Primary Porcine Kidney Cells Research Articles

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs.

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells

Porcine circovirus type 3 (PCV3) was first detected in aborted fetuses in 2015 when sows displaying clinical signs that looked like porcine dermatitis and nephropathy syndrome died suddenly. Primary porcine kidney cells were selected for both the isolation and propagation of PCV3 strain SNUVR181115 (GenBank accession number MK503331) as these cells were permissive to PCV3 infection. PCV3 did not produce cytopathic effect on infected monolayers, therefore PCV3 infection was confirmed by in situ hybridization with a PCV3 specific DNA probe. Electron microscopy was used to analyze cell culture for the presence of virus. The intracytoplasmic inclusion bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell. Virus replication peaked at 6th passage yielding titers close to 106 genomic copies of PCV3 per mL. PCV3 strain SNUVR181115 isolated from primary porcine kidney cells was highly conservative and was clustered with the Korean and Chinese strains. These results demonstrated that primary porcine kidney cells are useful for PCV3 isolation and replication.

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells.

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Sequences outside that of residues 93–102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. During 2010 and 2011, there was an epidemic of the Mya-98 lineage of the Southeast Asia (SEA) topotype in East Asia, including China. Changes in the FMDV 3A protein have been previously reported to be associated with the inability of FMDV to grow in bovine cells and cause disease in cattle. In this paper, we report the generation of a full-length infectious cDNA clone of FMDV O/SEA/Mya-98 strain O/GZSB/2011 for the first time along with two genetically modified viruses with deletion at positions 93–102 and 133–143 in 3A based on the established infectious clone. All the recombinant viruses grew well and displayed growth properties and plaque phenotypes similar to those of the parental virus in baby hamster kidney (BHK-21) cells, porcine kidney (PK-15) cells, and primary fetal porcine kidney (FPK) cells. While the recombinant viruses rvGZSB and rvSBΔ133–143 exhibited similar growth properties and plaque phenotypes with the parental virus in primary fetal bovine kidney (FBK) cells, the recombinant virus rvSBΔ93–102, containing deletion at positions 93–102 in 3A, grew at a slower rate and had a smaller plaque size phenotype in FBK cells than that of the parental virus. Therefore, the results suggest that the deletion at positions 93–102 of 3A protein does not affect FMDV replication efficiency in BHK-21, PK-15 and FPK cells, but affects virus replication efficiency in FBK cells, although, cannot alone account for the inability to replicate in bovine cells.

Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro: an initial characterization

BackgroundA good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney.ResultsWe carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics.ConclusionsOn the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells.

322 POTENTIAL OF PRIMARY PORCINE KIDNEY CELLS FOR THE GENERATION OF TRANSGENIC PIGS VIA SOMATIC CELL NUCLEAR TRANSFER

The generation of genetically tailored pig models for biomedical research using somatic cell nuclear transfer (SCNT) is an efficient and precise approach, whereas the outcome is crucially dependent on the source of nuclear donor cells. Especially for site-directed mutagenesis by homologous recombination, including the generation of single cell clones, the demands on the target cells are high. Different primary cells used for SCNT have been tested for their efficiency in SCNT experiments, but further characterisation of the specific cell types, their morphology, proliferation, lifespan, and stability of karyotype is mostly lacking. We have evaluated the potential of 2 primary porcine kidney cell lines (PKC) isolated from juvenile pigs by a simple collagenase digestion and culture in collagen-coated dishes as cell source for SCNT, including their morphology, proliferation capacity, transfection efficiency, and capacity to support full-term development of SCNT embryos after additive gene transfer or homologous recombination. Single cell clones generated by subcloning of PKC at passage 3 showed different morphologies, proliferation rates, and lifespan, indicating that PKC culture is a mixed population of different types of fibroblasts and/or other cells types. The PKC could be maintained in culture for up to 71 passages without signs of senescence and decreased proliferation, exhibiting a stable karyotype containing 74% normal chromosome numbers (2N = 38) determined from metaphase spreads. In contrast, porcine fetal fibroblasts (PFF) and porcine ear fibroblasts (PEF) could be not be passaged more than 20 times. The calculation of growth curves at passage 4 to 5 showed that PKC exhibited a higher proliferation rate with a population doubling time of 16.6 to 18.4 h compared with PFF (23.2. h) and PEF (32.9 h). Furthermore the determination of the developmental competence after SCNT using PKC at passage 4 in 3 independent experiments and in vitro cultivation for 7 days resulted in a higher blastocyst rate (21%) compared with that in PFF (9.1%) and PEF (4.3%). The comparison of different transfection methods (lipofection, nanofection, conventional electroporation, nucleofection), using an expression vector for green fluorescent protein (GFP), showed that the NucleofectorTM technology gave the best results with transfection efficiencies of 70 to 89%, high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of stress. So far, around 150 cloned piglets using 18 different gene constructs have been produced using stable transfected PKC after additive gene transfer and targeting of 3 different loci. These findings demonstrate that among the 3 tested types of donor cells, PKC, PFF, PEF, primary PKC have outstanding potential for the production of genetically modified pigs by SCNT. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.

Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

BackgroundSomatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination.ResultsPKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT.ConclusionThe high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

192 INDUCIBLE TRANSGENE EXPRESSION IN PIGS

Genetically modified pigs are important models for biomedical research. For certain applications, tightly controlled transgene expression is mandatory. For instance, expression of soluble receptor activator of the NFκB ligand (RANKL) is an established principle for inducing clinically relevant osteoporosis (Mizuno et al. 2002 J. Bone Miner. Metab. 20, 337–344). However, constitutive overexpression of RANKL is lethal; thus, an inducible system is required. A second example is the expression of porcine CTLA-4Ig to prevent co-stimulation of T-cells in pig-to-primate xenotransplantation. Because constitutive expression of porcine CTLA-4Ig causes immunosuppression in pigs (Phelps et al. 2009 Xenotransplantation 16, 477–485), an inducible expression system is desirable. The Tet-On system is used most widely. It is based on a tetracycline transactivator (TA) and a transactivator response element (TARE), which drives expression of the coding sequence for the transgene product of choice. Association of doxycyclin with TA allows binding to TARE, resulting in activation of transgene expression. Although widely used in mice, this system has not yet been established in pigs. Because consistently high expression of TA is required for a functional system, we first cloned an expression vector for TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). A floxed neomycin resistance cassette was linked to this vector to facilitate selection of stable clones after transfection of primary porcine kidney cells. The latter were used for nuclear transfer, and cloned embryos were transferred to synchronized recipient gilts, resulting in the birth of 5 CAG-TA transgenic piglets. A spectrum of organs and tissues was investigated by RT-PCR and Western blot for TA mRNA and protein expression. Primary kidney cells from all 5 TA transgenic piglets were transfected with TARE-RANKL and TARE-CTLA-4Ig constructs linked with a blasticidin resistance cassette to facilitate selection of double transgenic/transfected cell clones. These cell clones were individually stimulated with doxycyclin to identify cell clones with low background and high inducibility of expression of RANKL or CTLA-4Ig. For both combinations, several clones meeting these criteria were identified. Such cell clones were used for nuclear transfer to produce double-transgenic pigs. Application of doxycyclin (25 to 50 mg kg–1 of body weight/day) to a TA/TARE-RANKL transgenic pig resulted in a marked increase in plasma RANKL concentrations. In addition, 4 TA/TARE-CTLA-4Ig transgenic piglets have been born, which will undergo doxycyclin stimulation experiments in the near future. In summary, CAG-TA transgenic pigs serve as a reliable basis for inducible transgene expression, as shown in the present study for RANKL and CTLA-4Ig. Studies to show the biological functionality of the transgene are currently underway. Supported by DFG (FOR793) and BFS (FORZebRA).

Infectivity of Porcine Circovirus 1 and Circovirus 2 in Primary Porcine Hepatocyte and Kidney Cell Cultures

Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome.

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein.

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.

Characterization of the Recombinant Proteins of Porcine Circovirus Type2 Field Isolate Expressed in the Baculovirus System

Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

Detection of early infection of swine vesicular disease virus in porcine cells and skin sections. A comparison of immunohistochemistry and in-situ hybridization

Sensitive methods are required to study the early pathogenesis of swine vesicular diseases (SVD). Therefore, two new methods, immunohistochemistry (IHC) and in-situ hybridization (ISH), were developed and tested for their specificity and sensitivity. With these methods the SVD virus (SVDV) infection in cytospins of primary porcine kidney cells and in frozen skin sections was investigated. Both IHC and the ISH showed a specific cytoplasmic staining, but the IHC detected more infected cells than the ISH. Furthermore, both IHC and ISH were able to detect SVDV in skin sections 4.5 h after infection. It is concluded that IHC is the most suitable and simplest method to identify cells and tissues that support the initial replication of swine vesicular disease virus. However, IHC can only be applied to frozen sections, whereas ISH can also be used in paraformaldehyde-fixed tissues.

Isolation of caliciviruses from skunks that are antigenically and genotypically related to San Miguel sea lion virus

Caliciviruses were isolated from feces of skunks imported from the north central United States to Canada. Virus isolation was accomplished using adenovirus-transformed human kidney (293) cells, swine testes and Vero cells. Plaque size variants were present, but there was no apparent difference in virus morphology by negative stain or immune electron microscopy. Pigs infected with skunk calicivirus had a slightly elevated body temperature at 3 days postinfection. Although the infected animals seroconverted, no overt clinical signs were observed. Purified infectious genomic skunk calicivirus RNA behaved exactly as San Miguel sea lion virus (SMSV) 1 and 4 genomic RNA in cell culture transfection studies. Of the cell types examined, only primary porcine kidney, 293 and Vero cells supported viral replication. No viral replication was detected in cells of bovine, equine, ovine, caprine or feline origin. The skunk caliciviruses contained a single capsid protein with a relative mobility similar to SMSV virus 1 and 4 capsid proteins. The capsid protein was positive by Western blot analysis with SMSV and vesicular exanthema of swine virus (VESV) antisera. Purified RNA from skunk calicivirus infected cells was subjected to reverse transcription followed by polymerase chain reaction. Nucleotide sequences were identified that had greater than 85% similarity to the 2C and RNA polymerase gene regions of SMSV 1 and 4 and VESV A48. Predicted amino acid sequences of these regions were greater than 95% similar and the partial coding sequence of the polymerase gene contained the YGDD sequence common to positive-strand RNA virus polymerases.

Evidence for a Porcine Respiratory Coronavirus, Antigenically Similar to Transmissible Gastroenteritis Virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.

The multiplication of transmissible gastroenteritis viruses in several cell lines originated from porcine kidney and effects of trypsin on the growth of the viruses.

Plaque formation, replication and related cytopathic function of 9 strains of transmissible gastroenteritis (TGE) virus were examined in primary cells and cell lines such as CPK, IB-RS-2, ESK, and PK-15 originated from porcine kidney and the effects of trypsin on the replication of TGE virus were examined in CPK cells. All strains produced a cytopathic effect and grew well in CPK cells as well as in primary porcine kidney cells. The effect of trypsin on the plaque formation was different from strains. The number of plaques produced by strains TO-163, Ukiha and Niigata increased from 2.6 to 3.52 times when trypsin was present in the medium during incubation at 37 degrees C for 1 hr after adsorption of the virus at 4 degrees C for 2 hr. The plaque sizes of TO-163, h-5, Ukiha and Niigata became larger from 1.4 to 1.7 times, when trypsin was present in the agar MEM overlay.

Characterization of a Parvovirus isolated from the diarrheic feces of a pig.

A small DNA virus was isolated from the feces of a sow with diarrhea and identified as a parvovirus on the basis of its properties. The virus replicated preferentially in cell cultures of swine origin, including primary porcine thyroid gland and kidney cell cultures in which the cytopathic effect developed. The virus agglutinated erythrocytes of guinea pig, mouse and human group O but not these of chicken. The growth of the virus was inhibited by 5-iodo-2'-deoxyuridine. The virus was resistant to ether and heating at 56 degrees C for 30 min and stable at pH 3.0. The buoyant density of the infectious particles was 1.40 g/ml in CsCl density gradient, and the virions were 27 nm in diameter by electron microscopy. The viral protein seemed to be separated into four polypeptides with molecular weights of 81k, 70k, 66k and 62k daltons respectively. Cross serum neutralization test demonstrated that the virus was antigenically different from porcine parvovirus as well as bovine and canine parvoviruses. These findings and the survey on neutralizing antibody distribution indicated indirectly that another parvovirus which could be antigenically distinguished from well-known porcine parvovirus had been widespread among swine in Japan.

Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus.

The Cowden strain of porcine group C rotavirus (pararotavirus) was adapted to serial passage in a continuous monkey kidney cell line (MA104). Key factors in its successful adaptation included use of virus passaged in primary porcine kidney cells as the initial inoculum, use of roller tubes, and addition of pancreatin to the maintenance medium. A cell culture immunofluorescence test was used to quantitate the virus at each passage level, since a possible cytopathic effect was obscured by the effects of pancreatin. The virus titers dropped after initial passage into MA104 cells but increased thereafter, with peak titers evident after 16 passages (10(7) immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotavirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culture-propagated group C rotavirus was identical to that of the gut virulent virus from which it was derived. The cell culture-passaged group C rotavirus also retained its infectivity for gnotobiotic pigs. No group A rotavirus was detected in the intestinal contents of the pigs or in cell culture fluids from group C rotavirus-inoculated monolayers with the two former techniques or the cell culture immunofluorescence test. This is the first verified report of serial propagation of a non-group A rotavirus in a continuous cell line.

Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures.

A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures. Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful. A low-pH medium (pH 6.8) was also ineffective in virus propagation. Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium. The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation. Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation. Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16. Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13. The infected cells became separated, rounded, and detached, forming holes in the cell monolayer. Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy. Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.

Serial propagation of porcine group C rotavirus (pararotavirus) in primary porcine kidney cell cultures.

A porcine group C rotavirus was adapted to serial propagation in roller tube cultures of primary porcine kidney cells with high concentrations of pancreatin. Infected cells were identified by immunofluorescence staining of cell monolayers. Only group C rotavirus particles were observed in culture supernatants by immune electron microscopy.