Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Enhanced Detection of Western Equine Encephalitis Virus Plaque Variants in Crowded Cultures and Plaque Progeny—Potential Use in Genetic Studies

Enhanced detection of large-plaque variants of Western equine encephalitis virus in the presence of large numbers of small-plaque Western encephalitis virus was achieved by using higher bicarbonate concentrations in the agar-overlay medium and increasing concentrations of CO(2) in an enclosed environment during the time plaques were developed with neutral red. By this technique we were able to detect large-plaque mutants in cloned small-plaque suspensions for the first time and to show an increase in detectable plaque progeny in plaques developed by neutral red. Further increase in the number of progeny per plaque was determined by assaying extracts of agar overlaying cultures containing known numbers of plaques. The significance of these findings for potential use in mutational studies is discussed.     

Phenotypic and genotypic characterization of the neurovirulence and neuroinvasiveness of a large-plaque attenuated Japanese encephalitis virus isolate

The virulent phenotypes of Japanese encephalitis virus (JEV) can be divided into neuroinvasiveness (NI) and neurovirulence (NV). In this study, two JEV antigenic variants, CH2195LA (large-plaque, attenuated) and CH2195SA (small-plaque, non-attenuated), were passaged in suckling mice by intracerebral inoculation. Viruses at passage two and four were characterized in terms of NV and NI in weaning mice, as well as their in vitro growth characteristics in six cell lines. Following two brain-brain passages in mice, the attenuated variant CH2195LA was found to significantly restore the NV and NI by approximately 90% and 20-40%, respectively. The increased titers in THP-1 monocytic cells but not IMR-32 and Neuro-2A neuroblastoma cells were more correlated with the phenotypic changes of NI and NV in mice. Entire genomic sequencing was further performed to demonstrate that 14 nucleotides were altered in the attenuated variant CH2195LA following four brain-brain passages in mice, giving 12 amino acid changes, in prM-73, prM-80, E-161, E-170, E-276, NS2A-136, NS2A-215, NS3-346, NS4A-128, NS4B-196, NS4B-197, NS4B-198. This study indicated a cluster of amino acids which is involved in NV and NI of the JEV for mice and, perhaps, for humans. Elucidating the molecular basis of virulence of flaviviruses can provide valuable information for live-attenuated vaccine development.     

Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness

Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.     

Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.     

Discrimination between Sialic Acid-Containing Receptors and Pseudoreceptors Regulates Polyomavirus Spread in the Mouse

Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.     

Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions.

The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.     

Site of Host Restriction of Simian Virus 40 Mutants in an Established African Green Monkey Kidney Cell Line

A previously described large-plaque morphology mutant of SV40 (SV-L) in primary African green monkey kidney (AGMK) cells has been shown to be restricted in the established AGMK line CV-1. The site of restriction is an early step in infection, involving the virus particle. A block in penetration or uncoating has been tentatively proposed. Restriction is observed in a number of other established lines, including monkey, human, and mouse. Independent large-plaque mutants were isolated in AGMK and their properties were compared with SV-L (temperature restriction, host restriction, and virion antigen). They can be separated into two classes. Both are restricted on CV-1, though to a lesser degree than SV-L. Further-more, both are coat mutants as determined by immunological analysis of their virions, suggesting a relationship between coat mutants associated with large-plaque morphology in AGMK and host restriction in established cell lines. Temperature restriction is seen with one but not the other of the mutants.     

Markers of Rubella Virus Strains in RK13 Cell Culture

When tested on RK(13) cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK(13) culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK(13) cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK(13) cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the pH of the medium during plaque formation.     

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.     

Evidence of expanded host range and mammalian-associated genetic changes in a duck H9N2 influenza virus following adaptation in quail and chickens

H9N2 avian influenza viruses continue to circulate worldwide; in Asia, H9N2 viruses have caused disease outbreaks and established lineages in land-based poultry. Some H9N2 strains are considered potentially pandemic because they have infected humans causing mild respiratory disease. In addition, some of these H9N2 strains replicate efficiently in mice without prior adaptation suggesting that H9N2 strains are expanding their host range. In order to understand the molecular basis of the interspecies transmission of H9N2 viruses, we adapted in the laboratory a wildtype duck H9N2 virus, influenza A/duck/Hong Kong/702/79 (WT702) virus, in quail and chickens through serial lung passages. We carried out comparative analysis of the replication and transmission in quail and chickens of WT702 and the viruses obtained after 23 serial passages in quail (QA23) followed by 10 serial passages in chickens (QA23CkA10). Although the WT702 virus can replicate and transmit in quail, it replicates poorly and does not transmit in chickens. In contrast, the QA23CkA10 virus was very efficient at replicating and transmitting in quail and chickens. Nucleotide sequence analysis of the QA23 and QA23CkA10 viruses compared to the WT702 virus indicated several nucleotide substitutions resulting in amino acid changes within the surface and internal proteins. In addition, a 21-amino acid deletion was found in the stalk of the NA protein of the QA23 virus and was maintained without further modification in the QA23CkA10 adapted virus. More importantly, both the QA23 and the QA23CkA10 viruses, unlike the WT702 virus, were able to readily infect mice, produce a large-plaque phenotype, showed faster replication kinetics in tissue culture, and resulted in the quick selection of the K627 amino acid mammalian-associated signature in PB2. These results are in agreement with the notion that adaptation of H9 viruses to land-based birds can lead to strains with expanded host range.     

Genetic variation occurring on the genome of an in vitro insertion mutant of poliovirus type 1.

An insertion sequence of 72 nucleotides prepared from a polylinker sequence of plasmid pUC18 was introduced at nucleotide position 702 of the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of poliovirus type 1 by using an infectious cDNA clone of the virus strain. The insertion mutant thus obtained showed a small-plaque phenotype compared with that of the parent virus. Apparent revertants (large-plaque variants) were easily generated from the insertion mutant. Nucleotide sequence analysis was performed on the revertant genomes to determine the mutation(s) by which the plaque size of the parent virus was regained. Some large-plaque variants lacked genomic sequences including all or a part of the insertion sequence. A computer-aided search for secondary structures with respect to the deletion sites detected possible supporting sequences which provided fairly stable secondary structures at the deletion sites. This result was consistent with our supporting sequence-loop model which had been proposed as a new copy-choice model for the generation of genetic rearrangements occurring on single-stranded RNA genomes (S. Kuge, I. Saito, and A. Nomoto, J. Mol. Biol. 192:473-487, 1986). The other large-plaque variants had point mutations at any one of three positions of an AUG existing in the insertion sequence. A small-plaque phenotype was observed when an AUG codon was inserted in frame or out of frame with regard to the initiation site of viral polyprotein synthesis. Our data strongly suggest that an AUG sequence in this genome region is deleterious for efficient poliovirus replication.     

Host influence on the characteristics of Venezuelan equine encephalomyelitis virus

Heydrick, Fred P. (Fort Detrick, Frederick, Md.), Ralph F. Wachter, and Henry J. Hearn, Jr. Host influence on the characteristics of Venezuelan equine encephalomyelitis virus. J. Bacteriol. 91:2343-2348. 1966.-Alterations in plaque size, virulence, and lipid content of Venezuelan equine encephalomyelitis (VEE) virus were examined for possible interrelationships among these properties during 10 serial passages in embryonated eggs, suckling mice, chick embryo fibroblasts, and L cells. The chick embryo host maintained the same large-plaque and virulence properties of the virus through 10 passages as seen in the original seed. Passage of virus in either L cells or chick fibroblasts rapidly produced populations that were, in the main, intermediate with respect to plaque size and virulence. Passage of virus in suckling mouse brain yielded populations that were intermediate with respect to plaque size only. The nature of the lipid of the virus, in terms of the ratio of petroleum ether-soluble to -insoluble lipid, changed after only one passage in all systems except in chick embryos. Nine additional serial passages failed to enhance these changes in viral lipid, suggesting that the decrease in the large-plaque and virulence properties was not directly associated with changes in lipid content.     

Strong inclination toward transition mutation in nucleotide substitutions by poliovirus replicase

A viable insertion mutant of the Sabin strain of type 1 poliovirus was constructed. The mutant carried an insertion sequence of 72 nucleotides at nucleotide position 702 in the 5' non-coding region (742 nucleotides long) of the genome of the Sabin strain. This mutant showed a small-plaque phenotype, as compared with the parental virus. Indeed, the final yield of the mutant in a single cycle of infection was tenfold fewer than that of the parental virus. Many large-plaque variants that are easily generated from the insertion mutant appeared to regain efficient viral replication and have single nucleotide changes. All nucleotide changes observed were limited to within three nucleotides of an AUG sequence in the insertion sequence. The result indicates strongly that the AUG sequence itself in this genome region functions in reducing the plaque size of the parental Sabin type 1 virus. The insertion mutant with a small-plaque phenotype may be the first in vitro mutant of poliovirus whose viability is lowered only by a primary sequence inserted into the 5' non-coding region of the genome. Base substitutions to alter the AUG sequence should largely be the result of errors of the virus-specific replicase, since variants with base substitutions must be subject to only minimum selection pressure. Accordingly, nucleotide sequence analysis of the genome region containing the AUG sequence was performed on a number of genomes of large-plaque variants to investigate types of nucleotide substitutions caused by characteristic errors in RNA replication. Only one transversion mutation was detected in the genomes of 44 independently isolated large-plaque variants with single base changes in the AUG sequence. This result suggests strongly that transition mutations occur predominantly as nucleotide substitutions caused by characteristic errors of poliovirus replicase.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis

Theiler's murine encephalomyelitis viruses are picornaviruses that can infect the central nervous system. The DA strain produces an acute polioencephalomyelitis followed by a chronic demyelinating disease in its natural host, the mouse. The ability of DA virus to induce a demyelinating disease renders this virus infection a model for human demyelinating diseases such as multiple sclerosis. Here we describe the generation and characterization of DA virus mutants that contain specific mutations in the viral capsid protein VP1 at sites believed to be important contact regions for the cellular receptor(s). A mutant virus with a threonine-to-aspartate (T81D) substitution in VP1 loop I adjacent to the putative virus receptor binding site exhibited a large-plaque phenotype but had a slower replication cycle in vitro. When this mutant virus was injected into susceptible mice, an altered tropism was seen during the acute stage of the disease and the chronic demyelinating disease was not produced. A virus with a threonine-to-valine substitution (T81V) did not cause any changes in the pattern or extent of disease seen in mice, whereas a virus with a tryptophan substitution at this position (T81W) produced a similar acute disease but was attenuated for the development of the chronic disease. A change in amino acids in a hydrophobic patch located in the wall of the pit, VP1 position 91, to a hydrophilic threonine (V91T) resulted in a profound attenuation of the acute and chronic disease without persistence of virus. This report illustrates the importance of the loop I of VP1 and a site in the wall of the pit in pathogenesis and that amino acid substitutions at these sites result in altered virus-host interactions.     

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.     

Determinants in the 5'' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.     

Recombinant protein rVP1 upregulates BECN1-independent autophagy,MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration

The monocyte/macrophage is critical for regulating immune and antitumor responses. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus induces apoptosis and inhibits migration/metastasis of cancer cells. Here, we explored the effects of rVP1 on macrophages. Our results showed that rVP1 increased LC3-related autophagosome formation via WIPI1 and WIPI2 in a BECN1-independent manner. rVP1 treatment increased macrophage migration that was attenuated by knockdown of ATG5, ATG7, WIPI1 or WIPI2 and was abolished when both WIPI1 and WIPI2 were depleted. Treatment of macrophages with rVP1 increased matrix metalloproteinase-9 (MMP9) activity and phosphorylated mitogen-activated protein kinase 1/3 (MAPK1/3), two major mediators of cell migration. Knockdown of WIPI1, WIPI2, ATG5 and ATG7 but not BECN1 attenuated the rVP1-mediated increase in MAPK1/3 phosphorylation and MMP9 activity. These results indicated that rVP1 upregulated autophagy, MAPK1/3 phosphorylation and MMP9 activity to promote macrophage migration, which was dependent on WIPI1, WIPI2, ATG5 and ATG7 but not BECN1.     

Strain-dependent structural variants of herpes simplex virus type 1 ICP34.5 determine viral plaque size,efficiency of glycoprotein processing,and viral release and neuroinvasive disease potential

The ability of certain strains of herpes simplex virus type 1 (HSV-1) to cause encephalitis or neuroinvasive disease in the mouse upon peripheral infection is dependent on a combination of activities of specific forms of viral proteins. The importance of specific variants of ICP34.5 to neuroinvasive disease potential and its correlation with small-plaque production, inefficient glycoprotein processing, and virus release were suggested by comparison of ICP34.5 from the SP7 virus, originally obtained from the brain of a neonate with disseminated disease, and the tissue culture-passaged progeny of SP7 (SLP5 and SLP10) and the KOS321 virus. SLP5, SLP10, and KOS321 are attenuated and exhibit a large-plaque phenotype, including efficient glycoprotein processing and viral release. We show that expression of the KOS321 ICP34.5 protein in cells infected with SP7 or ICP34.5 deletion mutants promotes large plaque formation and efficient viral glycoprotein processing, while expression of the SP7 ICP34.5 protein decreases efficiency of viral glycoprotein processing. In addition, a recombinant virus, 4hS1, with the SP7 ICP34.5 gene replacing the KOS321-like ICP34.5 gene in the SLP10a background, rescues the small-plaque phenotype and neuroinvasive disease. The major difference in the ICP34.5 gene product is the number of Pro-Ala-Thr repeats in the middle region of the protein, with 18 for SP7 and 3 for KOS321. Strain-dependent differences in the ICP34.5 protein can therefore alter the tissue culture behavior and the virulence of HSV-1.