**1. Introduction**

586 A Bird's-Eye View of Veterinary Medicine

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Members of the family *Paramyxoviridae* are pleomorphic enveloped viruses that contain a single-stranded, non-segmented RNA genome of 13-19 kilobases (Lamb and Parks, 2007). These viruses have been isolated from many species of avian, terrestrial and aquatic animals around the world. Some members of the family *Paramyxoviridae* are well known human (measles, mumps, respiratory syncytial virus and various human parainfluenza viruses) and animal (rinderpest virus, canine distemper virus and Newcastle disease virus [NDV]) pathogens, while the pathogenic potential of many others is still unknown. The family is divided into two subfamilies, *Paramyxovirinae* and *Pneumovirinae*. Subfamily *Paramyxovirinae* is further divided into five genera: *Rubulavirus, Respirovirus, Henipavirus*, *Morbillivirus* and *Avulavirus*. Subfamily *Pneumovirinae* is divided into two genera: *Pneumovirus* and *Metapneumovirus* (Mayo, 2002).

All paramyxoviruses that have been isolated to date from avian species are placed in the genus *Avulavirus* except for avian metapneumoviruses which are placed in the genus *Metapneumovirus* due to antigenic differences and differences in genetic organization*.* The avian paramyxoviruses (APMVs) that comprise genus *Avulavirus* have been divided into nine different serotypes (APMV-1 through -9) based on haemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays (Alexander and Collins, 1984). APMV-1 comprises all strains of NDV and is the most characterized serotype due to the severity of disease caused by virulent NDV strains in chickens. (Alexander, 1980a). The complete genome sequences and the molecular determinants of virulence have been determined for representative NDV strains (Krishnamurthy and Samal, 1998; de Leeuw and Peeters, 1999; Huang et al., 2004; Panda et al., 2004; Rout and Samal, 2008). As a first step in characterizing the other APMV serotypes, complete genome sequences of one or more representatives of APMV serotypes 2 to 9 were recently determined, expanding our knowledge about these viruses (Chang et al., 2001; Kumar et al., 2008; Nayak et al., 2008; Subbiah et al., 2008; Paldurai et al., 2009; Samuel et al., 2009; Xiao et al., 2009).

NDV causes respiratory, neurological or enteric disease in birds. Among poultry, chickens are the most susceptible while geese and ducks are the least susceptible (Wakamatsu et al.,

Pathogenicity of Avian Paramyxovirusserotype-3 in Chickens and Turkeys 589

APMV-3 strains parakeet/Netherlands/449/75 and turkey/Wisconsin/68 (obtained from National Veterinary Service Laboratory, Ames, Iowa, USA) was propagated in 9-day-old specific pathogen free (SPF) embryonated chicken eggs via allantoic route of inoculation. The allantoic fluids from infected embryonated eggs were collected 96 h post-inoculation and virus titer was determined by hemagglutination (HA) assay with 0.5% chicken RBC. The virus titer in infected tissue samples was determined by the tissue culture infective dose (TCID50) method and by plaque assay in chicken embryo fibroblast (DF-1) cells (ATCC, Manassas, VA, USA) (Reed and Muench, 1938). For the plaque assay, DF-1 cell monolayers in 12 well plates were infected with different dilutions of tissue homogenates. The tissue homogenates were allowed to adsorb for 1 h at 37 °C, washed with phosphate-buffered saline (PBS), and then overlaid with 1 mL DMEM containing 0.8% (wt/vol) methylcellulose. The virus plaques in the DF-1 cell monolayer were visualized 96 h PI after staining with 1%

The pathogenicity of the APMV-3 strains Netherlands and Wisconsin was determined by two standard pathogenicity tests. These included the mean death time (MDT) in 9-day-old embryonated SPF chicken eggs and the intracerebral pathogenicity index (ICPI) in 1-day-old

MDT value was determined following the standard procedure (Alexander, 1998). Briefly, a series of 10-fold (10−6 to 10−12) dilutions of fresh infective allantoic fluid in sterile PBS were made and 0.1 mL of each dilution was inoculated into the allantoic cavities of five 9-day-old embryonated SPF chicken eggs, which were then incubated at 37 °C. Each egg was examined three times daily for 7 days, and the times of embryo deaths were recorded. The minimum lethal dose is the highest virus dilution that caused death of all the embryos. MDT is the mean time in hours for the minimum lethal dose to kill all inoculated embryos. The MDT has been used to characterize the NDV pathotypes as follows: velogenic (less than

For determining ICPI value, 0.05 mL of 1:10 dilution of fresh infective allantoic fluid (28 HA units) of each virus was inoculated into groups of 10 one-day-old SPF chicks via the intracerebral route. The birds were observed for clinical symptoms and mortality every 8 h for a period of 8 days. At each observation, the birds were scored as follows: 0, healthy; 1, sick; and 2, dead. The ICPI is the mean score for all of the birds in the group over the 8-day period. Highly virulent NDV (velogenic) viruses give values approaching 2 and avirulent

1-day-old and 2-week-old SPF chickens (Charles River, North Franklin, Connecticut, USA) and 1-day-old and 2-week-old turkeys (Murry-McMurry hatchery, Webster City, Iowa, USA) were housed in positive pressure isolators in our BSL-2 facility. Birds confirmed to be negative for APMV-3 specific antibody by HI assay were further selected for pathogenesis

60 h), mesogenic (60 to 90 h), and lentogenic (more than 90 h) (Alexander, 1980b).

NDV (lentogenic) viruses give values close to 0 (Alexander, 1998).

**2.3 Pathogenesis assessment in chickens and turkeys** 

**2. Methods used in the study** 

**2.1 Viruses and cells** 

crystal violet.

**2.2 Pathogenicity index tests** 

SPF chick (Alexander, 1998).

2006). NDV strains are classified into low virulent (lentogenic), moderately virulent (mesogenic), and highly virulent (velogenic) pathotypes based on severity of disease produced in chickens. In contrast very little is known about pathogenicity of APMV-2 to -9 in chickens and turkeys. APMV-2 has been shown to cause mild disease and drop in egg production in chickens and turkeys (Warke et al., 2008). APMV-6 and -7 have been associated with respiratory disease in turkeys (Shortridge et al., 1980; Saif et al., 1997). APMV-4, -8 and -9 have been isolated from different species of birds but the clinical signs of the disease in those birds were not apparent (Alexander et al., 1983; Gough and Alexander, 1984; Stallknecht et al., 1991; Maldonado et al., 1995; Capua et al., 2004). Recently, experimental infection of 1-day-old chicks with APMV-2, -4 and -6 showed viral infection in gastrointestinal tract, respiratory tract and pancreas (Warke et al., 2008).

APMV-3 has been isolated from wild and domesticated birds in different parts of the world (Tumova et al., 1979b; Alexander, 1980b; Macpherson et al., 1983; Alexander, 2003). Recently, APMV-3 was isolated from ostrich, indicating a wide host range for the virus (Kaleta et al.). The virus has been isolated from diseased turkeys associated with coughing, nasal discharge and swelling of the infra-orbital sinus (Redmann et al., 1991). APMV-3 has been associated with encephalitis and high mortality in caged birds (Tumova et al., 1979a). The virus causes acute pancreatitis and central nervous system (CNS) symptoms in *Psittacine* and *Passerine* birds (Beck et al., 2003). APMV-3 also infects chickens at an early age, with evidence of stunting growth that may be more marked in broiler chicken breeds (Alexander and Collins, 1982a). In terms of pathogenicity to domestic poultry birds, APMV-3 probably is second in importance to NDV. The exact economic impact of APMV-3 infection in poultry industry is not known. This is partly because the pathogenicity of APMV-3 in poultry is not well studied. There is a high degree of amino acid sequence variation between APMV-3 and APMV-1 (NDV), but by the HI test there is low level of cross reaction between APMV-3 and APMV-1 serum samples, which often leads to misdiagnosis of APMV-3 as APMV-1.

APMV-3 strain Wisconsin was first isolated from a turkey in 1968 in Wisconsin (Tumova et al., 1979b). APMV-3 strain Netherlands was isolated from a parakeet in 1975 in the Netherlands and is the prototype for the entire serotype (Alexander and Chettle, 1978). Initially, these two viruses were considered as two different strains of APMV-3 based on cross HI test using monoclonal antibodies (Anderson et al., 1987; Anderson and Russell, 1988). Recently, reciprocal cross HI and cross neutralization assays using post infection serum from chicken indicated that these two strains are antigenically distinct, although the difference was modest (Kumar et al., 2010b). However, complete genome sequence analysis revealed substantial genome-wide nucleotide and amino acid sequence differences that are consistent with the two strains representing distinct antigenic subgroups (Kumar et al., 2008; Kumar et al., 2010b). We have performed experimental infection of APMV-3 strains Wisconsin and Netherlands in 9-day-old embryonated chicken eggs, 1-day-old chicks and turkeys, and 2-week-old chickens and turkeys in order to investigate their tropism and pathogenicity. Birds were infected by the oculonasal route and the viral tropism and replication efficiency were evaluated by quantitative virology and immunohistochemistry of a wide range of possible target organs. In addition, a separate group of 1-day old chicks were infected intracerebrally to evaluate the potential of these viruses to replicate in neuronal tissue.
