Serological studies on virions and polyhedron protein of a nuclear polyhedrosis virus of the cabbage looper, Trichoplusia ni.

Improved purification procedures were employed to isolate polyhedron protein and enveloped virus particles (virions) from a nuclear polyhedrosis virus (NPV) of the cabbage looper, Trichoplusia ni, to study the serological relationship between these two components. Polyhedra were digested in sodium carbonate-saline buffer, pH 11, for 1 hr at room temperature and centrifuged at 40,000 g for 30 min to crudely separate the polyhedron protein from the virions. Polyhedron protein was purified by Sepharose 6B column chromatography and virions were purified by Ficoll density gradient centrifugation and Sepharose 2B column chromatography. Antiserum was prepared by injecting rabbits with highly purified intact polyhedra, polyhedron protein, or virions. When partially purified antigens were used in Ouchterlony double diffusion in agarose gels, multiple precipitin lines were formed and polyhedron protein cross-reacted with virions. However, the highly purified polyhedron protein and virions reacted only with their homologous antisera and no cross-reactions were visible. Immunoelectrophoresis showed that virions and polyhedron protein had significantly different mobilities and reacted only with their homologous antisera. It was therefore concluded that no serological relationship exists between highly purified polyhedron protein and virions of NPV of T. ni.     

Stability and immunogenicity of empty particles of foot-and-mouth disease virus

Summary Three strains of foot-and-mouth disease virus were shown to contain significant amounts of naturally occurring 75 S, empty particles as well as the infectious, 140 S full particles. One of these strains — A Pando (1970) — was studied in detail.The empty particles from this virus strain were shown to have an observed sedimentation coefficient of 67S in 0.04m phosphate buffer; they were labile in SDS, non-infectious and probably RNA-free and, on heating, they broke down to 12 S subunits as did the 140 S particles. The empty particles differed from the full particles in their polypeptide composition since they contained VP₀, but there was no evidence for a diminished content of VP₄.The 75 S particles were shown to be present in significant amounts and to be stable to AEI inactivation. At 4° C they were stable for at least two years. In guinea pigs they were as immunogenic as the 140 S particles. The antisera raised against the 75 S particles had the same serological specificity in neutralization tests as sera prepared against the 140 S particle. It was concluded that the 75 S particles from the A Pando (1970) strain of FMD virus may provide as important a contribution as 140 S particles to the immunogenicity of inactivated vaccines prepared from this virus strain.With 5 Figures     

A new ilarvirus isolated from Viola × wittrockiana and its detection in pansy germoplasm by qRT-PCR

An infectious agent was transmitted mechanically from samples of Viola spp. showing white mosaic and leaf deformation to Nicotiana benthamiana. dsRNA extracted from the N. benthamiana plants migrated as four specific bands that were absent in non-inoculated plants. Sequence analysis of cDNA clones generated from the second-smallest dsRNA showed the greatest similarity to the RNA3 of prune dwarf virus (PDV) (genus Ilarvirus, family Bromoviridae). However, because of differences in molecular, biological, and serological properties between this virus isolate and PDV, a new ilarvirus species, named “Viola white distortion associated virus” (VWDaV) is proposed. Specific oligonucleotides and a TaqMan^® probe were designed for diagnostic purposes. The possible association between the virus and the original white distortion symptoms is discussed.     

Serological relationships of an iridescent virus (type 25) recently isolated from Tipula sp. with two other iridescent viruses (types 2 and 22).

The isolation, purification, and serological properties of a 130-nm iridescent virus (IV), type 25, from Tipula sp. is reported. The polypeptide profile of this isolate was different from that of another small Dipterous iridescent virus-IV22 isolated from Simulium sp. However, these viruses were indistinguishable when compared by serum neutralization, gel immunodiffusion, complement fixation, tube immunoprecipitation, and immune electron microscopy. Iridescent virus type 2 isolated from Sericesthis pruinosa was shown to be distinct from IV22 and IV25 by all of these techniques.     

A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus.

The chemical and serological properties of the full, naturally occurring empty and artificially produced empty particles of foot-and-mouth disease virus, serotype A(subtype 10, strain 16) have been studies. The full 146S particles comprised the virus RNA, three polypeptides (VP1 to VP3) mol. wt. about 30 X 10-3, one polypeptide (VP4) mol. wt. about 13-5 X 10-3, and a small amount of a polypeptide (VPo) mol. wt. about 43 X 10-3. The naturally occurring 75S empty particles contained no RNA and much less VP1 and VP4 than were found in the fall particles. However they contained a much greater proportion of VPo than the full particles. Dialysis of purified full particles against tris-EDTA, pH 7-6, produced artificial 75S empty particles which contained only a small amount of RNA and no VP4; otherwise the polypeptide composition was similar to that of the full particles. Immunological and serological tests showed that the full particles were antigenically similar to the naturally occurring empty particles but distinct from the artificial empty particles. The latter particles, however, had serological properties similar to those of the 12S protein subunit of the virus. Both the full and naturally occurring empty particles attached efficiently to susceptible cells, whereas the artificial empty particles attached only to a limited extent. The results are related to the function of the individual polypeptides of the virus particle and compared with published work on other picornaviruses.     

Sequencing of the Tamus red mosaic virus genome: further evidence that it is a distinct species of the genus Potexvirus

In 1993, a virus causing red mosaic and leaf distortion has been isolated from black bryony (Tamus communis) in Italy. Based on particle properties and serology, the virus was assigned to the genus Potexvirus and named Tamus red mosaic virus (TRMV), pending a genome sequence. The original Italian TRMV isolate was submitted to the DSMZ plant virus collection (PV-0397). To confirm the taxonomic status of the virus, the entire genome sequence was determined comprising 6,495 nucleotides excluding the poly(A)-tail. Five putative open reading frames (ORFs) in an arrangement typical for potexviruses were predicted. TRMV is closely related to but distinct from Clover yellow mosaic virus and Allium virus X. In addition to previous morphological and serological characterization, the results presented in this study further reinforce the classification of TRMV as a distinct virus species of the genus Potexvirus.     

Comparative studies on polyribosomal, nonpolyribosome-associated and viral 42 S RNA from BHK 21 cells infected with Semliki Forest virus.

Virus-specific RNA sedimenting at about 42 S on sucrose density gradients can be isolated from polyribosomes of BHK-21 cells infected with Semliki Forest virus and from intracellular nonpolyribosome-associated ribonucleoprotein particles sedimenting at about 55 S. These RNA species have been compared to the 42 S RNA of Semliki Forest virus particles by three different techniques: Infectivity, sedimentation on sucrose density gradients in formamide, and translation into virus-specific protein in vitro. The results of these experiments have shown that all three RNA species consist of intact, unbroken molecules, that they are equally infectious and that they contain the mRNA sequences for the viral core protein (the structural protein associated with the viral RNA in the virus particle). Some implications of these results concerning the possible utilization of the different 42 S RNA species for assembly of virus particles and the translation of the polyribosome-associated 42 S RNA in vivo are discussed.     

Kotonkan and Obodhiang viruses: African ephemeroviruses with large and complex genomes

Kotonkan virus (KOTV) and Obodhiang virus (OBOV) are rhabdoviruses that were isolated from arthropods in Africa and formerly classified as lyssaviruses. KOTV causes clinical bovine ephemeral fever in cattle; the ecology and pathogenicity of OBOV is poorly understood. In this paper, we report the complete genome sequences of KOTV and OBOV, their gene expression profiles, and their serological and phylogenetic relationships to other rhabdoviruses. The 15,870 nt KOTV genome (3′-l-N-P-M-G-G_NS-α1-α2-β-γ-δ-L-t-5′) is similar to that of bovine ephemeral fever virus but encodes an additional protein (δ) that shares homology with the pleckstrin homology domain of coactivator-associated arginine methyltransferase. The 14,717 nt OBOV genome (3′-l-N-P-M-G-G_NS-α1-α2-β-L-t-5′) is similar to that of Adelaide River virus from which it is distinguishable serologically. In each virus, all ORFs, except α1 and α2, are transcribed as monocistronic mRNA. Genetic and serological data indicate that KOTV and OBOV should be classified as new species in the genus Ephemerovirus.     

A mutant of chikungunya virus isolated from a line of Singh's Aedes albopictus cells by plaque formation on virus-sensitive cloned cells obtained from another Singh's A. albopictus cell line

A chikungunya (CHIK) virus-resistant line of Singh's Aedes albopictus (SAAK) cells released spontaneously two kinds of plaque-forming agents which could be distinguished by the plaque size on a sensitive clone C6/36 of another A. albopictus cell line (SAAR). Release of the large plaque (Lp) and the small plaque (sp) viruses into the culture medium occurred at different rates when the SAAK cells were subcultured. The Lp virus was removed from the SAAK cells by subculturing or cloning in the presence of anti-CHIK serum. Such Lp(-) cells continued to produce the sp virus, but were permissive for CHIK virus. The plaque-purified Lp virus, but not sp virus, interfered with the growth of CHIK virus in the C6/36 cells. The Lp virus resembled in physicochemical and serological properties CHIK virus grown in C6/36 cells. However the Lp virus was not pathogenic for suckling mice, was heat labile, and showed higher plating efficiency on the C6/36 cells than on BHK21 cells. The data indicate that the Lp virus is a mutant of CHIK virus and is responsible for the CHIK virus resistance of the SAAK cells.     

Properties of the nucleocapsids of a virus isolated from Oryctes rhinoceros.

Nucleocapcids were isolated from purified particles of “Oryctes” virus (a candidate Baculovirus) by treatment with Nonidet P-40. The nucleocapsids were homogeneous in size and density and contained the viral DNA as well as eight of the twelve virus particle polypeptides resolved on polyacrylamide gels. The nucleocapcids were serologically related to nucleocapcids of another Baculovirus (a nuclear polyhedrosis virus from Spodoptera littoralis), although the “Oryctes” nucleocapcids were morphologically distinct in carrying a novel tail-like projection at one end of the particle.     

Antigenic relationships between strains of tobacco mosaic virus.

The serological relationships between six strains of tobacco mosaic virus were studied with antisera from 40 rabbits bled at regular intervals during at least 8 mo. The extent of cross reactivity between strains was expressed by a serological differentiation index (SDI) equal to the difference between homologous and heterologous titers denoted as Neg Log₂. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. Average SDI values calculated from a large number of bleedings taken from different animals agreed closely with the corresponding SDI values calculated from reciprocal serological tests. The intraclass correlation coefficient between two sets of SDI values obtained in reciprocal serological tests was r′ = 0.95. A correlation coefficient of r = 0.75 was calculated between the serological relatedness expressed as SDI values and the extent of sequence homology in the coat protein of different strains.     

Further characterization of Mengo subviral particles: a new hypothesis for picornavirus assembly.

The “50S particle” found in Mengo virus-infected L cells (P. W. K. Lee, E. Paucha, and J. S. Colter, 1978, Virology 85, 286–295) has been further characterized. Its sedimentation coefficient has been estimated to be 53 S from centrifugal analysis in sucrose density gradients. When, during the isolation of 53 S particles, the KCI concentration in the suspending buffer is increased to 150 mM or higher, some of the particles are converted to structures having a significantly larger sedimentation coefficient. A similar conversion of 53 S to more rapidly sedimenting particles occurs when the former are centrifuged to equilibrium in a CsCI density gradient. The sedimentation coefficient of this new particle has been estimated to be 75 S. Molecular weight determinations of the previously described 14 S particles and of the 53 and 75 S particles by means of Sepharose 4B exclusion chromatography suggest that the molecular compositions of these particles are (?αγ)₅, (?αγ)₂₅, and (?αγ)₅₀, respectively. Based on this information and the previously reported evidence suggesting a precursor role for the 53 S particles, a new hypothesis regarding the mechanism of Mengo virus assembly has been proposed. In this model, the viral RNA interacts with either a 75 S particle or two 53 S particles to form a complex represented by RNA[(?αγ)₅]₁₀, before assembly is completed by the addition of two 14 S subunits.     

Some observations on the binding properties of alfalfa mosaic virus to polystyrene and its significance to indirect ELISA

Summary The adsorption and retention properties of native (unfixed) and glutaraldehyde-fixed alfalfa mosaic virus (AMV) antigens to the polystyrene of ELISA plates were studied using [³⁵S]-labelled virus preparations. It was shown that adsorption was a temperature-dependent, relatively slow process which varied between different AMV isolates. The amount of virus antigen adsorbed was dependent on the type and pH of the suspending buffer. Although native virus antigen adsorbed very efficiently at high pH when the particles had dissociated, significant amounts also adsorbed at pH 7.0, or lower. However, glutaraldehyde-fixed virus particles which retained their integrity even at pH as high as 9.6, adsorbed much more efficiently than native virus antigen above pH 9.0, but hardly at all around pH 7.0. The wide variation in adsorption of AMV antigen to microtitre plates under even slightly different conditions had significant influence on ELISA readings, which calls for extreme caution in interpreting serological results from indirect ELISA when antigen is used to coat the microtitre plates.Professor R. I. B. Francki passed away in November 1990.     

Characterization of two morphologically distinct top component a particles from alfalfa mosaic virus.

Top component a particles from alfalfa mosaic virus were isolated from the supernatant fluid as well as from the precipitate after treatment of virus solutions with 0.03 M MgSO₄. Both preparations were tested for electron microscopic appearance, electrophoretic mobility in 3% polyacrylamide gels, RNA species, RNA content, and particle weight. The number of coat protein subunits was determined. The particles are very different in shape but identical or very similar in the other properties investigated. The particles insoluble in 0.03 M MgSO₄ are structurally related to larger bacilliform particles of the virus, and the particles soluble in 0.03 M MgSO₄ are spheroidal. Apparently the top component a nucleocapsid can be assembled in two different ways.     

Purification and antigenicity of three isolates of barley yellow dwarf virus

An isolate (RPV) of barley yellow dwarf virus transmitted specifically by Rhopalosiphum padi and an isolate (PAV) transmitted nonspecifically by both R. padi and Macrosiphum avenae were purified by procedures previously found satisfactory for another isolate (MAV) transmitted specifically by M. avenae. As with MAV, infectivity of RPV and PAV samples removed from sucrose gradient columns was associated with a dense polyhedral particle about 30 nm in diameter in shadowed preparations. No differences in sedimentation rate (sedimentation coefficient 115–118 S) among the 3 virus isolates were detected in parallel sucrose gradient centrifugation tests.     

Inclusion of MERS‐spike protein ELISA in algorithm to determine serologic evidence of MERS‐CoV infection

The Centers for Disease Control and Prevention (CDC) algorithm for detecting presence of serum antibodies against Middle East Respiratory Syndrome coronavirus (MERS‐CoV) in subjects with potential infections with the virus has included screening by indirect ELISA against recombinant nucleocapsid (N) protein and confirmation by immunofluorescent staining of infected monolayers and/or microneutralization titration. Other international groups include indirect ELISA assays using the spike (S) protein, as part of their serological determinations. In the current study, we describe development and validation of an indirect MERS‐CoV S ELISA to be used as part of our serological determination for evidence of previous exposure to the virus.

     

Effective chemical virus inactivation of patient serum compatible with accurate serodiagnosis of infections

ObjectivesHighly pathogenic viruses such as EBOV are a threat to routine laboratory workers. Inactivation procedures with Triton X-100 0.1% and/or heat are currently recommended, but have unknown effects on the accuracy of serological testing. Furthermore, virus inactivation by Triton X-100 0.1% was shown to be ineffective in serum. This study aimed to demonstrate virus inactivation in serum by Triton X-100 1% and maintained accuracy of serological testing.MethodsA panel of 19 serological tests was run on patient serum samples after treatment with Triton X-100 1%, 0.1%, and 0.1% + heat inactivation at 60°C for 1 h. Mean differences between measurements (bias) were calculated applying the Bland–Altman method. To determine effectiveness of virus inactivation, herpes simplex virus 1 (HSV-1) was spiked into medium containing 90% or 1% serum, and treated with Triton X-100 0.1% or 1%. Infectious titres were then determined on Vero cells.ResultsSerological measurements showed good agreement between controls and samples treated with Triton X-100 0.1% and 1%, with an estimated bias of 0.6 ± 9.2% (n = 258) and –0.1 ± 18.6% (n = 174), respectively. Discordant qualitative results were rare. Conversely, heat inactivation alone and combined with Triton X-100 0.1% triggered a bias of 17.5 ± 66.4% (n = 200) and 37.9 ± 79.8% (n = 160), respectively. Triton X-100 1% completely inactivated HSV-1 in 1% and 90% serum while Triton X-100 0.1% failed to do so in 90% serum.ConclusionsUnlike heat inactivation, Triton X-100 1% enabled accurate serological testing and completely inactivated HSV-1 in serum. This simple method could allow safe routine serological diagnostics in high-risk patients.     

Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.