Revista de Salud Animal
versión ISSN 0253-570X
Rev Salud Anim. v.29 n.1 La Habana ene.-abr. 2007
MULTIPLICATION OF A TRANSMISSIBLE GASTROENTERITIS VIRUS CUBAN ISOLATE IN DIFFERENT CELL LINES
MULTIPLICACIÓN DE UN AISLAMIENTO CUBANO DEL VIRUS DE LA GASTROENTERITIS TRANSMISIBLE EN DIFERENTES LÍNEAS CELULARES
Edisleidy Rodríguez*, A. Betancourt** and Maritza Barreras*
* Grupo de Virología Animal, Centro Nacional de Sanidad Agropecuaria (CENSA), Apartado 10, San José de las Lajas, La Habana, Cuba. E-mail: firstname.lastname@example.org; ** Facultad de Medicina Veterinaria, Universidad Agraria de La Habana (UNAH), Cuba
In this study we report a wide range of cells from different species, in which a Cuban TGE isolate multiplies. Both primary cultures and continuous cell lines from different species were used. We found out that 11 from the 13 kinds of cultures assessed were sensitive to the Cuban isolate, which not only showed an expected affinity to cells from porcine origin, but also it was able to replicate and produce a cytopathic effect in cells from bovine, hamster, monkey and quail origin.
Key words: transmissible gastroenteritis virus; porcine coronavirus; TGE; cellular multiplication; cytopathic effect.
En esta investigación reportamos un amplio rango de células de diferentes especies donde se multiplica un aislado cubano del virus de TGE, para lo cual se utilizaron tanto cultivos primarios como líneas celulares continuas, originadas de distintas especies animales. Se determinó que 11 de los 13 tipos de cultivos evaluados fueron susceptibles a la multiplicación del aislado cubano, el cual no mostró afinidad única por las células de origen porcino, ya que fue capaz de multiplicarse y producir efecto citopático en células de bovino, hámster, mono y codorniz.
Palabras clave: virus de la gastroenteritis transmisible del cerdo; coronavirus porcino; TGE; multiplicación celular; efecto citopático.
Transmissible gastroenteritis of pigs (TGE) is a highly contagious disease, caused by a virus of the Coronaviridae family, Coronavirus genus (8). This disease is characterized by profuse diarrhoeas and vomiting (9), besides high mortality and lethality in suckling piglets (19, 13). Due to the serious losses it provokes in the pig industry, this disease is included in the list of disease of obligatory declaration of the World Organization for Animal Health (OIE) (11).
Coronavirus, as many animal viruses, is characterized by a restricted host range and tissue tropism (5, 6). Enjuanes and Cavanagh (7) report the cell lines: ST, PK 15 and LLC-PK1 (all from pig origin) to be permissible for TGE virus multiplication.
However, there are some reports of multiplication of this virus in canine and feline tissues (2).
Although some viruses are able to be multiplied in cells of a wide range of animal species, many of them are restricted to infect cells of their natural host species, since they require the presence of specific receptors for the virus attachments and penetration into the cells. TGEv entry into the host cells is mediated by the S glycoprotein through interactions with porcine aminopeptidase N (pAPN), which is the cellular receptor (15, 10).
APN, also called CD13, is a 150- to 160- Kda type II glycoprotein that is a membrane peptidase, which is expressed in cell surface of epithelial cells of kidney, intestine and respiratory tract; granulocytes, monocytes, fibroblasts, endothelial cells, cerebral pericytes, at the blood-brain barrier and synaptic membranes in the Central Nervous System (CNS) (21).
In Cuba, TGE outbreaks with an epizootic presentation were reported (18), from which a virus isolate was obtained and cloned by plaque assay that is being currently characterized. Here we report the ability of this isolate to multiply in a wide range of cell lines of both mammal and bird origin.
MATERIALS AND METHODS
Virus: The 266c Cuban isolate of TGEv used throughout this study was obtained from piglets experimentally infected with homogenised intestinal tissue of sick animals (1) and cloned by plaque assay. Stock virus was passage 3 times in swine kidney cells (SK6) after cloning, with an infective titter of 106.5 infective doses in tissue cultures (IDTC50/ 0.1 ml).
Cells: We used both primary cultures and continuous cell lines from different species (Table 1).
Cell inoculation: 200 ìl of the viral stock were inoculated into each cell monolayer (previously washed with PBS) and 300 ìl of DMEM plus treated with 10 ìg/ml trypsin and 20 mM Hepes (12) (DMEM HT). After incubation at 37ºC for 1 h, the cell sheets were overlaid with DMEM without trypsin.
Viral Neutralization: BHK-21 cell line was used for neutralizing the cytopathic effect with three monoclonal antibodies: 1DB12 (INGENASA, S.A, Spain), TGE25 c9.3c and TGE45 a8.f10.c6 (gently donated by the Dr. Osorio, University of Nebraska), all of them, against the S protein of the TGEv.
RESULTS AND DISCUSSION
With the exception of Fibroblast of Chicken Embryo (FCE) and MDCK cell line, the rest of the cells showed susceptibility to the multiplication of the virus evaluated (Table 2). The cytopathic effect found was characterized by rounded refringent cells, syncytia, detachment and lysis, plus final monolayer destruction in complete agreements with previous reports by Enjuanes and Cavanagh (7), who described the effect associated to this virus in swine cells. Uninfected cells were included which showed no effect.
Cowen and Braune (3) assessed the susceptibility of the QT-35 cell line regarding the multiplication of TGE virus. In this case no effect was found, thus showing that this cell line was not sensitive to the virus. However, our Cuban isolate did multiply in this cell line reaching the total destruction of the monolayer at 24 hours post-infection (pi) (Table 2).
Delmas et al. (4) accomplished the characterization of a surface molecule used by the virus to bind and enter the host cell. They report that aminopeptidase N acts as a membrane receptor suitable for viral attachment and penetration. These authors carried out recombinant aminopeptidase N expession in cells that under normal circumstances are not sensitive to TGEv multiplication. They used BHK-21 and MDBK cell lines, which have also been shown to be sensitive to the Cuban isolate multiplication, producing a complete cytophatic effect at 24 hours pi (Table 2, Figure 1).
Benbacer et al. (2) developed a similar study with BHK-21 and MDCK cell lines. Viral multiplication was attained once transfected cell expressed the specific receptor on their surface (aminopeptidase N). They report MDCK cell line as nonpermissive, in agreement with our results (Table 2). Nonetheless, even though we achieved viral identification before inoculating the different cell cultures, a viral neutralization assay was performed in BHK-21 cells with 3 monoclonal antibodies specifically reacting with viral protein S, wich showed a complete neutralization.
Coronaviruses attach to the host cells through the spike (S) glycoprotein. TGEv entry into the cells is also mediated by the S glycoprotein through interactions with porcine aminopeptidase N (pAPN), which is the cellular receptor. Aminopeptidase N also serves as the receptor for human, canine and feline coronaviruses (16, 17). These viruses have shown marked species specificity in receptor utilization, as Human Coronavirus (HCV-229E) can utilize human but not porcine APN, while TGEv can utilize porcine but not human APN. Thus, receptor specificity appears to be important determinant of the species specificity of HCV-229E and TGEv infection (20). Interestingly, while porcine and human aminopeptidases showed species specificity, the feline aminopeptidase (fAPN) seems to serve as a receptor for feline, canine, porcine and human coronaviruses (14).
The ability of nonfeline group coronaviruses to use fAPN as a receptor has important implications for the evolution of this group of viruses. These RNA viruses can undergo rapid evolution by recombiation. Possibly recombination between different coronaviruses could ocurr in a cat that simultaneously infected with feline coronavirus (FeCV) or feline infectious peritonitis virus (FIPV) and another coronavirus in group I. Recombinants between different coronaviruses could have properties different from either parents and might show altered host range, tissue tropism, antigenicity, and/ or virulence, possibly resulting in emergence of a new disease (20).
Genetic alterations in either the virus or host cells can change the dynamics of virus-cell interaction. Retrospective studies showed that a domain of the spike protein encoded by S gene nucleotides (nt) 1518 to 2184 is efficiently recognized by pAPN (cell receptor), and transfection of pAPN to nonpermissive cells makes them susceptible to TGEv. In this study, they report that baculovirus-expressed polypeptides corresponding to amino-acids 522 to 744 of the spike protein were able to efficiently recognize pAPN (14).
According to the results obtained in this research, our isolate behaves in a different way as compared to previosly analyzed TGEv strains. This suggests possible changes at the genomic level, specifically in the region coding to the binding site virus-cell receptor. Therefore future studies will be aimed to accomplish a molecular study of the gene coding glycoprotein S.
1. Barrera Maritza, Díaz de Arce Heidy, Acevedo Ana María, Cuello Sandra, Rodríguez Edisleidy, Vega A et al Transmissible gastroenteritis in Cuba: experimental reproduction of the disease and molecular characterization of the virus. Spanish J Agric Res. 2005;3(3):305-312.
2. Benbacer L, Kut E, Besnardeau L, Laude H, Delmas B. Interspecies Aminopeptidase- N Chimeras Reveal Species-Specific Receptor Recognition by Canine Coronavirus, Feline Infectious Peritonitis Virus and Transmissible Gastroenteritis Virus. J Virol. 1997;71(1):734-737.
3. Cowen BS, Braune MO. The propagation of Avian Viruses in a continuous Cell Line (QT-35) of Japanese Quail Origin. Avian Dis. 1988;32:282-297.
4. Delmas B, Gelfi J, Sjostrom H, Noren O, Laude,H. Further characterization of aminopeptidase N as a receptor for coronaviruses. Adv Exp Med Biol. 1993;342:293-298.
5. Delmas B, Gelfi J, Vogel RL, Norén HS, Laude H. Aminopeptidase N is a major receptor for the enteropathogenic coronavirus TGEV. Nature. 1992;357:417-420.
6. Enjuanes L, Van Der Zeijst Molecular basis of transmissible gastroenteritis virus epidemiology. In, The Coronaviridae, edited by Stuart G. siddell, Plenum Press, N.Y. 1995.
7. Enjuanes L, Cavanagh D. Coronaviridae. In, "The Springer Index of Viruses". Eds. CA Tidona, G Darai, 2002;pp 272-280 University of Heidelberg, Alemania.
8. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Virus Taxonomy. Eighth Report of the International Committee on Taxonomy of Viruses. (eds). Academic Press, 2004;1162 pp.
9. Knipe DM, Howley PM. Fundamental Virology. Fourth Edition. Edited by Lippincott Williams & Wilkins. 2001;1395p.
10.Oh JS, Dae Sub Song DS, Yang JS, Song JY, Yoo HS, Jang YS et al Effect of soluble porcine aminopeptidase N on antibody production against porcine epidemic diarrhea virus. J Vet Sci. 2004;5(4):353-357.
11.OIE Animal Diseases Data. Diseases Notifiable to the OIE.2005 [On line]. Available in: http://www.oie.int/eng/maladies/en_classification.htm
12.Paton D, Ibata G, Sands J, Mcgoldrick A. Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus. J Virol Methods. 1997;66:303-309.
13.Rodák L, míd B, Nevoránková Z, Valíèek L, Smítalová R. Use of Monoclonal Antibodies in Blocking ELISA Detection of Transmissible Gastroenteritis Virus in Faeces of Piglets. J Vet Med Series B 52 2005(3),105-111.
14.Sanchez CM, Izeta A, Sánchez JM, Alonso S, Sola I, Balasch M. Targeted Recombination Demonstrates that the Spike Gene of Transmissible Gastroenteritis Coronavirus is a Determinant of Its Enteric Tropism and Virulence. J Virol. 1999;73(9):7607-7618.
15.Schneider-Schaulies J. Cellular receptors for viruses: links to tropism and pathogenesis. J Gen Virol. 2000;81: 1413-1429.
16.Schwegmann-Wessels C, Zimmer G, Laude H, Enjuanes L, Herrler G. Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins. J Virol. 2002;76:6037-6043.
17.Schwegmann-Wessels C, Zimmer G, Schröder B, Breves G, Herrler G. Binding of Transmissible Gastroenteritis Coronavirus to brush Border Membrane Sialoglycoproteins. J Virol. 2003;77(21):11846-11848.
18.Serrano DF. Transmissible gastroenteritis in Cuba. [en línea Mayo del 2003] Disponible en: [http://www.oie.int/eng/info/hebdo/ais_20.htm#Sec4] Disease Information. 16 (19). [Consulta: Marzo 22 del 2004].
19.Sestak K, Saif LJ. Porcine coronavirus. In: Trends in emerging viral infection of swine. Iowa State Press, Ames.2002;Pp. 321-330.
20.Tresnan DB, Levis R, Holmes KV. Feline Aminopeptidase N serves as a receptor for feline, canine, porcine and human coronaviruses in serogroup I. J Virol. 1996;70(12):8669-8674.
21.Wentworth D, Holmes K. Molecular Determinants of species Specificity in the Coronavirus Receptor Aminopeptidase N (CD13): Influence of N-linked Glycosylation. J Virol. 2001;75(20):9741-9752.
(Recibido 31-10-2005; Aceptado 20-8-2006)