Characterization of paramyxoviruses isolated from three snakes

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Virus Research 43 (1996) 77-83

Virus Research

Short communication

Characterization of paramyxoviruses isolated from three snakes Gary A. Richter a, Bruce L. Homer b, Sue A. Moyer c, Donna S. Williams d, Gail Scherba e, Sylvia J. Tucker a, Betty J. Hall b, Janice C. Pedersen f, Elliott R. Jacobson "'* ~Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32169, USA bDepartment of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 3216, USA ~Department of Pediatrics, College of Medicine, Univeristy of Florida, Gainesville, FL 32610, USA aElectron Microscopy Core Laboratory, ICBR, University of Florida, Gainesville, FL 32610, USA ~Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61801, USA fNational Veterinary Services Laboratories, Ames, 1A 50010, USA Received 6 September 1995; revised 22 February 1996; accepted 6 March 1996

Abstract

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm 3 to 1.18/cm 3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be R N A by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae. Keywords: Snake; Paramyxovirus; Characterization

Fer-de-lance virus ( F D L V ) , isolated f r o m vipers (Bothrops moojeni) in a v e n o m l a b o r a t o r y in Switzerland, was the first paramyxo-like virus identified in a reptile (Foelsch and Leloup, 1982;

* Corresponding author. Tel.: + 1 352 392 4700 ext. 5700; fax: + l 352 392 6125; e-marl: [email protected]. UFL.EDU

Clark et al., 1979). Since the initial characterization o f F D L V , there have been a variety o f publications concerning similar viral isolates recovered from snakes (Ahne and Neubert, 1989, 1991; A h n e et al., 1987; Blahak et al., 1991; J a c o b s o n et al., 1980, 1992; Potgieter et al., 1987) as well as f r o m a lizard (Ahne and Neubert, 1991). While most o f these viruses had biological and physical

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G.A. Richter et al. / Virus Research 43 (1996) 77 83

properties in common with FDLV, only two instances of antigenic cross-reactivity with known avian and mammalian paramyxoviruses have been reported (Blahak et al., 1991; Potgieter et al., 1987). We examined the physicochemical characteristics of one isolate from a snake and compared these features with those of two additional snake isolates, as well as with those of accepted members of the Paramyxoviridae. The first isolate, designated Neotropical virus (NTV; deposited as VR-1408, American Type Culture Collection (ATCC), Rockville, MD), was obtained from the lung tissue of a Neotropical rattlesnake (Crotalus durissus terrificus) that died in 1992 during an epizootic in the snake collection at the Memphis Zoo, Memphis, Tennessee. The second isolate, designated bush viper virus (BVV; ATCC VR-1409), was obtained from multiple tissues, including the lung, of a bush viper (Atheris sp.) that died with a proliferative pneumonia in 1993 at the Louisiana Purchase Zoo, Monroe, LA. The third isolate, designated Aruba Island rattlesnake virus (AIV), was provided by Dr. Donald Nichols at the National Zoological Park, Washington, DC. This virus was isolated from the lung and several other tissues of an Aruba Island rattlesnake (Crotalus unicolor). NTV was chosen as the archetype virus for complete physicochemical characterization since it was the easiest to propagate in cell culture. The three snake isolates were initially propagated in viper heart cells (ATCC, Rockville, MD) using previously described methodology (Jacobson et al., 1980). Subsequently, the isolates were adapted for growth in Vero cells (ATCC). Prior to infection, Vero cells were maintained in Dulbecco's Modified Eagles's Medium ( D M E M ) containing 25 m M Hepes buffer, 5% FBS, and 1% antibiotic/antimycotic solution at 37°C. Following infection, the FBS in the media was replaced with 0.3% bovine serum albumin (BSA), and the cultures were incubated at 30°C. Trypsin (5 /tg/ml) was added to facilitate the cleavage of the viral fusion protein, aiding in the spread of infection (Kingsbury, 1991; Nishikawa et al., 1981). Syncytia formation occurred with each of the isolates, but intracytoplasmic inclusions were not observed.

The three viral isolates were visualized by negative staining electron microscopy. BVV, the smallest of the three viruses, had pleomorphic spherical particles that ranged from 30-90 nm in diameter. The diameter of NTV was between 125 and 128 nm, while the largest, AIV (Fig. I(A)), ranged between 120 and 500 nm. In the intact AIV, and to some extent, NTV particles, a helical

J

Fig. 1. (A) Electron photomicrograph of a negatively stained virion purified from cell culture infectedwith the Aruba Island rattlesnake isolate. (B) Electron photomicrograph of a helical nucleocapsid strand released from a ruptured virion purified from cell culture infected with the Neotropical rattlesnake isolate. A drop of virus suspension was placed on a 300 mesh formvar/carbon coated copper grid and prepared negatively stained (Hayat, 1986). Grids were examined using a Zeiss electron microscope.

G.A. Richter et al. / Virus Research 43 (1996) 77 83

nucleocapsid could be seen. All preparations appeared to have viral particles with a lipid envelope containing glycoprotein peplomers. Numerous ruptured particles were visible in the NTV preparations, revealing extruded helical nucleocapsids whose outer diameter measured 20-21 nm (Fig. I(B)). The lengths of these nucleocapsids were difficult to accurately measure because of their irregular arrangement, but ranged from 940 to 1002 nm. Veto cell monolayers infected with any of the isolates were fixed in 2.5% glutaraldehyde, postfixed in OsO4, and ultrathin sections stained with uranyl acetate and lead citrate were examined by electron microscopy. Nucleocapsid strands were seen in the cytoplasm and adjacent areas of the cell membrane were thickened, with 'blebs' budding out into the extracellular space. While most of the virus particles appeared spherical in shape, some filamentous forms also were present. It was concluded, based upon negative staining and transmission electron microscopy, that the size and morphology of the snake viruses resembled that of paramyxoviruses. Polyclonal antibodies to NTV were raised in two rabbits. The immunogen consisted of pelleted and resuspended Veto cell-propagated NTV. The suspension was filtered (0.2 ~m), diluted 1:1 with sterile PBS, then mixed 1:1 with Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO.). The immunogen was injected intradermally in 0.1 ml aliquots at five sites in each rabbit and boosted at two weeks with a similar quantity of virus in Freund's incomplete adjuvant (Sigma Chemical Co.). Antisera to AIV and BVV were similarly raised in rabbits. To test for protein expression, individual monolayers of Vero ceils grown on plastic chamber slides were separately infected with each viral isolate. Approximately 7 days post-infection the cells were fixed in cold absolute methanol and were stained by a fluorescent-labeled antibody methodology using a 1:50 dilution of the antiNTV antibody (Potgieter and Aldridge, 1977). Using UV microscopy, Vero cells infected with NTV had dark nuclei surrounded by cytoplasm with extensive fluorescent staining. Staining was not observed within uninfected cells or with

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preimmune serum on infected cells. Thus, virus replication appeared to occur in the cytoplasm, suggestive of an R N A virus. Since members of several viral families are capable of hemagglutination, the ability of NTV, AIV, and BVV to agglutinate various erythrocytes was evaluated. The cells and media in flasks of virus-infected Vero cells with maximal syncytia formation were harvested and freeze-thawed twice (Wechsler et al., 1985). The hemagglutination titers of each virus were determined at 4°C as previously described (Carbrey et al., 1974). Chicken, guinea pig, sheep, and human Type-O erythrocytes were used. All three isolates consistently agglutinated chicken erythrocytes (Table 1) and equivalent HA titers were obtained with guinea pig erythrocytes. However, titers were reduced almost by half when using human type-O, and sheep erythrocytes. Rabbit erythrocytes were only slightly and inconsistently agglutinated by NTV. A hemagglutination inhibition (HI) assay was used to determine the presence of specific anti-viral antibodies based on the ability of the antisera to inhibit viral hemagglutination (Carbrey et al., 1974). Non-specific agglutinins from the viral antisera were removed by diluting the sera with PBS (1:4) and incubating the diluted sera with a 1:10 dilution of chicken erythrocytes at 22°C for 30 min then pelleting the erythrocytes. Two-fold dilutions (1:8- l:1024) of 0.025 ml of treated serum was made with a diluent containing 10 HA units of antigen. After incubation at room temperature for 15 min, 0.05 ml of a 0.5% suspension of chicken erythrocytes was added to each well. After 10 minutes of incubation at 22°C, the HI titer was read as the reciprocal of the highest dilution of serum that inhibited hemagglutination. To determine the relatedness of the three snake viruses to members of the Paramyxoviridae, antisera to known paramyxoviruses and to the three snake viral isolates were reacted with NTV. The antibodies tested were specific against Sendai (PI1; HI titer of 160), simian virus (PI2; HI titer of 320), hemadsorption virus 1 (PI3; HI titer of 1280), Roakin strain of Newcastle disease virus (NDV; PMV1; HI titer of 640), Long strain of respiratory syncytial virus (RSV: neutralization

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Table 1 Comparison of characteristics of paramyxoviridae genera to those of neotropical rattlesnake, aruba island rattlesnake, and bush viper viral isolates Isolate

Cytopathic effects

HemagglutinNeuraminidase Buoyantdensity ationa (g/cm3)

Viriondiameter (rim)

Capsiddiameter (nm)

Positive

Positive

1.18

125-188b

20-21 b

Syncytium, Positive lysis

Positive

1.15

120 500

15 16

Syncytium, lysis Syncytium, lysis Syncytium, lysis Syncytium, lysis Syncytium, lysis

Positive

Positive

1.13

30- 90

Positive

Positive

1.18 1.20

120 300

12 17

Negative

Positive

1.18-1.20

120- 300

12-17

Negative

Negative

1.18-1.20

120-300

12-17

Positive

Positive

1.18-1.20

100-600

12-18

Neotropical isolate Syncytium, ( Crotalus durissus lysis terrificus )

Aruba i s l a n d isolate (Crotalus unicolor )

Bush viper isolate (Atheris sp.)

Paramyxovirusc,a Morbillivirusc.d Pneumovirusc.d Rubulavirusd,e

ND

aUsing chicken red blood cells. bMeasurements made immediately following microscope calibration. CKingsbury (1991). dRima et al. (1995). eWolinsky (1996). ND, Not determined. titer of 10 000); (Braton Biotech, Rockville, M D ) and Snyderhill strain of canine distemper virus (CDV; virus neutralization titer of 160; courtesy of Dr. Max Appel, Cornell University, Ithaca, NY). In addition, A I V and BVV were tested against antisera to avian paramyxoviruses (PMV) l an parainfluenza virus (PIV) types 1, 2, 3, 4A, 4B, and 5 by the H I assay. Furthermore, rabbit antisera to the NTV, AIV, and BVV were tested for cross-reactivity to avian P M V types 1, 2, 3, 4, 6, 7, 8, and 9, and m a m m a l i a n PIV3. Each of six antisera specific for either PIV1, PIV2, PIV3, NDV, RSV, or C D V failed to inhibit the ability of N T V and AIV to agglutinate chicken erythrocytes. Moreover, only the hemagglutination ability of BVV was inhibited by antisera to avian paramyxoviruses PMV1 at a dilution of 1:8. No antisera to either avian or mammalian paramyxoviruses inhibited the hemagglutinating activity of AIV. Conversely, rabbit antisera to NTV, AIV, and BVV did not inhibit the hemagglutinating activity of the avian paramyxoviruses.

In addition, rabbit antisera to NTV, AIV, and BVV when tested against the PIV3 virus had H I antibody titers of 10, 40, and 20, respectively; however, the preimmune rabbit antisera also had comparable H I antibody titers. The reason for H I activity in the pre-immune sera is not known and seems to be an artifact, since reactivity in rabbit antisera to NTV, AIV, and BVV was not detected to PIV3 virus with either virus-neutralization or indirect fuorescent antibody tests (Beard, 1989). Virion neuraminidase activity is variously represented a m o n g the Paramyxoviridae genera. The presence of neuraminidase activity in each of the three partially purified viruses was determined in a fluorometric assay using 4-methylumbelliferyl N-acetylneuraminic acid ( M U N e u N A c ) as the substrate as described previously (Lambr'e et al., 1989; Yolken et al., 1980). Each of the NTV, BVV, and A I V crude preparations were positive for neuraminidase activity in this assay (Table 1). Neuraminidase activity was not detected in uninfected Vero cell extracts indicating the absence of

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this enzyme in the cells used to propagate the viruses. Neuraminidase activity was inactivated when the viruses were incubated at 75°C for 10 min. The ether and pH sensitivity was determined for NTV. Samples of virus treated with ether did not produce CPE in Vero cells or agglutinate chicken erythrocytes and were thus sensitive to this chemical. The pH of suspensions of NTV were either decreased to 5.0 with 0.1 N HC1 or increased to 12.2 by the addition of 0.1 N NaOH. An untreated virus suspension at pH 8.2 served as a control. The samples were incubated at 4°C overnight and then their pH was adjusted to approximately 8.0 before titration using the TCIDs0 assay (Reed and Muench, 1938). Vero cells infected with NTV after incubation of the virus at either pH 5.0 or pH 12.2 displayed noticeably less CPE when compared with cells infected with the pH 8.2 control. A similar reduction in HA titers was noted for the viruses following ether treatment or placement at either pH 5.0 or pH 12.2. Statistical analyses of the acidic versus control samples showed significance at the P < 0.05 level for both the T-test (P = 0.0004) and the Wilcoxon rank sum test (P = 0.05). These results together indicated the dependence of viral infectivity on an envelope. To determine their densities, NTV, AIV, and BVV were propagated in Vero cells until CPE was maximal (approximately 7 days). The cells and media were freezethawed three times, clarified by low speed centrifugation and then pelleted on a glycerin cushion at 47 800 g for 2 h at 4°C. The pelleted virus was then banded on a 20-60% (w/w) sucrose gradient by centrifugation at 107 170 g for 18 h at 4°C in the SW 41 rotor. Fractions (0.5 ml) were collected and the buoyant density, HA titer and protein concentration of each fraction was determined. Gradient purification of the crude virus preparations resulted in a single broad peak for each virus as determined by assaying for HA activity across the gradient. For the NTV, the fraction containing a peak HA titer of 1024 coincided with the highest protein concentration of (0.28 mg/ml) and a buoyant density of 1.18 g/cm 3. The AIV peak fraction had an HA titer of 128, a protein concentration of 0.21 mg/

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ml, and a buoyant density of 1.15 g/crn 3. The BVV peak fraction had an HA titer of 1024 with 0.14 mg/ml protein and a buoyant density of 1.13 g/cm 3. None of the fractions from the uninfected Vero cell control gradient had HA activity. To determine if the genome was RNA, actinomycin D (2 pg/ml, Gibco BRL, Grand Island, NY) and [3H]uridine (100 uCi/ml DuPont NEN, Wilmington, DE) were added to the medium of uninfected or NTV-infected Vero cells at 4 days post infection. After 24 h at 32°C, the cells and media were harvested, frozen, thawed, and clarified by low speed centrifugation. Virus was banded on a sucrose density gradient (as above) and acid precipitable counts (CPM) determined across the gradient. Two peaks of radioactivity were seen in NTV-infected Vero cells; the control showed minimal counts across the gradient. One peak of radioactivity corresponded to the characteristic density (1.18 g/cm 3) of spherical paramyxovirus particles. A second peak with a lower density likely represented the filamentous particle form seen budding from infected Vero cells by transmission electron microscopy (TEM). These data are consistent with the NTV snake virus containing a RNA genome. Viral proteins of the three snake isolates were radiolabeled in infected cells and cell extracts immunoprecipitated (Fig. 2). Compared with the noninfected cells (mock), six major virus specific bands could be identified for each virus (lanes AI, BV and NT). The NTV and AIV proteins had similar electrophoretic mobilities, and while the molecular weights of BVV proteins differed slightly, the anti-NTV serum still immunoprecipitated the BVV proteins. Based on their calculated molecular weights, the viral proteins were tentatively identified as the L, HN, NP, F0, P, and M proteins according to the analogous proteins found in other paramyxoviruses. Other less abundant proteins also were seen in infected cells and may represent truncated forms of structural proteins. Except for optimal growth at temperatures below that of avian and mammalian paramyxoviruses, the requirements for propagation in cell culture of the three snake viruses in our study were the same as for known members of this

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group. Morphogenesis, as seen by electron microscopy, and various physicochemical properties, including presence of an R N A genome, further supported categorization within Paramyxoviridae. The diameter of virions and length and diameter of nucleocapsids overlapped the reported range for paramyxoviruses (Kingsbury, 1966). The appearance of nucleocapsids of this

lflll L

P HN Fo,NP M

length also serves to rule out the possibility that N T V is an orthomyxovirus, as these viruses have segmented genomes 50-130 nm in length. The buoyant densities in sucrose of the NTV, AIV, and BVV isolates ranged from 1.13 to 1.18 g/cm 3 as compared with 1.19 g/cm 3 for F D L V and 1.18-1.20 g/cm 3 for m a m m a l i a n paramyxoviruses (Kingsbury et al., 1978). It is possible that the limits of these characteristics, defined mainly in m a m m a l i a n viruses, will change as additional isolates from lower vertebrates are identified. Attempts to establish the antigenic relatedness of NTV, AIV, and BVV to known paramyxoviruses were unsuccessful. Hemagglutination inhibition was not observed when the snake viruses were incubated with antisera to a wide variety of mammalian or avian paramyxoviruses. The lack of cross reactivity with members of all four genera of Paramyxoviridae illustrates that the snake viruses, while physicochemically similar, are antigenically distinct from other paramyxoviruses. However, the three snake viruses were antigenically similar to each other, since a rabbit polyclonal antibody produced against N T V inhibited the agglutination of chicken erythrocytes by NTV, AIV, and BVV and immunoprecipitated the proteins of all three viruses. Future molecular studies will be necessary to determine the structure of the virus genomes.

Acknowledgements Fig. 2. Analysis of radiolabeled virus proteins in NTV, AIV, and BVV infected cells. Vero cells (60 mm dishes) were infected with NTV, AIV or BVV and incubated until they displayed about 70% CPE. The infection media was aspirated, low methionine/cysteine media (1.5 ml) was added and the cells were radiolabeled with 100 pCi 35S-Translabel (ICN, Los Angeles, CA) for 16 h at 32°C. Cytoplasmic cell extracts were prepared in 1% NP-40 (300 pl) and samples (100 pl) were immunoprecipitated with anti-NTV antibody (2 pl), collected with Staphylococcus aureus (Cowan strain) and analyzed by 10% polyacryamide-SDSgel electrophoresis and fluorography (Kingsbury, 1966). Mock (Vero), NT, AI, and BV proteins are in lanes 1-4, respectively. Protein molecular weight standards in kilodaltons are indicated at the left and the tentative identification of viral proteins at the right.

The authors thank Dr. Max Appel, Cornell University, for providing canine distemper antiserum, Dr. Donald Nichols, National Zoological Park, for providing the Aruba Island Isolate, Dr. Gregory Erdos, Electron Microscopic Core Laboratory, University of Florida, for technical assistance, and Drs. William M. Schnitzlein, Paul Gibbs, Janet Y a m a m o t o , and Paul Klein, for manuscript review. This study was supported by a grant from the Audubon Institute, New Orleans, LA. Published as University of Florida, College of Veterinary Medicine Journal Series No. 453.

G.A. Richter et al. / Virus Research 43 (1996) 77 83 manuscript review. This study was supported by a grant from the Audubon Institute, New Orleans, L A . P u b l i s h e d as U n i v e r s i t y o f F l o r i d a , C o l l e g e o f V e t e r i n a r y M e d i c i n e J o u r n a l S e r i e s N o . 453.

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