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.

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.

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 <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 α-NiV N protein antiserum (20).

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 log₁₀ 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.

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 ≥40°) 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).

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; t₁₂ = 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.

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; t₁₂ = 3.2; difference between means: 10^1.1 [95% CI 10^0.3–10^1.8]). 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 (Technical Appendix) 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.