Incidence of amantadine-resistant influenza A viruses in sentinel surveillance sites and nursing homes in Niigata, Japan

We surveyed the incidence of amantadine-resistant influenza A viruses both at sentinel surveillance sites and at nursing homes, and verified their types of change by partial nucleotide sequence analysis of the M2 protein. Fifty-five influenza A viruses from 27 sentinel surveillance sites during six influenza seasons from 1993 to 1999, and 26 influenza A viruses from 5 nursing homes from 1996 to 1999 were examined for susceptibility to the drug by virus titration in the presence or absence of amantadine. While amantadine-resistant viruses were not found in sentinel surveillance sites, a high frequency of resistance (8/26, 30.8%) in nursing homes was observed. Resistant viruses can occur quickly and be transmitted when used in an outbreak situation at nursing homes, where amantadine is used either for neurologic indications or for influenza treatment. Eight resistant viruses had a single amino acid change of the M2 protein at residue 30 or 31. In vitro, all 11 sensitive viruses turned resistant after 3 or 5 passages in the presence of 2 microg/ml amantadine, and they showed an amino acid change at residue 27, 30, or 31. The predominant amino acid substitution in the M2 protein of resistant viruses is Ser-31-Asp (a change at 31, serine to asparagine). The results indicate that a monitoring system for amantadine-resistant influenza viruses should be established without delay in Japan.     

Studies on Parainfluenza Virus Infections among Infants and Children in Sendai

A cell line sensitive enough for the recovery of all parainfluenza viruses and free of simian virus contamination frequently occurring in monkey kidney cells was sought. The VERO cell obtained from African monkey kidney was found suitable for the initial isolation of types 1, 2 and 3 parainfluenza viruses, although the cells did not always allow the successive transfer. Mixed cultures of VERO and HEp-2 cells were also useful in the recovery of various respiratory viruses including parainfluenza viruses. The characteristics of hemagglutinins of parainfluenza viruses were examined, and type 2 parainfluenza and SV5 viruses agglutinated both guinea pig and green monkey erythrocytes at 36 C, whereas types 1 and 3 parainfluenza viruses agglutinated only guinea pig erythrocytes. Thus parainfluenza viruses were divided into two groups by the presence or absence of hemagglutinins for green monkey erythrocytes. Identification of these parainfluenza isolates, employing HI microtechnique was simple and reliable, even with the first passage harvest, when guinea pig erythrocytes were used and the test read at 36 C. Specific standard antisera for these parainfluenza viruses were prepared by immunizing chickens intravenously and bleeding within a short period. These type-specific antisera were useful for the identification of parainfluenza isolates by HI test.     

Sampling conditions for reliable routine detection by enzyme-linked immunosorbent assay of three ilarviruses in fruit trees

Enzyme-linked immunosorbent assay (ELISA) was used to test plum trees for prune dwarf (PDV), Prunus necrotic ringspot (NRSV) and apple mosaic (ApMV) viruses, cherry trees for PDV and NRSV, and apple trees for ApMV. Optimum conditions were determined for sampling in large-scale surveys for these viruses. All three viruses were detected throughout the growing season in individual samples of young leaves, or twigs with newly formed buds. However, when single infected leaves were combined with different numbers of healthy leaves, tests were most successful for all three viruses early in the growing season. PDV was detected in 1/40 (infected/total leaves) cherry leaves in April and May and 1/40 plum leaves until July, whereas NRSV was detected in 1/20 cherry leaves until July and 1/20 plum leaves until May. ApMV was detected in 1/20 apple or plum leaves until June but was detected less readily in mature leaves after June than either NRSV or PDV. There was no evidence of uneven distribution of virus infection in the trees. The viruses were detected in leaf samples kept for 8 wk at 3°C but freezing was less reliable for storage especially with ApMV. ApMV was detected in tests on plants held for several weeks at 25°C, and PDV and NRSV in plants held at 30°C.     

Effect of Temperature and Relative Humidity on Survival of Airborne Columbia SK Group Viruses

Three strains of the Columbia SK (Col-SK) group of viruses [Mengo, Maus Elberfeld (ME), and Col-SK viruses] have been studied in the airborne state. All three strains were found to give identical aerosol decay patterns at 16 or 26 C, when held at the same relative humidity (RH). During the first 5 min of aerosol storage time at 16 C, virus inactivation was RH-dependent, with survival maximal at either high (greater than 80%) or low (less than 5%) RH. After 5 min at 16 C, further inactivation, regardless of RH, was insignificant. At 26 C, the effect on survival of RH between 40 and 60% was even more pronounced than at 16 C, and continued after 5 min through 6 hr. Results of this study indicated that the inactivation of airborne Col-SK group viruses was similar to that of other ribonucleic acid (RNA) viruses, particularly poliovirus. Since members of the Col-SK group are picornaviruses, they may well serve as an aerosol model representative of small, ether-resistant, single-stranded RNA viruses.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

Effects of influenza, mumps, and western equine encephalitis viruses on fetal rhesus monkeys (Macaca mulatta).

Pregnant Rhesus monkeys were infected via instillation of influenza, mumps and western equine encephalomyelitis viruses respectively into the amniotic sacs at approximately 90 days gestation to determine if fetal infections would occur. Virus was recovered from fetal tissues after seven days in 100% of the exposed animals. Thus, the viruses are capable of causing fetal infection. Rhesus monkey fetuses were inoculated with influenza, mumps and WEE viruses by the direct intracerebral route at approximately 90 days gestation to determine possible teratogenicity of the viruses. influenza virus caused no malformations or measurable fetal effects. Mumps virus resulted in significant fetal mortality. WEE virus resulted in a 100% incidence of encephalitis and hydrocephalus. Thus, mumps and WEE viruses are teratogens in primates and are potential teratogens of man.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus

A number of factors affect the infectivity of retroviruses. The effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus (MoMuLV) was determined in this work. The ecotropic MoMuLVs were found to remain infectious at a narrow pH range from 5.5 to 8.0. Our experiments indicated that the viruses were inactivated swiftly at lower or higher pH. Within 5 min of exposure to pH 4 about 95% of the viruses lost infectiousness. The viruses were completely inactivated after exposure to pH < 3 or pH >11 for 5 min. The inactivation of MoMuLV was irreversible. Electron microscopy revealed that ecotropic MoMuLV remained round-shaped at pH between 7.0 and 5. They became irregular with a convex head at pH < 4. At pH 2, virtually all virion particles were penetrated by stains, causing the accumulation of heavy metals inside the particles. The penetration of heavy metal inside the particles indicated the disassociation of the lipid bilayer of the viruses at low pH. A FACS-based screening strategy for selecting high-titer retrovirus producing cell lines is also presented in this report.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater.

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

SOME EFFECTS OF HIGH TEMPERATURE ON THE SUSCEPTIBILITY OF PLANTS TO INFECTION WITH VIRUSES

When plants were kept at 36°C. for some time before inoculation, their susceptibility to infection by five mechanically transmissible viruses was greatly increased. When kept at 36° after inoculation, fewer local lesions were produced than at lower temperatures, but the effects of the post-inoculation treatment differed with different viruses. Tomato spotted wilt and tobacco mosaic viruses multiply in plants at 36°, and the post-inoculation treatment reduced the local lesions they caused to numbers that varied between 10 and 90% of the control; these two viruses also have large thermal coefficients of heat inactivation. By contrast, tobacco necrosis, tomato bushy stunt and cucumber mosaic viruses, were much affected by post-inoculation treatment, lesion formation being completely prevented by exposure to 36° for a day or more. These three viruses appear not to multiply in plants at 36°, and although they have high thermal inactivation points, they have small temperature coefficients of thermal inactivation.
The extent to which lesion formation was affected by pre- or post-inoculation exposure of plants to 36° depended not only on the length of the treatment, but also on the physiological condition of the plants.
The symptoms of infected plants changed considerably if kept at 36°. At 36° Nicotiana glutinosa , inoculated with tobacco mosaic virus, gave chlorotic local lesions instead of necrotic ones, and became systemically infected. When systemically infected plants were brought to ordinary glasshouse temperature, the infected tissues all collapsed and died in a day.     

Concentration of Viruses from Sewage and Excreta on Insoluble Polyelectrolytes

The concentration of viruses from sewage by adsorption on and elution from an insoluble cross-linked copolymer of maleic anhydride is described. Viruses either added to sewage or naturally contained in sewage were preferentially adsorbed to this polyelectrolyte at a pH range of 5.0 to 6.0 and were eluted at pH 8.0 to 9.0. In a 2-month survey of viruses in sewage in the spring (April to May 1968), when viruses are at low levels, efficient and economical detection of these agents was accomplished with the polyelectrolyte-concentration method. This method lends itself to the detection of viruses present in minute amounts in fecal samples, urine, sewage, and other natural waters. Large volumes of these fluids can be treated with the polymer described, and virus can be concentrated sufficiently for detection.     

Stability of feline caliciviruses in marine water maintained at different temperatures

Since human caliciviruses are responsible for viral gastroenteritis transmitted by contaminated foods and the viruses barely propagate in cell culture, feline caliciviruses were employed as a model for the measurement of their stability in marine water. Survival of four strains of feline calicivirus in marine water was measured when the seed viruses were diluted 1/10 with marine water and maintained at 4 degrees C, 10 degrees C, and 20 degrees C respectively. Among the virus strains studied, a considerable amount of infective viruses remained at 10 degrees C or lower temperature conditions even for a period of 30 days.     

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

A dendrogram of plant viruses.

To facilitate the recognition of plant viruses with similar characteristics a dendrogram of characterized viruses was constructed. The sequence of criteria included: type of nucleic acid; single or double stranded; presence or absence of lipid envelope; helical or nonhelical symmetry; and divided or single genome. Nonhelical RNA viruses with divided genomes were further divided into viruses with one or more than one capsid size. Those with one capsid size were subdivided into viruses with one or more than one sedimenting component. Nonhelical RNA viruses with a single genome were divided according to their RNA size, and their sensitivity to sodium dodecyl sulfate and ethylenediaminetetraacetic acid.     

The Physical State of Viral Nucleic Acid and the Sensitivity of Viruses to Ultraviolet Light

Ultraviolet light action spectra in the range 2250 to 3020 A have been determined for the plaque-forming ability of the following bacteriophage and animal viruses: T-2, ϕx-174, R-17, fr, MS2, 7-S, fd, vesicular stomatitis, vaccinia, encephalomyocarditis, reovirus-3, and polyoma. Absolute quantum yields for the plaque-forming ability of MS2, fr, fd, ϕx-174, and T-2 were determined over the range 2250 to 3020 A. Relative quantum yields for plaque-forming ability indicated that viruses with single-stranded nucleic acid were on the average ten times more sensitive to UV than double-stranded viruses. In addition for ten of the twelve viruses a relation existed between the shape of their action spectra and the stranded state of their nucleic acid. The ratio of the inactivation cross-section at 2650 A to that at 2250 A for these viruses was 1.0 for single-stranded viruses and 2.0 for viruses with double-stranded nucleic acid. The above relations were dependent on the stranded state of the nucleic acid not the ribose or deoxyribose form of the sugar present.     

Detection of Potato Viruses Y and A in Tubers by Enzyme-Linked Immunosorbent Assay after Natural and Artificial Break of Dormancy

The effects of natural and artificial break of dormancy on the detection of potato viruses Y (PVY) and A (PVA) in tubers of field-grown potato cultivars with primary and secondary infections were studied by enzyme-linked immunosorbent assay (ELISA). Both viruses were at low concentration and unevenly distributed in dormant -tubers. Four to six weeks after treatment with Rindite, maximum concentrations of PVY and PVA were detected at both tuber ends with generally higher values at the rose end than at the heel end. Slower and smaller virus increase and a retarded and/or limited virus translocation were observed in treated tubers of resistant cultivars. Consequently, detectability of PVY and PVA was higher in tubers of susceptible cultivars than in those of the resistant ones. Detection of both viruses was very difficult and unreliable in tubers after natural break of dormancy. The very low concentration of PVY and PVA in dormant tubers and their drastic multiplication in Rindite- treated tubers appear to be characteristic of potyviruses and are unique among the major potato viruses.     

Protective effect of cellular immunity in experimental Flavivirus infections

Experiments were conducted with the tick-borne encephalitis (TE) virus; confirmation of a protective action of cellular immunity in mice was obtained. Administration of sensitized splenocytes to the animals together with the virus was accompanied with an increase of their mean survival or with the reduction of mortality in comparison with control animals given nonimmune or destroyed cells. The protective action of the effector cells was not connected with the intensification of antibody formation in the recipients. A high specificity of cellular immunity was noted in experimental flaviviral infections. The presence of common antigens in the TE and Langat viruses was revealed with the acid of cross splenocyte migration inhibition test (CSMRT). There was also revealed a difference of these viruses from the viruses of yellow fever, Dengue type 2, or Sindbis. The results of studying of the specificity of cellular immunity in the CSMRT found confirmation in experiments with adoptive transfer of splenocytes. Cross protection was caused only by splenocytes sensitized to the TE and Langat viruses.     

Survival of enteric viruses adsorbed on glass microfibers during postal transport

The survival of enteric viruses (poliovirus type 1, Mahoney strain, and indigenous viruses of waste waters) has been studied after adsorption of the viruses (pH 3.5) on glass microfiber filters. After postal transport, the presence of the viruses was checked on the filters being soaked in a 3% beef extract solution (pH 7.5) either frozen or without protection against heat. Viruses were recovered at a rate of 59 to 65%. For qualitative studies, postal shipment of samples adsorbed on fiberglass may allow extension of a control system for enteroviruses in water.     

Isolation of virus strains from mosquitoes collected in Queensland, 1972-1976.

171,348 mosquitoes and 4,353 other arthropods collected at three centres in Queensland in 1972-1976 yielded 151 strains of 18 viruses. Culex annulirostris was the major source of virus isolation but 42 strains from Aedes normanensis indicate it to be a vector of importance. Ross River and Kokobera viruses were isolated at Kowanyama in the dry season, a finding of interest as being compatible with year-round survival in vector-vertebrate cycles. Culex fatigans has in part replaced Culex annulirostris in peridomestic breeding sites at Kowanyama; the infrequency of virus isolation from it suggests that this replacement may lower arbovirus infection rates. Twelve strains were identified as viruses antigenically distinct from any previously isolated in Australia or New Guinea: Ch16129, showed by the International Reference Centre for Arboviruses to be a previously undescribed member of the Simbu Group (Facey's Paddock virus), Ch16313 (Murweh), Ch19520 (Parker's Farm) and Ch19546 (little Sussex). The remaining strains were identified as viruses previously known in Australia, but included many new host or geographical records.