key: cord-0004995-a5pc1lb5 authors: Schat, K. A.; Myers, T. J. title: Cultivation of avian rotaviruses in chicken lymphocytes and lymphoblastoid cell lines date: 1987 journal: Arch Virol DOI: 10.1007/bf01310714 sha: 3cb4e0df9f98eb23f6c2f0189f23bbf37006addc doc_id: 4995 cord_uid: a5pc1lb5 Avian rotavirus isolates were used to infect normal chicken spleen cells, lymphoblastoid T cell lines transformed by Marke's disease virus, an avian leukosis virus-transformed B cell line, and a reticuloendotheliosis virus-transformed line, which is a pre-B, pre-T cell line. All five isolates tested were able to infect spleen cells and the three types of lymphoblastoid cell lines, suggesting that avian rotaviruses can infect both B and T cells. Splenic lymphocytes were considerably less susceptible to infection than chick kidney cells. Lymphoblastoid cell lines remained virus-positive during a 10-day culture period. Virus was neutralized by the addition of low dilutions of normal chicken serum and high dilutions of chicken anti-rotavirus serum. The isolation and cultivation of rotaviruses were problematic until the recognition that treatment with pancreatic proteolytic enzymes enhances viral replication in vitro (reviewed in 10). Primary epithelial cells or epithelial cell lines are most often used for in vitro cultivation of mammalian rotaviruses (reviewed in 10) . Similarly, avian rotaviruses are grown in either primary avian kidney- (20) or embryo liver cell (12) cultures or in the epitheliM cell line MA 104 (19) . In vivo studies on the pathogenesis of infection with rotavirus in mammals and birds confirmed that viral replication occurs mostly in the mature epithelial cells of the intestinal villi. Occasionally, macrophages with rotavirus particles have been observed in the lamina propria of rotavirusinfected calves (17) and virus antigens have been demonstrated in scattered lymphocytes in the mesentric lymph nodes of infected piglets (18) There is a paucity of information on replication of rotaviruses in lymphocytes. Only NOZAWA and FONSECA (13) have reported that human rotavirus can replicate in mitogen-stimulated peripheral blood lymphocytes. In this paper we report that several avian rotaviruses can infect both nonstimulated avian splenic lymphocytes and lymphoblastoid cell lines transformed by avian tumor viruses. The following avian rotaviruses were used: chicken isolates Ch-1 and Ch-2, turkey isolates Tu-1 and Tu-2 and the pheasant isolate Ph-I (20) . Virus stocks were prepared from infected chick kidney cell cultures (CKC) between passage levels 5 and 10 and stored at -80 ° C. The use of CKC cultures for rotavirus propagation has been described elsewhere (20) . Short-term spleen cell cultures were prepared from spleens obtained[ from 5-to 8-week-old, specific-pathogen-free chickens as described by CAL~EK et al. (4) . Briefly: spleens were collected aseptically and gently forced through a 60 ~m autoclavable screen (Tetco, Inc., Elmsford, NY). Cells were centrifuged over Ficoll-Paque (Pharmaeia, Inc, Piscataway, NJ). The splenic lymphocytes collected from the interface, were washed twice in PBS and counted. These cells consist mainly of T and B cells, macrophages and natural killer cells. Five × 106 spleen cells were cultured in 1 ml of LM-Hahn (LMH) n~mdium (3) in plastic tubes (=~ 2057, Falcon Plastics, Oxnard, CA) with caps loose at 41 ° C in a humidified atmosphere of 5 percent CO: in air. LMH-medium consists of equM parts of Leibovitz and McCoy medium (both from Gibco, Grand Island, NY) supplemented with 10 percent heatinactivated chicken serum and 8 percent fetal bovine serum (FBS), 5 percent tryptose phosphate broth, 0.0] mM 2-mercaptoethanol, 2 mM glutamine, t m~ sodium pyruvate and antibiotics. The use of chicken serum and FBS is essential for the cultivation of avian lymphoblastoid cells and tbr the short term (48 to 72 hours) cultivation of avian splenic lymphocytes. The following lymphoblastoid cell lines were used: i) Two Marek's disease virus-transformed cell lines: MDCC-CU 2 and CU 36 (2, 3), which are beth I a-expressing T cells (15) . ii) Two avian leukosis virus-transformed cell lines: LSCC-CU l0 (2) and RP-9 (14) , which are B cells, iii) One cell line transformed by reticuloendotheliosis virus: RECC-CU 60, which is a pre-T, pre~B cell line (D. WEI~STOCK, personal communication). Prior to infection of CKC, spleen cells or lymphoblastoid cell lines, the rotaviruses were treated with 5 ~g/ml trypsin for 1 hour at 37 ° C (19) . Lymphoblastoid cell lines were washed twice in PBS, and 1 × 106 cells/ml were resuspended in 0.4 ml PBS containing different concentrations of trypsin-treated rot~virus and incubated ~br 1 hour at 37 ° C. The cells were washed 1 to 6 times, placed in 1 ml of LMH and incubated at 38 ° or 41 ° C. Spleen cell cultures were infected tbllowing the same method, except that 5 x 106 cells per culture were used. CKC cultures, cultivated on glass coverslips were infected as described elsewhere (20) . Convalescent antisera against rotavirus were obtained from SPF-chickens infected with the Ch-2 strain of rotavirus. These sera were conjugated with fluorescein isothiocyanate (20) for immunofluorescence tests and also used in a virus-neutralization assay. Mouse monoclonal IgG anti-chicken IgM (~ chain) (MACIgm) (7), mouse monoclonal anti-chicken I a (MAIa) (11) , mouse monoclonal anti-chicken panlymphocy~ antigen (~LACLA), which detects immature B ceils prior to hatching, some macrophages and circulating T cells (C.-L. CHEN, personal communication) and a mouse monoclonal antibody against circulating T cells (MATs) (A. BENEDICT, personal communication) were used as previously described (16) . l~abbit IgG antimouse Ig (heavy and light chain, Miles-Yeda Ltd, Rehovot, Israel) (l~AM) and goat IgG antirabbit Ig/s conjugated with rhodamine (GARG-TRITC) (Cappel Laboratories, West Chester, PA) were used for indirect fluorescent antibody tests in conjunction with monoclonal antibodies. The numbers of infected cells in the lymphoblastoid and spleen cell cultures were determined as described by CALNEK et al. (4) for MDV-infected lymphocytes. Briefly: The number of live cells in each culture was estimated by counting cells in a hematoc~e counter using trypan blue dye exclusion. Cells were washed once in PBS, resuspended in 0.1 ml of PBS and 10 txl drop smears were air-dried on glass slides and acetone-fixed. Smears were then stained with FITC-conjugated rotavirus antibodies or with the goat antiserum against human rotavirus followed by FITC-conjugated swine anti-sheep IgG. The number of positive cells was enumerated using a Leitz fluorescence microscope with epiillumination. Supernatant fluids were harvested from lymphoblastoid cell line cultures, frozen at -70°C and assayed on CKC cultures for virus titers. Lymphocyte surface markers on rotavirus-infectcd spleen cells were detected following the technique described by CALNEK et al. (5) for MDV-infected lymphocytes. Briefly, spleen cells were washed in modified PBS (MPBS), containing 1 percent bovine serum albumin and 0.1 percent sodium azide, and then sequentially treated with the desired monoclonal antibody, RAM, and GARG-TRITC. Ten ~l drop smears were air-dried, fixed in acetone and stained with the FITC-conjugated chicken antirotaserum. The Leitz microscope was equipped with filters suitable for exciting either conjugate, and by switching from one to the other it was possible to determine whether or not a given virus antigenpositive cell had a given surface marker. Virus-neutralization assays were conducted using the Ch-2 rotavirus strain and heatinactivated sera from rotavirus-infected birds, while sera from SPF birds were used as a negative control. Aliquots of l04 TCIDs0 in 0.25 ml of trypsin-treated virus were mixed with 0.2 ml of appropriate se~-dm dilutions and incubated for 1 hour at 37 ° C. The virus-serum mixture was added to 106 washed and drained MDCC-CU 36 cells, incubated for an additional hour at 37 ° C, washed again and resuspended in LMH medium. Cells were examined by immunofluorescence test for virus replication at 24 hours of incubation. In vitro exposure of different cell lines to the trypsin-treated Ch-2 strain of rotavirus resulted in the establishment of infection in a dose-dependent fashion (Table 1) . Other avian rotavirus isolates were also able to infect lymphobIastoid celt lines (Table 2) . Cells with fluorescent cytoplasmic staining were present in infected but not in control, mock-infected cultures after staining with the rotavirus-speeific conjugate. Likewise, the use of the goat antirotavirus serum in an indirect FA assay showed positive cells in the infected, but not in the control cultures. Specific staining was absent when a normal goat serum, free of antibodies to rotavirus, was used. Splenic lymphocytes exposed to the trypsin-treated Ch-2 strain showed similar specific cytoplasmic staining as the infected lymphoblastoid cell 1 × I06 lymphoblastoid cells were infected with 4 × t06 TCIDs0 as determined by centt~fugation on CKC-eulture b Each culture was centrifuged, rcsuspended in I00 txI and 2 smears were made using 10 pl/smear Table 3 . lines. The number of infected cells was considerably lower in splenic tymphocymes than in the lymphoblastoid cell lines (Table 1 ). An increase in the number of positive cells was noticed at 48 hours compared to 24 hours of incubation at 41 ° C. However, there was no increase when the number of positive cells was expressed as a percent of the total number of cells (Table 3) . Cell lines remained positive for virus during a 10day-culture period, but the percent of virus positive cells decreased markedly aider 4 days (Table 4 ). Supernatant fluids collected from the infected lymphoblastoid cell lines at 8 and 10 days in culture were positive for cell-free virus. Titers were between 103 and 104 TCIDs0 for each cell line. Spleen cell cultures seemed to be less susceptible to infection than the lymphoblastoid cell lines. Spleen cells do not survive very well under these conditions and the percentage of viable cells was only 44, 12 and 12 at 48 hours for the 3 spleen cell preparations in Table 1 . In contrast, the lymphoblastoid cell lines were either stable or increasing in cell numbers at 48 hours (data not shown). The importance of trypsin treatment prior t~) infection and temperature of incubation were examined. Absence of treatment of rotavirus with tr3q~sin decreased the infection rate tenfold but infection could be established in lymphoblast~id cell lines with non-t~psin-treated ~drus. Due to the low level of infection, attempts were not made to infect splenic lymphocytes with nontrypsin-treated virus. Cultures were incubated at 38 and 41 ° C after infection with Ch-2 and smears were made at 24 and 48 hours. There were no differences detectable in infection rates between the two temperatures (data not shown). Spleen cells infected with the Ch-2 strain of rotavirus were examined at 48 hours postinfection for the presence of rotavirus antigen and specific surface markers. The following surface antigens were present on some but not all of the rotavirus-infected spleen cells: I a, ~ and CLA. The actual numbers of cells positive for both rotavirus and surface markers were quite low. Lymphocytes positive for rotavirus and T 3 were not detected. Uninfected spleen cells confirmed the presence of the surface markers. The incubation of trypsin-treated rotavirus with dilutions of SPF serum, free of antibodies against rotavirus, resulted in considerable inhibition of infection of MDCC-CU 36 (Table 5) . A dilution of 1:80 resulted in a reduc- Thus far, only NOZAWA and FONSECA (13) have reported that rotaviruses can replicate in lymphocytes albeit after activation with mitogens. This paper extends their results and describes that avian rotaviruses can certainly infect and perhaps replicate in splenic lymphoeytes in the absence of mitogens, but, it can not be excluded that the low percentage of positive cells were activated cells. The surface markers present on virus-positive spleen cells are compatible with B and T cells (I a, ~x and CLA). The finding that three different types of lymphoblastoid cell lines are susceptible to infection supports the concept that rot~viruses can infect both B and T lymphocytes. The question if cell-free infectious virus is indeed produced in lymphoblastoid cell lines is difficult to answer. The recovery of low levels of infectious virus at 8 and l0 days in culture (Table 4) suggest that it is possible. On the other hand it can not be excluded that some of the original inoeulum survived outside the cells. It is still unresolved how rotavirus enters a cell. Although the presence of receptors has been postulated, there is no conclusive proof for their existence (1). Infection of both CKC (10, 12) and lymphocytes requires activation of the virus by trypsin. However, the establishment of infection in lymphocytes is much less efficient than the infection of CKC: 4 × 10 ~ TCIDs0 in CKC infected between 200 and 1400 lymphoblasteid cells depending on the cell line. It is not clear if these differences are due to quantitative differences in expression of the putative receptors or that the lymphoblastoid cells lack certain enzymes important for viral replication. Clearly, the finding that lymphocytes and lymphoblastoid cells are susceptible to infection will be an important aspect in the search for a rotavirus receptor. The neutralization of rotavirus by normal SPF serum was not expected. The negative serum was obtained from our SPF flock of chickens free of antibodies to rotavirus as determined by indirect immunofluorescence. Sera from the departmental SPF flocks were also tested elsewhere and found to be free of antibodies to rotavirus (Dr. Stewart McNulty, Veterinary Research Laboratories, Belfast, personal communication). Virus neutralizing substances {VNS) have been described in the albumin fraction of FBS interfering with the replication of duck hepatitis virus (6) . LMH medium contains both chicken serum and FBS, buth the lymphoblastoid cells are washed prior to exposure with rotavirus. Apparently, the 1-hour incubation period with the chicken serum allows the VNS to interact with the trypsintreated virus. It will be of interest to determine if the albumin fraction is responsible for the neutralization of the virus. Rotavirus and coronavirus infections in animals. 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