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Rift Valley fever virus (RVFV) is transmitted among susceptible animals by mosquito vectors. Although the virus can be isolated from nasal and oral swabs of infected animals and is known to be highly infectious when administered experimentally via oral or respiratory route, horizontal transmission of the virus is only sporadically reported in literature. We considered that immunosuppression resulting from stressful conditions in the field may increase the susceptibility to horizontally transmitted RVFV. Additionally, we reasoned that horizontal transmission may induce immune responses that could affect the susceptibility of contact-exposed animals to subsequent infection via mosquito vectors. To address these two hypotheses, viremic lambs were brought into contact with sentinel lambs. One group of sentinel lambs was treated with the immunosuppressive synthetic glucocorticosteroid dexamethasone and monitored for signs of disease and presence of virus in the blood and target organs. Another group of contact-exposed sentinel lambs remained untreated for three weeks and was subsequently challenged with RVFV. We found that none of the dexamethasone-treated contact-exposed lambs developed detectable viremia, antibody responses or significant increases in cytokine mRNA levels. Susceptibility of immunocompetent lambs to RVFV infection was not influenced by previous contact-exposure. Our results are discussed in light of previous findings.

Vaccines based on nonspreading Rift Valley fever virus (NSR) induce strong humoral and robust cellular immune responses with pronounced Th1 polarisation. The present work was aimed to gain insight into the molecular basis of NSR-mediated immunity. Recent studies have demonstrated that wild-type Rift Valley fever virus efficiently targets and replicates in dendritic cells (DCs). We found that NSR infection of cultured human DCs results in maturation of DCs, characterized by surface upregulation of CD40, CD80, CD86, MHC-I and MHC-II and secretion of the proinflammatory cytokines IFN-β, IL-6 and TNF. Interestingly, expression of the most prominent marker of DC maturation, CD83, was consistently downregulated at 24 hours post infection. Remarkably, NSR infection also completely abrogated CD83 upregulation by LPS. Downregulation of CD83 was not associated with reduced mRNA levels or impaired CD83 mRNA transport from the nucleus and could not be prevented by inhibition of the proteasome or endocytic degradation pathways, suggesting that suppression occurs at the translational level. In contrast to infected cells, bystander DCs displayed full maturation as evidenced by upregulation of CD83. Our results indicate that bystander DCs play an important role in NSR-mediated immunity.

Replicon particles of Rift Valley fever virus, referred to as nonspreading Rift Valley fever virus (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8(+) T cells in vitro and that prophylactic and therapeutic vaccinations of mice with NSR encoding a tumor-associated CD8 peptide can control the outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines., Importance: Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Vaccination has been one of the most important interventions in disease prevention and control. The impact of vaccination largely depends on the quality and suitability of the chosen vaccine. To determine the suitability of a vaccine strain, antigenic matching is usually studied by in vitro analysis. In this study, we performed three in vitro test methods to determine which one gives the lowest variability and the highest discriminatory capacity. Binary ethylenimine inactivated vaccines, prepared from 10 different foot-and-mouth disease (FMD) virus serotype A strains, were used to vaccinate cattle (5 animals for each strain). The antibody titers in blood serum samples 3 weeks postvaccination (w.p.v.) were determined by a virus neutralization test, neutralization index test, and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). The titers were then used to calculate relationship coefficient (r1) values. These r1 values were compared to the genetic lineage using receiver operating characteristic (ROC) analysis. In the two neutralization test methods, the median titers observed against the test strains differed considerably, and the sera of the vaccinated animals did not always show the highest titers against their respective homologous virus strains. When the titers were corrected for test strain effect (scaling), the variability (standard error of the mean per vaccinated group) increased because the results were on a different scale, but the discriminatory capacity improved. An ROC analysis of the r1 value calculated on both observed and scaled titers showed that only r1 values of the liquid-phase blocking ELISA gave a consistent statistically significant result. Under the conditions of the present study, the liquid-phase blocking ELISA showed less variation and still had a higher discriminatory capacity than the other tests.

The Bluetongue virus serotype 8 (BTV-8) strain, which emerged in Europe in 2006, had an unusually high ability to cause foetal infection in pregnant ruminants. Other serotypes of BTV had already been present in Europe for more than a decade, but transplacental transmission of these strains had never been demonstrated. To determine whether transplacental transmission is a unique feature of BTV-8 we compared the incidence and pathological consequences of transplacental transmission of BTV-8 to that of BTV-1. Nine pregnant ewes were infected with either BTV-8 or BTV-1. The BTV strains used for the infection were field strains isolated on embryonated chicken eggs and passaged twice on mammalian cells. Blood samples were taken to monitor the viraemia in the ewes. Four weeks after the infection, the foetuses were examined for pathological changes and for the presence of BTV. BTV-8 could be demonstrated in 12 foetuses (43%) from 5 ewes (56%). %). BTV-1 was detected in 14 foetuses (82%) from 6 ewes (67%). Pathological changes were mainly found in the central nervous system. In the BTV-8 group, lympho-histiocytic infiltrates, gliosis and slight vacuolation of the neuropil were found. BTV-1 infection induced a severe necrotizing encephalopathy and severe meningitis, with macroscopic hydranencephaly or porencephaly in 8 foetuses. In our experimental setting, using low passaged virus strains, BTV-1 was able to induce transplacental transmission to a higher incidence compared to BTV-8, causing more severe pathology.

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 10(6.3) TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

Background: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity., Methodology/principal Findings: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited., Conclusions/significance: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB (476)R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB (476)R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB (476)S variant into a His residue did not affect virulence. However, the (710)His in the C1.1.1/CoBrB virus, together with adaptive mutations (276)R, (476)R, and (477)I in E(rns), resulted in reduced virulence in pigs. These results indicated that mutations in E(rns) and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E(rns) may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the (710)L-->H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E(rns) (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553-9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser(476) and an HS-dependent recombinant virus (S-RT virus) with Arg(476) were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E(rns), E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg(476) was mutated back to Ser(476). Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E(rns) S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.

The 3' untranslated region (UTR) is an important element that determines the level of recombinant protein expression via baculovirus vectors. Previous work using chloramphenicol acetyl transferase as reporter has shown that p10-promoter based baculovirus vectors with the authentic p10 3' UTR resulted in higher expression levels than vectors carrying an SV40 early terminator, as part of a lacZ selection cassette. To examine whether a similar increase in expression levels could be obtained for baculovirus-expressed glycoproteins, the classical swine fever virus E2 antigen was used as a model. With the authentic p10 3' UTR higher levels of E2 transcript were found than in the presence of the SV40 terminator. This higher number of transcripts was accompanied by elevated levels of intracellular, non-glycosylated E2 protein. However, the levels of intracellular glycosylated forms of E2 and of extracellular E2 were similar for both type of terminators. These results show that translation of the recombinant mRNA is not the rate limiting step in the expression of glycoproteins, but the downstream processing and secretion of the translation products.

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.

An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein E(rns) of classical swine fever virus (CSFV) was used to rescue CSFV E(rns) deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of E(rns) from this cell line were indistinguishable from those of CSFV E(rns). Two E(rns) deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of E(rns) (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of E(rns) antibodies, due to a deletion of 66 amino acids in E(rns). The E(rns) deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by E(rns)-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that E(rns) from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) E(rns) in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.

The 5'-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5'-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

Envelope glycoprotein E(rns) of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E(rns) are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E(rns) was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E(rns), the mutated proteins had lost their RNase activity. The mutated proteins reacted with E(rns)-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E(rns). This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E(rns) (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E(rns) gene and by neutralizing polyclonal antibodies directed against E(rns), indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E(rns) plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

Pure preparations of envelope glycoproteins E(rns) and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV). Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 microg/ml E(rns). After removal of the virus no E(rns) was needed in the overlay medium (growth medium) to maintain this level of inhibition. In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 microg/ml E2 was only achieved when E2 was added to the overlay medium. When E2 was omitted, a maximum of 50% inhibition was achieved. This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface. Treatment with 100 microg/ml E(rns) released these particles from the cell surface. Furthermore, E(rns) bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by E(rns). These results demonstrated that E(rns) and E2 interacted with different cell surface receptors. Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor. BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV E(rns), whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of E(rns) plays a role in host tropism.

Conventionally, the genus Pestivirus of the family Flaviviridae has been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). To date, BDV and BVDV have been isolated from different species, whereas CSFV seems to be restricted to swine. Pestiviruses are structurally and antigenically closely related. Envelope glycoprotein E2 is the most immunogenic and most variable protein of pestiviruses. We cloned E2 genes of many different pestivirus strains, including those from a deer and a giraffe. The E2 genes were transiently expressed, characterized with monoclonal antibodies, sequenced, and compared. Based on these data, we can delineate six major groups within the Pestivirus genus. Four groups correspond to defined genotypes, whereas the two other groups could be new genotypes within the Pestivirus genus. One group comprises CSFV strains isolated from swine. A second group consists of BDV strains Moredun, L83, and X818, which have been isolated from sheep, and strain F from swine. A third group contains strain BD78 from sheep, strain 5250 from swine, and strain 178003 from cattle. On the basis of E2, these viruses are very similar to BVDV strains associated with acute severe outbreaks of bovine viral diarrhea, so-called type 2 BVDV. The fourth group consists of BVDV strains originating predominantly from cattle. This BVDV group can be divided into two subtypes or subgroups BVDV Ia and Ib: BVDV Ia contains viruses from the United States, such as like NADL and Oregon, and some others, such as 150022 and 1138 from Europe. Subgroup BVDV Ib contains strain Osloss and several Dutch isolates. The fifth and sixth "groups" could be proposed as two new genotypes and contain strains Deer and Giraffe, respectively., (Copyright 1997 Academic Press.)

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.

Envelope glycoprotein E2, formerly called E1 or gp51-54, of classical swine fever virus (CSFV) expressed in insect cells protects swine from classical swine fever. Monoclonal antibodies directed against epitopes of domains B and C and subdomain A1 are neutralizing. The domains are located on two structural antigenic units in a proposed model of the antigenic structure of E2. One unit consists of nonconserved antigenic domains B and C and the other contains highly conserved antigenic domain A. We produced several mutant E2 proteins by use of the baculovirus expression system. Two selected mutants were E2 proteins in which one of the two structural antigenic units, unit B/C or unit A, was deleted. The protective capacity of the mutant E2 proteins was investigated in an immunization experiment in pigs. Titres of the neutralizing responses in pigs immunized with mutant E2 proteins were all comparable with that of intact E2. These vaccinated pigs were protected against an intranasal lethal CSFV challenge, indicating that the immune response induced by one structural antigenic unit of E2 can protect pigs against classical swine fever.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain. Furthermore, we showed that the sequences at the 5' and 3' termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins Erns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames (ORFs) that encode putative viral proteins. ORFs 2 to 7 were cloned in plasmids downstream of the Sp6 RNA polymerase promoter, and the translation of transcripts generated in vitro yielded proteins that could be immunoprecipitated with porcine anti-LV serum. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and antipeptide sera were raised in rabbits. Antisera that immunoprecipitated the in vitro translation products of ORFs 2 to 5 and 7 were obtained. Sera containing antibodies directed against peptides from ORFs 3 to 7 reacted positively with LV-infected alveolar lung macrophages in the immunoperoxidase monolayer assay. Using these antipeptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5. The E protein in virus particles contains one or two N-glycans that are resistant to endo-beta-N-acetyl-D-glucosaminidase H. This finding indicates that the high-mannose glycans are processed into complex glycans in the Golgi compartment. The protein composition of the LV virions further confirms that LV is evolutionarily related to equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.

Three groups of 150 SPF chickens were spray-vaccinated with live Newcastle disease La Sota-type vaccine (clone 30) at one day of age, and another three groups were NDV spray-vaccinated at 10 days of age. In each of the two series of NDV-vaccinated groups, one group also received at day-old 10(5) TCID50 of chicken anaemia virus (CAV) also and another group 10(5) TCID50 of CAV plus a low dose of virulent Marek's disease virus (MDV). After one week, chickens of the groups which had been NDV-vaccinated and CAV-infected at day-old, with or without MDV, showed severe respiratory distress, conjunctivitis, drooping wings and ruffled feathers. After two weeks, wet and inflamed eyes were observed. After three weeks the respiratory problems were overcome, but the entire group showed retarded growth as compared with the group which had received NDV vaccine only. The 'respiratory sounds' were milder in the chickens NDV-vaccinated at 10 days of age, about 10% of the chickens showing retarded growth. Mortality in CAV-infected chickens which had received NDV vaccine at day-old was above 30% at 4 weeks of age, and between 15 and 20% when NDV had been administered at the age of 10 days, and was 5% in the two NDC vaccine control groups. Decreased haematocrit levels were measured in all four CAV-infected groups at 14 days of age. In serum samples collected for 6 weeks at weekly intervals from chickens of the six groups, no differences were observed between HI antibody titres against NDV virus. Thus, dual infection with CAV and live NDV vaccine did not impair the humoral immune response against attenuated Newcastle disease vaccine.

Envelope glycoprotein E1 (gp51 to gp54) is the most antigenic protein of hog cholera virus or classical swine fever virus (CSFV). Four antigenic domains, A to D, have been mapped on E1 with a panel of monoclonal antibodies (MAbs) raised against CSFV strain Brescia. The boundaries of these domains have been established by extensive studies on binding of MAbs to transiently expressed deletion mutants of E1 (P. A. van Rijn, E. J. de Meijer, H. G. P. van Gennip, and R. J. M. Moormann, J. Gen. Virol. 74:2053-2060, 1993). In this study, we used neutralizing MAbs of domains A, B, and C to isolate MAb-resistant mutants (MAR mutants) of CSFV strain Brescia and Chinese vaccine strain ("C"). The E1 genes of MAR mutants were cloned in a eukaryotic expression vector, and the effects of MAR mutations on epitopes were studied with a panel of 19 MAbs by immunostaining of COS1 cells transiently expressing these mutant E1s. Except for the MAR mutation Cys-->Arg at position 792, which abolished binding of all MAbs of domains A and D, amino acid substitutions affected only MAbs belonging to the same domain as the MAb used to select the MAR mutant. However, a MAR mutation in a particular domain did not per se abolish binding of all MAbs recognizing that domain. Furthermore, MAR mutants possessed conservative as well as nonconservative amino acid substitutions. To investigate the significance of a secondary structure for the binding of MAbs, all cysteine residues in the N-terminal antigenic part of E1 were mutated to serine. We found that the cysteines at positions 693 and 737 were essential for binding by MAbs of domains B and C, whereas those at positions 792, 818, 828, and 856 appeared to be essential for the binding of most MAbs of domains A and D. These results fully comply with the previously proposed two-unit structure of the N-terminal half of E1. One unit consists of antigenic domains B and C, whereas the other unit consists of the highly conserved domain A and domain D. We conclude that the first six cysteines are critical for the correct folding of E1. A model of the antigenic structure of E1 is presented and discussed.

Four antigenic domains (A, B, C and D) on envelope glycoprotein E1 (gp51-54) of hog cholera virus strain Brescia have been specified by using 13 monoclonal antibodies (MAbs) that recognize non-conserved and conserved epitopes. It was shown that the non-conserved epitopes map to the N-terminal half of E1 by analysis of chimeric E1 proteins of strains Brescia and C. Conserved epitopes, however, could not be mapped using this approach. Here we describe mapping of both conserved and non-conserved epitopes on E1 by the use of an extensive set of single and double deletion mutants of E1 of strain Brescia. Deletion mutants were transiently expressed in COS1 cells and analysed by immunostaining with the 13 MAbs directed against strain Brescia and four MAbs directed against strain C. All MAbs bound to the N-terminal half of E1, i.e. amino acids 690 to 866 encoded by the sequence of strain Brescia. Domain B and one epitope in domain C are located between residues 690 and 773. Other epitopes in domain C are located on an extended region, i.e. between residues 690 and 800. Conserved epitopes of domain A are mapped between residues 766 and 866, whereas the only non-conserved epitope in this domain is located between residues 766 and 813. Domain D, represented by one MAb, is located in the same region as this non-conserved epitope of domain A, i.e. between residues 766 and 800. The results suggest the presence of two distinct antigenic units on E1, one consisting of domains B and C and the other consisting of domain A.

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.

During the replication of Lelystad virus in alveolar lung macrophages, a 3'-coterminal nested set of six subgenomic RNAs (RNA2 to RNA7) is formed. These contain a common leader sequence derived from the 5' non-coding region of the genomic RNA. In this study, the sequence of the junction sites, i.e. the sites where the leader sequence joins to the body of the subgenomic RNA, was determined for all six subgenomic RNAs. For each subgenomic RNA, six to nine cDNA clones were isolated by means of reverse transcription and PCR. The nucleotide sequence at the junction site was identical for all eight cDNA clones derived from subgenomic RNA4. However, heterogeneity was observed in the nucleotide sequence surrounding the junction sites of the cDNA clones derived from subgenomic RNAs 2, 3, 5, 6 and 7. This heterogeneity suggests that the fusion of the leader to the body of the subgenomic RNA may be imprecise. The junction sites of the six subgenomic RNAs had a conserved sequence motif of six nucleotides (UCAACC or a highly similar sequence). The distance between the junction site and the translation initiation codon of the downstream open reading frame varied from nine to 83 nucleotides.

In an earlier report, we described the construction of the genetically engineered pseudorabies virus strain 2.4N3A which does not express glycoprotein gI. Although this strain showed a strongly reduced virulence in 10-week-old seronegative pigs, it could still cause severe disease or death in 3-day-old piglets. To attenuate the strain further, we constructed mutants with a deletion in the viral thymidine kinase gene. One mutant strain, designated 783, has a deletion of 19 base pairs and was shown to be highly immunogenic and safe for vaccination of pigs against pseudorabies virus.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.

The nucleotide sequences of two different rat lens gamma-crystallin cDNA clones, pRL gamma 2 and pRL gamma 3, have been determined. pRL gamma 3 contains the complete coding information for a gamma-crystallin of 173 amino acids whereas pRL gamma 2 is incomplete in that it lacks the codons for the first three amino acids of a separate but very homologous gamma-crystallin of identical length. Both rat gamma-crystallins are homologous to the known amino acid sequence of bovine gamma-crystallin II which is only a single amino acid longer. The length of the region downstream the coding sequence to the A-A-T-A-A-A polyadenylylation signal sequence is 40 nucleotides in each clone. In pRL gamma 3 the poly(A) signal sequence is followed at 14 nucleotides by a remnant of the poly(A) tail which indicates that this clone contains a complete 3' noncoding region. pRL gamma 2 has only seven nucleotides following this signal sequence and no poly(A) tail, suggesting an incomplete 3' end. The cDNA clones show an overall nucleotide sequence homology of 85%. The mutual homology at the amino acid level is 73% whereas their amino acid homology with bovine gamma-crystallin II is about 70%. The nucleotide sequence of each clone also reveals a high intragenic homology and seems to be duplicated in itself. We suggest that the gamma-crystallin genes have arisen by multiple duplications of a primordial gene which consisted of about 120 nucleotides.

A human genomic cosmid clone, pHcos gamma-1, has been isolated containing two closely linked gamma-crystallin genes, oriented in the same direction. The sequence of these genes and their 5' and 3' flanking regions has been determined. The coding regions of both genes are interrupted by two introns. The first introns (94 and 100 bp, respectively) are located in the 5' region of the genes. The second introns (2.82 and 0.95 kb, respectively) divide the genes into two halves, each encoding a structural domain of the gamma-crystallin protein. The coding regions of the two genes show 80% homology. Due to a mutation in the splice acceptor site of the second intron of the first gene, the coding region of its third exon is 3 bp longer than that of the second gene. In the flanking regions several conserved sequence elements were found, including those elements that are known to be necessary for the correct expression of eukaryotic genes. The flanking and intronic regions of the genes contain 'simple sequence' DNA and Alu repeats. The Alu repeats are usually clustered, contain truncated elements, and are often located near simple sequence DNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.

To provide access to crystallin-specific DNA sequences, we have constructed plasmid clones bearing duplex DNA sequences complementary to crystallin mRNAs isolated from rat lens. Optimization of the cDNA reaction conditions enabled us to fractionate three double-stranded (ds) cDNA groups. Molecular cloning of dC-tailed ds cDNAs into the Pst I site of dG-tailed pBR322 yielded crystallin-specific clones of each group. By means of positive hybridization selection and translation, recombinant plasmids containing cDNA sequences coding for rat lens polypeptides from alpha-, beta-, and gamma-crystallins could be identified. The established cDNA clones have been used for a blot-hybridization analysis to map the crystallin mRNAs from which they originated. Both procedures revealed a high degree of homology between the gamma-crystallin sequences. From the beta-crystallin class, the beta H-specific cDNA coding for the beta B1a polypeptide was obtained. The alpha A-chain clone did not show any cross-hybridization to the alpha B-chain mRNA despite the existence of 60% homology between the corresponding gene products. As this clone hybridized to both alpha A2 and alpha AIns mRNAs, sequence analysis was applied for further characterization. The results showed that the cloned cDNA corresponds to the alpha A2 sequence exclusively.

The rat beta B1-crystallin gene is 13.6 kilobases long and contains six exons. The coding region of the gene is divided over five exons. Each functional entity of the protein is encoded by a separate exon except for the carboxyl-terminal extension, which shares the last exon with the fourth protein motif. Exon 2, encoding the amino-terminal extension of the protein, contains two direct repeats with an overall homology of 68% to the rat brain identifier sequence. A copy of the brain identifier sequence is also found in the 3'-flanking region of the gene. The start site of the mRNA was located by S1 nuclease mapping and analysis of the RNA sequence. The 5' end of the gene was shown to be a 27-base-pair noncoding exon, which is separated from the translation start site by 1.36 kilobases of intronic DNA. The 5'-flanking sequence of the beta B1 gene is highly homologous to that of a gamma-crystallin gene.

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.