Page 191 - Avian Virology: Current Research and Future Trends

184 | Kibenge et al.

1999). However, due to the innate advantages of the continuous Genome structure and organization
cell lines, their use to grow avian reovirus has practical benefits. The avian reovirus genome was initially determined to consist
Sahu and Olson, (1975) examined the replications of different of RNA by using DNA inhibitors (Hieronymus et al., 1983),
avian orthoreovirus strains in bovine (MDBK), human (Chang and susceptibility to DNase or RNase digestion (Spandidos and
C, Hep-2, HeLa), murine (L929), canine (MDCK) and primate Graham, 1976). The genome was shown to contain ten linear
(LLC-MK2, VERO) cells and observed cytopathic effects (CPE) double-stranded (ds) RNA segments (Sekiguchi et al., 1968;
only in VERO cells but not with all virus strains tested. Barta et al. Glass et al., 1973; Spandidos and Graham, 1976; Gouvea and
(1984) tested the sensitivity of seven mammalian and two chicken Schnitzer, 1982; Murphy et al., 1999), and its segments were
cell lines to the WVU2937 avian reovirus strain and found out divided into three size classes, designated L (large), M (medium)
that the virus replicated successfully in six of the lines tested after and S (small) in sucrose gradients (Spandidos and Graham,
some adaptation, but VERO was found to be the most sensitive 1976), or by analysis of the migration pattern of the viral genome
among mammalian cell lines. Wilcox et al. (1985) were able to using sodium dodecyl sulfate-polyacrylamide gel electrophore-
adapt only one of six Australian avian reoviruses (strain RAM-1) sis (SDS-PAGE) (Rekik et al., 1990). There are three segments
to VERO cells, although Hussain et al. (1981) had found all three of the L-class (L1, L2, L3), three of the M-class (M1, M2, M3)
of the Australian strains they examined to produce CPE in VERO and four of the S-class (S1, S2, S3, S4) (Spandidos and Graham,
cell line. Nwajei et al. (1988) managed to adapt all the 22 avian 1976; Rekik et al., 1990; Benavente and Martínez-Costas, 2007;
orthoreovirus strains tested by them in VERO cells, however, all Yun et al., 2013). The overall genome size is 23,492 base pairs
of these strains had been passaged in avian embryonic cell cul- (bp) for the avian orthoreovirus 138 isolate and 23,493 bp for the
tures before testing on VERO cells. Further they were unable to 176 isolate (Xu and Coombs, 2009), and each RNA segment is
directly isolate avian orthoreoviruses in VERO cells from faecal present in equimolar proportion in virions, representing one copy
or joint material already shown to contain avian reovirus by chick of each genome segment per virion (Murphy et al., 1999). The
embryo liver passage. Accordingly, they concluded that VERO approximate molecular weight range of each genomic segment
6
6
cells were unsuitable for the primary isolation of avian reoviruses group is 2.5–2.7 × 10 , 1.3–1.8 × 10 , and 0.71–1.2 × 10 daltons
6
from field material. They suggested that the virus strains differ in for L, M and S segments, respectively (Spandidos and Graham,
their tropism towards specific tissues and that differences exist in 1976; Lozano et al., 1992). The migration patterns of dsRNA
the sensitivity among batches of VERO cells from various sources segments of avian orthoreovirus genomes were heterogenous
and these may be exaggerated by the use of different media. In within the same serotype as well as among different serotypes
accordance with this suggestion Jones and al Afaleq (1990) dis- (Rekik et al., 1990). In addition to the ten genomic segments,
covered that there were significant differences in titre of the four the avian reovirus particle contains many small, adenine rich,
avian reovirus strains propagated in VERO cells using different single-stranded oligonucleotides (Spandidos and Graham,
growth media. According to the studies on adaptation of the avian 1976). Similarly, for all members of family Reoviridae, the posi-
reovirus strain R-85 to VERO, BHK- 21 and MDBK mammalian tive strands of each double-stranded segment have 5′/- terminal
cell lines, VERO cells turned out to be the most susceptible demethylated type 1 Cap structure ( GpppGm 2’OH ), (Miura
m7
(Georgieva and Jordanova, 1999), yielding more rapid syncytial et al., 1974), and the negative strands have phosphorylated 5′/
7.2
type CPE and higher virus titre of 10 TCID /ml. Therefore, termini but the 3’/termini of both positive and negative strands
50
it was concluded that VERO cells could be effectively utilized lack poly (A) tails (Murphy et al., 1999). All reovirus genome
as a standardized system for propagation on avian reoviruses. segments sequenced so far, including those of avian reoviruses,
Many studies revealed, among the many established continuous have conserved 5′- and 3′-terminal nucleotide sequences of vari-
mammalian cell lines tested, only VERO cells supported the able length, which are useful for reovirus classification (Duncan,
replication of certain strains of avian orthoreoviruses (Barta et al., 1999; Chen et al., 2011a; Thimmasandra et al., 2015). Genome
1984; Wilcox et al., 1985; Nwajei et al., 1988; Jones and al Afaleq, segment re-assortment occurs readily in cells co-infected with two
1990). In contrast to avian origin cell cultures, mammalian cell avian reovirus isolates sharing the same packaging signals (Attoui
lines like VERO have further advantages such as the convenience et al., 2012) producing reassorted progeny viruses that contain
for use and no risk of contamination with vertically transmitted genome segments from the two parental viruses (Benavente and
avian reoviruses. However, the CPE produced in VERO cells is Martínez-Costas, 2007).
characterized by focal areas of cell fusion (syncytia) (Wilcox et The gene coding assignments in avian reoviruses are sum-
al., 1985; Nwajei et al., 1988; Simoni et al., 1999), and the virus marized in Table 6.1. The ten segments encode at least eight
was so highly cell associated (Wilcox et al., 1985; Nwajei et al., structural proteins (λA, λB, λC, µA, µB, µBC, µBN, σC, σA, and
1988) that it required up to 10 days PI to obtain maximum CPE σB) and four non-structural proteins (µNS, p10, p17, and σNS)
(Wilcox et al., 1985). In addition, infected cells had to be frozen (Shmulevitz et al., 2002; Benavente and Martínez-Costas 2007).
and thawed at least four times before the virus could be passaged All the genome segments are monocistronic except S1 segment
successfully. In contrast, in chicken cell cultures, avian reoviruses which is tri-cistronic (Attoui et al., 2012). However, in certain
required only 4 days incubation to produce CPE and one freeze– strains of classical Muscovy reovirus (classical MDRV), S4 seg-
thaw cycle to release virus from infected cells (Robertson and ment is bi-cistronic whereas S1 is monocistronic (Ma et al., 2012;
Wilcox, 1984). Wang et al., 2012; Yun et al., 2012).