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Vaccine 29 (2011) 1222–1227

Contents lists available at ScienceDirect

Vaccine

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ights and shades on an historical vaccine canine distemper virus,he Rockborn strain

. Martellaa,∗, M. Blixenkrone-Møllerb, G. Eliaa, M.S. Lucentea, F. Cironea, N. Decaroa,. Nielsenb, K. Bányaic, L.E. Carmichaeld, C. Buonavogliaa

Department of Veterinary Public Health, University of Bari, ItalyDepartment of International Health, Immunology & Microbiology, The Panum Institute, Faculty of Health Sciences, University of Copenhagen, DenmarkVeterinary Medical Research Institute, Hungarian Academy of Sciences, Hungária krt. 21, H-1143 Budapest, HungaryCornell University, Ithaca, NY, USA

r t i c l e i n f o

rticle history:eceived 25 February 2010eceived in revised form3 November 2010ccepted 1 December 2010vailable online 15 December 2010

eywords:anine distemper virusog

a b s t r a c t

Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual viru-lence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptibleanimal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts ofBritain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirustype-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstand-ing, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe thanother CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s.By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercialvaccine (Candur® SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly

accineemagglutininenotype

used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDVstrains 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 indicatethat 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

the field.

Canine distemper virus (CDV) is an enveloped single-strandedegative RNA virus belonging to genus Morbillivirus in the Paramyx-viridae family [51]. The disease caused by CDV has been known forenturies and severe CDV-related epidemics were described in the7th century in Southern America, Spain, Italy, England and Russia5]. The introduction of the live modified CDV vaccines in the 1950snd their extensive use has greatly helped keep under control theisease [1,3,21]. However, CDV-related disease in the world canineopulation seems to have increased in the last decades and bothporadic cases and large outbreaks of CDV disease in vaccinated

nimals have been reported [6,15–17,32,46]. This has been relatedither to the use of inappropriate vaccines [16] or to increasingllegal commercial sale of pups [35].

∗ Corresponding author at: Dipartimento di Sanità Pubblica e Zootecnia, Univer-ità di Bari, S.p. per Casamassima Km 3, 70010 Valenzano, Bari, Italy.el.: +39 080 4679805; fax: +39 080 4679843.

E-mail address: v.martella@veterinaria.uniba.it (V. Martella).

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.12.001

© 2010 Elsevier Ltd. All rights reserved.

Initial attempts to produce immunization tools against CDVwere made in the 1920s by Puntoni and by Laidlaw and Dunkin,using either formalin inactivated brain material of infected dogs, orparallel administration of infectious brain material and hyperim-mune sera. In 1939 a vaccine (Green’s ‘Distemperoid’ vaccine) wasdeveloped using a CDV isolate (strain Onderstepoort) made froma disease outbreak among North American ranched foxes in the1930s. The strain was attenuated by 50 serial passages in ferret [20]but residual virulence was observed. The Distemperoid vaccine wasfurther attenuated by 130 serial passages in chorio-allantoic mem-brane of embryonated hen’s eggs [23,24] but it was found to looseimmunogenicity 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 currentlyavailable vaccines.

In the 1950s, extensive epidemics caused by CDV occurred inSweden 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 [45]. The virus was stillpathogenic after 36 serial passages on primary dog kidney cells,

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ut became ‘completely attenuated’ after 56 serial passages [44];t was found to loose immunogenicity by the 70th passage. Theaccine was distributed globally after 1962 and proved to elicitolid immunity and protection to vaccinated animals. In the early980s, post-vaccinal encephalitis was reported in dogs from var-

ous parts of Britain after administration of a particular batch ofombined CDV Rockborn strain/canine adenovirus type-1 vaccine,lthough incrimination of the Rockborn strain was subsequentlyetracted [13,14]. This report noted analogous observations maden 1974 and 1978 [4,27] and led to the opinion that the Rockborntrain was less attenuated and less safe than other CDV vaccines, e.g.nderstepoort strain. Suspected cases of post vaccinal encephali-

is were also reported in 1995 in USA in dogs vaccinated with aolyvalent vaccine containing the strain Rockborn. Consequently,he Rockborn strain was withdrawn from several markets after the

id 1990s [18].The H glycoprotein is a key protein for CDV as the virus uses

his protein for attachment to the signaling lymphocyte activationolecule (SLAM) receptor (CD150) [49] on the cell membrane and

n adequate host immune response against the H protein may pre-ent CDV infection [53]. Comparative studies of CDV strains haveevealed that the H gene is subjected to higher genetic/antigenicariation than other CDV genes and it appears as the most suit-ble target to obtain epidemiological information [7,22,31,36,40].n spite of its historical importance, only limited sequence infor-

ation is available for the Rockborn strain, and the sequence of thegene has not been determined. Sequence information on the H

ene is available for the avianized strains Onderstepoort and Led-rle [11]. Sequence information is also available for the pathogenicnyder Hill virus [9], but not for the ‘attenuated Snyder Hill virus’,hat seems to be no longer available in the market. Based on the

gene, the vaccine strains Ondestepoort, Lederle and Snyder Hillppear to be genetically related to each other (<4% aa variation)ut more distantly related (>8% aa variation) to the CDV strainspreading worldwide [8,12,22,26,30,34,37,40]. Taking advantage ofhis genetic/antigenic diversity, several diagnostic assays have beeneveloped that are able to distinguish between the Onderstepoorttrain and field CDV strains [31,40–42,50,36].

In order to fill this information gap, the sequence of the H genef a laboratory passage (46th) of the vaccine CDV strain Rockborn,vailable from the James Baker Institute, was determined. In addi-ion, the sequence of commercially available CDV vaccines wasetermined and compared.

. Materials and methods

.1. Virus origin

The 46th laboratory passage of the vaccine CDV strain RockbornRockborn-46th) was available at the James Baker Institute labora-ories. The virus was re-amplified by two serial passages on VEROells and viral growth was monitored by visualization of the cyto-athic effect and by an indirect immunofluorescence (IF) assay. Theaccine Candur® SH+P (Hoechst Rousell Vet GmbH) (batch number12684) that contains the strain Rockborn (Rockborn-Candur), waslso analysed.

Two additional vaccines (A and B) commercialized from twoifferent companies were also analysed. For those vaccines, infor-ation on the CDV strain was not specified on the labels. By

sing an RT-PCR genotyping assay with primers specific for vari-

us CDV genotypes (America-1, Arctic-like, European, Asia-1 andsia-2) [36] vaccines A and B were found not to contain America-1

Onderstepoort-like) CDV strains. Therefore, the sequence of the Hene was determined in order to establish firmly which CDV strainas included in the vaccine formulation.

9 (2011) 1222–1227 1223

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 forRNA extraction. The RNA was extracted using the RNeasy Kit(Qiagen, GmbH, Germany) according to the manufacturer’s instruc-tions.

Reverse transcription and PCR amplification of the H gene of CDVwas achieved as previously described [37]. To obtain a PCR prod-uct for sequencing of the H gene of the Italian strains, 0.5 �l each ofprimers CDV-F8 and CDV-R8 (50 pmol/�l) [40] was added to a totalof 49 �l of the reaction mixture containing 0.2 mM of each dNTP,1.2 mM MgSO4 and 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 immediatelysubjected to PCR amplification in a single-step protocol, usingSuperScript One-Step RT-PCR kit (Invitrogen – Life Technologies,Milan, Italy). Reverse transcription was carried out at 48 ◦C for60 min, followed by denaturation of the reverse transcriptase at95 ◦C for 2 min. Amplification was conducted by a temperaturecycling protocol consisting of 35 cycles of 30 s of denaturation at94 ◦C, 1 min of primer annealing at 55 ◦C, and 1 min of extensionat 68 ◦C, followed by 10 min of the final extension phase at 68 ◦C.To obtain PCR products suitable for sequencing, the inner primerpair RH-3 and RH-4 [40] was used to amplify in a nested PCR thecomplete H-gene. About 0.5 �l each of primers RH-3 and RH-4(50 pmol/�l) was added to a total of 49 �l of the reaction mixtureconsisting of 0.5 �l of a 1:100 dilution of the first-round PCR prod-uct, 0.25 �l of TaKaRa LA Taq (5 U/�l; Takara Bio Inc., Japan), and5 �l of 10× PCR buffer, 8 �l of 2.5 mM dNTP mixture, and 35.75 �lof distilled H2O. Amplification was conducted by a temperaturecycling protocol consisting of 25 cycles of 1 min of denaturationat 94 ◦C, 2 min of primer annealing at 50 ◦C, and 2 min of exten-sion at 68 ◦C, followed by 2 min of the final extension phase at68 ◦C.

1.3. Sequencing

The RH3–RH4 PCR products were purified with the Wizard PCRPreps DNA Purification System (Promega, Madison, WI). Then, theDNA was used as template for direct sequencing. The DNA wassequenced by using the conserved primers RH3 and RH4 and spe-cific primers designed according to an overlapping strategy. Thesequences were assembled using Bioedit software package ver-sion 2.1 [25] and compared to cognate sequences in the geneticdatabases using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) andFASTA (http://www.ebi.ac.uk/fasta33) web-based programs. Thegene H sequences of the strains Rocborn-46th and Rockborn-Candur is available under accession numbers GU810819 andGU266280.

1.4. Phylogenetic analysis

Nucleic acid sequences were aligned with a selection of CDVH sequences by using Mega 4.0 software package [48]. The treewas inferred using the Kimura two-parameter model and theneighbor-joining method. Statistical significance of the phylogenywas estimated by bootstrap analysis over 1000 pseudoreplicatedata sets.

2. Results and discussion

The H gene of the strain Rockborn-46th was 1824 nt long andcoded for a 607 aa-long polypeptide. Eight conserved potentialN-glycosylation sites were found at residues 19, 149, 309, 391,422, 456, 587 and 603. The virus Rockborn-46th differed in a

1224 V. Martella et al. / Vaccine 29 (2011) 1222–1227

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ig. 1. Phylogenetic tree based on the full-length H gene displaying the genetic relatootstrap values lower than 80% are not shown. In the box, a sub-tree generated us

nique aa change, 544-K → T (nt 1631-A → C) from the vaccineirus Rockborn-Candur. By FASTA analysis, the virus had the high-st identity (99.6% nt and 99.3% aa) to a CDV strain detected from aesser Panda (Ailurus fulgens) in China (AF178039). In addition, theirus was found to match (>99.0% nt identity in the full-length Hene) a canine virus (strain 25259, accession AY964114) detected inissouri 2004 [43] and to be virtually identical (100% nt) in a 995-nt

ong fragment to a canine strain detected in Japan 1991 (strain 6083,ccession AB191310) [50] and in a 800-nt long fragment to canineiruses (99.9–100% nt) detected in Austria 2007 (strain 2727-07nd 2730-07, accessions GQ214370 and GQ214371, respectively).y phylogenetic analysis, using a selection of CDV strains repre-

entative of the various CDV lineages, Rockborn-like viruses werehown to be genetically related to America-2 viruses and distantlyelated to America-1 CDVs (Fig. 1). The strain Rockborn-46th pas-age displayed 93.0% nt and 91.7% aa identity to the vaccine strainnderstepoort, prototype of the America-1 lineage.

ps between the strain Rockborn and a selection of CDV strains of various genotypes.385-nt fragment of the H gene (nt 416–800 of the H gene) is shown.

Interestingly, the CDV strains from vaccines A and B were alsofound to contain Rockborn-like viruses. Vaccine A differed in onlytwo residues, 160-R → K and 524-T → K, from strain Rockborn-46thwhile 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 theRockborn-Candur strain, while the change 589-T → P in vaccineB disrupted the N-587-glycosylation site (Table 1). Assuming thatthe viruses likely came from different virus passages and batches,it is tempting to speculate that those differences may have beenaccumulated during cultivation on cell cultures. Accordingly, thelaboratory 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 strainRockborn-46th; in contrast, vaccine B displayed 4 mutations, withthe change 589-T → P, altering a potential N-linked sugar bindingsite. By solving the crystal structure of measles virus H protein,

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a morbillivirus closely related to CDV, sugar moieties have beenseen to mask massively the protein surface and to help the homod-imeric structure of the H protein to maintain correct orientation[29]. Residues of the putative receptor-binding site are strategi-cally positioned in an unshielded area on the top of the protein,where neutralizing epitopes have also been mapped to [38,47], andtend to be highly conserved among morbilliviruses. Accordingly,changes in the sugar moieties can mask or expose epitopes on theH protein but functional constraints tend to conserve the antigenicmonotypic profile of morbilliviruses [29].

An alternative hypothesis to explain the presence of Rockborn-like viruses in vaccines A and B is that they are variants of a fieldRockborn-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 withthe databases, at least five Rockborn-like sequences were identi-fied. Scattered mutations were identified in the full-length H genesequences between the virus Rockborn-46th and the Chinese LesserPanda virus (7 nt and 4 aa changes) and the USA strain 25259 (18nt and 8 aa changes) (Table 1). Due to the consistent number ofmutations, the origin of these viruses may not be unequivocallyattributed to Rockborn-based vaccines and a possible explanationis that Rockborn-like viruses are still circulating in the field orthat animalised vaccine-derived viruses were introduced in unre-lated geographical settings on several occasions and circulated inthe area in different carnivores, taking advantage of the broadhost range of CDV. Also, Ondestepoort-like CDVs, identified inthe 1930s and long believed to be extinct in the field, have beenidentified from outbreaks in wildlife animals in United States andhave been reported occasionally in dogs from Ireland, Korea andPoland [28,32,34,46], suggesting vaccine-induced disease, eventu-ally followed by animalisation of vaccine viruses in some animalpopulations, or persistence of America-1 CDVs in certain ecologicalsettings.

Conversely, exploring the databases, three CDV sequences (oneJapanese and two Austrian viruses) were found to match (nearly100% nt) the sequence of strain Rockborn-46th. Such high geneticidentity is much more consistent with a hypothesis of residualvirulence of vaccine viruses. This phenomenon appears to be mod-ulated by host factors, such as age, genetic constitution, state ofimmune competence and/or immuno-suppression due to concomi-tant diseases/infection as well as by virus-intrinsic factors, i.e.the grade of virus attenuation. Vaccine-related disease has beenreported on several occasions in dogs [4,14,18,23,27,33,39]. Pupsbecoming ill within 2–3 weeks after administration of CDV vaccineare always suspected to have developed vaccine-induced diseaseand this may result in disputes involving veterinary practition-ers, pet owners and commercial pet shops. In most suspectedcases, however, it has been difficult to establish whether the dis-ease was induced by the vaccine virus or by field viruses infectingpups shortly before or after vaccine administration. This issue wasnot ruled out firmly in older studies, since appropriate diagnos-tic tools were not available. Therefore, in some circumstances themagnitude of this phenomenon may have been overrated. Dur-ing a 2-year-surveillance in Italy, we found all suspected casesof CDV vaccine-induced illness or death to be caused by CDVfield strains [36], thus suggesting that residual virulence is infre-quent.

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 andgrown for vaccine passage cultures on Madin Darby Canine Kidney(MDCK) cells.

In most cases, increased virulence of the dog-kidney-cell-attenuated CDV was suspected to have been triggered by the

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ontemporaneous presence in the vaccine formulations of liveodified canine adenovirus type-1. Under experimental condi-

ions, re-acquisition of virulence by the kidney-cell-attenuatedockborn virus has been demonstrated in vivo after six sequentialassages in dogs and in vitro after ten passages in primary dog lungacrophages, but the molecular bases of this phenotypic changeere not investigated [2]. Along with the H protein, other viral pro-

eins appear to regulate CDV replication and dissemination in thenfected host [52], thus complicating a dissection of the molecular

echanisms behind attenuation of CDV virulence.In conclusion, these findings indicate that (i) Rockborn-like vac-

ines are still available in the market; (ii) Rockborn-like viruses maye recovered from dogs or other carnivores with distemper dis-ase, suggesting residual virulence of some vaccine batches and/orhe circulation of analogous viruses in some ecological niches. Thenformation provided with this study will be paramount to dis-olve shades that have been accumulating over the years on anistorically important CDV strain and to develop and/or update dif-

erential diagnostic tools able to identify promptly vaccine-relatedisease in dogs.

cknowledgments

This work was granted by the project “Gastroenteriti virali deiarnivori domestici – Fondi di Ateneo 2008, “Infezioni virali del canecarattere zoonosico – Fondi di Ateneo 2009” and by grants from

he Italian Ministry of Health, Ricerca corrente 2009, project IZS VE1/09 RC “Definizione di una procedura validata per la selezione diani per programmi di Interventi Assistiti dagli Animali (IAA)”.

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