Effect of relative humidity on the airborne survival of rotavirus SA11.

Rotavirus SA11, suspended in tryptose phosphate broth with 2.5 mg of rhodamine B per ml, was aerosolized (Collison nebulizer) into a rotating drum, and the aerosols were held at 20 +/- 1 degree C with the desired relative humidity (RH). An all-glass impinger with tryptose phosphate broth was used to collect 1-min (5.6-liter) samples of air from the drum. The virus was found to survive best at medium (50 +/- 5%) RH, where its half-life was nearly 40 h. The half-life of the virus at the low (25 +/- 5%) RH level was about 9 h. Even at 72 h of aerosol age, 45 and 21% of the infectious virus remained detectable in the air at the medium and low RH levels, respectively. The high (80 +/- 5%) RH level was found to be the least favorable to the survival of the virus, since 50% of the infectious virus became undetectable within 2 h of aerosolization. In a separate experiment at the midrange RH, 3% of the infectious virus was detectable in the drum air after 223 h (9 days) of aerosol age. Rotaviruses could, therefore, survive in air for prolonged periods, thus making air a possible vehicle for their dissemination.     

Effect of relative humidity on the airborne survival of rhinovirus-14

Rhinovirus-14, suspended in tryptose phosphate broth supplemented with uranine (physical tracer) and an antifoam, was aerosolized by use of a Collison nebulizer. The aerosols were held in a rotating drum with the relative humidity at either the low (30 +/- 5%), medium (50 +/- 5%), or high (80 +/- 5%) level at 20 +/- 1 degrees C. An all-glass impinger was used to recover the virus from the air in the drum, with the first air sample being collected after a 15-min period of aerosol stabilization. Subsequent air samples were withdrawn at 2, 4, 8, and 14 h after stabilization of the aerosol. At the low and medium relative humidity levels, the infectivity of the airborne virus was rapidly lost and less than 0.25% could be detected in the first air sample. At the high RH level, however, the airborne virus had a half-life of 13.7 +/- 1.91 h and nearly 30% of the input infectious virus could be detected in the drum air even after 24 h of aerosolization. These findings suggest that under certain environmental conditions, notably high relative humidity, air may act as a vehicle for the spread of rhinovirus infections.     

Effect of relative humidity, atmospheric temperature, and suspending medium on the airborne survival of human rotavirus

The Wa strain of human rotavirus, grown in MA-104 cells, was suspended either in tryptose phosphate broth or feces from a case of rotaviral diarrhea. It was then aerosolized into a rotating drum using a Collison nebulizer. The drum air was sampled using an all-glass impinger containing tryptose phosphate broth as collecting fluid. At 20 +/- 1 degree C, the virus aerosolized from tryptose phosphate broth was found to survive best at 50 +/- 5% relative humidity, where its half-life was 44.2 +/- 6.3 h. At 30 +/- 5% and 80 +/- 5% relative humidity, the half-life of the virus was 24.5 +/- 3.5 and 3.8 +/- 1.0 h, respectively. At 6 +/- 1 degree C, the airborne survival of the virus at the mid and low relative humidity levels was further enhanced, but at the high relative humidity it remained very similar to that seen at 20 +/- 1 degree C. When aerosols of fecally suspended human rotavirus were held at 20 +/- 1 degree C with 50 +/- 5% relative humidity, nearly 80% of the airborne virus particles remained infectious even at the aerosol age of 24 h. These findings may help in our understanding of the epidemiology of rotaviral infections.     

Effects of temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne Gumboro vaccine virus

Survival of airborne virus influences the extent of disease transmission via air. How environmental factors affect viral survival is not fully understood. We investigated the survival of a vaccine strain of Gumboro virus which was aerosolized at three temperatures (10°C, 20°C, and 30°C) and two relative humidities (RHs) (40% and 70%). The response of viral survival to four metrics (temperature, RH, absolute humidity [AH], and evaporation potential [EP]) was examined. The results show a biphasic viral survival at 10°C and 20°C, i.e., a rapid initial inactivation in a short period (2.3 min) during and after aerosolization, followed by a slow secondary inactivation during a 20-min period after aerosolization. The initial decays of aerosolized virus at 10°C (1.68 to 3.03 ln % min(-1)) and 20°C (3.05 to 3.62 ln % min(-1)) were significantly lower than those at 30°C (5.67 to 5.96 ln % min(-1)). The secondary decays at 10°C (0.03 to 0.09 ln % min(-1)) tended to be higher than those at 20°C (-0.01 to 0.01 ln % min(-1)). The initial viral survival responded to temperature and RH and potentially to EP; the secondary viral survival responded to temperature and potentially to RH. In both phases, survival of the virus was not significantly affected by AH. These findings suggest that long-distance transmission of airborne virus is more likely to occur at 20°C than at 10°C or 30°C and that current Gumboro vaccination by wet aerosolization in poultry industry is not very effective due to the fast initial decay.     

Gene protein products of SA11 simian rotavirus genome

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.     

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.     

Structural polypeptides of simian rotavirus SA11 and the effect of trypsin

Analysis of purified simian rotavirus has shown that it contains fewer structural polypeptide classes than previously reported. Two polypeptides (molecular weights, 62,000 and 28,000) commonly found in purified rotaviruses were, in fact, produced by cleavage of a larger structural polypeptide (molecular weight, about 88,000) by trypsin, which is usually employed to increase the yield of rotaviruses in tissue culture. Trypsin-uncleaved, double-shelled rotaviruses are probably composed of only five polypeptide classes; three in the inner layer, and two in the outer layer.     

Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.     

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

Effect of relative humidity and temperature on the survival of Clausenia josefi (Hymenoptera: Encyrtidae)

The survival of adults of Clausenia josefi was investigated as a function of temperature and relative humidity. Natural severe climatic conditions were simulated and the adaptability of the parasites to such conditions was determined.

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Résumé Clausenia josefi, parasite interne de Planococcus vitis, cochenille qui attaque la vigne, a été trouvé dans la zone aride du sud d'Israël (Negev). La moyenne de la température maximum en été (Juillet-Août) est dans cette région de 34° et l'humidité relative moyenne à 14h00 est de 40%. Le début de l'été (Mai-Juin) est la période la plus sévère, quant à l'instabilité des conditions climatiques. L'apparition saisonnière de C. josefi est caractérisée par 2 pics, un premier en Juin et un second, plus petit, en Octobre-Novembre. L'objet de cette étude était de déterminer les effets de la température et de l'humidité sur la longévité des adultes de Clausenia josefi et d'expliquer les raisons de la réduction de ses populations en été.A 28°, des humidités relatives comprises entre 20% et 80% n'ont aucun effet mesurable sur la survie des adultes de C. josefi (Fig. 1). L'effet d'une baisse d'humidité relative (20%) est plus prononcé pour des durées d'exposition de 2h et plus à de hautes températures (43°) (Fig. 3). A 43°, l'augmentation de l'humidité relative provoque un accroissement marqué de la survie (Fig. 4).Les conditions extrêmes dans les vignobles en Mai-Juin, même pendant de courtes périodes de temps, pourraient affecter les populations de ce parasite entomophage. Le fait que les populations de l'hôte sont à un maximum entre Mai et Juin, masque probablement les effets défavorables des conditions extrêmes sur l'entomophage, de sorte que le pic de ses populations apparaît à ce moment. Puisque les conditions climatiques sont moins extrêmes en Juillet et Août, la réduction des populations du parasite pendant ces mois n'est pas due aux effets climatiques mais plutôt à la réduction des populations de son hôte.

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Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan. Israel. 1972 Series, No. 2226-E.     

Synthesis of the outer-capsid glycoprotein of the simian rotavirus SA11 in Escherichia coli

The major outer layer protein, VP7, of the simian rotavirus SA11 has been synthesized in Escherichia coli, under the control of the lac promoter, as a fusion polypeptide with beta-galactosidase (beta Gal). The viral protein in the hybrid polypeptide is missing its N-terminal hydrophobic region and 26 amino acids (aa) at its C-terminus; it is flanked at both ends by beta Gal sequences. We have purified the hybrid 145-kDa protein by affinity chromatography using a column specific for beta Gal. Unexpectedly, a second protein of 118-kDa was also specifically bound to the column. N-terminal aa sequence analysis of these two proteins showed that the 145-kDa protein represented the expected fusion product, whereas the 118-kDa protein was apparently the result of initiation of translation at an internal site close to the 3' end of the viral sequence, in the chimeric mRNA. Each of the two polypeptides represented about 2 to 3% of the total protein of the recombinant-plasmid-carrying bacteria. When a bacterial lysate enriched for the hybrid polypeptides was injected into mice, it induced neutralizing antibodies to SA11 rotavirus.