Discussion
In the present study, one hundred and sixteen aMPV-B partial G gene
sequences, obtained during routine diagnostics in different European
Countries in the last few years, were analysed by sequence and
phylogenetic analyses in order to molecularly characterize them.
The G gene, which harbours mutations at variable positions between
aMPV-B strains, was conveniently amplified by the routine diagnostic
RT-PCR protocol employed in the study, and its sequencing proved useful
for epidemiological purposes. Furthermore, its variability can give an
indication of whether vaccine or field strains are present, making the
differentiation relatively easy and cheap to perform.
Live attenuated aMPV vaccines are widely administered to prevent disease
in turkeys and chickens, and the recovery of vaccine-derived strains is
not unusual both in vaccinated or in unvaccinated flocks (Banet-Noach et
al., 2009; Lupini et al., 2011; Chacon et al., 2011; Cecchinato et al.,
2013a; Listorti et al., 2014; Arafa et al., 2015; Bayraktar et al.,
2019; Andreopoulou et al., 2019).
The 40% of the aMPV-B strains detected in the present study were
classified as vaccine-derived strains, being phylogenetically related
and showing high nucleotide identity with live commercial vaccine
strains licensed in Europe. As expected, vaccine-derived strains formed
separate clusters depending on the vaccine strain of origin and were
detected in all tested European countries.
A large part of the vaccine-derived strains analysed in the present
study was detected in homologous-vaccinated birds, from approximately
two to four weeks after vaccination, and only occasionally in
unvaccinated birds. Reversion to virulence of aMPV subtype A or B has
been previously demonstrated (Catelli et al., 2006; Brown et al., 2011,
Cecchinato et al., 2014). Therefore, the detection of vaccine-derived
strains closely related to the applied vaccine, concurrently with
respiratory signs, could be reliably linked to the vaccine reversion to
virulence.
The detection of the strain aMPV/B/France/GuineaFowl/1060/18 in
unvaccinated guinea fowls is noteworthy, since its partial G gene
sequence shared 100% nucleotide identity with the vaccine strain 11/94.
It could be speculated that the vaccine virus could had been introduced
as a contaminant by personnel, fomites, vehicles movement or airborne
from neighbouring premises. The field veterinarian reported the presence
of a turkey farm at approximately 500 meters from the guinea fowl one.
Vaccinal strain 11/94 was applied in the turkey flock and the use of the
litter from the turkey farm for fertilization of the surrounding crops
was reported. The ability of vaccine-derived aMPVs to spread beyond the
administration site is well known and it has been proven by Lupini et
al. (2011) following a turkey rhinotracheitis outbreak caused by aMPV
subtype A in unvaccinated turkeys.
The remaining 60% aMPV-B viruses analysed in the present study were
classified as field strains since they clustered separately and showed a
low nucleotide identity with vaccines and vaccine-derived strains.
The phylogenetic tree reconstructed with European sequences showed that
the virus has continued to evolve from its first appearance in the ’80s.
In fact, more recently detected field strains belonged to clades
phylogenetically distant from the older field strains, confirming the
previously-reported aMPV tendency to evolve over time (Cecchinato et
al., 2010).
Unlike vaccine-derived strains, field strains tended to cluster
according to their geographic origin, with few exceptions. Distinct
clusters were observed for French, Italian, Romanian and Spanish
strains, yet some Italian and Greek field isolates clustered closely
together, indicating a potential transmission route between these two
countries (Tucciarone et al., 2017; Andreopoulou et al., 2019).
The molecular epidemiology of aMPV within each country was analysed in
detail reconstructing country-related phylogenetic trees. Heterogeneous
field strain populations seemed to co-exist within single European
countries, with the only exception of Romania, where all identified
strains were part of just one clade. This last finding could be
explained by keeping into account that all the processed samples came
from different sites within the same company in which viral circulation
could be compartmentalized (Franzo et al., 2020).
French, Italian, and Spanish trees showed a rather heterogeneous field
strain population, as the strains fell into more than one cluster,
divided in several sub-clusters. The heterogeneity was further deduced
from the within-group mean p-distance values. The highest value,
indicating the highest heterogeneity of nucleotide sequence, was
observed for Italian field strains, presumably because of the larger
number of sequences available over a broader time period and coming from
different commercial poultry companies.
Furthermore, time-related clustering was visible for Italian and Spanish
field strains, as strains detected in the last few years tend to form
separate clusters or sub-clusters.
As a common finding, recent field aMPV-B strains, belonging to different
and distant phylogenetic clades, were detected in the presence of
respiratory signs in vaccinated flocks. Due to the G gene variability
observed in recent aMPV-B strains, and knowing that the encoded surface
G glycoprotein is a key antigen for vaccine-induced immune protection
(Naylor et al., 2007) and can evolve in order to avoid vaccine-induced
immunity (Catelli et al., 2010; Cecchinato et al., 2010), re-assessment
of protection conferred by commercially available vaccines against
currently circulating aMPV field strains might be necessary to improve
disease control strategies.
The obtained phylogenetic data revealed that aMPV-B strains clustered
together irrespective of the host species where the viruses had been
detected. A recent experimental challenge study showed that both
chickens and turkeys are susceptible to aMPV-B infection with the same
virus isolate (Brown et al., 2019). Moreover Cecchinato et al. (2018)
reported an outbreak of respiratory disease in guinea fowls caused by
aMPV-B strain identical to the ones circulating in the surrounding
turkey flocks. Therefore, no evidences are currently available to
support a host-specific adaptation of aMPV variants.
As a final remark, the tree reconstructed on European and extra-European
strains further confirmed the geographic clustering of field strains. As
a rule, field strains grouped together for country location and time
period, suggesting a local evolution tendency of the virus that might
have taken place after a single introduction event. The majority of
recently detected European field strains were located in a big subtree
indicating a certain genetic similarity and supporting the wide
circulation of a quite homogeneous aMPV subtype B clade in European
countries, with the only exception of two clusters of
phylogenetically-distant French and Spanish strains.
In conclusion, the molecular characterization of aMPV subtype B and the
differentiation between vaccines and field strains through G gene
sequence analysis can be a useful tool towards a correct diagnosis and
should be routinely applied in order to better address the control
strategies. In this respect, current vaccine research is focused on
reducing the issues connected to live attenuated vaccine reversion to
virulence or the selection of potentially virulent subpopulations
(Franzo et al., 2015). Therefore, considering the associated risks,
further efforts should be directed at improving administration and
biosecurity measures, in order to reduce their prolonged circulation and
spreading.
Several research groups have attempted to develop efficacious, more
stable and safer next-generation vaccines (Qingzhong et al., 1994;
Tarpey et al., 2001; Kapczynski and Sellers, 2003; Kapczynski, 2004;
Chary, Njenga, & Sharma, 2005; Yu et al., 2013; Hu, Roth, Zsak, & Yu,
2017) but, despite this effort, live vaccines still provide the greatest
protection after homologous challenge.
Promising reverse genetics systems for aMPV have been developed in
recent years for A, B and C subtypes (Naylor et al., 2004; Yu et al.,
2010; Laconi et al., 2016) other than being an exceptional tool for the
study of the virus properties (Brown et al., 2011), can be used for the
development of rationally modified aMPV vaccines (Naylor, Lupini, &
Brown, 2010) or recombinant vaccines (Falchieri et al., 2013) expressing
foreign genes.
Conflict of Interest Statement
The authors declare no conflict of interest.