Infection by echoviruses 1 and 8 depends on the alpha 2 subunit of human VLA-2.

Anti-VLA-2 antibodies protected HeLa cells from infection by echoviruses 1 and 8 but not from infection by other echovirus serotypes. Echoviruses 1 and 8 bound to and infected nonpermissive hamster cells transfected with the alpha 2 subunit of human VLA-2. These results indicate that the human alpha 2 subunit is critical for infection by echoviruses 1 and 8 but that other echovirus serotypes must bind receptors other than VLA-2.     

Co-circulation and evolution of polioviruses and species C enteroviruses in a district of Madagascar

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5'-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3'-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3D(pol) coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.     

Polioviruses, Coxsackieviruses, and Echoviruses: Comparison of the Genomes by RNA Hybridization

Hybridization of single-stranded RNA from virions of human enteroviruses with denatured double-stranded RNA from infected cells indicates that a minimum of about 5% of the genome is shared by these viruses. Polynucleotide sequence relationships, furthermore, are consistent with the biologic classification into polioviruses, coxsackieviruses groups A and B, and echoviruses. In general, about 30 to 50% of the nucleotide sequences are shared by different serotypes of virus within each of these major groups, whereas among different groups less than 20% homology is observed. Coxsackievirus B4 appears to be more closely related to echoviruses than to group A coxsackieviruses, whereas polioviruses are only distantly related to any of the other agents.     

Optimal conditions for plaque assay of echoviruses in human embryonic lung fibroblast cells

The present paper gives optimal conditions for plaque assays tn human embryonic lung fibroblast cells with 45 echoviruses including 11 fresh isolates, covering 31 serotypes. All the viruses tested except echovirus type 23 produced plaques at a high titer in the same cells by the methods described in this communication. Plaque formations by a number of strains were markedly enhanced in size or number or both by the addition of polycation.     

Survival of enteroviruses and coliform bacteria in a sludge lagoon.

Enteroviruses associate with aerobically and anaerobically digested sludge were determined before the addition of the sludge to a sludge lagoon. The fate of sludge-associated viruses was followed during detention of sludge in the lagoon and after application of sludge to land for disposal. While digested sludge was being added to the lagoon, enteroviruses were readily detected in grab samples of sludge from the lagoon. Sludge-associated viruses dropped to low or undetectable levels after disposal of sludge on land and during periods when addition of digested sludge to the lagoon was suspended. Changes in the levels of fecal coliforms in the lagooned sludge paralleled changes in the numbers of enteroviruses. Enteroviruses were not detected in water from deep wells located on the sludge disposal site or near the lagoon. During the initial part of the study, poliovirus serotypes accounted for greater than 90% of the viruses identified. Later, poliovirus serotypes comprised less than 40% of the virus isolates, and echoviruses and Coxsackieviruses were the most common enteroviruses identified.     

A retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of Marseille, France (1985-2005)

Human enteroviruses (HEV) are frequent human pathogens and, associated in particular with large outbreaks of aseptic meningitis. Here, we have compiled a database of clinical HEV isolates from the Public Hospitals of Marseille, from 1985 to 2005. Amongst 654 isolates that could be characterized by complete sequencing of the VP1 gene, 98% belonged to species HEV-B; the most frequently isolated serotypes were Echovirus E30, E11, E7, E6 and E4. The high incidence of E30 and the recent emergence of E13 are consistent with reports worldwide and peak HEV isolation occurred mostly in the late spring and summer months. The proportion of echoviruses has decreased across the years, while that of coxsackieviruses has increased. Stool (the most frequent sample type) allowed detection of all identified serotypes. MRC5 (Human lung fibroblasts) cell line was the most conducive cell line for HEV isolation (84.9% of 10 most common serotype isolates, 96.3% in association with BGM (Buffalo green monkey kidney cells)). Previous seroneutralization-based serotype identification demonstrated 55.4% accuracy when compared with molecular VP1 analysis. Our analysis of a large number of clinical strains over 20 years reinforced the validity of VP1 serotyping and showed that comparative p-distance scores can be coupled with phylogenetic analysis to provide non-ambiguous serotype identification. Phylogenetic analysis in the VP1, 2C and 3D regions also provided evidence for recombination events amongst clinical isolates. In particular, it identified isolates with dissimilar VP1 but almost identical nonstructural regions.     

Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1-9 in chickens and ducks

Avian paramyxovirus (APMV) serotypes 1-9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2-9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2-9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2-9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2-9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1-9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.     

Evidence for frequent recombination within species human enterovirus B based on complete genomic sequences of all thirty-seven serotypes

The species Human enterovirus B (HEV-B) in the family Picornaviridae consists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5'-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strain's sequence shifted, indicating potential recombination points. These points are distributed in the 5'-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein.

Mutations in the 5' untranslated regions (5'-UTRs) of all three serotypes of the Sabin vaccine strains are known to be major determinants of the attenuation phenotype. To further understand the functional basis of the attenuation phenotype caused by mutations in the 5'-UTR, we studied their effects on viral replication, translation, and the interaction of the viral RNA with cell proteins. A mutation at base 472 (C472U), which attenuates neurovirulence in primates and mice, was previously found to reduce viral replication and translation in neuroblastoma cells but not in HeLa cells. This mutation reduced cross-linking of the poliovirus 5'-UTR to polypyrimidine tract-binding protein (pPTB) in neuroblastoma cells but not in HeLa cells. These defects were absent in a neurovirulent virus with C at nucleotide 472. When C472U and an additional mutation, G482A, were introduced into the 5'-UTR, the resulting virus was more attenuated, had a replication and translation defect in both HeLa cells and neuroblastoma cells, and cross-linked poorly to pPTB from both cell types. A neurovirulent revertant of this virus (carrying U472C, G482A, and C529U) no longer had a replication defect in HeLa and SH-SY5Y cell lines and cross-linked with pPTB to wild-type levels. The results suggest that the attenuating effects of the mutation C472U may result from an impaired interaction of the 5'-UTR with pPTB in neural cells, which reduces viral translation and replication. Introduction of a second mutation, G482A, into the 5'-UTR extends this defect to HeLa cells.     

Immunity to poliomyelitis in adult population of Moscow region. Role of vaccine and wild polioviruses in its formation

Assessment of immunity to poliomyelitis in adults from 8 towns of Moscow region was conducted. Low levels of population immunity against some serotypes of poliovirus in several towns have been found. At the same time, these levels were high and very high in other towns. Measurement of levels of strain-specific antibodies to vaccine and wild polioviruses demonstrated wide circulation of wild polioviruses during past decades which had significant influence on formation of immunity. Substantial number of non-immune adults represents favorable conditions for circulation of vaccine polioviruses after cessation of vaccination, which, in its turn, could result in reestablishment of their neurovirulent properties and subsequent reemergence of poliomyelitis.     

Two separate envelope regions influence induction of brain disease by a polytropic murine retrovirus (FMCF98).

The major determinants involved in neurological disease induction by polytropic murine leukemia virus FMCF98 are encoded by the envelope gene. To map these determinants further, we produced four chimeras which contained neurovirulent FMCF98 envelope sequences combined with envelope sequences from the closely related nonneurovirulent polytropic virus FMCF54. Surprisingly, two chimeric viruses containing completely separate envelope regions from FMCF98 could both induce neurological disease. Clinical signs caused by both neurovirulent chimeras appeared to be indistinguishable from those caused by FMCF98, although the incubation periods were longer. One neurovirulence determinant mapped to the N-terminal portion of gp7O, which contains the VRA and VRB receptor-binding regions, while the other determinant mapped downstream of both of the variable regions. Western blot (immunoblot) analyses and immunohistochemical staining of tissue sections indicated that the variations in neurovirulence of these viruses could not be explained by differences in either the quantitative level or the location of virus expression in the brain.     

Recombination in circulating enteroviruses

Recombination is a well-known phenomenon for enteroviruses. However, the actual extent of recombination in circulating nonpoliovirus enteroviruses is not known. We have analyzed the phylogenetic relationships in four genome regions, VP1, 2A, 3D, and the 5' nontranslated region (NTR), of 40 enterovirus B strains (coxsackie B viruses and echoviruses) representing 11 serotypes and isolated in 1981 to 2002 in the former Soviet Union states. In the VP1 region, strains of the same serotype expectedly grouped with their prototype strain. However, as early as the 2A region, phylogenetic grouping differed significantly from that in the VP1 region and indicated recombination within the 2A region. Moreover, in the 5' NTR and 3D region, only 1 strain of 40 grouped with its prototype strain. Instead, we observed a major group in both the 5' NTR and the 3D region that united most (in the 5' NTR) or all (in the 3D region) of the strains studied, regardless of the serotype. Subdivision within that major group in the 3D region correlated with the time of virus isolation but not with the serotype. Therefore, we conclude that a majority, if not all, circulating enterovirus B strains are recombinants relative to the prototype strains, isolated mostly in the 1950s. Moreover, the ubiquitous recombination has allowed different regions of the enterovirus genome to evolve independently. Thus, a novel model of enterovirus genetics is proposed: the enterovirus genome is a stable symbiosis of genes, and enterovirus species consist of a finite set of capsid genes responsible for different serotypes and a continuum of nonstructural protein genes that seem to evolve in a relatively independent manner.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants.

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.     

Genetic determinants of dengue type 4 virus neurovirulence for mice.

Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.     

Agglutination of African Primate and Rodent Erythrocytes by Adenoviruses, Reoviruses, and Enteroviruses

African nonhuman primate and rodent erythrocytes were tested for agglutination by adenoviruses, reoviruses, and enteroviruses. Squirrel erythrocytes were agglutinated by reovirus serotypes and adenovirus types 3, 11, 16, and 21. Adenoviruses also agglutinated brazza monkey erythrocytes to the same titers as those obtained with either rhesus or grey monkey cells. Prototype reovirus types 1 and 2 agglutinated grey monkey erythrocytes to much lower titers than either squirrel or human group O red cells. Among the enteroviruses tested, only echovirus types 7 and 12 agglutinated grey, red-tail, brazza, and rhesus monkey erythrocytes. The specificity of agglutination of squirrel, grey, and brazza monkey erythrocytes by reoviruses, echoviruses, and adenoviruses, respectively, was confirmed by hemagglutination-inhibition tests. The titers obtained were similar to those obtained with erythrocytes usually used in these tests. Erythrocytes of bush babies, potto unstriped grass mice, swamp rat, rusty-nosed rat, bush rat, harsh-furred mice, soft-furred rat, and giant rat were not agglutinated by adenoviruses, reoviruses, or enteroviruses.     

Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles.

The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.     

Sequence Determination of the (+) Leader RNA Regions of the Vesicular Stomatitis Virus Chandipura, Cocal, and Piry Serotype Genomes

Using 3'-end-labeled genome probes, cells infected with vesicular stomatitis virus Chandipura, Cocal, and Piry serotypes were shown to contain (+) leader RNAs of approximately 50 nucleotides in length. The nucleotide sequence of the leader RNA regions of these genomes was determined and compared with the previously reported sequences of both the (+) and (-) leader RNA regions of other vesicular stomatitis virus serotypes. Regions of strong conservation of nucleotide sequence among the various vesicular stomatitis virus serotypes suggest those nucleotides thought to be involved in control functions during vesicular stomatitis virus replication.     

Survival of polioviruses and echoviruses in Acanthamoeba castellanii cultivated in vitro

Cultures of Acanthamoeba castellanii, kept at room temperature in sterile Bacto-Casitone (Difco) medium, were artificially infected with vaccination poliovirus strains type 1 and type 3 and with echovirus type 4 and echovirus type 30. No remarkable virus cumulation was observed on the surface or inside amoeba cells during the observation period of 21 days. Amoeba-adsorbed viruses were impossible to remove by repeated washings. Virus neutralization with specific antisera showed that enteroviruses were most probably present only on amoeba surfaces. In contrast to amoeba-free virus suspension, echoviruses bound to amoebae and their cell pulp persisted even after 52 to 75 days. However, the tested amoeba species played only the role of a solids-like carrier in this survival of echoviruses.