**3. Pathogenicity of avian paramyxovirus serotype-3**

The mean death time (MDT) for APMV-3 strain Netherland was 112 hr while that of APMV-3 strain Wisconsin was > 168 hr. The intracerebral pathogenicity index (ICPI) value for APMV-3 strain Netherlands was 0.39, while the ICPI for APMV-3 strain Wisconsin was zero, consistent with a lentogenic virus. Although, the ICPI and MDT values of strain Netherlands were higher than those of strain Wisconsin; their values indicate that both the APMV-3 strains are lentogenic viruses. These results indicated that APMV-3 strain Netherlands and Wisconsin are probably not highly pathogenic to chickens, similar to lentogenic NDV strains (Kumar et al., 2010a).

In order to evaluate the ability of APMV-3 to replicate in neuronal tissue, 1X103 PFU of each APMV-3 strain was inoculated intracerebrally into 1-day-old chicks. Our results showed that neither of the two APMV-3 strains produced any clinical signs nor did they kill any chicks during 5 DPI. Both of the APMV-3 strains were isolated from the brain homogenates of all of the chicks on days 1 to 5 day post inoculation, grew with similar kinetics, and reached similar maximum titers (Kumar et al., 2010a). Interestingly, while both the APMV-3 strains replicated more slowly than moderately virulent NDV, they achieved a similar final titer compared to NDV, but without causing noticeable neurological disease and with no mortality (Kumar et al., 2010a).

Infection of either APMV-3 strain in 1-day-old chickens and turkeys resulted in mild clinical signs that included altered gait, respiratory distress, dullness, ruffled-feathers, loss of appetite and weight loss. The other visible signs included diarrhea that was evident in 1 day-old chickens and turkeys at 4 DPI. The neurological signs were more evident in 1-dayold chicks and turkeys infected with APMV-3 strain Netherlands as compared to strain Wisconsin. In contrast, infection of 2-week-old chickens and turkeys with the APMV-3 strains did not result in any apparent clinical signs of disease. APMV-3 infection did not kill any of the 1-day-old or 2-week-old chickens and turkeys. The most remarkable finding upon postmortem examination of birds was enlargement of the pancreas with focal necrosis in the 2-week-old chickens and turkeys (Fig. 1). The foci of necrosis were distributed along the entire length of pancreas and the extent of necrosis was similar in both chickens and

experiments and housed in negative pressure isolators. The 1-day-old and 2-week-old chickens and turkeys, in groups of 12 for each species and age, were infected with 0.1 mL (103 PFU) per bird of APMV-3 strain Netherlands or Wisconsin through the occulonasal route. Infections with the different strains were performed at separate times to avoid cross infections. An additional six birds of each species/age group remained as uninfected controls and sacrificed 14 day post infection (DPI) after collection of serum. Birds were provided with food and water *ad libitum* and monitored daily for any visible signs and symptoms twice daily. Three birds from each infected group were euthanized on 3, 5, 7 and 14 DPI by rapid asphyxiation in a CO2 chamber. The birds were swabbed orally and cloacally just before euthanasia. The following tissue samples were collected both for IHC and for virus isolation: brain, trachea, lung, spleen, kidney and pancreas. In addition serum samples were collected on day 14 when the three remaining birds in each group and the control birds were euthanized. Seroconversion was evaluated by HI assay

The mean death time (MDT) for APMV-3 strain Netherland was 112 hr while that of APMV-3 strain Wisconsin was > 168 hr. The intracerebral pathogenicity index (ICPI) value for APMV-3 strain Netherlands was 0.39, while the ICPI for APMV-3 strain Wisconsin was zero, consistent with a lentogenic virus. Although, the ICPI and MDT values of strain Netherlands were higher than those of strain Wisconsin; their values indicate that both the APMV-3 strains are lentogenic viruses. These results indicated that APMV-3 strain Netherlands and Wisconsin are probably not highly pathogenic to chickens, similar to

In order to evaluate the ability of APMV-3 to replicate in neuronal tissue, 1X103 PFU of each APMV-3 strain was inoculated intracerebrally into 1-day-old chicks. Our results showed that neither of the two APMV-3 strains produced any clinical signs nor did they kill any chicks during 5 DPI. Both of the APMV-3 strains were isolated from the brain homogenates of all of the chicks on days 1 to 5 day post inoculation, grew with similar kinetics, and reached similar maximum titers (Kumar et al., 2010a). Interestingly, while both the APMV-3 strains replicated more slowly than moderately virulent NDV, they achieved a similar final titer compared to NDV, but without causing noticeable neurological disease and with no

Infection of either APMV-3 strain in 1-day-old chickens and turkeys resulted in mild clinical signs that included altered gait, respiratory distress, dullness, ruffled-feathers, loss of appetite and weight loss. The other visible signs included diarrhea that was evident in 1 day-old chickens and turkeys at 4 DPI. The neurological signs were more evident in 1-dayold chicks and turkeys infected with APMV-3 strain Netherlands as compared to strain Wisconsin. In contrast, infection of 2-week-old chickens and turkeys with the APMV-3 strains did not result in any apparent clinical signs of disease. APMV-3 infection did not kill any of the 1-day-old or 2-week-old chickens and turkeys. The most remarkable finding upon postmortem examination of birds was enlargement of the pancreas with focal necrosis in the 2-week-old chickens and turkeys (Fig. 1). The foci of necrosis were distributed along the entire length of pancreas and the extent of necrosis was similar in both chickens and

(Alexander, 1980b).

**3. Pathogenicity of avian paramyxovirus serotype-3** 

lentogenic NDV strains (Kumar et al., 2010a).

mortality (Kumar et al., 2010a).

turkeys. Gross examination of other organs including the brain, trachea, liver, kidney, spleen and lung showed normal tissue morphology with no noticeable gross lesions.

Fig. 1. Gross morphology of pancreas collected from 2-week-old turkeys infected with APMV-3 strain Netherlands (a) or Wisconsin (b): comparison with pancreas from uninfected controls (left panels) reveals necrotic foci along the length of the infected organ.

The brain, trachea, lung, spleen, kidney and pancreas showed the presence of viral antigen upon inoculation. Although APMV-3 strain Wisconsin replicated less efficiently in 2-weekold chickens, the titer of virus in lungs was somewhat higher than that of strain Netherlands, suggesting that it might be more respirotropic, whereas strain Netherlands appeared to be more neurotropic (Kumar et al., 2010a).

In general, infected turkeys had virus replication in fewer organs and for shorter duration than chickens of same age groups. Strain Netherlands was isolated from all of the tested organs except for the pancreas and kidneys in turkeys. Strain Wisconsin was isolated from all of the tested organs in turkeys. Results also suggested that turkeys are less susceptible to infection with either APMV-3 strain compared to chickens (Kumar et al., 2010a).

All the chickens and turkeys infected with either strain seroconverted 14 days post infection confirming virus replication. Extensive immunofluorescence was observed in the brain, trachea, lung and pancreas, suggesting extensive virus replication in these organs (Kumar et al., 2010a). The study also suggested that the epithelial lining in the lungs and trachea are more privileged site for virus replication.
