NiV type and   Euthanasia,
ferret no.         dpi

 Bangladesh
     B1             7
     B2             7

     B3             7

     B4             7

     B5             8

     B6             9

     B7             8

     B8             8

  Malaysia
     M9             7

    M10             7

    M11             5

    M12             8

    M13             9

    M14             8

    M15            10

NiV type and
ferret no.        Resp
               ([dagger])

 Bangladesh
     B1             -
     B2            +

     B3            +

     B4            +

     B5             -

     B6            +

     B7             -

     B8            +

  Malaysia
     M9            +

    M10            +

    M11            NA

    M12             -

    M13            +

    M14             -

    M15             -

NiV type and
ferret no.        Neuro

 Bangladesh
     B1             -
     B2            +/-

     B3            +/-

     B4            +

     B5            +

     B6            +

     B7            +

     B8            +

  Malaysia
     M9            +

    M10            +

    M11            NA

    M12            +

    M13            +/-

    M14            +

    M15            +

NiV type and
ferret no.       Hemorr
                ([double
                dagger])
 Bangladesh
     B1             -
     B2             -

     B3             -

     B4             -

     B5             -

     B6             +

     B7             -

     B8             -

  Malaysia
     M9             +

    M10             +

    M11            NA

    M12             +

    M13             +

    M14             +

    M15             +

NiV type and   Criteria for
ferret no.     euthanasia

 Bangladesh
     B1        Obtundation
     B2        Respiratory +/- mild
               neurologic signs

     B3        Respiratory +/-
               neurologic signs

     B4        Respiratory signs,
               neurologic signs,
               and obtundation
     B5        Neurologic signs and
               obtundation
     B6        Respiratory signs,
               neurologic signs,
               hemorrhage, and
               obtundation

     B7        Neurologic signs

     B8        Respiratory signs,
               neurologic signs,
               and obtundation
  Malaysia
     M9        Respiratory signs,
               neurologic signs,
               hemorrhage, and
               obtundation
    M10        Respiratory signs,
               neurologic signs,
               hemorrhage, and
               obtundation
    M11        NA

    M12        Neurologic signs and
               severe hemorrhage

    M13        Respiratory signs,
               hemorrhage, and
               obtundation

    M14        Neurologic signs,
               hemorrhage, and
               obtundation

    M15        Neurologic signs,
               hemorrhage, and
               obtundation

NiV type and
ferret no.     Clinical signs

 Bangladesh
     B1        Severely obtunded; hunched posture
     B2        Hunched posture; possible mild neurologic
               disease (agitation); sneezing; >10% reduction in
               body weight ([section])
     B3        Possible mild neurologic disease (continuous
               licking, smacking lips); dehydration; ([paragraph])
               vomiting;
               rapid deterioration in clinical condition after
               sampling at 7 dpi
     B4        Fine tremors/myoclonus of forelimbs; nasal
               discharge

     B5        Hind limb myoclonus/paresis, ataxia; dehydration;
               periorbital/facial/ventral neck edema
     B6        Forelimb myoclonus; sneezing; mucoid nasal
               discharge; reduced feces production;
               periorbital/facial edema; hemorrhage of oral
               mucosa at euthanasia time point; >10% reduction
               in body weight
     B7        Myoclonus and muscular spasm affecting the tail,
               ataxia; ventral neck edema
     B8        Myoclonus of the flanks, ataxia, hind limb
               paralysis; vomiting; ventral neck edema

  Malaysia
     M9        Severe ataxia, facial and hind limb tremors, head
               tilt and torticollis (left); sneezing; nasal discharge;
               facial edema; hemorrhage of rectal mucosa at
               euthanasia
    M10        Dyspnea with prolonged expiration phase; mild
               ataxia; reduced feces production; facial and
               ventral neck edema; hemorrhage from nose and
               mouth at euthanasia
    M11        Euthanasia at 5 dpi for humane reasons; no
               evidence of clinical disease associated with NiV
               infection
    M12        Spastic paralysis of right forelimb, rhythmic
               myoclonus of right trunk, ataxia; sneezing; nasal
               discharge; facial edema; extensive cutaneous
               petechial hemorrhages and facial bruising
    M13        Mild neurologic disease (hind limb paresis) at 6
               dpi but not apparent at euthanasia; nasal
               discharge; facial edema; inappetence; >10%
               reduction in body weight
    M14        Hunched posture; spastic paralysis of hind limbs,
               fine muscular fasciculations over flanks, ataxia;
               hunched posture; dehydration; nasal discharge;
               cutaneous petechial hemorrhages; >10%
               reduction in body weight
    M15        Sporadic hind limb myoclonus; recumbency;
               nasal discharge; cutaneous petechial
               hemorrhages and hemorrhage from mouth; >10%
               reduction in body weight

* NiV, Nipah virus; dpi, days postinfection; resp, respiratory
involvement; neuro, neurologic involvement; hemorr, hemorrhage;
+/-, vague clinical signs that might indicate neurologic
involvement; NA, not applicable.

([dagger]) Increased respiratory effort and/or rate unless
otherwise stated under clinical disease.

([double dagger]) Cutaneous hemorrhages or frank hemorrhage from
oral, nasal, and rectal mucosa.

([section]) Based on weight data collected before experimental
infection.

([paragraph]) Based on the observation of reduced skin turgor at
physical examination.

Table 2. Viral RNA and virus isolation results from
ferrets experimentally infected with Nipah virus,
Bangladesh strain *

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 1   Dpi 2
B1
  NW              -
  OS              -
  RS              -
  Blood           -
B2
  NW              -
  OS              -
  RS              -
  Blood           -
B3
  NW              -
  OS              -
  RS              -
  Blood           -
B4
  NW              -
  OS              -
  RS              -
  Blood           -
B5
  NW                      -
  OS                      -
  RS                    Indet
  Blood                 Indet
B6
  NW                      -
  OS                      -
  RS                      -
  Blood                   -
B7
  NW                      -
  OS                      -
  RS                      -
  Blood                  +/+
B8
  NW                      -
  OS                      -
  RS                      -
  Blood                   -

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 3
B1
  NW            Indet
  OS              -
  RS            Indet
  Blood         +/ ([double dagger])
B2
  NW             +/-
  OS              -
  RS              -
  Blood           -
B3
  NW             +/-
  OS              -
  RS              -
  Blood         Indet
B4
  NW             +/-
  OS              -
  RS              -
  Blood           -
B5
  NW
  OS
  RS
  Blood
B6
  NW
  OS
  RS
  Blood
B7
  NW
  OS
  RS
  Blood
B8
  NW
  OS
  RS
  Blood

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 4
B1
  NW
  OS
  RS
  Blood
B2
  NW
  OS
  RS
  Blood
B3
  NW
  OS
  RS
  Blood
B4
  NW
  OS
  RS
  Blood
B5
  NW             +/-
  OS             +/+
  RS              -
  Blood           -
B6
  NW              -
  OS              -
  RS              -
  Blood           -
B7
  NW             +/-
  OS             +/+
  RS              -
  Blood           -
B8
  NW              -
  OS              -
  RS              -
  Blood           -

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 5
B1
  NW            +/- ([double dagger])
  OS            - ([double dagger])
  RS            - ([double dagger])
  Blood         - ([double dagger])
B2
  NW            +/-
  OS            +/-
  RS            -
  Blood         +/+
B3
  NW            - ([double dagger])
  OS            - ([double dagger])
  RS            - ([double dagger])
  Blood         - ([double dagger])
B4
  NW            +/-
  OS            +/-
  RS            -
  Blood         -
B5
  NW
  OS
  RS
  Blood
B6
  NW
  OS
  RS
  Blood
B7
  NW
  OS
  RS
  Blood
B8
  NW
  OS
  RS
  Blood

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 6
B1
  NW
  OS
  RS
  Blood
B2
  NW
  OS
  RS
  Blood
B3
  NW
  OS
  RS
  Blood
B4
  NW
  OS
  RS
  Blood
B5
  NW            +/+
  OS            +/-
  RS            -
  Blood         +/+
B6
  NW            +/- ([double dagger])
  OS            +/+ ([double dagger])
  RS            - ([double dagger])
  Blood         +/+ ([double dagger])
B7
  NW            +/+
  OS            +/+
  RS            -
  Blood         +/-
B8
  NW            - ([double dagger])
  OS            +/- ([double dagger])
  RS            - ([double dagger])
  Blood         +/+ ([double dagger])

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 7
B1
  NW            +/+
  OS            +/-
  RS            +/-
  Blood         +/([section])
B2
  NW            +/+
  OS            +/-
  RS            +/-
  Blood         +/([section])
B3
  NW            +/-
  OS            +/-
  RS            +/+
  Blood         +/([section])
B4
  NW
  OS
  RS
  Blood
B5
  NW
  OS
  RS
  Blood
B6
  NW
  OS
  RS
  Blood
B7
  NW
  OS
  RS
  Blood
B8
  NW
  OS
  RS
  Blood

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 8
B1
  NW
  OS
  RS
  Blood
B2
  NW
  OS
  RS
  Blood
B3
  NW
  OS
  RS
  Blood
B4
  NW
  OS
  RS
  Blood
B5
  NW            +/+
  OS            +/+
  RS            +/-
  Blood         +/+
B6
  NW            NS
  OS            NS
  RS            NS
  Blood         NS
B7
  NW            +/+ ([double dagger])
  OS            +/- ([double dagger])
  RS            +/+ ([double dagger])
  Blood         +/+ ([double dagger])
B8
  NW            +/+
  OS
  RS            +/+
  Blood         +/+

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 9
B1
  NW
  OS
  RS
  Blood
B2
  NW
  OS
  RS
  Blood
B3
  NW
  OS
  RS
  Blood
B4
  NW
  OS
  RS
  Blood
B5
  NW
  OS
  RS
  Blood
B6
  NW            +/+
  OS            +/-
  RS            +/-
  Blood         +/-
B7
  NW
  OS
  RS
  Blood
B8
  NW
  OS
  RS
  Blood

                Virus in shedding samples and
  Ferret no.,   blood over time, RNA/virus
    sample      isolation ([dagger])
                Dpi 10
B1
  NW
  OS
  RS
  Blood
B2
  NW
  OS
  RS
  Blood
B3
  NW
  OS
  RS
  Blood
B4
  NW
  OS
  RS
  Blood
B5
  NW
  OS
  RS
  Blood
B6
  NW
  OS
  RS
  Blood
B7
  NW
  OS
  RS
  Blood
B8
  NW
  OS
  RS
  Blood

* Dpi, days postinfection; NW, nasal wash; -, Nipah virus not
detected by reverse transcription PCR (RT-PCR) for N gene;
therefore, virus isolation was not attempted for this sample;
empty cells represent days on which sampling was not
scheduled for that animal; Indet, mean cycle threshold value >39.1
(the defined cutoff value for positive samples); +/-, virus
detected by RT-PCR but negative by virus isolation; +/+, sample
positive by RT-PCR and virus isolation; OS, oral sample; RS,
rectal sample; NS, not sampled.

([dagger]) RNA detection by RT-PCR.

([double dagger]) Pyrexia first detected.

([section]) Virus isolation not attempted for this sample.

Table 3. Viral RNA and virus isolation results from
ferrets experimentally infected with Nipah virus,
Malaysia strain *

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 1
M9
  NW                       -
  OS                       -
  RS                       -
  Blood                    -
M10
  NW                       -
  OS                       -
  RS                       -
  Blood                    -
M11
  NW                       -
  OS                       -
  RS                       -
  Blood                    -
M12
  NW
  OS
  RS
  Blood
M13
  NW
  OS
  RS
  Blood
M14
  NW
  OS
  RS
  Blood
M15
  NW
  OS
  RS
  Blood

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 2
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW                      +/+
  OS                       -
  RS                       -
  Blood                    -
M13
  NW                       -
  OS                       -
  RS                       -
  Blood                    -
M14
  NW                       -
  OS                       -
  RS                       -
  Blood                    -
M15
  NW                       -
  OS                       -
  RS                       -
  Blood                    -

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 3
M9
  NW                      +/-
  OS                      +/+
  RS                       -
  Blood                    -
M10
  NW
  OS                       -
  RS                       -
  Blood                    -
M11
  NW
  OS                       -
  RS                       -
  Blood                    -
M12
  NW
  OS
  RS
  Blood
M13
  NW
  OS
  RS
  Blood
M14
  NW
  OS
  RS
  Blood
M15
  NW
  OS
  RS
  Blood

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 4
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW             +/+ ([double dagger])
  OS              - ([double dagger])
  RS              - ([double dagger])
  Blood           - ([double dagger])
M13
  NW                      +/-
  OS                       -
  RS                       -
  Blood                    -
M14
  NW                      +/-
  OS                       -
  RS                       -
  Blood                    -
M15
  NW                      +/-
  OS                       -
  RS                       -
  Blood                    -

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 5
M9
  NW             +/- ([double dagger])
  OS              - ([double dagger])
  RS             +/+ ([double dagger])
  Blood          +/- ([double dagger])
M10
  NW             +/+ ([double dagger])
  OS              - ([double dagger])
  RS             +/- ([double dagger])
  Blood           - ([double dagger])
M11
  NW
  OS
  RS
  Blood
M12
  NW
  OS
  RS
  Blood
M13
  NW
  OS
  RS
  Blood
M14
  NW
  OS
  RS
  Blood
M15
  NW
  OS
  RS
  Blood

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                        Dpi 6
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW                      +/-
  OS                     Indet
  RS                      +/-
  Blood                   +/+
M13
  NW             +/+ ([double dagger])
  OS              - ([double dagger])
  RS             +/+ ([double dagger])
  Blood           - ([double dagger])
M14
  NW             +/+ ([double dagger])
  OS             +/- ([double dagger])
  RS              - ([double dagger])
  Blood          +/+ ([double dagger])
M15
  NW                      +/+
  OS                      +/-
  RS                      +/-
  Blood                   +/+

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 7
M9
  NW                      +/+
  OS                      +/-
  RS                      +/-
  Blood                   +/+
M10
  NW                      +/+
  OS                      +/+
  RS
  Blood                   +/+
M11
  NW
  OS
  RS
  Blood
M12
  NW
  OS
  RS
  Blood
M13
  NW
  OS
  RS
  Blood
M14
  NW
  OS
  RS
  Blood
M15
  NW
  OS
  RS
  Blood

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 8
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW                      +/+
  OS
  RS                      +/+
  Blood                   +/+
M13
  NW                      NS
  OS                      NS
  RS                      NS
  Blood                   NS
M14
  NW                      +/+
  OS                      +/+
  RS
  Blood                   +/+
M15
  NW             +/- ([double dagger])
  OS              - ([double dagger])
  RS                 +/([section])
  Blood          +/+ ([double dagger])

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                         Dpi 9
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW
  OS
  RS
  Blood
M13
  NW                      +/+
  OS                      +/+
  RS                      +/-
  Blood                   +/+
M14
  NW
  OS
  RS
  Blood
M15
  NW
  OS
  RS
  Blood

  Ferret    Virus in shedding samples and
  no.,        blood over time, RNA/virus
  sample         isolation ([dagger])
                        Dpi 10
M9
  NW
  OS
  RS
  Blood
M10
  NW
  OS
  RS
  Blood
M11
  NW
  OS
  RS
  Blood
M12
  NW
  OS
  RS
  Blood
M13
  NW
  OS
  RS
  Blood
M14
  NW
  OS
  RS
  Blood
M15
  NW                      +/+
  OS                      +/+
  RS
  Blood                   +/+

* Dpi, days postinfection; NW, nasal wash; -, Nipah virus not
detected by reverse transcription PCR (RT-PCR) for N gene;
therefore, virus  isolation was not attempted for this
sample; empty cells represent days on which sampling
was not scheduled for that animal; +/-, virus detected
by RT-PCR but negative by virus isolation; +/+, sample
positive by RT-PCR and virus isolation; OS, oral sample;
RS, rectal sample; Indet, mean cycle threshold value
>39.1 (the defined cutoff value for positive samples);
NS ,not sampled

([dagger]) RNA detection by RT-PCR.

([double dagger]) Pyrexia first detected.

([section]) Virus isolation not attempted for this
sample.

Table 4. NiV in environmental samples after experimental
infection of 15 ferrets *

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 1
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    -                      -
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      -
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 2
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    -                      -
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      -
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 3
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    -                      -
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      3.6 ([double dagger])
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 4
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    -                      -
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      -
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 5
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    NS                     NS
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      -
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 6
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    -                      -
  2 (B3, B4)    -                      -
  3 (B5, B6)    -                      -
  4 (B7, B8)    -                      -
Malaysia
  5 (M9)        -                      -
  6 (M10, 11)   -                      -
  7 (M12, 13)   5.1 ([double dagger])  -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 7
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    5.1 ([double dagger])  -
  2 (B3, B4)    5.2 ([double dagger])  4.1
  3 (B5, B6)    4.1                    3.4
  4 (B7, B8)    3.6                    4.3
Malaysia
  5 (M9)        4.9 ([double dagger])  -
  6 (M10, 11)   4.9 ([double dagger])  -
  7 (M12, 13)   -                      -
  8 (M14, 15)   -                      -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 8
  (ferret no.)  U F

Bangladesh
  1 (B1, B2)    NA                     NA
  2 (B3, B4)    NA                     NA
  3 (B5, B6)    4.6 ([double dagger])  -
  4 (B7, B8)    -                      4.9 ([double dagger])
Malaysia
  5 (M9)        NA                     NA
  6 (M10, 11)   NA                     NA
  7 (M12, 13)   4.9 ([double dagger])  -
  8 (M14, 15)   4.6 ([double dagger])  -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 9
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    NA                     NA
  2 (B3, B4)    NA                     NA
  3 (B5, B6)    5.3 ([double dagger])  4.6
  4 (B7, B8)    NS                     -
Malaysia
  5 (M9)        NA                     NA
  6 (M10, 11)   NA                     NA
  7 (M12, 13)   -                      -
  8 (M14, 15)   3.6                    -

  Virus         [log.sub.10] NiV
  strain,       copies/mL ([dagger])
  cage no.      Dpi 10
  (ferret no.)  U                      F

Bangladesh
  1 (B1, B2)    NA                     NA
  2 (B3, B4)    NA                     NA
  3 (B5, B6)    NA                     NA
  4 (B7, B8)    NA                     NA
Malaysia        NA                     NA
  5 (M9)        NA                     NA
  6 (M10, 11)   NA                     NA
  7 (M12, 13)   NA                     NA
  8 (M14, 15)   -                      -

* NiV, Nipah virus; dpi, days postinfection; U, urine;
F, feces; -, negative; NA, not applicable because cage
was empty after euthanasia of ferrets; NS, no sample available.

([dagger]) Calculated from standard curve generated
for NiV N gene copies by reverse transcription PCR.
Samples with mean cycle threshold <39.1 (based on
duplicate reactions) were defined as NiV positive.
([double dagger]) Sample was also NiV positive
by virus isolation.

Table 5. Histopathologic and immunohistochemical
findings in major systems of ferrets infected
with NiV from Bangladesh or Malaysia *

                               No. animals with
                                lesion/antigen/
                             vasculitis ([dagger])

                                NiV-Bangladesh,
System, predominant lesion           n = 8
Respiratory
  Acute rhinitis                     7/5/1
  Acute bronchoalveolitis            8/8/1
Lymphoid
  Lymphadenitis                      8/8/1
  Splenic necrosis                   7/7/1
Renal, glomerular necrosis           8/6/0
Hepatic, focal hepatic               8/7/0
necrosis
Central nervous
  Meningitis                         0/4/0
  Encephalitis                       0/7/0

                               No. animals with
                                lesion/antigen/
                             vasculitis ([dagger])

                                 NiV-Malaysia,
System, predominant lesion           n = 7
Respiratory
  Acute rhinitis                     6/7/0
  Acute bronchoalveolitis            7/7/4
Lymphoid
  Lymphadenitis                      7/7/0
  Splenic necrosis                   6/7/1
Renal, glomerular necrosis           6/7/1
Hepatic, focal hepatic              1t/2/0
necrosis
Central nervous
  Meningitis                         2/5/1
  Encephalitis                       0/3/0

* NiV, Nipah virus.

([dagger]) No. animals in which the predominant
lesion was observed/antigen staining was observed
in that organ or tissue/vasculitis was present in that
organ or tissue. For all animals, vasculitis was
associated with antigen staining in blood vessel
endothelium or tunica media. ([double dagger])
This animal also had cholecystitis with associated viral
antigen.
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