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White spot syndrome virus (WSSV) is a significant threat to the aquaculture sector, causing mortality among crabs and shrimps. Currently available diagnostic tests for WSSV are not rapid or cost-effective, and a new detection method is therefore needed. This study demonstrates the development of a biosensor by functionalization of magnetosomes with VP28-specific antibodies to detect WSSV in seafood. The magnetosomes (1 and 2 mg/ml) were conjugated with VP28 antibody (0.025-10 ng/µl), as confirmed by spectroscopy. The magnetosome-antibody conjugate was used to detect the VP28 antigen. The binding of antigen to the magnetosome-antibody complex resulted in a change in absorbance. The magnetosome-antibody-antigen complex was then concentrated and brought near a screen-printed carbon electrode by applying an external magnetic field, and the antigen concentration was determined using impedance measurements. The VP28 antigen (0.025 ng/µl) bound more efficiently to the magnetosome-VP28 antibody complex (0.025 ng/µl) than to the VP28 antibody (0.1 ng/µl) alone. The same assay was repeated to detect the VP28 antigen (0.01 ng/µl) in WSSV-infected seafood samples using the magnetosome-VP28 antibody complex (0.025 ng/µl). The WSSV in the seafood sample was also drawn toward the electrode due to the action of magnetosomes controlled by the external magnetic field and detected using impedance measurement. The presence of WSSV in seafood samples was verified by Western blot and RT-PCR. Cross-reactivity assays with other viruses confirmed the specificity of the magnetosome-based biosensor. The results indicate that the use of the magnetosome-based biosensor is a sensitive, specific, and rapid way to detect WSSV in seafood samples., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cyprinid herpesvirus 3 (CyHV-3) is the pathogenic agent of koi herpesvirus disease (KHVD) afflicting common carp and koi (Cyprinus carpio L.) populations globally. As described previously, proteomic analyses of purified CyHV-3 particles have shown that at least 46 structural proteins are incorporated into CyHV-3 virions; among these ORF136 may encode a putative envelope protein. In this study, Western blotting analysis showed that a specific band with the predicted molecular weight of 17 kDa was detected both in purified virions and envelope components using a rabbit anti-ORF136 polyclonal antibody. Indirect immunofluorescence assay with confocal laser scanning microscopy indicated that the ORF136 protein was distributed in the cytoplasm of CCB cells infected with CyHV-3 and transfected with a pVAX1-ORF136 plasmid. Furthermore, immunogold electron microscopy confirmed that ORF136 protein localized to the CyHV-3 envelope.

The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds.

A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.

White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360-11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process.

We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.

It was recently reported that disease severity increased during the 1997 Cuban dengue 2 virus epidemic and it was suggested that this might be explained by the appearance of neutralization resistant escape mutants. We investigated these observations and ideas by sequencing 20 dengue 2 virus isolates obtained during the early (low case fatality rate) and the late (high case fatality rate) phases of the outbreak. Our results showed total conservation of the E gene sequence for these isolates suggesting that the selection of envelope gene escape mutants was not the determinant of increased disease severity. Alignment of these sequences with those available in GenBank, followed by Maximum likelihood phylogenetic analysis generated a tree, which indicated that our isolates are closely related to the virus that circulated in Venezuela in 1997/98 and subsequently in Martinique in 1998. This "American/Asian" genotype has therefore gradually dispersed across the Caribbean region during the past 5 years.

A cDNA library of short random fragments derived from four of the five genes of the rabies virus genome has been used to isolate genetic suppressor elements (GSEs) expressed intracellularly that inhibit rabies virus replication. Two nucleotide fragments, one from the rabies virus nucleocapsid protein (N) gene and the other from the phosphoprotein (P) gene, have been identified as inhibitors of rabies virus replication in cell culture. The N cDNA fragment is expressed in sense-orientation and could produce a dominant negative protein affecting virus replication. The P cDNA fragment is expressed in the inhibitory antisense direction. Inhibition of rabies virus replication was detected in cell culture using an ELISA for detection of rabies virus glycoprotein expression on the cell surface and immunofluorescence for detection of intracellular rabies virus N expression. Both the sense and antisense GSEs, because of their targeted inhibition of rabies virus replication, have possible uses in rational design of antiviral compounds for treatment of rabies. This approach could be applied to any virus, particularly to those that lyse their target host cell.

The gamma2 human herpesvirus-8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) ORFs 22 and 47 are counterparts to glycoproteins gH and gL, respectively, that are conserved among the members of herpesviruses. To define HHV-8 gH and gL, rabbit polyclonal antibodies were raised against GST-gH and GST-gL fusion proteins. Anti-gL and anti-gH antibodies reacted with the surface of virus carrying BCBL-1 cells. Both antibodies immunoprecipitated the HHV-8 envelope associated 120 kDa and 41-42 kDa proteins. In transfected COS-1 cells, gH was expressed as an endo-H sensitive 110 kDa glycoprotein, which was absent on the surface of the cells. However, after co-transfection with gL, gH was detected as an endo-H resistant 120 kDa glycoprotein, and was expressed on the surface of the cells. Non-covalent complex formation between gH and gL was detected in the transfected COS-1 cells. Anti-gH and anti-gL antibodies neutralized HHV-8 infectivity in the absence of complement, individually and more efficiently together. However, virus binding to the target cells was not inhibited. These studies suggest that HHV-8 gL is required for gH processing and expression on the cell surface membranes, and gH/gL complex plays an important role in the post-binding step of HHV-8 infection.

Human Cytomegalovirus (HCMV) UL73 encodes for a polymorphic structural glycoprotein, gpUL73(gN), conserved among herpesviruses. This study analyzed the intracellular and intraviral localization of gpUL73 by immunoelectron-microscopy comparing the reactivity of two different antibodies. We found that gN is an envelope component of the mature viral particle with at least a portion exposed at the virus surface and another at the internal side of the envelope. Furthermore, gpUL73 is also present in the matrix of dense bodies and "black holes". These results, as well as immunoblotting analysis, suggest that the two antibodies recognize different forms, fully processed or unprocessed, of gpUL73-gN.

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the recently recognized Arteriviridae family within the genus Arterivirus, order Nidovirales, which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Mature viral particles are composed of an envelope 50-72 nm in diameter, with an isometric core about 20-30 nm enclosing a linear positive-stranded RNA genome of approximately 15 kb. The virions are assembled by the budding of preformed nucleocapsids into the lumen of the smooth endoplasmic reticulum and/or Golgi apparatus. The mature virions are then released by exocytosis. The viral genome contains eight open reading frames (ORFs) which are transcribed in cells as a nested set of subgenomic mRNAs. The ORF1a and ORF1b situated at the 5'end of the genome represent nearly 75% of the viral genome and code for proteins with apparent replicase and polymerase activities. The major structural proteins consist of a 25 kDa envelope glycoprotein (GP5), an 18-19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs 5, 6 and 7, respectively. The N protein is the more abundant protein of the virion and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. Four to five domains of antigenic importance have been identified for the N protein, a common conformational antigenic site for European and North American strains being localized in the central region of the protein. In cells and virions, both M and GP5 occur in heterodimeric complexes linked by disulfide bonds. The expression products of ORFs 2 and 4 are also incorporated into virus particles as additional minor membrane-associated glycoproteins designated as GP2 and GP4, with M(r) of 29 and 31 kDa, respectively. The structural nature of the ORF3 product, a highly glycosylated protein with an apparent M(r) of 42 kDa, is still being debated, in view of the apparently conflicting data on its presence in virus particles. Nonetheless, the GP3 of North American and European strains has been shown to be antigenic, providing protection for piglets against PRRSV infection in the absence of a noticeable neutralizing humoral response. Pigs exposed to the native form of GP5 by means of DNA immunization develop specific neutralizing and protecting antibodies. The GP5 is involved in antigenic variability, apoptosis, and possibly antibody-dependent enhancement phenomena. The GP4 also possesses antigenic determinants that trigger the immune system to produce neutralizing antibodies. Each of the PRRSV structural proteins carries common and type-specific antigenic determinants that permit the ability to differentiate between European and North American strains. The potential use of the PRRSV structural proteins in subunit recombinant-type vaccines is also discussed.

The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp 145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection.

Equine herpesvirus 1 glycoprotein D (EHV-1 gD) expressed constitutively in mammalian cell lines had similar electrophoretic mobility to gD produced in EHV-1 infected cells but lacked a possibly complexed higher molecular weight form seen in the latter. Recombinant gD was N-terminally cleaved at the same site as gD in EHV-1 infected cells and expression was associated with enhanced levels of cell-cell fusion, indicating a role for EHV-1 gD in cell-to-cell transmission of virus.

The predicted amino acid sequence and secondary structures of S1 of the spike protein (S) of infectious bronchitis viral (IBV) strains from Europe, the U.S.A., and Japan were compared. An antigenic determinant that was highly conserved in both the primary amino acid sequence and secondary structure of all strains was identified between amino acid positions 240 to 255. A synthesized peptide corresponding to this region was found to react with all polyclonal antisera examined from various IBV strains and with one monoclonal antibody (MAb), 9B1B6, out of nine known to react with the S of Gray. The specificity of the interaction with MAb 9B1B6 was confirmed by competitive ELISA using bound and unbound peptide. Interestingly, the previously described epitope for 9B1B6 had been characterized as cross-reactive with several strains of IBV, as conformation-independent but reacting only with intact whole S, and as associated with the functional integrity of other epitopes, including neutralizing epitopes on the S protein. The apparent critical functional and structural nature of this highly immunogenic determinant suggests a potential contribution in developing protective, cross-reactive subunit vaccines to IBV.

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26-27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer < 1:10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.

We have constructed a recombinant baculovirus expressing the rubella virus E2 (42-45 KDa) and C (34 KDa) proteins. Sf9 cells infected with recombinant virus were able to synthesize and process the two proteins coded by a unique precursor gene. By immunoblot and immunoprecipitation analysis with polyclonal and monoclonal antibodies, a precursor polyprotein (66 KDa) and two other proteins migrating with an apparent molecular weight of 42 KDa and 36 KDa were recognized as E2 glycoprotein and C protein, respectively. The recombinant E2 protein appeared to be glycosylated since it was susceptible to tunicamycin. The results indicate that the RV polyprotein coding for E2 and C is expressed and proteolytically cleaved in insect cells. This baculovirus expression system provides a useful alternative approach for the production of rubella virus antigens and should allow the purification of large quantities of the RV proteins for further biochemical and immunological studies.

Three glycoproteins of bovine herpesvirus-1 (BHV-1) other than glycoproteins gI, gIII, and gIV were identified by monoclonal antibody (MAb) analyses. Monoclonal antibodies were obtained by immunization of mice with either BHV-1 envelope or virus infected cells, from which the glycoproteins gI, gIII, and gIV were removed by immunoaffinity. In the latter immunization procedure mice were tolerized either against normal cellular antigens with or without glycoproteins gI, gIII, gIV, and nucleocapsid. From 154 anti-BHV-1 hybridomas isolated, 39 MAbs precipitated a 108K glycoprotein. Two other glycoproteins of respectively 42K and 93K were precipitated each by one MAb. These three glycoproteins were detected in infected cell lysate. Nine anti-108K glycoprotein MAbs neutralized BHV-1 infectivity and three non-neutralizing MAbs were able to reduce plaque development when virus was grown in the presence of these MAbs. It is therefore suggested that this glycoprotein is involved in viral entry into the cell and in cell-to-cell spread of the virus.

Haemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.

Monoclonal antibodies directed against a surface glycoprotein of the bovine herpes virus type 2 (BHV-2, bovine herpes mammillitis virus) recognize also determinants of the major glycoprotein gB of the human herpes simplex virus type 1 (HSV-1). Cross-reacting antigens of the virions and in infected cells were localized with immunocytochemical methods, immunofluorescence as well as pre-embedding and cryoultramicrotomy immune electron microscopy. All antibodies stain to different degrees cell free BHV-2 and HSV-1 virions. In the cell two predominant staining patterns could be observed indicating that expression of epitopes is dependent upon the cell compartment: (i) staining of cytoplasmic membranes and enveloped particles within membrane systems and (ii) staining of intranuclear antigens. Antibodies tagging intranuclear antigens react with moderately dense material or with the periphery of nucleocapsids. This unexpected result is interpreted in terms of two hypotheses: (1) presence of common epitopes on two entirely different herpesvirus proteins conserved in HSV-1 and BHV-2 and (2) transport of gB or its precursor into the nucleus.

Monospecific antisera to HN and F glycoproteins of Newcastle disease virus were prepared, and their effects on the biological activities of the virus were investigated. Anti-HN serum inhibited hemagglutinating and neuraminidase activity, as well as hemolysis. Anti-F serum had no effect on hemagglutination or neuraminidase but inhibited hemolysis and virus-induced cell fusion. Anti-HN serum was highly neutralizing, while neutralization by anti-F serum was very inefficient in conventional plaque reduction tests, although both sera were estimated to contain comparable amounts of antibody reacting with the virus as indicated by complement fixation and immuno-diffusion tests. The neutralizing activity of anti-F serum was greatly enhanced by the addition of anti-IgG serum or fresh guinea pig serum, whereas that of anti-HN serum was little enhanced. Anti-HN serum incorporated in the agar overlay suppressed the development of plaques to some degree, while anti-F serum had little effect. The combination of anti-HN and anti-F sera resulted in a marked decrease in the number and size of plaques, demonstrating the synergistic effect of the two species of antibody in the containment of the spread of viral infection.

Ecotropic wild mouse retrovirus (1504 M)-induced neurogenic paralytic disease has been studied in inbred strains of mice. The major criterion for the successful transmission of the disease in the laboratory strains of mice is inoculation of high titer ecotropic virus in FV-1n strains of mice at newborn stage (less than or equal to 1 day old), Hybridization studies using 1504 M viral cDNA as probe indicate that in nonparalyzed mice, the inoculated virus replicates primarily in spleen tissue, whereas virus replication is evident in both spleen and central nervous system (CNS) tissue of paralyzed mice. Our studies on virus gene expression indicate that both viral gag gene product p30 and env gene product gp70 are expressed in brain, spinal cord and spleen tissues of paralyzed mice. Together, these results indicate that inoculation of neurotropic wild mouse virus into FV-1n strains of newborn laboratory mice is necessary for the establishment of infection in CNS tissue leading to virus replication and expression and resulting in the paralytic disease.

The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particles sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Bovine viral diarrhea virus (BVDV) was concentrated and purified by a combination of ultrafiltration, hydroextraction using polyethylene glycol and affinity chromatography. A lectin from Crotalaria juncea that has an affinity for galactose was used in the affinity chromatography. Virions of BVDV with classic envelopes were observed by electron microscopy. Four major proteins with estimated molecular weights of 75,000, 66,000, 54,000, and 26,000 were identified in sodium dodecyl sulfate--polyacrylamide gel electrophoresis slab gels. The proteins of 75,000 and 54,000 were glycoproteins as shown by staining with dansyl hydrazine.

Immunoelectron microscopy was used to detect the distribution of some dengue-2 virus proteins in infected Vero and Aedes albopictus (C6/36) cells. It was found that the envelope protein (GP 60) was located in clumps on the surface of plasma membrane, and accumulated very little in the infected cytoplasm. However no envelopment of dengue-2 virus nucleocapsids through the plasma membranes was observed. In contrast, the NS 3 (P 67) protein was distributed throughout the whole cytoplasm. No specific association of this protein with the proliferated virus-induced structures was seen. The NS 1 (GP 46) protein showed almost similar distribution as the NS 3 but its quantity appeared to be lower. The NS 3 and NS 1 distribution patterns observed supported the results of immunofluorescent staining but results on E protein were not consistent in both methods.

Indirect immunofluorescence with rabbit antisera was used to probe the intracellular locations of the antigens of envelope, prM (precursor to structural protein M) and the nonstructural glycoproteins NS 1 (formerly described as NV 3 or SCF) specified by the flaviviruses dengue-2 and Kunjin. Perinuclear staining in various types of foci was prominent for all antigens, and the distribution was influenced by whether cells were fixed with acetone or formaldehyde. Staining of Golgi-like masses or inclusions by anti-envelope sera occurred regularly and prominently in cells infected and stained with homologous anti-envelope antibodies; in the cross reactions, such staining was largely absent, especially in dengue-2 infected cells in which it was replaced by many small circular foci scattered throughout the cytoplasm. Anti-NS 1 also stained large perinuclear inclusions and small cytoplasmic foci, but the distribution of these was dissimilar to that observed with anti-envelope sera. Anti-prM appeared to contain a mixture of antibodies of different specificities, evident at different dilutions, possibly because of different cytoplasmic locations of prM and its cleavage products. All antisera produced small discontinuous foci on the plasma membrane of unfixed infected cells; antigens of NS 1 were sometimes prominent on the surface of acetone-fixed cells.

We have prepared four monoclonal antibodies to rubella virus E1 glycoprotein. Three nonoverlapping antigenic sites were delineated on E1 protein by competitive binding assays. Antibodies binding to one site were characterized by high hemagglutination inhibition (HI) titer but poor neutralizing activity. The addition of antiglobulin conferred neutralizing activity. Antibodies directed to two other antigenic sites had modest hemolysis inhibition but little or no HI and neutralizing activities. The addition of antiglobulin markedly augmented HI activity but had little effect on neutralizing activity. Epitopes defined by three antibodies were conserved among four rubella virus strains examined.

The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293-1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with 3H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the 3H-DFP was subjected to sequence analysis. The amino acids determined from the NH2-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that 3H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.

Four geographic variants (topotypes) of yellow fever (YF) virus from Africa and South America previously determined by RNA oligonucleotide mapping were analyzed for their structural, antigenic and virulence characteristics. The electrophoretic migration mobility and carbohydrate content of the envelope protein E characterized YF virus strains of South America. The NS 1 protein of South American and Central African YF virus strains was not precipitated by anti-NS 1 monoclonal antibodies (MAB) that precipitated NS 1 of West African strains. No distinction could be made among the YF virus strains on the basis of the neutralizing capacity of available MAB. South American, but not African YF virus strains were virulent for 8-day-old mice upon intraperitoneal inoculation. The results permitted characterization of topotypes of Central Africa and South America but failed to differentiate YF strains from West Africa.

Undenatured rubella virus envelope glycoproteins released by Tween-ether and trypsin treatment have been separated by Fast Protein Liquid Chromatography. Red cell adsorption located the haemagglutinin on VPI and its 13K cleavage product.

Analysis of highly purified fish Lymphocystis Disease Virus (LDV), strain Leetown NFH, by three different methods, namely periodic Acid Schiff reaction, radiolabelling with tritiated fucose and N-acetyl-D-glucosamine and staining with three lectins, indicated that ten glycoproteins were associated with the virus structure. Six of them were detected by all of the three methods, three by both radiolabelling and lectin staining but only one by the lectin technique. Localization of these glycoproteins at the surface or inside the virion is discussed.