Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Analysis of the microRNA expression profiles in feline kidney cell line infected with feline panleukopenia virus.

MicroRNAs (miRNAs) play crucial roles in post-transcriptional regulation of gene expression in many biological processes. Feline panleukopenia virus (FPV) is a highly infectious pathogen that can cause severe disease in pets, economically important animals and wildlife. In this study, miRNAs associated with FPV infection were identified using high-throughput sequencing. Our results showed that 673 known miRNAs and 278 novel miRNAs were identified and 57 significantly differential expression miRNAs were found post-FPV infection in feline kidney cell line. Stem-loop qRT-PCR was applied to validate the expression of the randomly selected miRNAs; the results were consistent with the sequencing data. Furthermore, the target genes of differential expression miRNAs were analyzed and predicated by GO and KEGG pathway. Altogether, our analysis provides a potential link between miRNA expression and the pathogenesis of FPV infection.

The propagation of feline panleukopenia virus in micro-carrier cell culture and use of the inactivated virus in the protection of mink against viral enteritis.

Using microcarrier cell culture for the production of virus antigen, a formalin-inactivated feline panleukopenia virus vaccine was evaluated for protection of mink against specific mink enteritis virus infection. The vaccine showed a good immunogenic effect in mink when used either alone or in combination with Clostridium botulinum type C-toxoid and/or Pseudomonas aeruginosa vaccine. A single vaccination induced persistent immune responses for periods of at least 1 year, as evaluated by ELISA and challenge tests. Neither immunological interference between vaccine constituents nor adverse reactions were observed.

Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan.

Canine parvovirus type 2 (CPV-2) and feline panleukopenia (FLP) virus (FPLV) are well known and ubiquitous diarrhea-causing pantropic viruses. A "new" antigenic variant of CPV-2 (designated as CPV-2a) has been also prevalent among dogs in Japan. In the present study, 24 canine and 8 feline isolates collected during 1987-1991 were compared with 17 CPV-2 or CPV-2a and 7 FPLV strains that had been characterized previously. Genomic properties were determined by the restriction cleavage patterns of amplified genes encoding the capsid proteins VP1 and VP2 by the polymerase chain reaction. Antigenic properties were determined by hemagglutination-inhibition assay with monoclonal antibodies against an FPLV strain. Growth characteristics in feline CRFK and canine MDCK cells were also examined. Genomic and antigenic properties of the canine isolates were relatively invariable with one exceptional isolate, C27, which was recovered from a typical clinical case of parvovirus infection but possessed properties similar to FPLV rather than CPV-2 and CPV-2a. All isolates from FPL cases possessed the same genomic and antigenic properties as those of reference FPLVs isolated in the 1970s, but three of five strains isolated from the feces of clinically healthy cats were likely to be of canine origin because they possessed very similar properties to CPV-2a. Although species-specificity of these novel isolates could not be determined definitely, the results indicate a possibility that transmission of parvovirus has occurred between these two animal species.

Use of modified live feline panleukopenia virus vaccine to immunize dogs against canine parvovirus.

Modified live feline panleukopenia virus (FPLV) vaccine protected dogs against canine parvovirus (CPV) infection. However, unlike the long-lived (greater than or equal to 20-month) immunity engendered by CPV infection, the response of dogs to living FPLV was variable. Doses of FPLV (snow leopard strain) in excess of 10(5.7) TCID50 were necessary for uniform immunization; smaller inocula resulted in decreased success rates. The duration of immunity, as measured by the persistence of hemagglutination-inhibiting antibody, was related to the magnitude of the initial response to vaccination; dogs with vigorous initial responses resisted oronasal CPV challenge exposure 6 months after vaccination, and hemagglutination-inhibiting antibodies persisted in such dogs for greater than 1 year. Limited replication of FPLV in dogs was demonstrated, but unlike CPV, the feline virus did not spread to contact dogs or cats. Adverse reactions were not associated with living FPLV vaccination, and FPLV did not interfere with simultaneous response to attenuated canine distemper virus.

[The growth of attenuated strains of canine parvovirus, mink enteritis virus, feline panleukopenia virus, and rabies virus on various types of cell cultures]. / Rast atenuovaných kmenov parvovírusu psov, vírusovej enteritídy noriek a panleukopénie maciek a vírusu psinky na rozdielnych druhoch bunkových kultúr.

The growth characteristics were studied in the attenuated strains of canine parvovirus CPVA-BN 80/82, mink enteritis virus MEVA-BN 63/82 and feline panleucopenia virus FPVA-BN 110/83 on the stable feline kidney cell line FE, and in the attenuated canine distemper virus CDV-F-BN 10/83 on chicken embryo cell cultures (KEB) and cultures of the stable cell line VERO. When the FE cultures were infected with different parvoviruses in cell suspension at MOI 2-4 TKID50 per cell, the first multiplication of the intracellular virus was recorded 20 hours p. i. In the canine parvovirus, the content of intracellular and extracellular virus continued increasing parallelly until the fourth day; then, from the fourth to the sixth day, the content of extracellular virus still increased whereas that of intracellular virus fell rapidly. In the case of the mink enteritis virus the release of the virus into the culture medium continued parallelly with the production of the cellular virus until the sixth day. In the case of the feline panleucopenia virus the values concerning free virus and virus bound to cells were lower, starting from the second day p. i. When KEB or VERO cultures were infected in cell suspension with the canine distemper virus at MOI about 0.004 per 1 cell, the replicated intracellular virus was first recorded in the KEB cultures five hours after infection but in the VERO cultures only 20 hours after infection, with a timely release of the virus into the culture medium in both kinds of tissue. In the KEB and VERO cultures the highest values of infection titres were recorded on the fourth day p. i., the course of virus multiplication on the cells being parallel with its release into the culture medium.

Parvovirus (feline panleucopaenia virus) plaque formation.

A plaque assay was developed for feline parvovirus (FPV; feline panleucopaenia virus) in a feline embryo (FEmb) cell line. Higher numbers and larger diameter plaques were obtained with a) seeding rates of 0.7 X 10(5) and 1.5 X 10(5) cells cf. 3 X 10(5) and 6 X 10(5) cells/well of 35 mm diameter, b) synchronised cells infected at the G1-S interface cf. nonsynchronised cells and c) 5 to 6 days incubation post inoculation. The plaque assay was standardised by using serum deprivation for 24 hours to synchronize cells, a seeding rate of 1.5 X 10(5) cells/35 mm diameter well, inoculation of virus 16 hours post seeding followed by 5 days incubation. The standardised assay gave consistent, reproducible results. A dose-response curve using the assay showed a linear, 45 degrees slope, relationship between plaque forming units and virus dilution which further verified the sensitivity and reliability of the assay. Plaques produced by "wild" type and plaque purified virus were invariably non uniform in diameter; diameter of plaques in fact followed a normal frequency distribution under standard assay conditions.

A plaque assay for feline panleukopenia virus.

Plaque formation with representative strains of feline panleukopenia virus (FPV) has been obtained using a permanent line of feline kidney cells under agarose overlay. FPV-infected cells appear as white plaques after neutral red staining. Plaque size is determined by the extent of cell division in the infected monolayer. FPV assay by the plaque procedure is rapid and gives infectivity titres which exceed those determined by the common inclusion body and immunofluorescent assays of FPV by a factor of about 100 and 10, respectively. Moreover, the plaque assay offers an effective means for the quantification of neutralizing antibodies in feline sera as well as for the detection of heat-stable substances in bovine sera which strongly interfere with replication of the virus.

Studies on the growth of feline panleukopaenia virus in cell cultures.

The growth of feline panleukopaenia virus was examined in relation to various cell growth curves of secondary feline kidney cells. In cell cultures with a high mitotic index, the manifestation of virus infection, expressed as percentage number of cells with inclusion bodies, was correspondingly high. In cultures with low mitotic index, the percentage number of cells with inclusion bodies was low. A cytopathic effect was seen by inoculation of virus with a titre of 10-3-5TCID-50/ML. This effect was most pronounced using cell cultures seeded in a quantity of 1 million cells per ml to 500,000 cells per ml with inoculation made during the first 24 hours after seeding when the mitotic index was high. Both the cytopathic effect and the occurrence of inclusion bodies were of transient nature. The peak of infection, both with regard to the cytopathic effect and the percentage number of cells with inclusion bodies, was seen three days after inoculation.

Characterization of the stage(s) in the virus replication cycle at which the host-cell specificity of the feline parvovirus subgroup is regulated in canine cells.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription.

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.

Feline parvovirus propagates in cat bone marrow cultures and inhibits hematopoietic colony formation in vitro.

Feline parvovirus (FPV) causes leukopenia in naturally infected cats. We investigated the mechanism of hematopoietic depression by this virus in feline bone marrow cultured in vitro. In suspension cultures we demonstrated FPV propagation and replication using DNA molecular hybridization. Viral RNA and DNA were observed by in situ hybridization in about 10% of marrow cells at day 3. Granulocytes and their precursors were virtually absent from infected cultures after six days. Infected cells showed viral capsid protein predominantly in nuclei by immunofluorescence. In clonal assays, FPV most efficiently inhibited hematopoietic colony formation by myeloid progenitor cells (CFU-GM), but erythroid colony formation (BFU-E and CFU-E-derived) was also depressed in the presence of virus. Inhibition of colony formation could be abrogated by physical inactivation of the virus or preincubation with specific neutralizing antibodies. Recombinant human colony stimulating factors GM-CSF and G-CSF supported feline myelopoiesis in progenitor assays, and FPV completely inhibited factor dependent colony formation.