Virus Research 80 (2001) 67 – 74

www.elsevier.com/locate/virusres

Identification and molecular characterization of 18

paramyxoviruses isolated from snakes
J. Franke a,*, S. Essbauer b, W. Ahne a, S. Blahak c
a
Institute of Zoology, Fishery Biology and Fish Diseases, Faculty of Veterinary Medicine, Ludwig-Maximilians-Uni6ersity Munich,
Kaulbachstrasse 37, D-80539 Munich, Germany
b
Institute of Medical Microbiology, Infectious and Epidemic Diseases, WHO-center of Comparati6e Virology,
Faculty of Veterinary Medicine, Ludwig-Maximlians-Uni6ersity Munich, Veterinärstr. 13, D-80539 Munich, Germany
c
State Veterinary Office, Westernfelderstrasse 1, D-32788 Detmold, Germany

Received 23 March 2001; received in revised form 4 July 2001; accepted 10 July 2001

Abstract

Viral agents from 18 different snake species (families Colubridae, Viperidae, and Crotalidae) showing respiratory
symptoms and neuronal disease were identified as paramyxoviruses by typical cytopathogenic effect (CPE), electron
microscopy, and hemagglutination inhibition. Detailed molecular characterization of the viruses was performed by
partial L- and F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) and sequencing, nucleotide
and amino acid sequence alignment, and phylogenetic analysis (PHYLIP). RT-PCR of the partial L-gene (566 nt) was
successful for all 18 viruses; amplicons of the partial F-gene (918 nt) could be obtained in 16 cases. F- and L-sequence
alignment revealed similarities to Fer de Lance virus (FDLV) ranging from 79 to 88% on a nucleotide basis, and 94
to 99% on an amino acid basis. Phylogenetic analysis of the ophidian paramyxoviruses resulted in three clusters for
the L-gene sequence and corresponding clusters for the F-gene sequence, indicating no species specificity. We analyzed
the F-protein of the snake paramyxoviruses, which proved to have an identical conserved motif of heptad repeat A and
predicted a furin cleavage site. This uniformity distinguishes the snake virus group from the other type species of the
subfamily Paramyxo6irinae. For further classification, we aligned the sequences of the ophidian paramyxoviruses and
members of the Paramyxo6iridae, such as Sendai virus (genus Respirovirus), mumps virus (genus Rubulavirus),
measles virus (genus Morbillivirus), human respiratory syncytial virus (genus Pneumovirus) (Van Regenmortel and 10
co-authors, 2000) and Hendra virus, which have recently been suggested as type species of the genus Henipavirus
(Wang et al., 2000). Maximum sequence similarity was found to the partial L-gene of Sendai virus, with 56%
nucleotide and 61% amino acid identity. The FDLV and Sendai virus cluster in the phylogenetic analysis of L- and
F-protein regarding the Paramyxo6irus type species and Hendra virus and show the closest relationship. Regarding
the biological properties, the antigenic distance, and particularly the low homology of available sequences, we propose
a new genus for the reptilian paramyxoviruses within the Paramyxo6iridae. © 2001 Elsevier Science B.V. All rights
reserved.

Keywords: Paramyxoviridae; Reptilian paramyxovirus; F-protein; L-protein; snake

* Corresponding author. Tel.: 49-89-2180-2968; fax: 49-86-280-5175.

E-mail address: julia.margarete.franke@campus.lmu.de (J. Franke).

0168-1702/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 7 0 2 ( 0 1 ) 0 0 3 5 3 - 7
68 J. Franke et al. / Virus Research 80 (2001) 67–74

The first epizootic disease among snakes caused
by a paramyxovirus (Fer de Lance virus, FDLV)
was reported 25 years ago in a serpentarium in
Switzerland (Foelsch and Leloup, 1976; Clark et
al., 1979). Since then, several paramyxovirus-like
particles have been isolated from snakes, lizards,
and turtles. Ophidian paramyxoviruses (OPMV)
have been identified in, e.g., Boidae, Elapidae,
Colubridae, Crotalidae, and Viperidae (Essbauer
and Ahne, 2001). Paramyxovirus infection shows
clinical signs such as hemorrhagic and prolifera-
tive pneumonia, pancreatic necrosis, and central
nervous system symptoms (Jacobson, 1986).
Viruses isolated from reptiles were shown to be
antigenically related, but they are distinct from
mammalian and avian paramyxoviruses (Clark et
al., 1979; Ahne and Neubert, 1989; Richter et al.,
1996).
We investigated 18 viral agents isolated between
1989 and 1999 from various snake species of the
Colubridae, Viperidae, and Crotalidae families in
Germany (Table 1). The viruses were compared
with the previously studied paramyxovirus iso-
lated from a tropical climber adder (Elaphe oxy-
cephala; Gono-GER85), (Ahne et al., 1987, 1999)
and with the as yet unassigned FDLV (ATCC VR
895) (Van Regenmortel and 10 co-authors, 2000).
The viruses were propagated in iguana heart cell
monolayers (IgH2, ATCC Cl-108) at 28 °C, using
Minimal Essential Medium supplemented by 10%
fetal bovine serum as described previously (Ahne
et al., 1999). One week after infection, cell cul-
tures showed cytopathic effects (CPE) consisting
of large syncytia formation, followed by cell de-
tachment from the monolayer and lysis of cells.
Infected cell cultures were frozen and thawed for
harvesting of the viruses. After removal of the cell
debris, viral particles were pelleted by ultracen-
trifugation at 100 000× g for 2 h at 4 °C and
negatively stained for investigation by electron
microscopy (Zeiss EM 109). Samples revealed
pleomorphic, spherical, and tubular-shaped envel-
oped particles. Virions were 190– 480 nm in di-
ameter. Some disrupted particles had released the

Table 1
Host and accession number (GenBank) of snake viruses used in this study

Host Virus L-gene F-gene Acc. number

nt aa nt aa

Bothrops atrox FDLV-79 100 100 100 100

Elaphe oxycephala Gono-GER85 88 97 88 99 AF349404
Crotalus cerastes CrotCe-96 88 97 87 99 AF349411
*Atheris squamigera AtheSq-96
*Crotalus spec. CrotX2-96
*Crotalus enyo CrotEn-96
Elaphe guttata ElaGut-91 87 96 n.a. n.a. AF349408
Crotalus spec CrotX1-96 86 96 n.a. n.a. AF349405
Trimeresus fla6omaculatus TrimFl-89 81 94 79 95 AF349409
Pituophis melanoleucus PituMe-89 81 96 80 97 AF349406
*Lampropeltis mexicana LampMe-89
*Vipera palaestinae VipPal-89
*Agkistrodon bilineatus AgkiBi-89
Elaphe situla ElaSit-92 80 96 79 97 AF349410
Bitis nasicornis BitisN-96 80 96 79 97 AF349407
Cerastes cerastes CeraCe-98 79 95 79 97 AF351137
*Bitis caudalis BitisC-98
*Atheris squamigera AtheDe-98
*Crotalus durissus CrotDu-98
*Crotalus durissus CrotDu-99

Virus code includes the year of isolation. Nucleotide (nt) and amino acid (aa) sequence identity (%) of the investigated ophidian
paramyxoviruses to the FDLV. Isolates in boxes originate from the same outbreak or collection. Viruses with identical nucleotide
sequences are marked with *. RNA of two isolates could not be amplified (n.a.) with F-gene specific RT-PCR.
 J. Franke et al. / Virus Research 80 (2001) 67–74
Fig. 1. Electron micrograph of negatively stained paramyx-
ovirus-like particle exhibiting pleomorphism. Several particles
have a spherical shape and released nucleocapsid. Bar: 50 nm.
helical nucleocapsid (Fig. 1). The size and mor-
phology of the viral particles were consistent with
those seen in the family Paramyxo6iridae (Van
Regenmortel and 10 co-authors, 2000).
To investigate the antigenic relationship, the 18
OPMV were tested for hemagglutination at room
temperature by using 0.5% chicken erythrocytes.
Hemagglutination titers ranged from 1/64 to 1/
256. Furthermore, hemagglutination was inhibited
by using a rabbit antiserum against the isolate
Gono-GER85 (Ahne et al., 1987).
For molecular characterization of the 18
OPMV, we performed a nested reverse transcrip-
tion polymerase chain reaction (RT-PCR) target-
ing the partial L- and F-genes. Viral RNA was
isolated from 400 ml supernatant of infected tissue
culture showing CPE. Isolation of RNA was car-
ried out by using the RNeasy Mini Kit (QIAGEN
GmbH, Hilden, Germany). Primers targeting the
L-gene of FDLV were used as described by Ahne
69
et al. (1999). We designed primers for the partial
F-gene sequence by alignment of the FDLV
(ATCC VR 895) (kindly provided by G. Kurath,
Western Fisheries Research Center, Seattle,
USA), of the human parainfluenza virus-1 (HPIV-
1) (M22347), and of the Sendai virus (M30202)
sequences. Three primers were used for semi
nested RT-PCR: F1 (5%-GCA CAG ATA ACN
GCG GGG ATT GC-3%), F3 (5%-TAG TNC CTC
TGA GGG ACA CCA TA-3%), and the reverse
complementary primer F2 (5%-GCA GCN ACA
CAA TTG GCT ATG AC-3%). RT- PCR condi-
tions were modified according to the method de-
scribed by Pfeffer et al. (1997). Each RT-PCR
sample contained 100 pmol primers, 5 ml viral
RNA, 200 mmol dNTP (Promega GmbH,
Mannheim, Germany), 10 ml Taq extension buffer
(10×), 50 ml dithiothreitol, 1 unit RNasin
(Promega GmbH, Mannheim, Germany), 2.5
units Taq polymerase (GibcoBRL, Life Technolo-
gies GmbH, Karlsruhe, Germany), and 1 unit
reverse transcriptase (RAV-2, Amersham Phar-
macia, Buckinghamshire, UK). The first-strand
synthesis was carried out at 45 °C for 30 min.
After denaturation at 94 °C for 30 s, 30 PCR
cycles were performed at 94 °C for 30 s, at 55 °C
for 45 s, and at 72 °C for 45 s, followed by 4 min
at 72 °C. In a second round, 2 ml first amplifica-
tion product and 48 ml reaction mixture as de-
scribed above but without reverse transcriptase
and RNasin were amplified under the same condi-
tions, except for the annealing temperature, which
was increased to 55 °C. The PCR products were
analyzed by gel electrophoresis and visualized by
ethidium-bromide staining and UV light. The
RNA of all 18 isolates and Gono-GER85 was
amplified to fragments of approximately 566 nu-
cleotides (nt) as expected for the partial L-gene
sequence (Fig. 2). Amplification of the RNA from
16 out of the 18 reptilian viruses and Gono-
GER85 resulted in the expected 918 nt for the
partial F-gene sequence (data not shown). Purified
amplicons were sequenced on both strands with
the corresponding L- or F-gene primers (sequenc-
ing service: GATC, Konstanz). Nucleotide se-
quences obatined were submitted to GenBank
(Table 1). Out of the 16 isolates, for both the L-
and F-gene sequences, six different nucleotide se-
70 J. Franke et al. / Virus Research 80 (2001) 67–74

quences could be revealed. Two additional nucle-
otide sequences were obtained for the L-gene;
these could not be amplified in the F-gene specific
RT-PCR (Table 1). Nucleotide sequences of
CrotCe-96, AtheDe-96, CrotX2-96, and CrotEn-
96 proved to be identical (AF349411). Identity
could also be shown for PituMe-89, LampMe-89,
VipPal-89, and AgkiBi-89 (AF349406) and Cer-
aCe-98, BitisC-98, CrotDu-98, and CrotDu-99
(AF351137). The viruses with identical sequences
originated from the same collection or outbreak
and occurred in the same year (Table 1), al-
though, virus CrotDu-99 was isolated from the
offspring of a snake infected with the identical
virus CrotDu-98.
Nucleotide sequences were aligned by using
CLUSTALW multiple sequence alignment (Hig-
gins, 1994) and compared with the sequence of
FDLV. Partial L-gene sequences revealed similar-
ity to FDLV ranging from 79% (CeraCe-98,
BitisC-98, AtheDe-98, CrotDu-98, and CrotDu-
99) to 88% (Gono-GER85, CrotCe-96, AtheDe-
96, CrotX2-96, and CrotEn-96). For the F-gene,
similarity of nucleotides to FDLV ranged from
79% (TrimFl-89, ElaSit-92, BitisN-96, CeraCe-98,
BitisC-98, AtheDe-98, CrotDu-98, and CrotDu-
99) to 88% (Gono-GER85) (Table 1).
In the paramyxoviruses, the F-protein precursor
(F0) is cleaved to the functional F1 and F2
proteins by cellular proteases, e.g., furin. A highly
conserved domain, the heptad repeat A, is located
close to the cleavage site, which encompasses the
fusion receptor (Scheid and Choppin, 1974; Klenk
and Garten, 1994) (Fig. 3). We analyzed this
F-protein region of the OPMV and compared
with the type species of the subfamily Paramyx-
o6irinae (Fig. 3). These were obtained from Gen-
Bank as Sendai virus (M30202), genus
Respiro6irus, measles virus (E04903), genus Mor-
billivirus, Hendra virus (AF017149), proposed
genus Henipa6irus (Wang et al., 2000) and mumps
virus (AF143383), genus Rubula6irus. Obtained
nucleotide sequences of partial F-genes were
translated, and the amino acid sequences were
aligned (Higgins, 1994). The eight nucleotide se-
quences of the OPMV resulted in three different
amino acid sequences for the partial F-protein
(Fig. 3, 41–3). The Gono-GER85 and the

Fig. 2. Agarose gel (2%) electrophoresis of RT-PCR products of the 19 snake paramyxoviruses exhibits the 566 nt of the partial
L-gene. Cell culture supernatant was used as a negative control. Gono-GER85 was used as a positive control (marker: 100 bp
ladder).
 J. Franke et al. / Virus Research 80 (2001) 67–74 71

Fig. 3. Schematic diagram of the precursor fusion protein of a paramyxovirus. The positions of signal sequence, cleavage site
hydrophobic fusion peptide, heptad repeat A (conserved domain), heptad repeat B, transmembrane domain, and cytoplasmic tail are
shown. The region investigated in this study covering the cleavage site up to the heptad repeat A was aligned with eight ophidian
paramyxoviruses and four representatives of Paramyxo6irinae genera (Bellini et al., 1998). Identical amino acids are shown as dots,
identical amino acids in all sequences are marked with *. The snake viruses differ in four amino acids in the complete region (1 – 3
). The conserved domain is 100% identical. Alignment of the ophidian paramyxoviruses with the Paramyxo6irinae and Hendra
virus shows 100% similarity to Sendai virus in the conserved domain.

CrotCe-96 sequence proved to be identical to the
FDLV sequence (Fig. 3, 41). Recently, a partial
F-gene paramyxovirus sequence was obtained
from the venom glands of a Fer de Lance viper
(Bothrops jararaca) (Azevedo et al., 2001). This
sequence (AF251500) showed 84% nucleotide
identity, 87% amino acid identity, and 94% simi-
larity to the corresponding sequence of the FDLV
(Bothrops atrox, USA), which was used in this
study.
The investigated viruses proved to have the
same TSAQITAGIAL motif as a conserved do-
main in the F-protein; this is identical to the
conserved F-motif published for Sendai virus
(M30202), but is different from that of measles
virus by one amino acid, from Hendra virus by
three amino acids, and from mumps virus by four
amino acids (Fig. 3). The fusion cleavage site of
Morbilli-, Rubula-, and Pneumoviruses are highly
basic and contain R-X-R/K-R consensus se-
quenses that are required for cleavage by furin
(Hosaka et al., 1991). The Respiroviruses (Sendai
virus) do not have this furin-cleavage site and use
different host cell proteases (Scheid and Choppin,
1974). All the investigated partial F-proteins of
snake viruses reveal a R-X-R/K-R consensus mo-
tif indicating furin usage in F-protein cleavage.
Further studies should establish the functionality
of the OPMV F-protein.
72 J. Franke et al. / Virus Research 80 (2001) 67–74
The obtained snake virus and FDLV nucleotide
sequences were used for phylogenetic analysis.
Calculations were carried out by means of the
phylogeny inference package (PHYLIP), version
3.5c (Felsenstein, 1993). Estimation of phyloge-
nies for maximum parsimony analysis of the par-
tial L- and F-gene was performed by using 100
Fig. 4. Phylogenetic tree of FDLV and the investigated ophid-
ian paramyxoviruses. Analysis was carried out by using
PHYLIP and maximum DNA parsimony. Values on each
branch represent% bootstrap support (100 × bootstrap). The
Sendai virus was used as the outgroup. (a) Phylogenetic analy-
sis based on the eight different partial L-gene nucleotide
sequences (566 nt) of the investigated ophidian paramyx-
oviruses and FDLV revealed three clusters (A, B, C). (b) Six
different partial F-gene nucleotide sequences (918 nt) of the
investigated ophidian paramyxoviruses and FDLV resulted,
after phylogenetic analysis, in two clusters (A, B).
bootstrap values. The nucleotide sequence of
Sendai virus was added as an outgroup (Fig. 4a,
b). The L-gene sequences revealed three clusters
(Fig. 4a: A, B, C). However, analysis of the
F-gene resulted in two clusters (Fig. 4b: A, B).
The two isolates forming cluster C (Fig. 4a) in the
L-gene tree (ElaGut-91, CrotX1-96) could not be
Fig. 5. Phenogram showing the genetic relationship of FDLV
and the type species of the genera of Paramyxo6iridae, the
measles, mumps, Sendai, HRS viruses, and Hendra virus.
Analysis, based on a 549 amino acid (F-protein) and a 700
amino acid (L-protein) PHYLIP calculation, was performed as
described in Fig. 4 without using an outgroup. Analysis based
on a part of the L-protein (a) and F-protein (b) shows the
classification of the paramyxoviruses. The FDLV and Sendai
virus are grouped together (D).
 J. Franke et al. / Virus Research 80 (2001) 67–74
Table 2
Comparison of sequences of representative species of Paramyxo6iridae to FDLV shows similarity (%) to FDLV based on nucleotide
(nt) and amino acid (aa) sequences
Genus Virus
Respirovirus Sendai virus
Rubulavirus Mumps virus
Morbillivirus Measles virus
Henipavirus Hendra virus
Pneumovirus Human respiratory syncytial virus
The F- and L-gene (nt) and protein (aa) sequence were analyzed concerning identity (id) and similarity (sim).
amplified in the F-gene-specific RT-PCR. This
distinct formation of nucleotide sequence suggests
too great a sequence divergence to allow the
binding of F-gene primers. Host specificity could
not be shown in the phylogenetic tree generated
either with partial L-gene sequences or with par-
tial F-gene sequences. The same assumption was
found during the study of L- and HN-gene se-
quences from paramyxoviruses of reptiles isolated
in North America and Europe (Ahne et al., 1999).
To date, OPMV are not included in the present
taxonomy of viruses. Only FDLV is listed as an
unassigned species of the family Paramyxo6iridae
(Van Regenmortel and 10 co-authors, 2000). Re-
garding the classification, we analyzed, for the
first time, the detailed sequence relationship of
partial L-gene and F-gene sequences of OPMV.
FDLV was compared with the type species viruses
of each genus within the paramyxovirus family
(Van Regenmortel and 10 co-authors, 2000) and
with Hendra virus (AF017149). A comparison of
nucleotide sequences of the F- and L-genes and
proteins of Sendai, mumps, measles, Hendra and
HRS viruses revealed the low similarity of FDLV
to these species. The least similarity of partial
L-gene sequences was detected to HRSV with 32%
in the nucleotide sequence and 29% in the amino
acid sequence, and the highest similarity was
found to Sendai virus with 56% in the nucleotide
sequence and 61% in the amino acid sequence.
For the F-gene, the similarity of nucleotide se-
quence to HRSV was 17 and 42% for Sendai
virus. The amino acid sequence alignment showed
a similarity of 22% for HRSV and 28% for Sendai
73
L-gene F-gene Acc. number
nt aa id/sim nt aa id/sim
56 61/75 42 28/50 M30202
53 44/62 26 23/45 AF143383
52 58/78 40 27/49 E04903
49 48/79 39 27/62 AF017149
32 29/51 17 22/48 HRU27298
virus (Table 2). The low homology of available
sequences suggests that the OPMV are only dis-
tantly related to the established genera of
Paramyxo6iridae. Viruses within each genus of the
subfamily Paramyxo6irinae share at least 40% se-
quence identity (Yu et al. 1998). Therefore, we
propose that FDLV is a type species of a new
genus within the Paramyxo6iridae.
Phylogenetic analysis, based on a 549 amino
acid (F-protein) and a 700 amino acid (L-protein)
PHYLIP calculation, showed that the FDLV clus-
tered with Sendai virus, genus Respirovirus (Fig.
5a, b: D). The results indicate a relationship of
reptilian paramyxoviruses with Respiroviruses,
despite their use of different cellular proteases for
protein modification (Scheid and Choppin, 1974).
Gono-GER85 was previously shown to lack
antigenic relationships with the Sendai, mumps,
measles, influenza-A, and influenza-B viruses
(Ahne et al., 1987; Ahne and Neubert, 1989).
Furthermore, several OPMV were found to be
antigenically distinct from mammalian and avian
paramyxoviruses (Clark et al., 1979; Ahne and
Neubert, 1989; Richter et al., 1996). A further
difference between mammalian, human, and bird
paramyxoviruses and the reptilian paramyx-
oviruses is their temperature optimum (Mayr et
al., 2000). Sequence homologies and antigenic and
biological properties of reptilian viruses indicate
that they cannot be assigned to the existing
genera, and that they might represent a distinct
evolutionary lineage within the subfamily
Paramyxo6irinae. In conclusion, we propose a
new genus for the reptilian paramyxoviruses.
74 J. Franke et al. / Virus Research 80 (2001) 67–74
Acknowledgements
We are very grateful to G. Kurath, Western
Fisheries Research Center, Seattle, USA, for
providing the complete FDLV L- and F-gene se-
quences used in this study. We thank our col-
leagues Dr F. Just and Dr M. Pfeffer for
assistance and the critical discussion. This work
was supported by a grant from the DFG (AH
18/16-1).
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