Calf Kidney Cells Research Articles

CHARACTERISTICS OF THE EQUINE RHINOPNEUMONIA VIRUS AK-2011

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks, and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0 lg TCID50/cm3. Sequencing and polymerase chain reaction (PCR) analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPE) induced by the AK-2011 virus stain (dilution 101) in calf trachea and horse kidney cell cultures were stable from the 1st to 10th passages, with biological activity of 5.75-6.00 lg TCID50/cm3. CPE caused by the virus were apparent on days 2-3, further developed intensively, and extended to 60-80% of the cell monolayer on days 5-7. In calf kidney, sheep kidney, green monkey kidney, and bovine kidney cell cultures, the same changes were observed 1-2 days later. The changes were slow, and by day 7-10 CPE extended to no more than 30-50% of ​​the cell monolayer. An attenuated strain AK-2011 of equine rhinopneumonitis virus was obtained. It was considered a candidate for the manufacture of a vaccine for equine rhinopneumonitis. Thus, the attenuated strain AK-2011 of the equine rhinopneumonitis virus was characterized.

ISOLATION AND IDENTIFICATION OF ROTAVIRUS FROM NEONATAL CALVES

Bovine rotavirus was successfully isolated in vero cell line and secondary calf kidney cell culture from faecal samples of newly bom calves affected with diarrhoea. The isolated viruses were cultivated from cell culture produced cytopathogenic effect which consisted of cytoplasmic granulation degeneration and detachment of infected cells from monolayer cell culture. The rotavirus was identified by indirect immunofluorecent test (IFT) and neutralization test (NT) by using reference hyperimmunser.

AK-2011 strain for the development of a vaccine against equine rhinopneumonitis.

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0lg TCID50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 101 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months.

Study of isolate of Infectious rynotracheitis virus identified in the acute course of the disease

Isolation of the virus from biological material from a two-month-old calf with pathology of the respiratory system from a herd with a morbidity rate of 48% was performed. After detection the presence of IRT antigens in the lungs of the dead animal, the pathogen was isolated on a continuous culture of calf kidney cells, where a characteristic cytopathic effect was observed. The genetic material of the bovine herpesvirus type 1 (Bovine herpesvirus-1, BHV-1) was identified by polymerase chain reaction in the test sample. The virus isolate was adapted to continuous cell cultures of calf kidneys, sheep kidney, cow embryo lung and calf trachea, and the most suitable biological system was determined, where adsorption and reproductive properties of the virus were more pronounced. It was found that the highest titer of infectious activity of BHV-1 isolate (6.1 lg TCD50/cm3) was obtained on continuous culture of lung embryonic cells of a cow embryo after its reproduction during three consecutive passages (observation period)

Design and study of immunogenicity of virus vaccine against viral pneumoenteritis of calves

The immunogenicity of a virus vaccine against viral pneumoenteritis in young cattle was studied in laboratory animals. The results of the selection of optimal vaccine strains to study the effects of different inactivants on infectious rhinotracheitis virus, viral diarrhea, parainfluenza-3, respiratory syncytial virus, rotavirus and coronavirus are presented. The accumulation of avirulent vaccine virus strains was carried out using established virological methods on transplanted cell cultures of MDBC (calf kidney cells) and SPEV (fetal pig kidney cells). Theotropine and formalin were used as inactivating agents to develop inactivation regimes for vaccine strains - components of the experimental vaccine. The antigenic activity of attenuated strains of infectious rhinotracheitis virus, viral diarrhea, parainfluenza-3, respiratory syncytial virus, rotavirus and coronavirus on white mice and calves and the level of specific antibodies in the sera of guinea pigs immunized with vaccines using different adjuvants were studied. Avirulent strains of viruses were used in the design of a new virus vaccine against viral pneumoenteritis: infectious rhinotracheitis (IBR-VBF-VHAVM No. 404); diarrhea (VDVBF-VHAVM No. 406); parainfluenza-3 (PG-VBF-VHAVM No. 403); respiratory syncytial virus (RSV-VBF-VHAVM No. 405); rotavirus (RTV-VBF-VHAVM No. 401) and coronavirus (CV-VBFVHAVM No. 407). The selected vaccine strains are non-reactive and induce active production of antiviral antibodies in sufficiently high titres in both laboratory animals (white mice) and farm animals (cattle). The most effective inactivating agents are 0.1% theotropine and 0.2% formalin. Two types of oil-based adjuvants, ISA 15 and ISA 25, were used to select the optimal adjuvants for the design of the virus vaccine. Adjuvant ISA 15 was used at 15% of the antigen quantity, ISA 25 at 25%. The adjuvant IZA 15 at a concentration of 15% is the optimum adjuvant for the preparation of an experimental virus vaccine against viral pneumoenteritis in young cattle.

COMPARATIVE STUDY BETWEEN ROTA VIRUSES ISOLATED FROM DIARRHEATIC INFANT BABIES AND NEWLY BORN CALVES

Rota viruses were isolated in vero continuous cell line from infant babies and newly borne calves affected by diarrhea after treatment in every passage with 10 mg/ml of trypsin and adding 0.5 mg/ml of trypsin in the maintenance media. The isolated viruses induced distinctive and progressive type of cytopatheic effect in infected cells and highest titer was 2x10 6. TCID,50/0.1 for bovine viral isolate. The isolated 50/0.1 for human viral isolate and 2x10 » viruses were identified by indirect fluorescent antibody technique by using reference calf rotavirus antisera. Comparative study were conducted on both human and bovine viral isolate including growth in different cell culture, vero cell line was very sensitive to support growth of both viruses than secondary embryonic calf kidney cell culture, lamb testes and primary embryonic chicken fibroblast cell culture. Both viruses induced morphologically similar kind or plaques in vero cell line but plaqes formed by human isolate were larger in size about 1.5 - 2.5 mm in diameter than those of bovine isolate 1.5 - 2 mm. Cross reactive viral antigens were detected between the isolated viruses in indirect fluorescent antibody technique but not in serum neutralization test by using reference bovine rotavirus antisera.

Ізоляція коронавірусу ВРХ в культурах клітин

One of the most common viruses in the world that causes disease in cattle is the bovine coronavirus (BCoV). This virus is the causative agent of respiratory and gastrointestinal infections in newborn calves, resulting in significant economic losses in both dairy and meat farming. Considering the complex epizootic situation with the coronaviruses in the world and partial antigenic affinity of BCoV with coronaviruses of other species of animals and humans, the isolation of new strains of coronaviruses, their identification and optimization of cultivation conditions becomes extremely important and relevant. The aim of our research was to determine the features of methods of isolation of bovine coronavirus and to select methods for its cultivation in cell culture in order to obtain the virus with the highest titers of infectious activity. Isolation of BCoV was performed in monolayers of MDBK and the primary-trypsinized calf kidney culture cells, using 20 samples collected from calves with clinical signs of respiratory or/and gastrointestinal disease. 16 samples were positive for BCoV by means of Real-Time PCR test. Up to fifth serial passage, only 4 of these isolates presented typical syncytial cytopathic effect. It has been experimentally established that the continious calf kidney cell culture line (MDBK) and the primary-trypsinized calf kidney culture (CK) are suitable for BCoV isolation and accumulation. The infectious titer of bovine coronavirus at the level of the fifth passage in the cultures of MDBK and CK cells reached 5.54 ± 0.16 lg TCD50/ml and 5.59 ± 0.14 lg TCD50/ml, respectively. However, due to the high cost of obtaining primary-trypsinized cell cultures, this isolation method may be unacceptable to most pharmaceutical companies and laboratories. Also after 5 serial passages, the viral material was again examined in Real-Time PCR to confirm the isolation of BCoV - the study of 4 samples with a characteristic syncytial CPE had a positive result in Real-Time PCR. However, of the Real-Time PCR-positive 12 samples, the virus could not be isolated in continuous cell cultures of MDBK and Vero, as well as in primary-trypsinized cattle lung and kidney cell cultures. This fact may indicate the presence of different strains of BCoV circulation in farms in our country. Further research is planned to be focused on optimizing the methods and modes of BCoV strains isolation, as well as to identify and study the cultural properties of new strains of BCoV circulating in Ukraine. We will also continue the study of the obtained viral isolate for the subsequent development of tools for the diagnosis and immunoprophylaxis of coronavirus infection in veterinary medicine.

Isolation of Bovine leukemia virus from cows with persistent lymphocytosis in Iraq

This is the first study to report on the isolation of bovine leukemia virus (BLV) from peripheral blood mononuclear cells of two cross bred cows in Iraq. The cattle were seropositive by ELISA when selected while being surveyed for the detection of BLV. Among six cows, two were cases of persistent lymphocytosis (PL). Cytopathology was characterized by the formation of multinucleated giant cells (syncytia) and cytoplasmic vacuoles. Moreover, the viruses produced clear plaques on the monolayer of the primary fetal calf kidney (FCK) cells. Inhibition of plaque formation by BLV-antisera suggested a diagnosis of BLV, which was further confirmed by PCR. Cells infected with the isolates were positive to a monoclonal antibody against the viral gp51 trans-membrane glycoprotein by immunocytochemistry. Both isolates replicated and induced cytopathic effects in bovine and human cell line cultures. Phylogenetic analysis based on partial gp51 env gene sequences revealed that Iraqi strain highly homogenous with Turkey strain (100%) and had 1% distance value with other world strains. In conclusion, this present study found that BLV-infected cattle with PL can be a source for viral isolation, and the cytopathological features of the virus infection are arranged and differ depending on the cell type. This is the first study to report on the isolation of the EBL virus in Iraq, and it provides the basis for further studies about a BLV Iraqi strain that can help control this disease.

Визначення цитотоксичності антимікробних препаратів і дезінфектантів на культурі клітин нирки теляти

Goal. To study the cytotoxicity of certain antibacterial preparations used in the treatment of infectious diseases of the urinary system, as well as some disinfectants for calf kidney cell culture. Methods. Cytological, toxic effects test on primary calf kidney cell culture. Determination of the maximum permissible concentrations of antibiotics, antiseptics, and disinfectants that do not yet lead to degeneration of the cell monolayer of calf kidney cells. Results. Tests of some drugs that are often used in veterinary medicine show that the least toxic to calf kidney cells of all tested antibacterial agents are Oxymicol 20 (active substance — oxytetracycline base), Norsulfazole sodium, and Farmazin 50 (active substance — tylosin). Conclusions. These drugs or similar with the same active substances should be used in the treatment of infectious diseases of the kidney and urinary system of cattle and other animals. The possibility of using the method of cultivation and use as test objects of primary cultures of eukaryotic cells in the selection of the optimal composition of therapeutic and prophylactic drugs or disinfectants for veterinary medicine. The obtained results of cytotoxicity can help veterinarians in choosing antimicrobials and disinfectants in terms of their wide range on the market.

CULTURAL PROPERTIES OF BOVINE RESPIRATORY SYNCYTIAL VIRUS STRAIN AB1908

Cattle respiratory diseases are some of the most spread pathologies that can cause economic damage, resulting from fi nancial losses and costs of treatment and diagnostics. One of the major factors contributing to respiratory pathology development is bovine respiratory syncytial infection. The analysis of serological testing, performed by the FGBI “ARRIAH” Reference Laboratory for Cattle Diseases in 2017–2018, showed that respiratory syncytial virus seroprevalence in animals of dairy farms is 60%. Herewith, it was noted that the most susceptible animals to this infection are calves under one year of age. The eff ectiveness of bovine respiratory syncytial infection control measures depends on timely diagnosis; that is why reliable and accurate diagnostic tools are needed, including optimal techniques of virus isolation from pathological material. For successful virus isolation from clinical samples, it is necessary to adhere strictly to optimal parameters of this agent cultivation. This paper presents data on study of bovine respiratory syncytial virus strain AB 1908 cultural properties. The tests performed showed that a continuous bovine turbinate (BT) cell line, continuous bovine fetal trachea (FBT) cell line and continuous bovine calf kidney (RBT) cell line are sensitive for cultivation of this agent and can be used to prepare viral suspension, needed for further research. Virus titre in BT cell culture was 4.33 ± 0.16 – 4.66 ± 0.12 lg TCID50/ cm3, in RBT cell culture – 4.33 ± 0.33 – 4.7 ± 0.36 lg TCID50/cm3 and in FBT cell culture – 4.13 ± 0.20 – 4.78 ± 0.17 lg TCID50/cm3. The following virus cultivation optimal parameters were also determined during this study: the age of the culture for virus inoculation should be 1–2 days and multiplicity of inoculation should be 0.1 TCID50/cell.

Short communication. First report in Cuba of bovine coronavirus detection in a winter dysentery outbreak

Bovine coronavirus (BCoV) infection causes epidemics of acute diarrhea in calves and winter dysentery (WD) in adult cattle. The disease in adult cattle can cause a decrease in milk production resulting in serious economic losses. In Cuba, BCoV infections have not been previously reported. During 2004, many outbreaks of enteric disease have occurred in adult cattle from thirteen dairy farms in the central and western part of the island. The clinical features of the outbreaks resembled those of WD, such as decrease in milk production and, 24 hours later, diarrhea, sometimes bloody, that lasted until the animals recovered in about 7-15 days. Laboratory confirmation of BCoV infection was provided by hemagglutination test (HAT) in 24 samples from four dairy farms from three provinces; RT-PCR assays confirmed the presence of BCoV in three of these samples. Cell culture isolation in secondary calf kidney cells was obtained from four pools of fecal diarrheic samples from each dairy farm. It was remarkable that the disease was also observed during summer. Studies of molecular characterization of the viral strain are in progress.

A mutant of Asia 1 serotype of Foot-and-mouth disease virus with the deletion of an important antigenic epitope in the 3A protein

Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed animals. The availability of a vaccine for differentiating infected from vaccinated animals (DIVA) is crucial for the control and eradication of Foot-and-mouth disease virus (FMDV). Because traditional inactivated vaccines may contain trace nonstructural proteins interfering with the DIVA, we hypothesized that mutant FMDV with deletion of immunodominant epitopes may be valuable. Our previous study has generated a full-length cDNA clone (pBSAs) of FMDV serotype Asia 1 isolated in China. In this study, a B-cell epitope was identified in the 3A region of a nonstructural protein (NSP) by anti-FMDV cattle sera. Furthermore, we generated recombinant FMDV (rvAs-3A14D) by selectively deleting 14 amino acids (position 91-104) in the 3A region of the NSP. Following in vitro transcription and transfection in BHK-21 cells, we successfully rescued the rvAs-3A14D from BHK-21 cells. Characterization of the rvAs-3A14D revealed that the infectivity, antigenicity, and replication kinetics in BHK-21 cells and virulence in mice of the rvAs-3A14D were similar to that of its parent virus. Notably, the mutant rvAs-3A14D only replicated well in BHK-21 but did poorly in primary calf kidney cells. These data suggest that the recombinant FMDV with deletion of this epitope in the NSP may be potentially used as a candidate inactivated vaccine. Therefore, the application of the marker vaccine and differential diagnostic tests may open a promising new avenue for the development of a vaccine for DIVA.

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

Foot-and-mouth disease virus (FMDV) serotype Asia 1 is one of the most predominant endemic serotypes in China. Our previous study has generated a full-length cDNA clone (pBSAs) of an Asia 1 serotype FMDV (As1/CHA/05) isolated from bovine. To further study the properties of this virus, a mutant in the 3A region of the cDNA clone (pBSAs-3A10D), containing the deletion at position 93-102 of the 3A protein of As1/CHA/05, was generated by PCR and cloning. After synthesis of RNA in vitro and transfection, the recombinant rvAs-3A10D virus was recovered from BHK-21 cells. Characterization of the rvAs-3A10D revealed that the infectivity, immunoreactivity, and replication kinetics in BHK-21 and PK-15 cells and virulence in mice of the rvAs-3A10D were similar to that of its parent virus. Notably, while wild-type and recombinant viruses containing full-length sequence of the 3A replicated well in primary calf kidney cells, the mutant rvAs-3A10D failed to replicate in primary calf kidney cells in vitro. Apparently, the full-length sequence of 3A in As1/CHA/05 is a necessary component for its replication in calf kidney cells. The availability of this 3A deletion infectious cDNA clone may help in further investigating the virulent determinants of FMDV and potentially developing FMDV vaccines.

Observations on lumpy skin disease in local Egyptian cows with emphasis on its impact on ovarian function

The present study monitored reproductive and health aspects of local cows in Egypt following natural infection with Lumpy skin disease virus (LSDV). The study was carried out on 640 mature Egyptian native cows reared at Lower Egypt. Cows were clinically examined and genital organs were scanned by ultrasonography and ovarian function was recorded. Blood samples were collected for virus isolation and propagation on calf Kidney cells, electron microscopy (EM) and for PCR assay. Moreover, progesterone level (ELISA), protein electrophoretic pattern (Polyaccrylamide Gel Electrophoresis), immunoglobulin concentrations (Radial Immune Diffusion) and some trace element values (Atomic Absorption Spectrophotometry) were determined in the serum. Results indicated that LSDV had typical cytopathic effect and plaque. EM showed that size of LSDV size ranges from 300 – 350 nm with crescent or ovoid shape. PCR assay indicated the presence of a band at 192 bp which belonged to viral attachment protein encoding gene. Epidemiological studies revealed that 25.47% of the examined cows were positive for LSDV with obvious year, age and seasonal variations. LSDV infected cows showed fever, become completely of food with skin nodules. Serum progesterone level was low (P < 0.01) during both follicular and luteal phases of the estrous cycle and 92.64% of infected cows had inactive ovaries. Infected cows showed obvious (P < 0.01) low albumin and high g globulin values as well as low concentrations of copper and iron (P < 0.01) in their serum as compared to non infected cow-mates. It was concluded that PCR is a useful method for rapid diagnosis of LSDV. Cows infected with LSDV mostly suffering from cessation of ovarian activity mainly due to poor body condition. Key words: LSDV, Cow, Ovary, PCR, infection, immunoglobulin.

Reaction Pattern in Vertebrate Cells (RiV): May the anti‐RiV ELISA Detect a Pathologic Cell Proliferation? (First Results)

AbstractRiV is mainly represented by characteristic exosome‐like particles with a diameter of 30 to 70 nm. RiV particle preparations (RiV‐PP) contain identified proteins. In the present paper, the authors started an evaluation of the hypothesis, that RiV‐reactive antibodies, detected in patient sera by an indirect ELISA, may be used for the diagnosis of pathologic cell proliferations. On the basis of the frequency distribution curve of about 500 blood donor sera, cut‐off values for RiV‐reactive IgG and IgM antibodies could be defined. Additionally it was found that 1.6 % of blood donor sera reacted strongly with an RiV‐PP (of calf kidney cells) for IgG antibodies. 29 % of cancer patient sera (n = 48), 17 % of 53 patients with endoprothetics and with lung disorders, 44.3 % of 115 patient sera of a doctor's practice and 28 % of 97 sera of 10 to 11 years old schoolchildren were positive in the anti‐RiV ELISA with IgG antibodies. About half of the selected group of schoolchildren contained different autoantibodies and some of these children displayed already insulin dependent diabetes mellitus (as shown by others). A correlation of our ELISA results with the other autoimmune antibody results was not found. Using RiV‐PP as an antigen complex, isolated from calf kidney cell cultures or from human FL cell cultures and HeLa cell cultures, results with small differences between human and animal RiV origin were obtained. Using the human RiV, the number of positive sera was not significantly lower. Generally, the anti‐RiV ELISA, using RiV‐PP from human cell cultures, detect autoantibodies against RiV‐specific proteins. This assay may detect, but not diagnose pathologic cell proliferation. The possibilities of RiV ELISA for diagnostics of defined autoimmune diseases, as well as further studies and the more meaningful RiV immunoblot are discussed.

RiV Particles are Heat Stable

The “reaction pattern in vertebrate cells” (RiV) is mainly represented by characteristic particles with a diameter of 50 ± 15 nm and smaller RiV-specific units probably as reaction products of the cells to specific stress. As vaccines they have been successfully applied against laryngeal papillomatosis and experimentally against foot-and-mouth disease of guinea pigs and mammary carcinoma of mice. Subsequently, the small-scale treatment of AIDS, cancer and hepatitis C was started in a single doctor's practice. RiV samples of calf kidney cell culture contain identified proteins and non-viral ribonucleic acid. Vaccine samples and samples for heat stability studies were produced from roller bottle cultivated cells after differential centrifugation and a short solvent treatment. The preparations were examined by electron microscopy, ELISA, protein determination, SDS-PAGE, immunoblot and UV spectroscopy. Since the efficacy of heated RiV (10 min at 100 °C) against the growth of balb/c mouse myeloma was higher than that of the non-heated sample, heated RiV samples were investigated by electron microscopy, immunoelectron microscopy (IEM) and ELISA to find a possible explanation for these results. The RiV particles were surprisingly heat-stable, after 10 (up to 60) min of boiling no differences were observed in morphology and diameter in comparison to the untreated sample. Furthermore, no difference was seen by IEM in the reaction of these RiV particles with an anti-RiV antiserum relative to the untreated sample. When two samples were compared by ELISA using the same antiserum, the reactivity of the heated samples decreased quickly during the first minutes of boiling, but the decrease became much slower later on. In conclusion, there seemed to be heat-sensitive as well as heat-stable reactive components within the RiV samples. The heat-stable component(s) should be associated with the RiV particles. Further studies are necessary to find out whether the positive results of the mouse myeloma experiment could also be obtained following the treatment of other cancers, AIDS and hepatitis C and also to answer the question, which of the known proteins are important for the therapeutic activities of RiV samples.

Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture.

Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR(136)) and FCS-2 (RAR/KR(109)). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR(109), RANN(109) or SANN(109), respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR(109) or the RANN(109) motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN(109) mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

Theileria annulata schizonts found in calf kidney cell culture.

Mononuclear cells infected with Theileria annulata schizonts were isolated at 4 different times from monolayer cultures of primary kidney cells derived from apparently healthy, 2 to 6 weeks old zebu calves. The presence of such an organism could interfere with the manufacturing procedure of rinderpest tissue culture vaccine if it is to be carried out in calf kidney cell culture.

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.

Detection of Mycoplasma Contamination in Primary Calf Kidney Cell Cultures

A total of 31 primary cell culture preparations of calf kidney including their processing stages (kidney, washing fluid, cell suspension, cell culture monolayer) were investigated for mycoplasmas by cultural methods. Mycoplasma (M.) arginini, was isolated from 8 out of 27 investigated samples of calf kidney from 3 out of 26 washing fluids, 5 out of 20 cell suspensions, and from 9 out of 21 cell culture monolayers. Furthermore, M. arginini was repeatedly found in throat swabs of the cell laboratory technicians. The results give indications as to the source and route of mycoplasma infections of primary cell cultures.