Salinity regulation of the interaction of halovirus SNJ1 with its host and alteration of the halovirus replication strategy to adapt to the variable ecosystem.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Viruses in Antarctic lakes.

Water samples collected from four perennially ice-covered Antarctic lakes during the austral summer of 1996-1997 contained high densities of extracellular viruses. Many of these viruses were found to be morphologically similar to double-stranded DNA viruses that are known to infect algae and protozoa. These constitute the first observations of viruses in perennially ice-covered polar lakes. The abundance of planktonic viruses and data suggesting substantial production potential (relative to bacteria] secondary and photosynthetic primary production) indicate that viral lysis may be a major factor in the regulation of microbial populations in these extreme environments. Furthermore, we suggest that Antarctic lakes may be a reservoir of previously undescribed viruses that possess novel biological and biochemical characteristics.

Porcine reproductive and respiratory syndrome: temperature and pH stability of Lelystad virus and its survival in tissue specimens from viraemic pigs.

We investigated the growth of Lelystad virus (LV) in porcine alveolar macrophages, the thermal and pH stability of the virus in cell culture medium, and its survival in tissue specimens from viraemic pigs. Lelystad virus grew to titres of 10(6) TCID50/ml, which were found at 40 h after virus inoculation when the macrophage cultures showed a cytopathic effect of approximately 40%. In culture medium at pH 7.5, LV was stable for prolonged periods of storage at -70 degrees C and -20 degrees C. At higher temperatures the half-life of LV was 140 h at 4 degrees C, 20 h at 21 degrees C, 3 h at 37 degrees C and 6 min at 56 degrees C. The half-life of LV, both at 4 degrees C and 37 degrees C, changed considerably when the pH of the medium was varied. At 4 degrees C and pH 6.25 a maximum half-life of 50 h and at 37 degrees C and at pH 6.0 a maximum half-life of 6.5 h was observed. However, increasing or decreasing the pH of the medium rapidly decreased the half-life of LV at both temperatures. Although, LV proved to be more stable at pH 6.00 than at pH 7.5, it did not replicate at pH 6.0. We also tested various tissue specimens from viraemic pigs for the presence of LV. The virus was detected in tonsils, lymph nodes, lungs, serum, and sporadically, albeit at low titres, in muscle tissue. The titre of virus in muscle tissue and organs was only minimally affected by storage for up to 48 h at 4 degrees C.

Growth and characterisation of human faecal astrovirus in a continuous cell line.

We report conditions for the growth of human faecal astrovirus in a continuous colonic carcinoma cell line (CaCo-2). Purified particles contained three polypeptides, one of which (24k) appeared loosely held on the exterior.

Cultivation and partial characterization of bovine astrovirus.

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.

Plaque quantitation and virus neutralization assays for human astroviruses.

Astroviruses, 28 nm-diameter, RNA-containing viruses which have been implicated in gastroenteritis can be cultivated in cell cultures containing trypsin, but do not show distinguishable cytopathic effects. However, with the 5 known astrovirus serotypes which we have been able to cultivate, 3 (types 1, 2, and 5) formed well-defined plaques in LLCMK2 cell cultures under an agar overlay containing trypsin. A virus neutralization assay based on plaque reduction was applied to these 3 serotypes. It was found that rabbit antisera prepared against individual serotypes neutralized virus type-specifically, and no cross-neutralization titers were obtained with any of the antisera to the 5 astrovirus serotypes. The type-specific neutralization observed agreed with the specificities seen by immunofluorescence (IF), whereas ELISA tests with the same antisera show cross-reactivity among all 5 serotypes. There was no virus neutralization detected with astrovirus monoclonal antibodies which were reactive with 5 serotypes by ELISA and IF. The results we have obtained permit quantitative techniques to be applied to epidemiological and biological studies of the human astroviruses.

Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink.

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.

Effect of Borna disease virus infection on athymic rats.

Homozygous athymic nude rats (rnu/rnu) infected intracerebrally with Borna disease virus produced relatively high titres of infectious virus in the central nervous system. However, no clinical signs of disease or pathological alterations could be found during a 100 day observation period. In contrast, heterozygous euthymic albino littermates (rnu/+), which were used as controls, reacted in a similar manner to immunocompetent Lewis rats. They developed behavioural alterations which coincided with encephalitis and retinitis. The results obtained confirm our previous concept that the genesis of Borna disease, at least in rats, is attributed to a cellular immune response.

Increase of virus yields and releases of Borna disease virus from persistently infected cells.

Borna disease virus grows to low titres in persistently infected cells with an infectious particle to cell ratio of 0.01 to 0.05. Inclusion of n-butyrate in the growth medium enhances infectivity yields up to 1 log. This effect is time and concentration dependent. In hypertonic medium with an excess of NaCl, KCl or Na2SO4 up to 50% of the total infectious virus yield is released from the cells. Released supernatant virus (buoyant density in sucrose rho = 1.22 g/cm3) is more heat stabile than cell-bound virus (rho = 1.18 g/cm3). The access to cell-free (released) virus opens new possibilities for the characterization of this neurotropic agent.

Replication of Borna disease virus in rats: age-dependent differences in tissue distribution.

There are age-dependent differences in the tissue distribution of Borna disease (BD) virus in rats infected intracerebrally. While in adult rats BD virus replication is restricted to neural cells, in neonatally infected rats infectious virus or viral antigens were found in the cells of most organs. The possibility that differences in the immune status between newborn and adult animals are responsible for different tissue susceptibility could be excluded.

Persistent, tolerant or subacute infection in Borna disease virus-infected rats.

The rabbit-adapted Borna disease (BD) virus strain V was passaged by intracerebral infection of 1-day-old Wistar rats. Infectivity titres reached 10(8) infectious units per gram of brain 4 weeks after infection. No clinical signs were evident. The persistent infection could be induced with adapted or field strains of BD virus. Strains were identified by neutralization tests. The virulence of the rabbit-adapted BD virus for the rat increased with rat passages. The 5th passage induced clinical symptoms in animals infected at 1 week of age or older. Between 20% and 50% of diseased rats died. Virus-specific antigen was detectable immunohistologically in neurons of rats infected at all ages. Animals inoculated at 1 or 2 months of age, but not the neonatal rats, showed signs of inflammation in the brain. Infected rats produced specific antibodies. In the older groups (infected at ages of 1 or 2 months), and especially in surviving animals, occasionally, neutralizing antibodies with high titres were found. Transfer of primed spleen cells resulted in subacute disease. These findings demonstrate that neonatal rats can acquire a persistent, tolerant infection and that expression of disease is mediated by immunological factors.

Propagation of Aleutian disease parvovirus in cell line CCC clone 81.

Aleutian disease parvovirus (ADV), mutant Gorham of the Utah-1 strain, was grown and comparatively assayed in feline cell lines CRFK and CCC clone 81 at 31.8 degrees C. The maximum virus titres as determined by a fluorescent focus assay were found to be about 10(5) FFU/ml in CRFK at day 6 p. i. and 10(6) FFU/ml at day 4 p. i. in CCC clone 81 cells. Shifting of the incubation temperature from 31.8 to 37 degrees C led to a reduced virus production after three passages. The synchronization of the CCC clone 81 cells by 1 X 10(-3) M hydroxyurea followed by infection with low (less than or equal to 0.8) multiplicities of infection (MOI) did not significantly influence the virus titres. Several mammalian cell lines such as MiCl 1 (S+L-), Mv1-Lu, 64F3 clone 7 and FEF or fish cell lines such as BB and CHSE 114 developed abortive infections after inoculation with the temperature-sensitive mutant Gorham of the ADV strain Utah-1 (ADV-G). Three new isolates designated ADV-Sl1--ADV-Sl3 were isolated from spleen and blood lymphocytes and bone marrow cells of ADV-infected mink and were adapted to grow in CCC clone 81 cells at 31.8 degrees C with virus titres between 10(4) and 10(4.7) FFU/ml. ADV particle populations varying in their bouyant density between 1.32, 1.36 and 1.43 g/ml were isolated from infected cells and culture supernatants. By protein blotting and immunodetection two major protein components with apparent M. W. of 85 and 75 KD and three minor polypeptides of 33, 28.9 and 27.5 KD were detected.

Pleural effusion disease in rabbits. Properties of the aetiological agent.

The size and heat sensitivity of Pleural effusion disease (PED) agent or virus (PEDV) propagated in rabbits were examined. The infectious particles were estimated to be between 25 and 50 nm by filtration. Residual infectivity of infectious serum was 0.1 per cent after heating at 56 degrees C for 4 hours. PEDV and the Stockholm agent appeared identical concerning pathogenic and immunogenic properties by infection experiments and protection tests in rabbits. Two of the three PEDV isolates were less pathogenic but appeared immunogenically identical to PEDV. The third isolate, obtained from the laboratory, which several years previously had supplied material for demonstration of the Stockholm agent, differed from PEDV in pathogenic and immunogenic properties. Serological examinations of paired rabbit sera did not indicate any antigenic relationship between PEDV and representative members of the two mammalian coronavirus antigenic groups. It is concluded that the aetiological agent of PED is a virus not belonging to the coronaviridae.

Aleutian disease in ferrets.

When 32 antibody-free ferrets were inoculated with the highly mink-virulent Utah-1 strain of Aleutian disease virus (ADV), most developed ADV antibody starting 15 days after infection, but the antibody titers were much lower than those seen in mink. Relatively small amounts of ADV were demonstrated in CRFK cell culture, using ferret spleen and lymph node homogenates only 4 to 10 days after experimental infection, but low-level viral persistence for 180 days was shown by mink inoculation. The ferrets inoculated with the Utah-1 strain of ADV did not develop elevated gamma globulin levels, but did have mild tissue lesions. Forty-two percent of a group of 214, approximately 1-year-old, recently pregnant, female ferrets were found to have antibody to ADV. An analysis of the serum proteins of the ferrets with ADV antibody showed that they had a significant, but mild, elevation of their serum gamma globulin. Serial ferret-to-ferret transmission of a ferret strain of ADV by inoculation of spleen homogenates was demonstrated, and some of these ferrets developed liver lesions. Mink inoculated with ferret ADV made antibody, but did not develop hypergammaglobulinemia or tissue lesions. Although both ferret and mink strains of ADV replicate and persist in the ferret, they fail to cause severe disease of the type usually seen in the closely related mink. Mink and ferret ADV strains appear to be biologically distinct.

Serial propagation of astrovirus in tissue culture with the aid of trypsin.

Astrovirus could be serially passed at least 13 times in primary human embryo kidney (HEK) cells when 10 micrograms/ml of crystalline trypsin was incorporated in a serum-free maintenance medium. In the presence of trypsin the virus was also passed and adapted to a continuous line of rhesus monkey kidney cells (LLCMK2) and primary baboon kidney (PBK) cells in which it was passed 25 and 16 times respectively, without evidence of diminishing infectivity. Attempts to adapt the virus to other cell lines (Vero, Hep II, MRC-5, BHK and HRT-18) were unsuccessful. After 11 passages in HEK cells, a titration of virus grown in different concentrations of trypsin showed that virus propagation was still trypsin-dependent.

Serological relationships between rotaviruses from different species as studied by complement fixation and neutralization.

Human, piglet, mouse, foal, lamb, calf and rabbit rotaviruses all infected, but could not readily be subcultured in LLC MK2 cells. Cells infected with mouse and calf rotaviruses reacted by indirect immunofluorescence (FA) with convalescent serum from children, piglets, mice, foals, lambs, calves or rabbits, taken after rotavirus infection. Human, calf, piglet, mouse and foal rotaviruses reacted with human, calf, mouse, foal and lamb convalescent serum by complement fixation (CF). It was not possible to distinguish between different rotaviruses by CF or FA. Neutralization tests, however, detected species-specific rotavirus antigens. Any virus was neutralized by a much higher dilution of homologous species convalescent serum than by any heterologous serum. With the exception of the mouse virus there was very little cross reaction. However, in sera with a very high neutralizing titre for the homologous virus the titre was proportionately raised against heterologous virus. It is, therefore, now possible to type to species an unknown rotavirus by a neutralization test in LLC MK2 cells using convalescent serum from each species.

Morphological, cytological and biological observations on viruses isolated from patients with subacute thyroiditis de Quervain.

New data on viruses isolated from patients with subacute thyroiditis de Quervain are reported. Characteristic morphological, cytological, some physico-chemical and biological features of the isolated viruses are described. A possible role of these viruses in human and animal health disorders is discussed. The isolated viruses remain unclassified so far.

Transmissible mink encephalopathy: infectivity of corneal epithelium.

Corneal epithelium from hamsters dying of transmissible mink encephalopathy contained a virus titer of 10-4.8 times the 50 percent lethal dose (10-4.8 LD50) per 0.05 milliliter when assayed as a cell suspension derived directly from the infected animal. After one passage in tissue culture, an equivalent concentration of cells contained only 10-0.8 LD50 per 0.05 milliliter.. It is concluded that corneal tissues are infectious; the infectivity may be mainly associated with free nerve endings. However, the most important immediate inference is that corneas from human beings affected with Creuzfeldt-Jakob disease are likely to be lethal if transplanted to healthy recipients.