Immunoperoxidase labeling of canine distemper virus replication cycle in Vero cells

An indirect immunocytochemical labeling technique, using horseradish peroxidase-conjugated antibody was used to detect the intracellular and surface membrane localization of canine distemper virus (R252-CDV) antigens during productive virus replication in infected Vero cells. Specific labeling of intracellular viral antigens was restricted to rough nucleocapsid aggregates. Surface membrane labeling correlated directly with the appearances both of virus-specific membrane spikes and buds and of mature virions. Syncytial cell formation was associated with labeled cytoplasmic nucleocapsid, but there was no evidence of productive CDV formation on surface membranes. The immunoperoxidase technique provided precise ultrastructural antigenic localization with concomitant preservation of excellent ultrastructural detail within single virus-infected cells during CDV replication cycle in vitro.     

Host immune-related gene responses against highly pathogenic avian influenza virus infection in vitro differ among chicken cell lines established from different organs

Highly pathogenic avian influenza virus (HPAIV) induces acute disease in chickens causing high mortality and morbidity and is a major threat to poultry industries in Southeast Asian countries. The mechanisms of disease manifestation and host innate immune responses against HAPIV in chickens are not well understood. In this study, we examined virus replication and host gene expressions in four chicken cell lines in vitro to elucidate the impact of host innate immune responses against viral replication. It was demonstrated that viral replication efficiencies were different depending on the cell line. The viral replication appeared to be affected by the basal expression of IFN related genes. The expression of immune-related genes against the viral infection also varied in a cell line dependent manner. In non-immune derived cell lines, but not in immune derived cell lines, the expression of the CCL5 and CCL20 genes were induced by HPAIV infection. Reverse genetics HPAIV, with internal genes from avirulent avian influenza, reduced virus replication and affected immune-related gene expression in a cell line dependent manner. These results suggest the possibility that differential immune responses in different cell types in local tissues could modulate the consequences of HPAIV infection in chickens.     

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.     

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.     

Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.     

In vitro cytopathogenicity and in vivo virulence of two strains of canine parainfluenza virus.

In vivo and in vitro properties of two strains of canine parainfluenza virus (CPIV) were investigated. One strain, designated CPIV(+), induced syncytial giant cell formation and cytolysis in vitro, whereas the second strain, CPIV(-), caused only a mild strand-forming cytopathic effect with few, small syncytial giant cells. Vero cells infected with CPIV(+) or CPIV(-) were 100% positive for CPIV antigen as determined by immunofluorescent staining; however, 100% of CPIV(+) and less than 10% of CPIV(-) infected cells were hemadsorption positive. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed no differences in electrophoretic mobility of viral polypeptides between both strains; however, in CPIV(-), reduced or absent synthesis of the putative HN and F1 proteins was observed. Isopycnic separation of CPIV(+) progeny virions showed a high proportion of viral particles with a buoyant density of 1.18 g/cm3. In contrast, CPIV(-) progeny virions had a heterogeneous density profile ranging from 1.08 to 1.18 g/cm3. Intracerebral infection of six ferrets with CPIV(+) resulted in moderate lymphocytic and histiocytic choroiditis, meningitis, and ependymitis, whereas CPIV(-) infection caused only mild to moderate inflammation. Immunohistologically, CPIV antigen was prominent in ependymal lining cells of the ventricles in CPIV(+)-infected ferrets and was reduced or lacking in CPIV(-)-infected ferrets (n = 6). Sham-injected ferrets (n = 6) did not have histologic lesions and no viral antigen was identified. The present findings suggest that certain changes in the activities of CPIV glycoproteins may lead to alterations of CPIV virulence in vivo.     

In vitro propagation of canine distemper virus: establishment of persistent infection in Vero cells

Primary cultures of bovine fibroblast (BF) and canine brain cells, persistently infected with virulent R252-canine distemper virus (CDV), were cocultured with African green monkey (Vero) cells. Transfer of persistent CDV from BF to Vero cells varied inversely with the in vitro passage level (age) of the CDV-infected BF cells. Successful transfer of CDV to Vero cells was signaled by the transient appearance of viral syncytia, rapid spread of viral antigen to all Vero cells in the culture, and by recovery of cell-free Vero-infectious virus in culture fluids. With time, viral cytopathic effects in Vero cells containing CDV disappeared, and the infected lines could not be distinguished from noninfected control Vero cells, except by immunoassay for viral antigen.     

In vitro infection studies with infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus.     

Infectious bursal disease virus: further characterization with evidence for a single-stranded RNA virus

Infectious bursal disease virus (IBDV) that had been adapted to grow and was then cloned in chick embryo fibroblast (CEF) cell culture was examined for its physicochemical properties, the cellular site of virus replication, and the nature of its viral RNA. The IBDV was an RNA virus, acid-stable, absolutely resistant to chloroform, and moderately thermolabile. It appeared to replicate only in the cytoplasm, as shown by virus-specific antigens restricted to the cytoplasm of infected cells. The viral RNA was composed of single-stranded RNA, as evidenced by flame-red fluorescence on acridine-orange staining and an absence of specific fluorescence in infected cells on immunofluorescent staining with antiserum specific for double-stranded RNA. The IBDV virion had a hexagonal outline with an average diameter of 62 nm and possessed a single layer of capsid composed of hollow capsomeres without envelope. The buoyant density as determined in a continuous sucrose gradient was 1.178 g/cm3. The IBDV was found to possess morphologial and physicochemical properties different from those of any established RNA virus group.     

Tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine‐derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine‐derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8). CDV replication‐induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV‐induced cancer therapy.     

In vitro antiviral efficacy of ribavirin against feline calicivirus, feline viral rhinotracheitis virus, and canine parainfluenza virus.

Ribavirin had marked in vitro activity against feline calcivirus, strain 255, and canine parainfluenza virus, but showed only slight antiviral effect on feline viral rhinotracheitis virus. Antiviral activity was manifested by partial to complete suppression of viral cytopathic effect and of viral replication, depending on concentration of ribavirin in the culture medium and dosage of viral inoculum. Concentrations of ribavirin as small as 3.2 microgram/ml and 1.0 microgram/ml showed some activity against feline calcivirus and canine parainfluenza virus, respectively.     

Replication of infectious bursal disease virus in macrophages and altered tropism of progeny virus

We serially passaged classical infectious bursal disease virus (cIBDV) and antigenic variant IBDV (vIBDV) in an avian macrophage cell line, NCSU cells, referred as mcIBDV and mvIBDV respectively and examined the in vitro and in vivo characteristics of the macrophage-adapted viruses. NCSU adapted viruses caused earlier destruction of NCSU cells than the unadapted viruses. Nitric oxide (NO) was detected earlier in cultures infected with mcIBDV and mvIBDV than in cultures infected with cIBDV and vIBDV. cIBDV and vIBDV were able to infect DF-1 cells, a chicken embryo fibroblast cell line, only after one replication cycle in NCSU cells. The genetic basis of altered tropism of progeny virus from NCSU cells infected cultures was not identified. No aa substitutions were observed in hypervariable region of VP2 of cIBDV and vIBDV passaged 1 time in NCSU cells whereas both mcIBDV and mvIBDV had multiple aa substitutions. To assess protective efficacy of mcIBDV and mvIBDV, embryonated chicken eggs were inoculated with mcIBDV and mvIBDV at embryonation day 18 (ED 18) and challenged with a virulent cIBDV at 3 weeks of age. mcIBDV and mvIBDV were immunogenic and generated antibody responses and provided 100% protection against cIBDV.     

Attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus

Kittens vaccinated with an avirulent biotype of the Black strain of feline infectious peritonitis virus (FIPV; given oronasally) developed both indirect fluorescent and virus-neutralizing antibodies, but were not protected against oronasal challenge exposure with virulent virus. In fact, kittens vaccinated with avirulent virus were more readily infected than were nonvaccinated cats. A proportion of kittens could be immunized to FIPV by giving sublethal amounts of virulent virus. This technique, however, was too inconsistent and hazardous to have clinical relevance. The results of these studies indicated that humoral immunity was not protective in FIPV infection. There was no correlation between fluorescent and virus-neutralizing antibodies and either disease or immunity. Immune serum from FIPV-resistant cats failed to passively protect susceptible animals against virulent virus given intraperitoneally or oronasally, and as expected, actually sensitized them to infection. It was concluded that cell-mediated immunity was probably responsible for protection.     

Suppression of influenza virus infection by the orf virus isolated in Taiwan

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.     

Replication of classical swine fever virus strains and isolates in different porcine cell lines

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.     

Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines.

Bovine T lymphoblast cell lines transformed by the protozoan Theileria parva were compared with bovine kidney (BK) and Vero cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapinised vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphoblasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Vero and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BK cells. It was also possible using the lymphoblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lymphoblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively little cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.     

Host cell modulation of foot-and-mouth disease virus procapsid synthesis

BHK21 (clone 13S) cells of high (BHK-SH) and low (BHK-SL) passage number were infected with foot-and-mouth disease virus (FMDV) subtypes A24, A25 and C3. While the amount of virus specific RNA produced in BHK-SH cells was 25% of that in BHK-SL cells and the virion production was 27% (C3) to 53% (A24) lower, the synthesis of viral proteins was comparable, associated with an accumulation of procapsids in BHK-SH cells. The results suggest that changes in viral infection pattern with increasing BHK21 cell passage number should be considered in FMDV vaccine production.     

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.     

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.