Alternative methods to determine infectivity of Tulane virus: a surrogate for human nororvirus.

Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV). The infectivity of a culturable virus was traditionally determined by plaque assay and/or 50% tissue culture infectious dose (TCID50) assay, both of which are time-consuming and labor-intensive. Molecular approaches, such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR, could be used for detection of the viral genome but yet fail to determine the infectivity of a virus. In this study, we evaluated different assays for determination of infectivity of Tulane virus (TV), a surrogate for HuNoV. The infectivity of TV was measured by RNase exposure assay, RT-PCR assays, cellular-receptor-mediated capture qRT-PCR assay, receptor-mediated in situ capture qRT-PCR assay, cell-culture-mediated amplification qRT-PCR, and confirmed by TCID50 assay. RNase exposure assay was only useful for measuring TV inactivation caused by heat. Short template RT-PCR assay did not reflect inactivation status of TV. Partial reduction in viral RNA signal could be measured by long-template RT-PCR only when TV was inactivated by thermal or chlorine treatments at full-inactivation levels. Cellular-receptor-mediated capture qRT-PCR exhibited low sensitivity and specificity for the evaluation of virus infectivity. The in situ capture qRT-PCR assay could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. The cell-culture-mediated amplification qRT-PCR could be used as an alternative method to rapidly determine the infectivity of TV.

Hydroponic Nutrient Solution Temperature Impacts Tulane Virus Persistence over Time.

Controlled environment agriculture (CEA), or indoor agriculture, encompasses non-traditional farming methods that occur inside climate-controlled structures (e.g., greenhouses, warehouses, high tunnels) allowing for year-round production of fresh produce such as leaf lettuce. However, recent outbreaks and recalls associated with hydroponically grown lettuce contaminated with human pathogens have raised concerns. Few studies exist on the food safety risks during hydroponic cultivation of leaf lettuce; thus, it is important to identify contributing risk factors and potential mitigation strategies to prevent foodborne transmission via hydroponically grown produce. In this study, the concentration of infectious Tulane virus (TV), a human norovirus surrogate, in hydroponic nutrient solution at 15 °C, 25 °C, 30 °C, and 37 °C was determined over a duration of 21 days to mimic the time from seedling to mature lettuce. The mean log PFU reduction for TV was 0.86, 1.80, 2.87, and ≥ 3.77 log10 at 15 °C, 25 °C, 30 °C, and 37 °C, respectively, at the end of the 21-day period. Similarly, average decimal reduction values (D-values) of TV at 15 °C, 25 °C, 30 °C, and 37 °C were 48.0, 11.3, 8.57, and 7.02 days, respectively. This study aids in the (i) identification of possible food safety risks associated with hydroponic systems specifically related to nutrient solution temperature and (ii) generation of data to perform risk assessments within CEA leaf lettuce operations to inform risk management strategies for the reduction of foodborne outbreaks, fresh produce recalls, and economic losses.

Norovirus surrogate survival on spinach during preharvest growth.

Produce can become contaminated with human viral pathogens in the field through soil, feces, or water used for irrigation; through application of manure, biosolids, pesticides, and fertilizers; and through dust, insects, and animals. The objective of this study was to assess the survival and stability of human noroviruses and norovirus surrogates (Murine norovirus [MNV] and Tulane virus [TV]) on foliar surfaces of spinach plants in preharvest growth conditions. Spinach plants were housed in a biocontrol chamber at optimal conditions for up to 7 days and infectivity was determined by plaque assay. Virus inoculation location had the largest impact on virus survival as viruses present on adaxial leaf surfaces had lower decimal reduction time (D values) than viruses present on abaxial leaf surfaces. Under certain conditions, spinach type impacted virus survival, with greater D values observed from survival on semi-savoy spinach leaves. Additional UVA and UVB exposure to mimic sunlight affected virus survival on adaxial surfaces for both semi-savoy and smooth spinach plants for both viruses. Human GII norovirus inoculated onto semi-savoy spinach had an average D value that was not statistically significant from MNV and TV, suggesting that these surrogates may have similar survival on spinach leaves compared with human noroviruses. An understanding of the behavior of enteric viruses on spinach leaves can be used to enhance growers' guidelines and for risk assessment with certain growing conditions.

Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers.

Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-ß inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that-like many other caliciviruses-does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.

Recovery of infectious virus by transfection of in vitro-generated RNA from tulane calicivirus cDNA.

Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5' end of the genome also hampered viral replication, suggesting that an authentic 5' end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.

Vectorial entry and release of hepatitis A virus in polarized human hepatocytes.

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes.

Surrogates for the study of norovirus stability and inactivation in the environment: aA comparison of murine norovirus and feline calicivirus.

Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values < 3 and > 9. FCV was more stable than MNV-1 at 56 degrees C, but both viruses exhibited similar inactivation at 63 and 72 degrees C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4 degrees C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.

Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

Variable sensitivity of a feline embryo cell line and of three kitten kidney cell cultures to feline herpes- and caliciviruses.

The number of plaques produced in a feline embryo (FEmb) cell line and in three independently derived kitten kidney (KK) cell cultures varied in a consistent and reproducible manner when each was inoculated with the same number of feline herpesvirus 1 (FHV1) plaque forming units (PFU); the three KK cells produced 2-9 times more plaques than FEmb cells. One of the three KK cells produced FHV1 plaques that were smaller in diameter than those FEmb cells. Each of the three KK cell cultures inoculated with the same number of FEmb cell PFU of a strain of feline calicivirus (FCV) produced different numbers of plaques; two of the three KK cell cultures produced 2-3 times more plaques than FEmb cells. The plaque diameter of FCV in the three KK cells was 30-50% smaller than the plaque diameter in FEmb cells.

Early interaction of canine calicivirus with cells is the major determinant for its cell tropism in vitro.

Canine calicivirus (CaCV) No. 48 strain isolated from a dog with fatal diarrhea is known to be able to replicate in MDCK and primary dog kidney cells. In this study, two new canine cell lines, MCM-B2 and MCA-B1, were determined to be permissive for CaCV No. 48, whereas other cell lines, including one canine cell line, A-72, were non-permissive. Flow cytometric analysis indicated that CaCV No. 48 binds efficiently to the permissive cells and to some degree also to Vero cells that are non-permissive for the virus, but does not bind to the other non-permissive cells tested. Both the permissive and non-permissive cells could be transfected with genomic RNA from CaCV No. 48, resulting in the appearance of CPE, production of capsid antigen and release of infectious progeny. These results suggested that the early interaction of the virus with cells, probably by binding to a virus receptor on the cell membrane, is the major determinant of CaCV No. 48 cell tropism in vitro.

Rabbit haemorrhagic disease: an investigation of some properties of the virus and evaluation of an inactivated vaccine.

An inactivated vaccine against rabbit haemorrhagic disease (RHD), developed and tested in our laboratory, is produced commercially by Bioveta, Ivanovice, Czechoslovakia. Rabbits developed full protection against infection 3 weeks after the administration of a single dose. Antibodies were detectable from day 5 after vaccination. Naturally acquired antibodies were demonstrated in some rabbits kept on commercial farms. The virus survived at least 225 days in an organ suspension kept at 4 degrees C, at least 105 days in the dried state on cloth at room temperature (around 20 degrees C), and at least 2 days at 60 degrees C, both in organ suspension and in the dry state. Experimental infection of rabbits younger than 2 months was successful in some animals. Hares, guinea pigs, white mice, golden and Chinese hamsters, chinchillas and hysterectomy-derived, colostrum-deprived piglets were resistant to infection.

Ice as a reservoir for pathogenic human viruses: specifically, caliciviruses, influenza viruses, and enteroviruses.

Hundreds of isolates of viable bacteria and fungi have been recovered from ancient ice and permafrost. Evidence supports the hypothesis that viral pathogens also are preserved in ice repositories, such as glaciers, ice sheets, and lake ice. Proof may depend upon narrowing the search by applying specific criteria, which would target candidate viruses. Such criteria include viral pathogens likely to occur in great abundance, likely to be readily transported into ice, and then participate in ongoing disease cycles suggestive of their having been deposited in and subsequently released from ice. Caliciviruses, influenza A, and some enteroviruses appear to satisfy all three criteria. Environmental ice appears to be an important abiotic reservoir for pathogenic microbes. World health and eradication of specific pathogens could be affected by this huge reservoir.

Comparative resistance of San Miguel sea lion virus and vesicular exanthema of swine virus to chemical disinfectants.

Two similar calici agents, San Miguel sea lion virus (SMSV) and vesicular exanthema of swine virus (VESV) are susceptible to the virucidal activity of disinfectants of differing formulation. Ten of 12 compounds were effective against six log10 plaque forming units (PFU) of SMSV in a 2-min exposure at 4, 25 and 37 degrees C. However, only seven of these 10 SMSV-positive compounds inactivated VESV under the same conditions of temperature and time. Two compounds were not effective against SMSV in a 20-min exposure at 4 and 25 degrees C, but were effective at 37 degrees C. Two of the three compounds not effective against VESV in a 20-min exposure at 4 and 25 degrees C were also effective at 37 degrees C. The test iodophor compound inactivated SMSV completely but had only minimal inactivating activity against VESV at the three temperatures tested.

Adaptation of the viral haemorrhagic disease virus of rabbits to the DJRK cell strain.

Liver emulsion of rabbits which had died of viral haemorrhagic disease (VHD) was inoculated onto DJRK cell culture. After two passages, specific cytopathic effect was observed. Immunofluorescence was found in the nucleus at the early stage of infection and later also in the cytoplasm. The virus propagated in cell culture at the fifth, tenth and sixteenth passages was found to cause typical VHD. Electron microscopy showed that there were numerous virions in the infected cells. The cultured virus, inactivated with formaldehyde, could elicit haemagglutination inhibition antibodies in the inoculated rabbits and protect them against the challenge of virulent VHD virus.

A strain of calicivirus isolated from lions with vesicular lesions on tongue and snout.

In December 1992, 17 African lions and 7 Siberian tigers in a Safari park in Japan became sick with characteristic clinical symptoms of acute vesicular formations on tongue and snout. The disease was highly contagious since all of these animals showed similar symptoms within two days after the onset of the first case. Swabs were taken from affected animals in rubbing tongues, snouts and some from rectums. Cytopathic viruses were isolated on CRFK cell culture by virological tests. The physicochemical property of a representative virus strain, named Arthur/L, isolated from a male lion was identified as a member of Caliciviridae. However, seroneutralization test indicated that this virus strain was antigenically distinct from Japanese isolates of feline caliciviruses used for comparison. Viral capsid proteins of the present isolate, Arthur/L, and of a feline calicivirus, strain FC7, were compared in an electrophoresis in SDS-PAGE gel. The major viral capsid polypeptide of them were proved to be significantly different in molecular weight. The polypeptide of FC7 was estimated to be ca. 63 KDa whereas that of Arthur/L consisted of 2 components of ca. 65 and 62 KDa. The viral proteins of these two strains were also proved to be distinct by an immunoblotting test.

Replication of feline herpesvirus and feline calicivirus in cell and organ cultures.

Virus titers from feline herpesvirus-infected feline tracheal organ cultures were higher than those from feline kidney cell cultures. The time for the development of peak viral yields was longer in organ culture as compared to cell cultures. Feline tracheal organ cultures infected with feline calicivirus produced minimal viral titers.

Isolation of feline syncytia-forming virus from oropharyngeal swab samples and buffy coat cells.

Thirteen of 40 female cats were found to be chronically infected with feline syncytia-forming virus (FeSFV). Attempts to isolate the virus from these cats by conventional methods were not successful. However, virus was isolated from oropharyngeal swab samples and buffy coat cells. A new method was used involving inoculation of actively dividing Crandell feline kidney cell cultures. Cultures were trypsinized 3 days after inoculation and, as a result, cytopathic effect was amplified and ability to detect the virus was enhanced. The FeSFV was detected in 93% (92/88) of the oropharyngeal swab samples and 100% (14/14) of the buffy coat cell specimens. Feline sera were tested by immunodiffusion for precipitating antibody against FeSFV antigen. There was 100% correlation between viral infection and the presence of precipitating antibody. Virus and antibody persisted in infected cats for the duration of this study (8 months for 5 of the infected cats). Urolithiasis was observed in 15 of 28 male cats. Although a direct relationship between FeSFV infection and urolithiasis was not established, most of these male cats (20 of 21) had antibody to FeSFV.

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.

Diseases produced by feline caliciviruses when administered to cats by aerosol or intranasal instillation.

Specific pathogen free cats were infected by two feline calicivirus isolates of different plaque type, an extra-large plaque (ep) former and a minute plaque (mp) former. A comparison was made of the disease produced when these isolates were administered by either aerosol or direct intranasal instillation. With both isolates, aerosol infection produced lesions and gave rise to virus replication throughout the respiratory tract. The effects of intranasal infection were more confined to the upper respiratory tract and oropharynx. By both routes of infection the disease produced by the mp virus was clinically and pathologically less severe than that produced by the ep virus.

[Disinfection of caliciviruses at 20 and 10 degrees C]. / Desinfektion von Caliciviren bei Temperaturen von 20 und 10 degrees C.

Five disinfectants, Venno FF super, Venno Vet 1 super, Venno Oxygen, M&Enno-Veterinär B neu und Neopredisan 135-1, were tested to evaluate their efficacy against caliciviruses at 20 and 10 degrees C. As model test virus served feline calicivirus type F9 (FCV F9). All disinfectants were tested according to Guidelines of the German Veterinary Association (DVG). The investigations were performed in suspension tests and germ carrier tests. The suspension tests were carried out without and with protein load. As protein was used foetal calf serum at the concentration of 40%. Venno FF super showed less protein dependence, however a considerable temperature dependence. This matter can be corrected by increase of concentration on 2%. Venno Vet 1 super was without protein especially effective. The losses on the effectiveness through low temperature and protein load can be annulled also here by increase of concentration. Venno Oxygen was more effective in the comparison to that here named both preparations. The effects of temperature can be corrected by extension of reaction time. The most effective preparation was M&Enno Veterinär B neu. The disinfection occurred at 20 degrees C with 0.5% solution within 120 min and at 10 degrees C with 1.0% solution within 60 min. The fifth disinfectant Neopredisan was in suspension tests without protein load and carrier tests with gauze at 20 and 10 degrees C relative convincing but in germ carrier tests with poplar wood, no complete disinfection could be achieved within tested concentrations and reaction times.