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and that AMPV-C and HMPV diverged from a common ances- residing in the trachea (especially in duck TOCs) can mask the
tor approximately 210 years ago (de Graaf et al., 2008b). These cilia making ciliostasis difficult to assess. Furthermore, it has been
data relating to the relationships between different metapneu- contended that the North American subgroup C viruses do not
moviruses make it easier to consider AMPV-A, B and D as type cause ciliostasis upon propagation (Cook et al., 1999). It is for
I metapneumoviruses and AMPV-C and HMPV as type II as has this reason that in ovo inoculation via the yolk sack of chicken or
been previously suggested (Brown et al., 2014). turkey embryos, followed by a number of blind passages in cell
culture, is to be considered. Both of these techniques are however
more laborious than direct isolation on cell culture. Cell types
Infectious agent often used for isolation and propagation of the virus include pri-
mary chick embryo fibroblasts (CEFs) and VERO and MA-104
Morphology cell lines derived from monkey kidney cells or quail tumour cell
Electron microscopic analysis shows AMPV in pleomorphic, line QT-35 (Giraud et al., 1986, 1987b; Chiang et al., 1998; Ben-
often bizarre shapes (Fig. 4.1) that are enveloped and covered nett et al., 2002).
with a fringe of projections 13–15 nm in length. The size of
spherical virus particles ranges from 50 to 600 nm in diameter; Genome structure and organization
however, long filamentous forms are also observed, and these can Avian Metapneumoviruses are enveloped viruses with single-
be up to 1000 nm in length. The nucleocapsid of AMPV has been stranded, negative-sense RNA genomes of 13,134 to 14,152
shown as a helical structure with a diameter of 14 nm and a pitch nucleotide bases in length. In general, it is the subgroup C viruses
of 7 nm consistent to that of RSV. that have the longer genomes, and this is mainly linked to the size
of their G gene (Toquin et al., 2003). Most AMPVs, like HMPVs
Propagation and pneumoviruses do not have a genome length that complies
Cell culture, tissue culture and in ovo culture systems are avail- with the ‘rule of six’ (Easton et al., 2004) unlike the closely related
able for the isolation and propagation of AMPV. Some of these paramyxoviruses. A graphic representation of the AMPV/HMPV
systems are laborious and some have been proposed to be unsuit- genome organization is shown in Fig. 4.2 together with RSV as
able for certain subgroups. For primary isolation, the technique a representative of the genus Pneumovirus. Fig. 4.2 clearly shows
of tracheal organ cultures (TOCs) (Cook et al., 1976) prepared that the general composition and organization of pneumovirus
from turkey, chicken or duck embryos have been widely used. and metapneumovirus genomes is very similar yet as mentioned
In this system ciliostasis is the indicator of virus growth how- briefly at the start of this chapter there are some differences when
ever, if tracheal rings are not meticulously prepared then mucus looking at the detail. Firstly, the metapneumovirus genomes

(A) (B) (C)

Figure 4.1 Electron micrographs (negative staining): avian metapneumovirus particles in Vero cell supernatant (Toquin, 1987). (A) Spherical
form; (B) filamentous forms; (C) disrupted particle with nucleocapsid protruding from the envelope.

Figure 4.2 Composition and organization of pneumovirus (RSV) and metapneumovirus (MPV) genomes.