The hemagglutinin neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells

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1 Virus Research 75 (2001) The hemagglutinin neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells Shaguna Seth 1, M.S. Shaila * Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore560012, India Received 28 August 2000; received in revised form 8 February 2001; accepted 8 February 2001 Abstract The genes coding for the surface glycoproteins hemagglutinin neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus Elsevier Science B.V. All rights reserved. Keywords: Peste des petits ruminants virus; Rinderpest virus; Hemagglutinin neuraminidase protein; Hemadsorption; Neuraminidase; Fusion promotion 1. Introduction The outer envelope of paramyxoviruses harbors two surface glycoprotein spikes, the hemagglutinin neuraminidase (HN) and fusion (F) proteins. These glycoproteins interact with the * Corresponding author. Tel.: / ; fax: address: shaila@mcbl.iisc.ernet.in (M.S. Shaila). 1 Department of Microbiology and Immunology, 3001 Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322, USA. host cell surface to initiate the infection process (Chanock and McIntosh, 1990). The hemagglutination activity (HA) of HN is responsible for virus attachment to cell surface receptors, as well as agglutination of erythrocytes while the neuraminidase activity cleaves sialic acid residues from the carbohydrate moieties of glycoproteins (Scheid and Choppin, 1974). The fusion protein, on the other hand, is involved in virus-induced cell fusion. Though HN proteins of paramyxoviruses exhibit both hemagglutinating as well as neuraminidase activities, the morbillivirus H /01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 170 S. Seth, M.S. Shaila / Virus Research 75 (2001) protein has been shown to possess only the hemagglutination activity (Choppin and Compans, 1975). Among the members of morbillivirus genus, only measles virus (MV) and peste des petits ruminants virus (PPRV) have been shown to exhibit hemagglutination activity (Varsanyi et al., 1984; Ramachandran et al., 1995). Though neuraminidase activity (NA) has not been demonstrated in Morbilliviruses, a model on the structure of paramyxovirus HN (Colman et al., 1983; Epa, 1997; Langedijk et al., 1997) proposed neuraminidase activity in morbillivirus HN proteins. Based on the model, the presence of NA was tested in infected cell extracts of rinderpest virus (RPV), MV, phocine distemper virus (PDV), canine distemper virus (CDV), dolphin mosaic virus (DMV) and PPRV using a variety of neuraminidase substrates (Langedijk et al., 1997). However, the results demonstrated low levels of neuraminidase activity with only bovine submaxillary mucin. Although the Paramyxovirus F protein seems to play a pivotal role in cell fusion, accumulated data suggest that a fusion promotion function of the homologous HN protein is required for cell fusion by most paramyxoviruses including human parainfluenza virus type 2 (PIV-2) (Hu et al., 1992), Newcastle disease virus (NDV) (Morrison et al., 1991) and MV (Wild et al., 1991). This cooperative interaction has been shown to be under a strict type-specific control (Hu et al., 1992; Heminway et al., 1994). In contrast, for simian virus 5 (SV5), the F protein can cause syncytium formation when expressed alone in CV- 1 cells (Paterson et al., 1985) and the coexpression of both HN and F proteins marginally increases the extent of the syncytium formation (Horvath et al., 1992). In this paper, we report the expression of PPRV HN and RPV H proteins from cloned cdnas in mammalian cells using a cytomegalovirus promoter driven expression system and demonstrate that the expressed proteins are biologically active. The biological activities of the transiently expressed recombinant proteins such as cell attachment and neuraminidase were evaluated. PPRV HN possessed hemagglutination activity, an attribute of cell attachment function of the virus, and neuraminidase activity while RPV H exhibited only NA activity. This is the first direct evidence of a morbillivirus HN protein expressed transiently at the surface of mammalian cells showing NA activity, which was thought to be absent in the members of the morbillivirus genus. 2. Materials and methods 2.1. Cells and iruses Vero and CV-1 cells were obtained from National Centre for Cell Science, Pune, India and were maintained in DMEM supplemented with 5% fetal calf serum (Gibco-BRL, USA) at 37 C. Vaccine strain of PPRV (Nig 75/1), provided by Dr A. Diallo, CIRAD-EMVT, France and a field isolate of PPRV (Ind/TN87/1) (Shaila et al., 1989) were employed in this study. A tissue culture adapted vaccine strain of RPV (RBOK) (Plowright and Ferris, 1957) was obtained from the Institute of Animal Health and Veterinary Biologicals, Bangalore, India. All these viruses were amplified in Vero cells Construction of recombinant plasmids The eukaryotic expression vector pcmx driven by a strong cytomegalovirus (CMV) promoter (a gift from Dr P.N. Rangarajan, Department of Biochemistry, Indian Institute of Science, Bangalore) was employed in cloning and expressing the HN/H genes. The PPRV HN gene of PPRV (Nig 75/1) was subcloned from pbacpak vector (carrying the full length clone 1.9 Kb of PPRV HN gene of PPRV (Nig 75/1), a gift of Dr A. Diallo, CIRAD-EMVT, France) as a BamHI/NotI, endfilled fragment into SmaI digested pgem-7zf vector yielding the clone pss96-1. The full length PPRV HN was released from pss96-1 plasmid as a KpnI/BamHI fragment and was directionally subcloned in KpnI/BamHI digested pcmx.pl2 (psshn). The RPV H clone (RBH 3.4) is a recombinant bluescript plasmid carrying the full length H gene of RPV (RBOK) vaccine strain (a gift from Dr T. Barrett, Institute for Animal Health, Pirbright, UK). parh carrying the full

3 S. Seth, M.S. Shaila / Virus Research 75 (2001) length cdna of RPV(RBOK) H was constructed in the laboratory of Dr P.N. Rangarajan, Department of Biochemistry, Indian Institute of Science, Bangalore employing the RPV H gene released from RBH 3.4 as PstI and XhoI fragment and cloned in PstI and SalI site of the pcmvintb plasmid Transfection of eukaryotic cells with the gene constructs CV-1 cells were plated on sterile cover-slips in 35 mm tissue culture dishes at a density of cells in 2 ml of DMEM supplemented with 5% FCS (Gibco-BRL, USA). When the cells were 70% confluent, medium was removed and cells were washed with phosphate buffered saline (PBS). Five microliters of Lipofectamine (2 mg/ ml) and 7 g of DNA were mixed in 500 l of OPTI-MEM medium (Gibco-BRL, USA) and incubated for 30 minutes at room temperature. This mixture was then added to the cells and the dishes were incubated at 37 C with 5% CO 2 inahumidified incubator for 4 h. The transfection mixture was then removed and 2 ml of complete DMEM was added to the dishes and were left for different time points at 37 C Electrophoresis and immunoblot analysis Proteins in infected/transfected cell extracts were separated by electrophoresis on a 10% SDSpolyacrylamide gel and then electrophoretically transferred to nitrocellulose membrane for Western blot analysis. Transferred proteins were probed with anti-hn hyperimmune serum raised in rabbits (Devireddy et al., 1998). Bound antibodies were identified by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin F(ab )2 fragments (Amersham, UK). Immunoreactive proteins were visualized using the ECL system (Amersham, UK) and Kodak X-Omat film Cell surface immunofluorescence CV-1 cells were transfected as described in Section 2.3. At 24 h post-transfection, cells were washed with PBS containing 0.1% sodium azide and processed for cell-surface immunofluorescence. Cells were then incubated with 1% BSA in PBS for 1 h at 37 C. This was followed by incubation with 100 l of 1:100 diluted rabbit hyperimmune sera raised against PPRV HN and RPV H (Devireddy et al., 1998) for 1 h at 37 C. The dishes were then washed thrice with PBS and then 100 l of 1:100 diluted goat anti-rabbit IgG- FITC conjugate (Boehringer Mannheim, Germany) was added and incubated at 37 C for1h. Cells were then washed with PBS thrice, mounted on slides in 50% glycerol and the slides were viewed under an Olympus fluorescence microscope Hemadsorption CV-1 cells grown on coverslips were transfected with psshn. At 24 h post-transfection, cells were incubated with 1% chicken RBCs for 30 min at room temperature (Ramachandran et al., 1995). Cells were gently rinsed with PBS, fixed with 90% acetic acid and 10% methanol for 10 min at 20 C and washed with PBS. Cells were subsequently stained with Giemsa (Gibco-BRL, USA). The stain was further treated with 0.1% acetic acid, washed with water and the coverslips were mounted on the slides in 50% glycerol. The slides were observed under the microscope for cells with rosette of erythrocytes. Mock transfected cells were used as control Neuraminidase assay At 24 h post-transfection, cells were washed with PBS, scraped and pelleted at 3000 rpm for 5 min at 4 C. The pelleted cells were lysed using 50 l of 0.5% Triton X-100 (Sigma, USA) in PBS for 10 min at room temperature. The cell debris was then removed by centrifugation at 3000 rpm for 5 min at 4 C. To 50 l of cell lysate, 100 l of phosphate buffer, ph 5.9 was added and incubated with different substrates at the specified concentrations for 18, 24 and 48 h at 37 C. The release of product, N-acetyl Neuraminic Acid (NANA) was estimated using a colorimetric method (Aymard-Henry et al., 1973). The assay

4 172 S. Seth, M.S. Shaila / Virus Research 75 (2001) was performed in triplicates and at least two independent experiments were carried out. The following substrates were used at the concentrations specified: fetuin (48 mg/ml), mucin type I-S from bovine submaxillary gland (25 mg/ ml), colominic acid (150 g/ml), N-glycolylneuraminic acid (150 g/ml), N-acetyl neuraminlactose (150 g/ml), 6 N-acetyl neuraminlactose (150 g/ml), transferrin (150 g/ml) and ganglioside (150 g/ml). All the substrates were obtained from Sigma, USA. 3. Results In order to study the biological activities of HN protein of PPRV, it was necessary to express PPRV HN protein in eukaryotic cells. This was achieved by employing the transient expression of transfected cdna construct in a eukaryotic expression vector under CMV promoter. At 24 h post-transfection, the expression of HN protein in CV-1 cells was analyzed using western blot analysis which showed the presence of a 68 kda protein band corresponding to recombinant HN protein (Fig. 1A). To see whether the transiently expressed HN protein is localized at the cell surface, cells transfected with psshn were analyzed for cell surface immunofluorescence 24 h after transfection. The results indicated a patchy localization of HN at the surface of transfected cells (Fig. 1B). The RPV H protein was also cloned and expressed transiently in a CMV driven expression vector and the recombinant protein was found to be localized at the surface of transfected cells. Hemadsorption assay was performed using CV- 1 cells transiently expressing PPRV HN protein or infected with PPRV (Nig 75/1). The cells were then observed for any adsorbed chicken RBCs at the cell surface and the cells transfected with psshn were found to exhibit hemadsorption activity similar to the virus infected cells (Fig. 2). This suggested that the transiently expressed HN protein was biologically active in terms of its ability to bind erythrocytes. However, cells expressing RPV H did not show any hemadsorption activity. Lysates from PPRV (Nig 75/1) infected and psshn transfected cells were tested for NA activity. The cell lysates were incubated with two different substrates, fetuin (48 mg/ml) and mucin (25 mg/ml) for 18, 24 and 48 h. The results (Fig. 3) indicated that the neuraminidase activity of HN protein of PPRV (Nig 75/1) from infected as well as transfected cell lysates was optimal after 24 h of incubation with fetuin as the substrate whereas mucin was found to be a poor substrate. To analyze the substrate specificity, lysates of cells infected with PPRV (Nig 75/1) and a field isolate of PPRV (Ind TN 87/1) as well as the transfected cell lysates were incubated with different substrates at the specified concentrations. The results suggested that the PPRV HN prefers substrates with -2,3 glycosidic linkages such as fetuin, N-glycolyl neuraminic acid and N-acetyl neuraminlactose showing significant neuraminidase activity as compared to transferrin and 6 N-acetyl neuraminlactose which have -2,6 glycosidic linkages (Fig. 4). The specific activity of the HN from the Indian isolate of PPRV was found to be higher for any substrate as compared to the vaccine strain of PPRV. Interestingly, PPRV Nig.75/1 cell lysate showed NA activity when 6 N-acetyl neuraminlactose and colominic acid were used as substrates whereas PPRV Ind TN87/1 infected cell lysate showed no activity with the same substrates despite the fact that TN 87/1 lysate showed higher specific activity than the vaccine strain when other substrates were used. However, the biological activity of recombinant HN paralleled that of its cognate viral HN in its preferences for various substrates. Similarly, when cell lysates from RPV H transfected CV-1 cells were incubated with the substrates and assayed for NA activity (Fig. 5), mucin and N-glycolyl neuraminic acid were found to be the preferred substrates for RPV H. 4. Discussion Viruses in the paramyxovirus genus exhibit hemagglutinating as well as neuraminidase activities (Choppin and Compans, 1975). These activities reside on a single polypeptide, designated as

5 S. Seth, M.S. Shaila / Virus Research 75 (2001) Fig. 1. Expression of recombinant PPRV HN and RPV H proteins in mammalian cells: A. Coomassie stained 10% SDS-PAGE analysis (a) and Western Blot Analysis of PPRV-HN using anti-hn hyperimmune serum raised in rabbits (b) Lane 1. Mock transfected cell lysate and Lane 2. psshn transfected cell lysate. B. Cell surface immunofluorescence to detect transiently expressed PPRV HN protein in CV-1 cells: After 24 h of transfection, cell surface expression of transiently expressed recombinant proteins was detected using anti-hn polyserum: (a) mock transfected; (b) psshn (PPRV HN) transfected; (c) parh (RPV H) transfected CV-1 cells. Fig. 2. Hemadsorption assay: at 24 h post-transfection, CV-1 cells ( ) were incubated with 1% chicken erythrocytes as described in Section 2 for 1 h at room temperature and observed microscopically: (A) mock transfected cells; (B) PPRV (Nig 75/1) infected cells (40 magnification); and (C,D) psshn transfected cells (40 and 100 magnifications).

6 174 S. Seth, M.S. Shaila / Virus Research 75 (2001) HN protein as shown in the case of SV5 (Scheid et al., 1972), parainfluenza virus (Chen et al., 1971) and NDV (Niikura et al., 1991). In the present work, recombinant PPRV HN protein transiently expressed in mammalian cells has been shown to possess both hemadsorption as well as neuraminidase activities. This would further, serve as a tool to study the structure function relationships of different domains of PPRV HN protein with respect to its biological activities. It has been shown earlier that PPRV infected cells exhibit hemadsorption activity and purified PPRV was able to agglutinate erythrocytes from a variety of sources (Wosu, 1991; Ramachandran et al., 1995) and this virus agglutinated chicken and monkey erythrocytes better than human, dog, goat or sheep erythrocytes (Hegde and Shaila, unpublished). The recombinant HN protein expressed at the surface of transfected cells was found to be in a biologically active form as shown Fig. 3. Neuraminidase activity of transiently expressed PPRV HN protein: the neuraminidase assay was carried out using infected and transfected cell lysates (1.5 mg/ml) followed by incubation with two different substrates, fetuin (48 mg/ml) and mucin (25 mg/ml) for 18, 24 and 48 h. The specific activity of neuraminidase represents nmoles of sialic acid released per minute per mg protein at 37 C. The background activity of the mock transfected cells were subtracted from the experimental values and plotted as shown: (A) PPRV-infected cells; (B) psshn transfected cells. Data represents results from two independent experiments performed in triplicates., Fetuin;, Mucin.

7 S. Seth, M.S. Shaila / Virus Research 75 (2001) Fig. 4. Substrate specificity of neuraminidase activity of PPRV HN protein: the infected cell lysates from two different strains of PPRV namely, Nig 75/1 and Ind/TN 87/1 and the psshn transfected cell lysates (1.5 mg/ml) were incubated with different substrates at the following concentrations: Fet, fetuin (48 mg/ml); Muc, mucin (25 mg/ml); 6 NA-, 6 N-acetyl neuraminlactose (150 g/ml); NA, N-acetyl neuraminlactose (150 g/ml); Col, colominic acid (150 g/ml); NGA, N-glycolyl neuraminic acid (150 g/ml); Tfr, transferrin (150 g/ml). The assay was performed as described in Section 2. The background activity of the mock transfected cells were subtracted from the experimental values and plotted as shown. Data represents results from two independent experiments performed in triplicates. PPRV Nig. 75/1; PPRV Ind.TN 87/1;, psshn transfected. by hemadsorption of chicken erythrocytes, thus signifying the presence of cell attachment activity in the transiently expressed protein exhibited in the absence of any other viral proteins. Unlike PPRV HN protein, the RPV H protein though expressed at the surface of transfected cells was not found to show hemadsorption activity. The recombinant HN protein expressed in CV- 1 cells also showed neuraminidase activity and it showed a preference for substrates with -2,3 glycosidic linkages. Similar results have been obtained with immunoaffinity purified HN protein from infected cells. (Shyam and Shaila, unpublished). Though studies conducted by Langedijk et al. (1997) showed a low level of neuraminidase activity in morbilliviruses with bovine submaxillary mucin as substrate, the results in this study demonstrate the substrate specificities of NA activity of RPV H and PPRV HN proteins in transiently transfected cells in the absence of any other viral protein. The differences observed in substrate specificity of HN from two isolates of PPRV namely, Nig 75/1 and Ind/TN87/1 suggest that the preference towards certain substrates could potentially contribute to the virulence associated with these viruses. In contrast, the RPV-H protein showed significant neuraminidase activity with mucin and N-glycolyl neuraminic acid, suggesting a difference in attachment activity and hence, receptor requirements of PPRV and RPV. Paramyxovirus-infected cellular monolayers are often characterized by the presence of multinucleate syncytia (Choppin and Scheid, 1980). However, with the notable exception of Simian virus 5 (Horvath et al., 1992; Ito et al., 1997; Paterson et al., 1985) the promotion of fusion by this group of viruses requires the cooperative efforts of two separate viral glycoprotein spikes, the HN and F protein (Lamb, 1993). Earlier work on other viruses such as bovine parainfluenza 3 virus, NDV, HPIV2, HPIV-3, mumps virus, CDV and measles virus have shown that the syncytia formation occurs only when F and HN are coexpressed together (Ebata et al., 1991; Morrison et al., 1991; Taylor et al., 1991; Wild et al., 1991; Hu et al., 1992; Cattaneo and Rose, 1993; Heminway et al., 1994). Therefore, it has been supposed that a virus type-specific functional interaction between homologous HN and F takes place during the fusion process.

8 176 S. Seth, M.S. Shaila / Virus Research 75 (2001) Fig. 5. Neuraminidase activity of transiently expressed RPV H protein: Different substrates such as fetuin (48 mg/ml), mucin (25 mg/ml), 6 N-acetyl neuraminic acid (150 g/ml), N-acetyl neuraminic acid (150 g/ml) and N-glycolyl neuraminic acid (150 g/ml) were used in this assay. The results are represented in specific activity which is nanomoles of sialic acid released per minute per milligram of protein at 37 C. The background activity of the mock transfected cells were subtracted from the experimental values and plotted as shown. Data represents results from two independent experiments performed in triplicates. The PPRV fusion protein has been shown to be biologically active as it brings about syncytium formation when expressed transiently in CV-1 cells as compared to another morbillivirus RPV fusion protein, which does not show fusion activity on its own (Seth and Shaila, unpublished). The coexpression of PPRV HN protein with PPRV F protein marginally increased the extent of fusion though the efficiency of fusion was found to be drastically enhanced (Seth and Shaila, unpublished). In conclusion, the recombinant PPRV HN when expressed in eukaryotic cells is biologically active showing hemadsorption, neuraminidase and fusion promotion activity in the total absence of other viral proteins. Acknowledgements We thank Dr T. Barrett, Institute for animal Health, Pirbright, UK and Dr A. Diallo, CIRAD- EMVT, France for kindly providing the full length cdna clones of RPV H, and PPRV HN. We thank Dr Richard W. Compans, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA for his valuable suggestions. SS was a Senior Research Fellow of Council of Scientific Research (CSIR), Government of India. References Aymard-Henry, M., Coleman, M.T., Dowdle, E.R., Laver, W.G., Schild, G.C., Webster, R.G., Influenza virus neuraminidase and neuraminidase-inhibition test procedures. Bull. WHO 48, Cattaneo, R., Rose, J.K., Cell fusion by the envelope glycoproteins of persistent measles viruses which caused lethal human brain disease. J. Virol. 67, Chanock, R.M., McIntosh, K., In: Fields, B.N. (Ed.), Virology, 2nd ed. Raven Press, New York, pp Chen, C., Compans, R.W., Choppin, P.W., Parainfluenza virus surface projections: glycoproteins with haemagglutinin and neuraminidase activities. J. Gen. Virol. 11, Choppin, P.W., Compans, R.W., In: Fraenkel-Conrat, H., Wagner, R.R. (Eds.), Comprehensive Virology, vol. vol. 4. Plenum, New York, pp Choppin, P.W., Scheid, A., The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Rev. Infect. Dis. 2, (Review). Colman, P.M., Varghese, J.N., Laver, W.G., Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 303,

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