Vaccine 29 (2011) 1222–1227
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Lights and shades on an historical vaccine canine distemper virus,
the Rockborn strain
V. Martellaa,∗, M. Blixenkrone-Møllerb, G. Eliaa, M.S. Lucentea, F. Cironea, N. Decaroa,
L. Nielsenb, K. Bányaic, L.E. Carmichaeld, C. Buonavogliaa
aDepartment of Veterinary Public Health, University of Bari, Italy
bDepartment of International Health, Immunology & Microbiology, The Panum Institute, Faculty of Health Sciences, University of Copenhagen, Denmark
cVeterinary Medical Research Institute, Hungarian Academy of Sciences, Hungária krt. 21, H-1143 Budapest, Hungary
dCornell University, Ithaca, NY, USA
a r t i c l e i n f o
Received 25 February 2010
Received in revised form
23 November 2010
Accepted 1 December 2010
Available online 15 December 2010
Canine distemper virus
a b s t r a c t
Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual viru-
animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of
Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus
ing, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than
By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial
vaccine (Candur®SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly
used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and
99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV
strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also,
Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate
that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggest-
ing cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in
© 2010 Elsevier Ltd. All rights reserved.
Canine distemper virus (CDV) is an enveloped single-stranded
centuries and severe CDV-related epidemics were described in the
17th century in Southern America, Spain, Italy, England and Russia
and their extensive use has greatly helped keep under control the
disease [1,3,21]. However, CDV-related disease in the world canine
population seems to have increased in the last decades and both
sporadic cases and large outbreaks of CDV disease in vaccinated
animals have been reported [6,15–17,32,46]. This has been related
either to the use of inappropriate vaccines  or to increasing
illegal commercial sale of pups .
∗Corresponding author at: Dipartimento di Sanità Pubblica e Zootecnia, Univer-
sità di Bari, S.p. per Casamassima Km 3, 70010 Valenzano, Bari, Italy.
Tel.: +39 080 4679805; fax: +39 080 4679843.
E-mail address: email@example.com (V. Martella).
Initial attempts to produce immunization tools against CDV
were made in the 1920s by Puntoni and by Laidlaw and Dunkin,
using either formalin inactivated brain material of infected dogs, or
parallel administration of infectious brain material and hyperim-
mune sera. In 1939 a vaccine (Green’s ‘Distemperoid’ vaccine) was
developed using a CDV isolate (strain Onderstepoort) made from
a disease outbreak among North American ranched foxes in the
further attenuated by 130 serial passages in chorio-allantoic mem-
brane of embryonated hen’s eggs [23,24] but it was found to loose
immunogenicity after 200 egg passages [10,19]. From the 1950s,
the egg-attenuated Onderstepoort strain has dominated the mar-
ket, especially in Europe, and is still contained in most currently
In the 1950s, extensive epidemics caused by CDV occurred in
Sweden in both vaccinated and unvaccinated dogs. Subsequently,
a canine isolate, strain Rockborn, was made on primary dog kid-
ney cells, and claimed to be attenuated . The virus was still
pathogenic after 36 serial passages on primary dog kidney cells,
0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
V. Martella et al. / Vaccine 29 (2011) 1222–1227
but became ‘completely attenuated’ after 56 serial passages ;
it was found to loose immunogenicity by the 70th passage. The
vaccine was distributed globally after 1962 and proved to elicit
solid immunity and protection to vaccinated animals. In the early
1980s, post-vaccinal encephalitis was reported in dogs from var-
ious parts of Britain after administration of a particular batch of
combined CDV Rockborn strain/canine adenovirus type-1 vaccine,
although incrimination of the Rockborn strain was subsequently
retracted [13,14]. This report noted analogous observations made
in 1974 and 1978 [4,27] and led to the opinion that the Rockborn
Onderstepoort strain. Suspected cases of post vaccinal encephali-
tis were also reported in 1995 in USA in dogs vaccinated with a
polyvalent vaccine containing the strain Rockborn. Consequently,
the Rockborn strain was withdrawn from several markets after the
mid 1990s .
The H glycoprotein is a key protein for CDV as the virus uses
this protein for attachment to the signaling lymphocyte activation
molecule (SLAM) receptor (CD150)  on the cell membrane and
an adequate host immune response against the H protein may pre-
vent CDV infection . Comparative studies of CDV strains have
revealed that the H gene is subjected to higher genetic/antigenic
variation than other CDV genes and it appears as the most suit-
able target to obtain epidemiological information [7,22,31,36,40].
In spite of its historical importance, only limited sequence infor-
mation is available for the Rockborn strain, and the sequence of the
H gene has not been determined. Sequence information on the H
gene is available for the avianized strains Onderstepoort and Led-
erle . Sequence information is also available for the pathogenic
Snyder Hill virus , but not for the ‘attenuated Snyder Hill virus’,
that seems to be no longer available in the market. Based on the
H gene, the vaccine strains Ondestepoort, Lederle and Snyder Hill
appear to be genetically related to each other (<4% aa variation)
but more distantly related (>8% aa variation) to the CDV strains
developed that are able to distinguish between the Onderstepoort
strain and field CDV strains [31,40–42,50,36].
In order to fill this information gap, the sequence of the H gene
of a laboratory passage (46th) of the vaccine CDV strain Rockborn,
available from the James Baker Institute, was determined. In addi-
tion, the sequence of commercially available CDV vaccines was
determined and compared.
1. Materials and methods
1.1. Virus origin
The 46th laboratory passage of the vaccine CDV strain Rockborn
(Rockborn-46th) was available at the James Baker Institute labora-
tories. The virus was re-amplified by two serial passages on VERO
cells and viral growth was monitored by visualization of the cyto-
vaccine Candur®SH+P (Hoechst Rousell Vet GmbH) (batch number
Two additional vaccines (A and B) commercialized from two
different companies were also analysed. For those vaccines, infor-
mation on the CDV strain was not specified on the labels. By
using an RT-PCR genotyping assay with primers specific for vari-
ous CDV genotypes (America-1, Arctic-like, European, Asia-1 and
Asia-2)  vaccines A and B were found not to contain America-1
(Onderstepoort-like) CDV strains. Therefore, the sequence of the H
gene was determined in order to establish firmly which CDV strain
was included in the vaccine formulation.
1.2. RNA extraction, reverse transcription and PCR amplification
A 140?l lysate of cells infected with strain Rockborn-
46th and 140?l of each vaccine suspension were used for
RNA extraction. The RNA was extracted using the RNeasy Kit
was achieved as previously described . To obtain a PCR prod-
uct for sequencing of the H gene of the Italian strains, 0.5?l each of
primers CDV-F8 and CDV-R8 (50pmol/?l)  was added to a total
of 49?l of the reaction mixture containing 0.2mM of each dNTP,
1.2mM MgSO4and 1?l of a mix of SuperScript II H-Reverse Tran-
scriptase and Platinum Taq HiFi (Invitrogen – Life Technologies,
Milan, Italy). The RNA was reverse transcribed and immediately
subjected to PCR amplification in a single-step protocol, using
SuperScript One-Step RT-PCR kit (Invitrogen – Life Technologies,
Milan, Italy). Reverse transcription was carried out at 48◦C for
60min, followed by denaturation of the reverse transcriptase at
95◦C for 2min. Amplification was conducted by a temperature
cycling protocol consisting of 35 cycles of 30s of denaturation at
94◦C, 1min of primer annealing at 55◦C, and 1min of extension
at 68◦C, followed by 10min of the final extension phase at 68◦C.
To obtain PCR products suitable for sequencing, the inner primer
pair RH-3 and RH-4  was used to amplify in a nested PCR the
complete H-gene. About 0.5?l each of primers RH-3 and RH-4
(50pmol/?l) was added to a total of 49?l of the reaction mixture
consisting of 0.5?l of a 1:100 dilution of the first-round PCR prod-
uct, 0.25?l of TaKaRa LA Taq (5U/?l; Takara Bio Inc., Japan), and
5?l of 10× PCR buffer, 8?l of 2.5mM dNTP mixture, and 35.75?l
of distilled H2O. Amplification was conducted by a temperature
cycling protocol consisting of 25 cycles of 1min of denaturation
at 94◦C, 2min of primer annealing at 50◦C, and 2min of exten-
sion at 68◦C, followed by 2min of the final extension phase at
The RH3–RH4 PCR products were purified with the Wizard PCR
Preps DNA Purification System (Promega, Madison, WI). Then, the
DNA was used as template for direct sequencing. The DNA was
sequenced by using the conserved primers RH3 and RH4 and spe-
cific primers designed according to an overlapping strategy. The
sequences were assembled using Bioedit software package ver-
sion 2.1  and compared to cognate sequences in the genetic
databases using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and
FASTA (http://www.ebi.ac.uk/fasta33) web-based programs. The
gene H sequences of the strains Rocborn-46th and Rockborn-
Candur is available under accession numbers GU810819 and
1.4. Phylogenetic analysis
Nucleic acid sequences were aligned with a selection of CDV
H sequences by using Mega 4.0 software package . The tree
was inferred using the Kimura two-parameter model and the
neighbor-joining method. Statistical significance of the phylogeny
was estimated by bootstrap analysis over 1000 pseudoreplicate
2. Results and discussion
The H gene of the strain Rockborn-46th was 1824 nt long and
coded for a 607 aa-long polypeptide. Eight conserved potential
N-glycosylation sites were found at residues 19, 149, 309, 391,
422, 456, 587 and 603. The virus Rockborn-46th differed in a
V. Martella et al. / Vaccine 29 (2011) 1222–1227
Fig. 1. Phylogenetic tree based on the full-length H gene displaying the genetic relationships between the strain Rockborn and a selection of CDV strains of various genotypes.
Bootstrap values lower than 80% are not shown. In the box, a sub-tree generated using a 385-nt fragment of the H gene (nt 416–800 of the H gene) is shown.
unique aa change, 544-K→T (nt 1631-A→C) from the vaccine
virus Rockborn-Candur. By FASTA analysis, the virus had the high-
est identity (99.6% nt and 99.3% aa) to a CDV strain detected from a
Lesser Panda (Ailurus fulgens) in China (AF178039). In addition, the
virus was found to match (>99.0% nt identity in the full-length H
accession AB191310)  and in a 800-nt long fragment to canine
viruses (99.9–100% nt) detected in Austria 2007 (strain 2727-07
and 2730-07, accessions GQ214370 and GQ214371, respectively).
By phylogenetic analysis, using a selection of CDV strains repre-
sentative of the various CDV lineages, Rockborn-like viruses were
shown to be genetically related to America-2 viruses and distantly
related to America-1 CDVs (Fig. 1). The strain Rockborn-46th pas-
sage displayed 93.0% nt and 91.7% aa identity to the vaccine strain
Onderstepoort, prototype of the America-1 lineage.
Interestingly, the CDV strains from vaccines A and B were also
found to contain Rockborn-like viruses. Vaccine A differed in only
while vaccine B differed in 4 residues, 160-R→K, 303-L→S, 544-
K→T and 589-T→P. The residue 544-T was also found in the
Rockborn-Candur strain, while the change 589-T→P in vaccine
B disrupted the N-587-glycosylation site (Table 1). Assuming that
the viruses likely came from different virus passages and batches,
it is tempting to speculate that those differences may have been
accumulated during cultivation on cell cultures. Accordingly, the
laboratory strain Rockborn 46th and the vaccine virus Rockborn-
Candur appeared to be similar cell passages of the virus, since
they differed only in a unique non-silent nt change. Similarly, vac-
cine A differed in only two aa residues from the laboratory strain
Rockborn-46th; in contrast, vaccine B displayed 4 mutations, with
the change 589-T→P, altering a potential N-linked sugar binding
site. By solving the crystal structure of measles virus H protein,
V. Martella et al. / Vaccine 29 (2011) 1222–1227
Nucleotide and amino acid changes in the H gene and protein of CDV strains Rockborn-46th, Rockborn-Candur and Rockborn-like viruses. Amino acid mutations are indicated in brackets.
a morbillivirus closely related to CDV, sugar moieties have been
seen to mask massively the protein surface and to help the homod-
imeric structure of the H protein to maintain correct orientation
. Residues of the putative receptor-binding site are strategi-
cally positioned in an unshielded area on the top of the protein,
tend to be highly conserved among morbilliviruses. Accordingly,
changes in the sugar moieties can mask or expose epitopes on the
H protein but functional constraints tend to conserve the antigenic
monotypic profile of morbilliviruses .
An alternative hypothesis to explain the presence of Rockborn-
like viruses in vaccines A and B is that they are variants of a field
Rockborn-like strain, circulating not only in Sweden in the 1950s,
but also in other geographical settings and isolated on more occa-
sions. By comparing the sequence of the virus Rockborn-46th with
the databases, at least five Rockborn-like sequences were identi-
fied. Scattered mutations were identified in the full-length H gene
Panda virus (7 nt and 4 aa changes) and the USA strain 25259 (18
nt and 8 aa changes) (Table 1). Due to the consistent number of
mutations, the origin of these viruses may not be unequivocally
attributed to Rockborn-based vaccines and a possible explanation
is that Rockborn-like viruses are still circulating in the field or
that animalised vaccine-derived viruses were introduced in unre-
lated geographical settings on several occasions and circulated in
the area in different carnivores, taking advantage of the broad
host range of CDV. Also, Ondestepoort-like CDVs, identified in
the 1930s and long believed to be extinct in the field, have been
identified from outbreaks in wildlife animals in United States and
have been reported occasionally in dogs from Ireland, Korea and
Poland [28,32,34,46], suggesting vaccine-induced disease, eventu-
ally followed by animalisation of vaccine viruses in some animal
populations, or persistence of America-1 CDVs in certain ecological
Conversely, exploring the databases, three CDV sequences (one
Japanese and two Austrian viruses) were found to match (nearly
100% nt) the sequence of strain Rockborn-46th. Such high genetic
identity is much more consistent with a hypothesis of residual
virulence of vaccine viruses. This phenomenon appears to be mod-
ulated by host factors, such as age, genetic constitution, state of
tant diseases/infection as well as by virus-intrinsic factors, i.e.
the grade of virus attenuation. Vaccine-related disease has been
reported on several occasions in dogs [4,14,18,23,27,33,39]. Pups
becoming ill within 2–3 weeks after administration of CDV vaccine
are always suspected to have developed vaccine-induced disease
and this may result in disputes involving veterinary practition-
ers, pet owners and commercial pet shops. In most suspected
cases, however, it has been difficult to establish whether the dis-
ease was induced by the vaccine virus or by field viruses infecting
pups shortly before or after vaccine administration. This issue was
not ruled out firmly in older studies, since appropriate diagnos-
tic tools were not available. Therefore, in some circumstances the
magnitude of this phenomenon may have been overrated. Dur-
ing a 2-year-surveillance in Italy, we found all suspected cases
of CDV vaccine-induced illness or death to be caused by CDV
field strains , thus suggesting that residual virulence is infre-
The vaccine strain Rockborn was suspected to retain resid-
ual virulence after several reports on vaccine-related disease
[4,14,18,27] and this was attributed to an intrinsic virulence of low
passage stocks established on primary dog kidney (DK) cells and
grown for vaccine passage cultures on Madin Darby Canine Kidney
In most cases, increased virulence of the dog-kidney-cell-
attenuated CDV was suspected to have been triggered by the
V. Martella et al. / Vaccine 29 (2011) 1222–1227
contemporaneous presence in the vaccine formulations of live
modified canine adenovirus type-1. Under experimental condi-
tions, re-acquisition of virulence by the kidney-cell-attenuated
Rockborn virus has been demonstrated in vivo after six sequential
passages in dogs and in vitro after ten passages in primary dog lung
macrophages, but the molecular bases of this phenotypic change
teins appear to regulate CDV replication and dissemination in the
infected host , thus complicating a dissection of the molecular
mechanisms behind attenuation of CDV virulence.
In conclusion, these findings indicate that (i) Rockborn-like vac-
be recovered from dogs or other carnivores with distemper dis-
ease, suggesting residual virulence of some vaccine batches and/or
the circulation of analogous viruses in some ecological niches. The
information provided with this study will be paramount to dis-
solve shades that have been accumulating over the years on an
ferential diagnostic tools able to identify promptly vaccine-related
disease in dogs.
This work was granted by the project “Gastroenteriti virali dei
a carattere zoonosico – Fondi di Ateneo 2009” and by grants from
the Italian Ministry of Health, Ricerca corrente 2009, project IZS VE
21/09 RC “Definizione di una procedura validata per la selezione di
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