determinants of persistence in canine distemper viruses

veterinary microbiology

Veterinary Microbiology 57 (1997) 83-93

Determinants of persistence in canine distemper viruses

Marianne Stettler, Karin Beck, Anita Wagner, Marc Vandevelde, Andreas Zurbriggen *

Institute of Animal Neurology. University of Beme, CH-3012 Beme, Switzerland

Received 28 May 1996; accepted 16 October 1996

Abstract

Viral persistence in the central nervous system is the driving force behind the chronic progressive disease caused by natural canine distemper virus (CDV) infection in dogs. Persistence of CDV is associated with non-cytolytic spread and impaired viral budding. Since budding is to a large extend dependent on the nucleocapsid- (N) and matrixproteins (M) of the virus, we analyzed the nucleotide- and deduced amino acid sequences of the corresponding genes of a spectrum of CDV strains, that differ with respect to virulence and persistence in vivo and in vitro. The wild type CDV (A75/17), which is capable of causing a persistent infection in vivo was compared to two tissue culture adapted CDV strains (passaged A75/17-CDV and Rockbom-CDV), which retain a residual virulence and the capacity to spontaneously persist in vitro. A modified distemper virus (Snyder Hill-CDV), which is neurovirulent but not capable of causing a persistent infection in vivo, and an avianized virus (Onderstepoort-CDV) which is completely apathogenic and spreads by budding in cell cultures were also examined. Differences were found in the C-terminal of the nucleocapsid protein, which - comparing the two extremes of the spectrum (wild A75/17-CDV and OP-CDV) - lead to changes of the predicted protein structure. Such changes could affect the budding process and thus play a role in persistence. Marked changes in the M-gene were found in its non-coding region: the nucleotide sequences of the SH-CDV and OP-CDV differed considerably from the other three strains. Moreover, an additional second open reading frame was detected in the ‘non-coding’ region of the M gene in the wild A75CDV, the two tissue culture adapted CDV strains and SH-CDV, but not in OP-CDV. The presence of this additional open reading frame correlated with the ability to cause a spontaneous persistent infection in vitro. Our findings support the notion that both N- and M-genes of CDV harbor determinants of viral persistence. 0 1997 Elsevier Science B.V.

Keywords: Canine distemper virus; Persistence; Nucleocapsid protein; Matrix protein

* Corresponding author.

0378-l 135/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-1 135(96)01281-3

84 M. Stettler et al. / Veterinary Microbiology 57 (1997) 83-93

1. Introduction

Members of the Morbilliviruses, such as measles and canine distemper (CDV) are capable of producing persistent infections of the central nervous system (CNS) leading to severe progressive neurologic disease. In distemper, persistence of viral antigen in the CNS stimulates the anti-viral inflammatory response which causes progressive tissue destruction (Vandevelde and Zurbriggen, 1995). Persistence is determined by host as well as viral factors (Zurbriggen et al., 1995; Miller et al., 1995). With respect to viral factors, persistence in canine distemper appears to be in part related to selective non-cytolytic viral spread from cell to cell without budding (Zurbriggen et al., 1995) presumably delaying immune recognition of the virus. Besides other abnormalities, budding is also impaired in subacute sclerosing panencephalitis (SSPE) in man, caused by persistent measles virus infection of the CNS (Billeter et al., 1994). Since budding is thought to be the usual way by which Morbilliviruses spread (Kingsbury, 1990) persistent CDV must be assembled in a way that differs from attenuated CDV strains in which the budding process has been studied. The Morbillivirus matrix (M) protein, which in SSPE is absent, defective or heavily mutated, plays a pivotal role in viral assembly (Peeples, 1991; Billeter et al., 1994). The budding process however also requires interaction of this protein with the nucleocapsid (N) protein (Ray et al., 1991). Thus, it is reasonable to postulate that viral persistence in distemper must be in part determined by the structure of the N- and M-protein of CDV. In order to understand mechanisms of viral assembly and thus persistence, it would be of interest to define the responsible molecular determinants.

In an attempt to correlate molecular properties of N and M of CDV with persistence, we sequenced the corresponding genes from a spectrum of CDV strains that differ from each other with respect to persistence. Wild CDV spontaneously establishes a persistent infection in primary dog brain cell cultures immediately following inoculation, as defined by lack of cytolysis and spread without budding. The wild type CDV (A75/17) which is capable of inducing a persistent CNS infection with a progressive demyelinating disease, was compared to distemper viruses that had been modified in various ways. Two strains (A75/17-adapted, RB-CDV) had been obtained by repeated passaging of wild CDV trough mammalian cells (Rockborn, 1958; Hamburger et al., 1991). Such strains have strongly reduced pathogenicity but exhibit residual virulence and retain the ability to cause a persistent infection in vitro (Hartley, 1974). Another strain (Snyder Hill) had been obtained trough repeated intracerebral passages of wild CDV in dogs (Gillespie and Rickard, 1956). This brain-passaged virus is virulent in dogs and produces encephalitis, but has lost the ability to cause a chronic infection with demyelination (Summers et al., 1984) although SH-CDV spontaneously establishes a persistent infection in vitro (Zurbriggen et al., 1987; Pearce Kelling et al., 1991). Finally, we studied a distemper virus (OP-CDV) that had been passaged in other species and subsequently in chicken-eggs (Appel and Gillespie, 1972). This avianized strain is completely apathogenic; it is neither capable of inducing neurologic disease nor a persistent infection in vitro. In contrast to other strains, it spreads by budding and cytolysis.

M. Stettler et al. / Veterinary Microbiology 57 (1997) 83-93 85

2. Methods

2.1. Viruses

2.1.1. A75/17-CDV A75/17-CDV, a virulent wild strain was isolated from a dog with spontaneous

distemper. Under experimental conditions, this virus causes a demyelinating disease and persistent infection of the CNS as seen in natural distemper (Summers et al., 1979; Summers et al., 1984). In our laboratory, the virus was propagated in lymphoid tissues of puppies and in primary dog brain cell cultures (DBCC), in which it retains virulence and ability to produce persistent infection and neurologic disease in vivo (Hamburger et al., 1991; Zurbriggen et al., 1984). A75/17-CDV, induces a spontaneously persistent (non-cytolytic, non-budding) infection in primary dog brain cell cultures (Zurbriggen et al., 1995). It does not grow in cell lines unless at least 12 serial blind passages are made (Table 1).

2.1.2. Vero cell adapted A75/17-CDV The virulent A75/17-CDV strain was adapted to Vero cells by 17 serial passages

(Hamburger et al., 19911, starting from infected lymphoid tissues. The adapted virus did no longer produce clinical disease in dogs upon experimental inoculation (Hamburger et al., 1991). However, in brain cell cultures, the passaged A75/17-CDV continued to produce a persistent (non-cytolytic, non-budding) infection. Also in Vero cells, the infection remained non cytolytic.

2.1.3. Rockborn (RB)-CDV Rockborn (RB)-CDV was originally isolated from a spontaneous case of distemper.

The virus had been attenuated by numerous repeated passages trough canine kidney cells (MDCK). The isolate used in this study was a gift from RhBne MCrieux company (Lyon) (Rockbom, 1958) where it is used in vaccine preparation. RB-CDV has a residual virulence since it can cause postvaccinal encephalitis under certain conditions (Hartley, 1974) and reverts quickly to virulence following few passages in primary canine macrophages (Appel, 1978). Similar to A75/17-CDV, this virus spontaneously establishes a persistent infection in brain cell cultures and also in cell lines.

Table 1 Comparison of the biological properties of the different CDV strains

A75/17 A75/17 adapted Rockbom Snyder Hill OP-CDV lab.

Virulence/disease + 7:b

a + a + -

Chronic demyelination + ?b - -

Persistence in DBCC + 7:b ?&

+ -

Persistence in vivo + - -

Spread in DBCC + + + ++ +++ Growth in cell lines - + + -/+ c +

a Residual virulence. b Not known. ’ Only small numbers of passages required.


2.1.4. Snyder Hill (SH)-CDV Snyder Hill (SH)-CDV, a virulent virus, was derived from a natural case of

distemper. The original isolate was then serially passaged trough direct intracerebral inoculation in dogs. After 35 passages, a strain was obtained which was thought to be more neurovirulent (Gillespie and Rickard, 1956). Several features of experimental SH-CDV infection are similar to the wild type CDV infection. However, SH-CDV causes a polioencephalitis and is no longer capable of inducing a demyelinating persistent infection in vivo (Summers et al., 1984). In brain cell cultures, SH-CDV spontaneously establishes a persistent infection but spreads much faster than A75/17- CDV (Pearce Kelling et al., 1990). SH-CDV does not grow in cell lines; adaptation however requires only 4-5 blind passages. The isolate used in this study was a gift from the Rh8ne MCrieux company (Lyon).

2.1.5. Onderstepoort COP)-CDV a This strain was derived from the so-called Green’s distemperoid virus (Green and

Carlson, 1945) which had been isolated from a natural distemper case and serially passaged in ferrets. The ferret-passaged virus was then adapted to chicken eggs and passaged in this system numerous times, after which it was called OP-CDV (Haig, 1948). OP-CDV is considered to be completely apathogenic and is used in vaccines (Appel and Gillespie, 1972). In contrast to all other strains, OP-CDV establishes a cytolytic infection in cultures and spreads by budding. It easily infects a variety of cell lines. The strain was a gift from the Swiss Federal Vaccine Institute, where it had been maintained and propagated in Vero cells.

2.1.6. OP-CDV b In addition to our own sequence of OP-CDV, we used the published nucleotide

sequence of OP-CDV (Sidhu et al., 1993; Bellini et al., 1986; Rozenblatt et al., 1985) for our comparative studies.

2.2. Complementary DNA (cDNA) clones

We produced cDNA clones complementary to the complete N-gene of the virulent A75/17-CDV and the laboratory OP-CDV, cDNA clones complementary to the C- terminal region of the N-gene starting at nucleotide 1101 of the adapted A75/17-CDV, RB-CDV and SH-CDV and clones corresponding to the complete M-gene of all CDV strains. A75/17-RNA was extracted from DBCC infected with this strain, as described previously (Zurbriggen et al., 1993). This RNA was used to synthesize cDNA using the avian myoblastosis virus reverse transcriptase AMV-RT (Pharmacia Biotech AB, Sollen- tuna). The resulting cDNA served as template for subsequent polymerase chain reaction (PCR). Primers were selected according to the published DNA sequences of the N- and M-gene of the OP-CDV (Sidhu et al., 1993; Bellini et al., 1986; Rozenblatt et al., 1985) and purchased from ANAWA (International Bioscience Park, Wangen/Zurich) and MWG-Biotech (Ebersberg). The primers were synthesized with an additional restriction enzyme recognition sequence at their 5’ ends including four protection bases to allow


directional cloning into the pUC19 plasmid vector. PCR amplified DNA was purified and then digested with the corresponding restriction enzymes. The digested DNA fragments were ligated into the multiple cloning site of the pUC19 vector and propagated in Escherichia coli.

A75/17 adapted-, RB- and OP-CDV laboratory strain RNAs were extracted from infected Vero cells. SH-CDV RNA was extracted directly from spleen tissue samples with the Rneasy’” Total RNA Kit (Qiagen AB, Base0 as described by the vendor. From these RNAs we produced cDNA clones as described above and propagated in Es- cherichia coli.

2.3. Nucleotide sequence

Sequence analyses were performed by the dideoxy chain termination method @anger et al., 1977) using the Sequenase kit Version 2.0 following the instructions given by the vendor (USB Lucema Chem AG, Lucerne). As gel we used a 6% polyacrylamide, 7M Urea solution as described (USB Lucema Chem AG). Sequencing was carried out with a Sequi-Gen DNA cell from Bio Rad (Bio Rad Laboratories AG, Glattbrugg). As template for sequencing denatured plasmid DNA was used with the universal sequencing primers for pUC19, and internal N- and M-primers. The N- and M-gene sequences were determined using three or more independent and overlapping clones spanning the same area. Each clone derived from different cDNA- and PCR-reactions and a separate sequencing reaction was carried out each time in both directions. Therefore all bases were at least sequenced six times.

2.4. Nucleotide and protein sequence analysis

The nucleotide sequence of the C-terminal region of the N- and the whole M-gene of the different CDV strains and the published sequence of the OP-CDV strain (Sidhu et al., 1993; Bellini et al., 1986; Rozenblatt et al., 1985) were compared to the sequence of the A75/17-CDV strain with the computer assisted program Align (Scientific and Educational Software). For the amino acid translation the computer assisted program Clone Manager (Scientific and Educational Software) was used, and the predicted amino acid sequences compared.

3. Results

3. I. Sequence analysis

The nucleotide and amino acid sequences of the virulent A75/ 17-, A75/17 adapted-, SH-, RB- and OP-CDV laboratory strain- N- (C-terminal) and M-gene were determined by cDNA sequence analysis. These sequences and a published sequence of the attenuated, non-pathogenic OP-CDV N- and M-gene were compared to the sequence of the virulent A75/17-CDV strain.

88 M. Stettler et al./ Veterinary Microbiology 57 (1997) 83-93

Table 2 Summary of the nucleotide and amino acid changes of the different CDV strains

AX/l1 A75/17 Rockbom Snyder OP-CDV OP-CDV adapted Hill lab. lit.

N nt (1101-1678) 0 6 37 43 49 N aa (351-523) 0 1 7 I 12 M nt 3’ non-cod. (l-32) 0 0 1 2 I M nt cod.reg. (33-1038) 2 15 40 54 59 M nt 5’ non-cod. (1039-1443) 0 13 58 81 85 M aa (l-335) 2 4 3 10 13 M’ (put.2. ORF nt 1223-1379) + + + + - -

3.1.1. Nucleocapsid protein (N-protein)

3.1.1.1. Nucleotide sequences. The nucleotide sequence alignment of the C-terminus (position 110 1 - 1678) of the N-gene revealed no differences in the adapted A75 / 17-CDV strain, 6 differences in the RB-, 37 in the SH-, 43 in the OP-CDV laboratory- and 49 in the published OP-CDV strain compared with the virulent A75/ 17-CDV strain (Table 2).

Al5/11 ( 351) A75/17 ad. ( 1) Rockborn ( 1) Snyder Hill ( 1) OP-CDV lab. ( 1) OP-CDV lit. ( 1)

Al5/17 1 401) A75/17 ad. ( 51) Rockborn ( 51) Snyder Hill ( 51) OP-CDV lab. ( 51) OP-CDV lab. ( 51)

A75/11 ( 451) A75/17 ad. ( 101) Rockborn ( 101) Snyder Hill ( 101) OP-CDV lab. ( 101) OP-CDV lab. ( 101)

Al5/1-l ( 501) A75/17 ad. ( 151) Rockborn ( 151) Snyder Hill ( 151) OP-CDV lab. ( 151) OP-CDV lab. ( 151)

NFGRSYFDPAYFRLGQEMVRRSAGKVSSALAAELGITKEEAQLVSEIASK .................................................. .................................................. .......................... ..T ..................... .................................................. G .................................................

TTEDRTIRAAGPKQSQITFLHSERSEVTNQQPPTINKRSENQGGDKYPIH .................................................. .................................................. ....... ..T.................A ...................... ....... ..T.................A ...................... ....... ..T.................A ......................

FSDERFPGYTPDVNSSEWSESRYDTQTIQDDGNDDDRKSMEAIAKMRMLT .................................................. ................ ..R ............................... ... ..L...................RI ....................... ... ..L....................I ....................... ... ..LL..........R.G......IV ......................

KMLSQPGTSEESSPVYNDRELLN ....................... ....................... .......... D ............ ........ ..DN......K .... ........ ..DN......K ....

Fig. 1. Aligned amino acid sequences of the C-terminus of the N-protein. Dots represent amino acid identity. Standard one-letter amino acid code.


3.1.1.2. Amino acid sequences. The deduced amino acid sequences of the whole N-protein revealed 11 modifications between the A75/17-CDV strain and our laboratory OP-CDV strain. Four of these changes occurred within the N-terminus (aa 1 - 159) and 7 in the C-terminus (aa 351-523). No amino acid changes were observed in the highly conserved middle part of the N-protein (aa 160-350). The comparison of amino

A75/11 A75/17 ad. Rockborn 1) Snyder Hill I 1) oP-CDV lab. ( 1) OP-CDV lit. ( 1)

MTEVYDFDQSSWDTKGSIAPILPTTYPDGRLVPQVRVIDPGLGDRKDECF .................................................. .................................................. ............................. ..I .................. .......... ..Y..................I .................. .......... ..Y..................I ..................

A75/17 ( 51) MYIFLLGIIEDNDGLGPPIGRTFGSLPLGVGRTTARPEELLKEATLLDIV Al5/17 ad. ( 51) ............................... ..A ................ Rockborn ( 51) ....... ..G ........................................ Snyder Hill ( 51) .................................................. OP-CDV lab. ( 51) ... ..M ......................................... ..M OP-CDV lit. ( 51) ... ..M ......................................... ..M

A75/17 A75/17 ( 101) ( 101) A75/17 ad. A75/17 ad. ( 101) ( 101) Rockborn Rockborn ( 101) ( 101) Snyder Hill ( 101) Snyder Hill ( 101) OP-CDV lab. OP-CDV lab. ( 101) ( 101) OP-CDV lit. OP-CDV lit. ( 101) ( 101)

A75/17 ( 151) A75/17 ad. ( 151) Rockborn ( 151) Snyder Hill ( 151) OP-CDV lab. ( 151) OP-CDV lit. ( 151)

A75/17 ( 201) Al5/17 ad. ( 201) Rockborn ( 201) Snyder Hill ( 201) OP-CDV lab. ( 201) OP-CDV lit. ( 201)

A75/1-l ( 251) A75/17 ad. ( 251) Rockborn ( 251) Snyder Hill ( 251) OP-CDV lab. ( 251) OP-CDV lit. ( 251)

A75/17 ( 301) A75/11 ad. ( 301) Rockborn 1 301) Snyder Hill ( 301) OP-CDV lab. ( 301) OP-CDV lit. ( 301)

VRRTAGVKEQLVFYNNTPLHILTPWKKVLTSGSVFSANQVCNAVNLIPLD . ..I .............................................. .W................S ............................... .................................................. ........................................ ..T ....... ........................................ ..T .......

IAQRFRWYMSITRLSDDGSYRIPRGMFEFRSRNALAFNILVTIQVEGDV .................................................. .................................................. .................................................. .................................................. ........................ ..V.................R .....

CSSRGNLSMFKDHQVTFMVHIGNFSRKKNQAYSADYCKLKIEKMGLVFAL .................................................. .................................................. ..................................................

D......G......A ................................... D......G....Y.A ...................................

GGIGGTSLHIRCTGKMSKALNAQLGFKKILCYPLMEINEDLNRFLWRLEC .................................................. .................................................. ......................................... ..s ...... ............................................. ..s .. ............................................. ..s ..

KIVRIQAVLQPSVPQDFRIYNDVIISDDQGLFKIL ................................... ............ ..K .................... ................ ..v ................ ................ ..v ................ ................ ..v ................

Fig. 2. Aligned amino acid sequences of the M-protein. Dots represent amino acid identity. Standard one-letter amino acid code.


acids of the variable C-terminal region of the N-protein (aa 35 l-523) is shown in Fig. 1. Compared with the virulent A75/17-CDV strain, the adapted A75/17-CDV had no amino acid changes, the RB-CDV 1, the SH-CDV 7, our laboratory OP-CDV 7 and the published OP-CDV 12 (Table 2).

3.1.2. Matrix protein (M-protein)

3.1.2.1. Nucleotide sequences. The nucleotide sequence differences of all CDV strains are summarized in Table 2. Within the non-coding 3’ region we found no changes in the adapted A75/17- and in the RB-CDV strain respectively, 1 in the SH-, 2 in the OP-CDV laboratory- and 7 in the published OP-CDV strain. The nucleotide sequence alignment to the coding region of the M-gene of the virulent A75/17-CDV strain revealed 2 differences in the adapted A75/17-CDV strain, 15 in the RB-, 40 in the SH-, 54 in the OP-CDV laboratory- and 59 in the published OP-CDV- strain. These nucleotide differences were dispersed over the whole length of this area. The non-coding 5’ region revealed no nucleotide differences in the adapted A75/17-CDV, 13 in the RB-, 58 in the SH-, 81 in the OP-CDV laboratory- and 85 in the published OP-CDV compared with the virulent A75/17-CDV strain.

3.1.2.2. Amino acid sequences. The M-gene encodes the M-protein with 335 amino acids. Most nucleotide alterations within the M-protein coding region were silent mutations at the third position of the triplet codon. The nucleotide differences in the M-coding region resulted in 2 amino acid modifications in the adapted A75/17-CDV-, 4 in the RB-, 3 in the SH-, 10 in the OP-CDV laboratory- and 13 in the published OP-CDV strain (Table 2; Fig. 2).

3.1.2.3. Putative second open reading frame in the M-gene. The virulent A75/17-, the adapted A75/17-, the RB- and the SH-CDV strains had an additional putative open reading frame (ORF) within the ‘non coding’ 5’ region, starting at position 1223 up to

; 4

2 7

!! A75/17 1) MKTAESNQFMPKSRLIIIGPS NVWMLGVLNFASRNLTLTIIPPRTCPISS Al5/17 ad. 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 Rockborn ( 1) . . . . . . . . . . . . . . . . . . ..R.A..........................:

:: Snyder Hill ( 1) ..I...K..LL.I.......R.E..P...S....I......L....L...

:: A75/17 ( 51) YH A75/17 ad. ( 51) . .

15 Rockborn ( 51) . . 16 Snyder Hill ( 51) H. 17 18

Fig. 3. Aligned amino acid sequences of the putative second ORF in the M-gene. Dots represent amino acid identity. Standard one-letter amino acid code.

M. Stettler et al./ Veterinary Microbiology 57 (1997) 83-93 91

1379. This putative ORF encoded a small protein of 52 amino acids. Amino acid alignments revealed no differences between the virulent and the adapted A75/17-CDV strains and 2 amino acid differences between RB- and A75/17-CDV. However, the SH-CDV differed markedly from the virulent A75/17-CDV in this area, containing 13 amino acid differences (Table 2; Fig. 3).

4. Discussion

The ability of CDV to cause a chronic demyelinating disease is due to viral persistence in the CNS. Viral persistence is probably multifactorial and depends on properties of the host but also on viral factors. Previous studies of measles and distemper have shown that persistence of Morbilliviruses is associated with viral spread and/or viral assembly (Zurbriggen et al., 1995). Since interactions between N- and M-proteins of Morbilliviruses play a central role in viral assembly (Billeter et al., 1994) we attempted to correlate molecular determinants of these proteins with the phenomenon of persistence of a spectrum of distemper viruses that had been manipulated in various ways.

Previous studies have shown that the variable region of the N-protein lies in the C-terminal which interacts with the M-protein. There were no or very few differences in the N-protein between wild A75/17-CDV, adapted A75/17-CDV and RB-CDV.

However, 5 amino acid differences clustered in the C-terminus of the N-protein of SH-CDV as compared to the wild A75/17 virus. It is possible that these changes could influence viral assembly, since SH-CDV, although capable of persisting in vitro, spreads much faster than virulent A75/17-CDV (Pearce Kelling et al., 1991). The faster spread may elicit a more efficient immune response than the wild type virus and be related to the observed loss of ability to persist in vivo (Summers et al., 1984).

Even more changes in the C-terminal, 5 of which at exactly the same positions as in SH-CDV, were found in the two OP-CDV isolates. Previous sequencing has shown that these differences in OP-CDV lead to significant conformational changes (Stettler and Zurbriggen, 1995). OP-CDV, an avianized strain, has completely lost its ability to cause disease and hence a persistent infection in its natural host. However, it has also lost the inherent potential of CDV for persistence in vitro; in contrast to all other strains investigated, OP-CDV spreads by budding and causes cytolysis (Zurbriggen et al., 1995; Zurbriggen et al., 1987). Moreover, both OP-CDV isolates also had a significant number of - albeit scattered - amino acid changes in the M-protein, which, in respect to the wild virus, were more pronounced than in the other manipulated strains.

It is reasonable to conclude that an accumulation of changes both in the N- and M-proteins appears to correlate with the loss of the ability to persist. However, marked changes also occurred in the non coding region of the M-gene. A surprising finding was the presence of a second open reading frame in the M-gene, coding for a putative protein of 52 amino acids in 4 of the 6 strains studied. The presence of the second open reading frame seems to correlate with the ability to cause persistence, since it was lacking in the 2 cytolytic OP-CDV isolates. Additional open reading frames within the M-gene have been reported in other Morbilliviruses (Bellini et al., 1986). So far, we could not prove


that a corresponding protein is really expressed. Antisera raised to a synthetic peptide from the predicted amino acid sequence failed to demonstrate this protein in infected cells in vivo and in vitro (data not shown). The presence of these putative ORFs within the M-gene in various Morbilliviruses indicates that there must be some evolutionary pressure in maintaining these ORFs and suggests a functional role of this sequence (Curran and Rima, 1988). Furthermore, the nucleotide sequence of the noncoding region of the M-gene differed dramatically between wild A75/17, adapted A75/17 and RB strains on one hand and the SH- and OP-CDV on the other. This high degree of variability has been found between different Morbilliviruses but not within various isolates of measles (Cut-ran and Rima, 1988; Bellini et al., 1986). The reason why such long non-coding regions are conserved within the Morbilliviruses is unclear. It has been suggested that this area may function as a controlling signal or stabilize viral RNA (Barrett et al., 1991; Bellini et al., 1986).

We have shown that changes in biological parameters induced by manipulation of wild CDV are associated with molecular changes in the N- and M-genes. Our findings presented in this study support the notion that both the N- and M-genes harbor molecular determinants of persistence. It is possible that persistence only occurs when different determinants interact, and it cannot be excluded that other viral genes also play a role. Nevertheless, sequencing studies help to search for molecular determinants of persistence and to localize regions of interest. Conclusive information will depend on the use of specifically modified viral recombinants.

Acknowledgements

The authors wish to thank Dr. R. Fatzer for critically reading the manuscript. Supported by the Swiss National Science Foundation (grants 32-33599.92 and 31- 29332.90) and the Swiss Multiple Sclerosis Society.

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