Inactivation of Indigenous Viruses in Raw Sludge by Air Drying

Air drying of raw sludge caused inactivation of indigenous viruses. A gradual loss of infectivity occurred with the loss of water until the solids content reached about 80%. A more rapid decline of viral infectivity occurred with further dewatering.     

Infectivity-destroying Effect of Humidity for Dried Coliphage T1

Infectivity of dried coliphage T1 has been measured as a function of humidity, temperature, and atmospheric pressure. Loss of infectivity by a factor of 10(4) was caused by water vapor of approximately 40 to 85% saturation when the microorganisms were kept for 3 days at 34 C in evacuated containers. At humidities below 40% and above 90% saturation, no loss of infectivity occurred. At a temperature of 24 C, the infectivity loss was 20-fold. When the virus preparation was kept at 34 C and atmospheric pressure, some loss of infectivity was also found at humidities below 40% and above 90% saturation. Damage to tail proteins or to the phage chromosome is considered as a possible explanation for the inactivation.     

Two year study on the infectivity of Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus

Ichthyophthirius multifiliis Fouquet (Ich) is a fish parasite that causes serious economic loss for aquaculture. A major difficulty in the maintenance of single isolates of Ich for research purposes is the loss of infectivity. After an unknown number of passages or infection cycles the Ich isolate loses its infectivity. This study determined the infectivity of an Ich isolate during 105 infection cycles in channel catfish Ictalurus punctatus over a 2 yr period. The mean percentage of fish infected by Ich, the infection levels and the time to trophont emergence were each compared after 4 cyclic periods: 1-25, 26-60, 61-90 and 91-105 Ich cycles. Results of this study demonstrated that Ich was significantly more infective (p < 0.05) at 1-25 than 26-105 cycles. Channel catfish were infected at a ratio of 1 infected fish to 8 naive fish at 1-25 and 26-60 cycles. A higher infection ratio occurred at 61-90 and 91-105 cycles. Trophont emergence was noted to be significantly longer at 91-105 compared to 1-25 cycles, during 7 and 5 d respectively, at 23.4 +/- 1.1 degrees C. The results of the present study indicate that the infectivity of I. multifiliis started to decrease after 25 infection cycles and was predominant in the single Ich isolate at 61-90 and 91-105 cycles.     

Survival of T3 Coliphage in Varied Extracellular Environments. I. Viability of the Coliphage During Storage and in Aerosols

The objective of this study was to determine the feasibility of using airborne T3 coliphage as a viral tracer in microbial aerosols. Although T3 coliphage was relatively stable when stored either at temperatures ranging from 21 to 37 C or in the frozen state at -20 C, there was a 2-log loss in infectivity when stored for 72 days at 4 C. Either agitation of stored coliphage suspensions held at 31 C or wide fluctuations in storage temperature produced an increased loss of infectivity. In the airborne state, freshly prepared coliphage and stored coliphage behaved similarly, with survival diminishing as the relative humidity (RH) was lowered. The greatest loss occurred during the first five min following aerosolization. The results showed that only under certain conditions of temperature and relative humidity can T3 coliphage be used as a satisfactory aerosol tracer.     

Persistence of Salmonella typhimurium on Fabrics

The objective of this study was to determine the feasibility of using airborne T3 coliphage as a viral tracer in microbial aerosols. Although T3 coliphage was relatively stable when stored either at temperatures ranging from 21 to 37 C or in the frozen state at -20 C, there was a 2-log loss in infectivity when stored for 72 days at 4 C. Either agitation of stored coliphage suspensions held at 31 C or wide fluctuations in storage temperature produced an increased loss of infectivity. In the airborne state, freshly prepared coliphage and stored coliphage behaved similarly, with survival diminishing as the relative humidity (RH) was lowered. The greatest loss occurred during the first five min following aerosolization. The results showed that only under certain conditions of temperature and relative humidity can T3 coliphage be used as a satisfactory aerosol tracer.     

Efficacy of chemical disinfectants against snakehead rhabdovirus

The susceptibility of snakehead rhabdovirus to treatment at 20°C with 5 commercially available disinfectants was examined. No reduction in virus infectivity occurred following exposure to 5 ppm malachite green for 6 hours. Treatment of infective cell culture fluids with 2% formalin resulted in > 99.9% reduction in virus titre within 5 minutes and complete inactivation within 30 minutes, but a negligible loss in infectivity after exposure to 0.025% formalin for 1 hour. Suspensions of virus in distilled water were completely inactivated within 5 minutes by 12.5 ppm chlorine, 50 ppm iodine, or a 1:2000 dilution of a peroxygen disinfectant. In the presence of serum in infective cell culture fluids, however, > 50 ppm chlorine was required to inactivate the agent and no measurable reduction in infectivity was observed following treatment with 500 ppm iodine for 30 minutes.     

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Plasmodium berghei: Adaptation of a mouse-adapted strain to the Mongolian jird (Meriones unguiculatus); infectivity and immunogenicity

A strain of Plasmodium berghei (K 173) was initially found to be almost noninfective but highly immunogenic for the Mongolian jird (Meriones unguiculatus. The parasite was adapted to this host through serial passage of infected blood. The adapted parasite is 97% lethal to jirds. During adaptation, antigenic changes or shifts in the antigenic profile were found to have occurred, as shown by differences in precipitins raised in rabbits by the original and the adapted strain, as well as by an increased preparent period indicative of an 100-fold loss of infectivity for the mouse after adaptation. Immunogenicity was found to depend upon the continued survival of some (noninfective) parasites in the host, and appears to be determined by antigens different from those responsible for infectivity. Vaccination with nonviable antigens led to the production of some protective antibody, but also to blocking antibody and the retardation of the development of immunity.     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

Enhancement of Parainfluenza 2 Virus Hemagglutinin

A comparison was made of the three previously described methods of enhancement of parainfluenza 2 virus hemagglutinin (HA): ultrasonic oscillation, adsorption to erythrocytes and elution (AE), and Tween-ether (TE) treatment. TE treatment resulted in the maximal elevation of HA titer, complete loss of infectivity, and the maximal loss of heat stability of HA. Ultrasonic oscillation caused the least elevation of HA titer, minimal loss of infectivity, and the least loss of heat stability of both HA and infectivity. The effect of AE remained intermediate. It is suggested that the changes produced by these three methods are varying degrees of disruption of virus particles. Growth studies on parainfluenza 2 virus are also presented.     

Origin of Reovirus Oligo(A)

Reovirus contains about 1,200 molecules per virion of oligo(A) of chain length 10 to 15 nucleotides in addition to the 10 double-stranded genome segments. Virions purified from infected BHK, HeLa, or L cells had similar amounts of oligo(A) of the same composition, indicating that it is a virus-specific product. Although conversion of virions to cores by chymotryptic digestion resulted in an almost complete loss of oligo(A) and a marked decrease in infectivity, the infectivity could be partially restored by adsorbing cores to cells in the presence of Kaopectate. Core-infected cells yielded virions that contained a normal complement of oligo(A). The results indicate that oligo(A) is not essential for infectivity or required as a primer/template for its own synthesis.     

Formalin inactivation of vesicular stomatitis virus impairs T-cell- but not T-help-independent B-cell responses.

The effects of formalin on the infectivity and immunogenicity of vesicular stomatitis virus (VSV) serotype Indiana were investigated. We found that formalin inactivation of VSV prevents infection of Vero cells in a concentration- and time-dependent manner, as shown by fluorometric cell analysis and inhibition of plaque formation. Inactivated VSV failed to induce significant cytotoxic T-lymphocyte responses in vivo or after restimulation in vitro. In contrast, the early immunoglobulin M (IgM) response, which is T help independent in the VSV system, was unaltered, suggesting normal antigenicity for and induction of B cells. However, no switch to IgG occurred, demonstrating failure of induction of T help. If cross-reactive T help was provided by previous infection with a second serotype of VSV (New Jersey), the IgG response was almost completely restored, confirming that the absence of IgG was due to lack of T help. A formalin-treated preparation of glycoprotein of VSV led to a delayed but otherwise normal IgG response, whereas treatment of VSV with UV light or beta-propiolactone reduced IgG titers to the same extent as did formalin. These results suggest that loss of infectivity and the ensuing lack of amplification of viral antigens of formaldehyde-inactivated VSV is the major factor impairing induction of specific T-helper cell responses.     

Mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.     

Characterization of an RNA-a protein complex isolated from the RNA bacteriophage M12.

The properties of an RNA-A protein complex isolated from the RNA bacteriophage M12 are described. The molar ratio of RNA to A protein in the complex is estimated to be 1:1. In sucrose gradients, the complex sediments like free RNA molecules. In contrast to RNA alone, which can only infect spheroplasts, the RNA-A protein complex infects intact Escherichia coli cells and produces infectious progeny particles like the original phage. Evidence is presented that the infection of the host cells by the complex takes place via F pili. All of the infectivity disappears if the ionic bonds of RNA to A protein in the complex are dissociated in 0.5 M sodium chloride buffer at 37 degrees C. Furthermore, the kinetics of complex dissociation and loss of infectivity are the same, implying that the binding of A protein to the RNA is a prerequisite for infectivity on intact host cells.     

High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Background

The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins.

Methods

Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols (<1 µM, 1–4 µM, and >4 µM aerodynamic diameters) adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay.

Results

Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed.

Conclusion

At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles <4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity >40% will significantly reduce the infectivity of aerosolized virus.     

Physical Studies on Pox Viruses: I. Inactivation of Vaccinia Virus Infectivity With Low-Energy Electrons

Vaccinia virus was irradiated in vacuo with low-voltage electrons of restricted ranges. It was found that the pock-forming ability of the virus was not decreased after bombardment with electrons penetrating 100 A beneath the virus surface. There was very slight reduction in titer with large doses of electrons penetrating 330 A, but a sudden marked drop in infectivity occurred after exposure to electrons penetrating 500 to 700 A. Electrons of higher energies, including those capable of penetrating the virus particle completely, did not produce significant further fall in infectivity titer. It is concluded that a highly radiation-sensitive unit essential for pock formation is situated 500 to 700 A beneath the surface of the virus particle, possibly in the form of a shell. The relation of this finding to the known structure of the virus and to other radiation data on the dimensions of the infectious unit is discussed.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.