Excretion of attenuated polioviruses in children vaccinated with live oral poliovirus vaccine

The excretion of attenuated polioviruses was studied in a group of nursery children vaccinated with 105TCD50 of each type of virus. The primovaccinated children were found to excrete type 1 poliovirus for 8 weeks, type 2 for 11 weeks after the vaccination with the type 1 + 2 bivaccine. Poliovirus type 1 as eliminated by 78% and type 2 by 98% of the vaccinees. The separately administered type 3 was detectable for 6 weeks and was isolated from 100% of the vaccinees. The highest per cent of children with type 1 excretion positivity was recorded at week 5, with type 2 positivity at week 1 and with type 3 positivity at week 2. The poliovirus excretion peaked early after the vaccination, the titres of the poliovirus type 2 were the highest. The children revaccinated next year with the type 1 + 2 bivaccine eliminated the respective types of virus 1 - 2 weeks; type 3 poliovirus was detectable for 6 weeks after revaccination and was excreted by the highest per cent of vaccines. The contact infections caused by the attenuated polioviruses developed in 9 from 22 children vaccinated previously. The excretion of polioviruses did not last longer than 1 week. The contact infections were most frequently caused by the poliovirus type 2. The examined children, particularly those vaccinated previously, turned out to excrete also other enteroviruses identified as Coxsakieviruses B 4 and B 5 and Echovirus 21. In the primovaccinated these viruses were isolated only from those with the negative excretion of polioviruses.     

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Aims:  To enhance the recovery of f₂ bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)₃-treated silica gel.
Methods and Results:  Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l⁻¹, pH 6·5, temperature 20∼30°C), the recoveries of Poliovirus I and f₂ from small-volume sewage (100 ml) were 110·76 ± 36·0% and 92·06 ± 8·65%, respectively ( P  < 0·05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85·65 ± 4·43% for f₂ and 88·73 ± 9·76% for poliovirus, significantly higher than the recoveries in the traditional methods ( P  < 0·05).
Conclusions:  PAC could enhance concentration efficiency of poliovirus and f₂ phage from sewage water.
Significance and Impact of the Study:  This method should significantly improve the recovery of viruses from sewage.     

Adsorption of Enteroviruses to Soil Cores and Their Subsequent Elution by Artificial Rainwater

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.     

Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland.

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Changes in population dynamics during long-term evolution of sabin type 1 poliovirus in an immunodeficient patient

The evolution of the Sabin strain of type 1 poliovirus in a hypogammaglobulinemia patient for a period of 649 days is described. Twelve poliovirus isolates from sequential stool samples encompassing days 21 to 649 after vaccination with Sabin 1 were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin 1 strain. Poliovirus isolates from the immunodeficient patient evolved gradually toward non-temperature-sensitive and neurovirulent phenotypes, accumulating mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. Analysis of plaque-purified viruses from stool samples revealed complex genetic and evolutionary relationships between the poliovirus strains. The generation of various coevolving genetic lineages incorporating different mutations was observed at early stages of virus excretion. The main driving force for genetic diversity appeared to be the selection of mutations at attenuation sites, particularly in the 5' noncoding region and the VP1 BC loop. Recombination between virus strains from the two main lineages was observed between days 63 and 88. Genetic heterogeneity among plaque-purified viruses at each time point seemed to decrease with time, and only viruses belonging to a unique genotypic lineage were seen from day 105 after vaccination. The relevance of vaccine-derived poliovirus strains for disease surveillance and future polio immunization policies is discussed in the context of the Global Polio Eradication Initiative.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Survival of enteric viruses adsorbed on electropositive filters.

Three viruses (poliovirus type 1, rotavirus SA-11, and bacteriophage f2) adsorbed on electropositive microporous filters survived at least 5 weeks at 4 degrees C. Poliovirus type 1 and bacteriophage f2 also survived at least 6 weeks at -20 degrees C. Rotavirus SA-11 was not recovered after 1 week at -20 degrees C. The stability of viruses adsorbed on electropositive filters may enable extensive monitoring of viruses in water.     

Viral pollution of the rivers in Toyama City

Viral pollution of the river water in Toyama City was surveyed during the two-year period from July 1979 to July 1981, and the ecology of viruses in the river water is discussed. Virus isolation from the river water samples, or from the water squeezed from cotton pads that were immersed in the stream for 3 days, was carried out by the "filter adsorption/elution" method. River waters were found to be contaminated with various species of enteric viruses, that is, poliovirus, echovirus, coxsackievirus, adenovirus, and reovirus. Poliovirus was isolated during the period immediately after the oral administration of polio vaccine, and coxsackie B virus was frequently isolated all year around. The enterovirus concentration in the river water was significantly high with a maximum of five plaque-forming units of coxsackie B2 virus per 250 ml. The species and type distribution of enteroviruses isolated from the river water coincided well with that of viruses isolated from inhabitants of Toyama Prefecture, with the exception of reovirus which was the largest population of virus species in the river water.     

Cleavage of VP1 and modification of antigenic site 1 of type 2 polioviruses by intestinal trypsin.

We have exposed 22 independent type 2 poliovirus isolates to human intestinal fluid and purified trypsin. In all cases the virus retained its infectivity, while polyacrylamide gel electrophoresis of viral proteins showed disappearance of the VP1 bands. Concomitantly, the viruses became resistant to antigenic site 1-specific monoclonal antibodies, indicating that the cleavage took place at the antigenic site 1. Sera from persons immunized solely with the inactivated poliovirus vaccine (IPV) neutralized intact type 2 polioviruses more readily than the corresponding trypsin-cleaved virus preparations. The ratio between the neutralization indices for the intact and trypsin-cleaved type 2 polioviruses was not significantly changed by a dose of trivalent oral poliovirus vaccine given to children previously immunized with IPV. These results indicate that while the antigenic site 1 of type 2 poliovirus is immunogenic in humans when IPV is used, the relative role of this antigenic site in human immunity appears to be less critical than that in the case of type 3 polioviruses. Before we obtained these results, only antigenic site 1 had been shown to be immunogenic in type 2 polioviruses.     

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water

The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3' end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.     

Poliovirus antigen chimeras.

Poliovirus, the aetiological agent of paralytic poliomyelitis, is arguably the best characterized of all animal viruses. Using recombinant-DNA technology, this information, together with the availability of infectious cDNA clones of the notably safe and efficacious live attenuated Sabin 1 vaccine strains of poliovirus, has enabled the creation of hybrid viruses (chimeras) possessing novel antigenicity. The potential applications of these 'epitope-presentation systems' include their use as immunogens, as antigens for serodiagnosis, and as vaccines.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.