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The NS and capsid genes determine the host range of porcine parvovirus.

Porcine parvovirus is an autonomous parvovirus which normally infects pigs and multiplies in porcine cells in vitro. In this report, we describe the properties of a variant designated P2, which has extended its host range to include canine cells. The variant was able to produce cytopathic effects (CPE) in canine cells, unlike the prototype NADL-2 strain. The variant also produced higher viral antigen and infectivity titers in canine cells than the NADL-2 strain, whereas both strains produced CPE and similar titers in porcine cells. Generation of recombinant plasmids between the P2 variant DNA and an infectious clone of NADL-2, and analysis of the properties of the virus stocks produced from these recombinant plasmids, indicated that two changes were necessary for this extension in the host range. One change was located in the nonstructural protein coding region and the other in the capsid coding region.

Amino Acid Sequence↗

Functional analysis of biologically distinct genetic variants of simian immunodeficiency virus isolated from a mandrill.

We examined the biological properties of two infectious clones of a simian immunodeficiency virus, SIVMND, which were designated as pMD121 and pMD122. Upon transfection into CD4-negative cells, pMD122 generated virions much less efficiently than pMD121. Likewise, the growth kinetics in CD4-positive cells of virus derived from pMD122 were remarkably delayed relative to those of virus from pMD121. The cytocidal activity of the MD122 virus was also low. A series of recombinant clones were constructed from pMD121 and pMD122 to determine the sequence responsible for the low virulence of the MD122 virus. The genetic determinant in pMD122 responsible for its properties was mapped to within a region (316 base pairs) encompassing the tat, rev, and env coding sequences. Sequence analysis revealed that the two clones differed by only one nucleotide in this region. A nucleotide substitution G (pMD121) to T (pMD122) altered an arginine codon to a serine codon in the first tat coding exon. Transient transfection experiments showed that the tat activity of pMD122 was about twofold less than that of pMD121. These findings indicate that small differences in tat activity can have a dramatic effect on the biological behavior of SIVMND.

Amino Acid Sequence↗

Vaccines and animal models for arboviral encephalitides.

Arthropod-borne viruses ("arboviruses") cause significant human illness ranging from mild, asymptomatic infection to fatal encephalitis or hemorrhagic fever. The most significant arboviruses causing human illness belong to genera in three viral families, Togaviridae, Flaviviridae, and Bunyaviridae. These viruses represent a significant public health threat to many parts of the world, and, as evidenced by the recent introduction of the West Nile virus (WNV) to the Western Hemisphere, they can no longer be considered specific to any one country or region of the world. Like most viral diseases, there are no specific therapies for the arboviral encephalitides; therefore, effective vaccines remain the front line of defense for these diseases. With this in mind, the development of new, more effective vaccines and the appropriate animal models in which to test them become paramount. In fact, for many important arboviruses (e.g. California serogroup and St. Louis encephalitis viruses), there are currently no approved vaccines available for human use. For others, such as the alphaviruses, human vaccines are available only as Investigational New Drugs, and thus are not in widespread use. On the other hand, safe and effective vaccines against tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) have been in use for decades. New challenges in vaccine development have been met with new technologies in vaccine research. Many of the newer vaccines are now being developed by recombinant DNA technology. For example, chimeric virus vaccines have been developed using infectious clone technology for many of the arboviruses including, WNV, JEV, and TBEV. Other successful approaches have involved the use of naked DNA encoding and subsequently expressing the desired protective epitopes. Naked DNA vaccines have been used for TBEV and JEV and are currently under development for use against WNV. The development of less expensive, more authentic animal models to evaluate new vaccines against arboviral diseases will become increasingly important as these new approaches in vaccine research are realized. This article reviews the current status of vaccines, both approved for use and those in developmental stages, against the major arboviral encephalitides causing human disease. In addition, research on animal models, both past and present, for these diseases are discussed.

Animals↗

Insights into the genetic basis for natural phenotypic resistance of human rhinoviruses to pleconaril.

Recent phylogenetic analyses of the deduced amino acid sequence of the major viral capsid protein (VP1) of all human rhinovirus (HRV) serotypes revealed two distinct species within the genus: species A (75 serotypes) and species B (25 serotypes). Pleconaril is a novel capsid inhibitor of HRVs. All 75 species A serotypes and 18 of the 25 species B serotypes are susceptible to inhibition by pleconaril in cell culture. The seven resistant serotypes are HRV-4, -5, -42, -84, -93, -97 and -99. We were interested in understanding the genetic basis for phenotypic resistance to pleconaril among these naturally occurring viruses. We compared the 25 amino acids of VP1 that comprise the drug-binding pocket of susceptible and resistant species B viruses. A consistent difference was observed at two positions: the vast majority of susceptible viruses had tyrosine and valine at VP1 residues 152 and 191, respectively (Y(152) and V(191)); all resistant viruses had phenylalanine and leucine at these positions (F(152) and L(191)). HRV-14, a pleconaril susceptible virus, has a drug-binding pocket amino acid composition that differs from the naturally resistant HRV-5 and HRV-42 only at these two positions. To gain further insight into the role of these specific residues in natural resistance to pleconaril, we substituted the amino acids at these two positions individually and in combination in an infectious clone of HRV-14 and tested the rescued virus for susceptibility to pleconaril and virion stability. The results indicate that substitution of V(191) to Leu in HRV-14 has a profound negative impact on drug susceptibility but that full resistance to pleconaril is only seen when combined with Phe at position 152 in a HRV-14 double variant (F(152), L(191)). These data identify L(191) in species B HRV as a potentially key residue in conferring significantly reduced susceptibility to pleconaril. These results may be useful in distinguishing naturally occurring viral resistance to pleconaril from treatment-emergent resistance.

Antiviral Agents↗

Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash.

A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.

Amino Acid Sequence↗

Functional analysis of HIV-1 vif genes derived from Japanese long-term nonprogressors and progressors for AIDS.

We analyzed the function of human immunodeficiency virus type 1 (HIV-1) vif gene from Japanese long-term nonprogressors (LTNPRs) and progressors (PRs) for acquired immunodeficiency syndrome (AIDS). We constructed a basic HIV-1 infectious clone, which facilitated the incorporation and evaluation of vif from infected individuals. Proviral reporter clones carrying vif from six Japanese LTNPRs and seven PRs were then generated and their in vitro growth kinetics were analyzed. The vif clones, which could confer infectivity on reporter viruses, were considered active, and the ratio of the active clones to the number of clones examined per individual was determined. For the majority of LTNPRs, there was no correlation between presence or absence of functional vif with long-term nonprogression for AIDS. There was one exception in which all the clones examined had inactive vif, suggesting a probable association of inactive vif with the nonprogression. All PRs with high viral load had a high ratio of active vif clones. Our results suggest that the presence of functional vif would influence HIV-1 infectivity and disease progression in infected individuals.

Amino Acid Sequence↗

The use of in situ hybridization and immunohistochemistry to study the pathogenesis of various Newcastle disease virus strains and recombinants in embryonated chicken eggs.

Avian paramyxovirus type 1, commonly referred to as Newcastle disease virus (NDV), is a serious pathogen of significant economic importance to the industry. To investigate the role of the fusion (F), hemagglutinin-neuraminidase (HN), and (P) phosphoprotein gene sequences in virulence, six strains of Newcastle disease virus (NDV) representing all pathotypes and seven recombinant strains created by reverse genetics were inoculated into 9-day-old chicken embryos. Tissues and chorioallantoic membranes (CAM) were harvested at 24-hour intervals post-inoculation. Riboprobe in situ hybridization and immunohistochemistry highlighted distinct tissue tropisms among the viruses. Presence of F and/or HN from virulent viruses inserted into lentogenic backbones caused dissemination of virus in a manner similar to wild type virulent viruses. Disruption of P gene decreased dissemination of velogeinic infectious clones. It is concluded that each of these genes contributes to pathogenicity.

Animals↗

Construction and biological properties of yellow fever 17D/dengue type 1 recombinant virus.

Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.

Amino Acid Sequence↗

Molecular characterization of attenuated Japanese encephalitis live vaccine strain ML-17.

The Japanese encephalitis (JE) zoonotic vaccine strain ML-17 was sequenced and compared to related JE virus strains to identify genomic attenuation markers. Relative to its parental strain, JaOH0566, 25 nucleotide alterations and 10 amino acid changes to, prM/M(2), NS2A(1), NS4B(3) and NS5(4) proteins were recorded. Both structural-gene changes were in the prM/M region (127Met-->Ile and 274Asn-->Thr). To study the effects of these prM/M changes, mutants bearing the changes were prepared using an infectious clone of JaOArS982 previously established at this lab. Compared with JaOArS982, mutant 127(Met-->Ile) showed marked reduction in murine neuroinvasiveness. Mutant 274(Asn-->Thr), showed slight reduction. Neither mutant recorded ML-17-equivalent attenuation, implying that prM/M changes need to combine with other recorded genomic differences to cause attenuation. Importantly, ML-17 with its unchanged E region, presents a possible backbone candidate for preparation of "E-replacement" type live attenuated flavivirus chimeric vaccines.

Animals↗

Identification of regions of the Beet mild curly top virus (family Geminiviridae) capsid protein involved in systemic infection, virion formation and leafhopper transmission.

Plant viruses in the genus Curtovirus (family Geminiviridae) are vectored by the beet leafhopper (Circulifer tenellus) and cause curly top disease in a wide range of dicotyledonous plants. An infectious clone of an isolate of Beet mild curly top virus (BMCTV-[W4]), associated with an outbreak of curly top in pepper and tomato crops, was characterized and used to investigate the role of the capsid protein (CP) in viral biology and pathogenesis. Frameshift mutations were introduced into the overlapping CP and V2 genes, and a series of CP alanine scanning mutations were generated. All mutants replicated in tobacco protoplasts or systemically infected plants, consistent with these gene products not being required for viral DNA replication. The CP frameshift mutant and most C-terminal alanine scanning mutants did not systemically infect Nicotiana benthamiana plants or form detectable virions, and were not leafhopper-transmitted. In contrast, most N-terminal alanine scanning mutants systemically infected N. benthamiana and induced disease symptoms, formed virions and were leafhopper-transmissible; thus, these substitution mutations did not significantly alter the functional properties of this region. One N-terminal mutant (CP49-51) systemically infected N. benthamiana, but did not form detectable virions; whereas another (CP25-28) systemically infected N. benthamiana and formed virions, but was not insect-transmissible. These mutants may reveal regions involved in virus movement through the plant and/or leafhopper vector. Together, these results indicate an important role for virions in systemic infection (long-distance movement) and insect transmission, and strongly suggest that virions are the form in which BMCTV moves, long distance, in the phloem.

Amino Acid Sequence↗

Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine.

Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine.

Amino Acid Sequence↗

Biological properties of chimeric West Nile viruses.

Recently, we have described a lineage 2 attenuated WN virus suitable for the development of a live WN vaccine. To design vaccine candidates with an improved immunogenicity, we assembled an infectious clone of the NY99 strain and created several chimeric constructs with reciprocal exchanges of structural protein genes between attenuated W956 and virulent NY99 and investigated their biological properties. Our data indicated that, while the growth rates of NY99 and chimeric viruses in tissue culture are determined primarily by properties of the structural proteins, determinants responsible for a highly cytopathic phenotype of NY99 or lack thereof for W956 are located within the nonstructural protein region of the WN genome. The high virulence of NY99 and the attenuated phenotype of W956 were found to be associated with determinants in the nonstructural region. Chimeric viruses carrying the NY99 structural proteins were attenuated in neuroinvasiveness and demonstrated an immunogenicity superior to W956.

Animals↗

Begomovirus genetic diversity in the native plant reservoir Solanum nigrum: Evidence for the presence of a new virus species of recombinant nature.

We examined the native plant host Solanum nigrum as reservoir of genetic diversity of begomoviruses that cause the tomato yellow leaf curl disease (TYLCD) emerging in southern Spain. Presence of isolates of all the species and strains found associated with TYLCD in this area was demonstrated. Mixed infections were common, which is a prerequisite for recombination to occur. In fact, presence of a novel recombinant begomovirus was demonstrated. Analysis of an infectious clone showed that it resulted from a genetic exchange between isolates of the ES strain of Tomato yellow leaf curl Sardinia virus and of the type strain of Tomato yellow leaf curl virus. The novel biological properties suggested that it is a step forward in the ecological adaptation to the invaded area. This recombinant represents an isolate of a new begomovirus species for which the name Tomato yellow leaf curl Axarquia virus is proposed. Spread into commercial tomatoes is shown.

Base Sequence↗

Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice.

Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.

Animals↗

Lack of infection of vertebrate cells by the densovirus from the maize worm Mythimna loreyi (MlDNV).

Several densoviruses have been used successfully in biological control of pests in the tropics. The densovirus from Mythimna loreyi (MlDNV) could also be an important tool in biological control of important pests. However, safety concerns remain as previous reports suggested that densoviruses may infect and transform L cells (from mouse). In this study, we show using molecular-biology tools that neither L nor other vertebrate cells support replication or transcription of densovirus, either after infection or after transfection. Quantitative PCR indicated no increase of viral DNA due to replication in vertebrate cells, in contrast to that in insect LD652 cells. After transfection, both the NS and VP mRNAs could be detected in LD652 cells but not in L cells. Moreover, the viral genome was excised from the plasmid after transfection of the infectious clone in LD652 cells, indicative of viral NS protein production, in contrast to L cells. The viral genome was able to integrate in the host chromosome of L cells after transfection, but not after infection. However, no viral transcription could be detected after integration.

Animals↗

Rescue of Ebola virus from cDNA using heterologous support proteins.

Using the infectious clone for Zaire ebolavirus, the functional specificity of viral proteins of the ribonucleoprotein complex in transcription/replication was investigated by substituting them with heterologous proteins derived from closely (Reston ebolavirus) and distantly related filoviruses (Marburgvirus). The data clearly demonstrated that transcription/replication are neither strictly species-specific nor genus-specific. Protein interactions between the nucleoprotein NP and the virion protein VP35 and the polymerase L and VP35 seemed to be the most critical steps. In contrast to previous data, viral proteins were able to target heterologous filovirus RNA. Together these results indicated that protein-protein interactions are more critical than protein-RNA interactions.

Animals↗

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus.

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].

Amino Acid Sequence↗

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity.

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.

Acylation↗