Contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model

The pathogenesis of three equine herpesvirus 1 (EHV-1) recombinants was assessed in a CBA mouse model. Sequences encoding the majority of glycoproteins I (gI) and E (gE) were deleted from the pathogenic EHV-1 strain RacL11 (L11ΔgIΔgE), and sequences comprising the 3859 bp deletion within the strain KyA U_S segment, which includes genes 73 (gI), 74 (gE), and 75 (putative 10 kDa protein 75), were re-inserted into attenuated KyA (KgI/gE/75). In addition, genes gE and 75 were inserted into KyA to generate the EHV-1 recombinant KgE/75. The insertion of the 3859 bp U_S segment was sufficient to confer virulence to KyA, as indicated by pronounced signs of clinical disease including substantial weight loss. A large plaque morphology was observed in cells infected with KgI/gE/75 compared with KyA, and a small plaque phenotype was observed in cells infected with L11ΔgIΔgE compared with RacL11. These data indicate that gI and/or gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell. The deletion of both gI and gE from the pathogenic RacL11 strain did not reduce clinical signs of disease in infected mice, but did decrease mortality compared with RacL11. Furthermore, the insertion of genes 74 (gE) and 75 into the vaccine strain KyA did not alter the attenuated phenotype of this virus. Finally, KgI/gE/75 and RacL11 elicited the production of the proinflammatory chemokines MIP-1, MIP-1β, and MIP-2 in the lungs of infected mice, while KyA did not, suggesting that gI and/or gI and gE contribute to the up-regulation of these mediators of inflammation. These findings show that gI, and/or gI and gE restore a virulent phenotype to the EHV-1 KyA strain, and indicate that virulence factors, in addition to gI and gE, contribute to the pathogenesis of the RacL11 strain.     

Skin virulence of attenuated pseudorabies virus (PRV) strains.

After intradermal inoculation of the attenuated pseudorabies virus (PRV) strains, Bartha, MK35 (gI+) and MK35-T-2 (gI-) to rabbits, they caused local inflammatory nodules with oedema reddiness and necrosis. The specifity of these reactions was confirmed by their inhibition with anti-PRV serum. The minimal dose that caused visible skin reaction was about 10(2) TCID50/ml for Bartha and MK35-T-2 (gI-) strains and over 10(4) TCID50/ml for the MK35 (gI+) strain. It is assumed that the established residual skin virulence can serve as an additional distinction marker for attenuated PRV strains.     

Formation of Pseudorabies virus glycoprotein E/I complex in baculovirus recombinant system

Glycoproteins E (gE) and I (gI) of Pseudorabies virus (PRV) form a non-covalently bound complex to which a number of functions have been attributed. The gE/gI complex formation was studied using a series of full-length and truncated forms of gE and gI expressed in baculovirus recombinant system. Both glycoproteins were truncated by stepwise removal of their C-terminal parts and their ability to form the complex was studied by radioimmunoprecipitation. It was found that N-terminal domains of gE and gI containing first 122 and 106 aa, respectively, were sufficient for the complex formation.     

Conversion of US3-encoded protein kinase gene from pseudorabies virus in a diploid gene located within inverted repeats by genetic recombination between the viral genome isomers.

The pseudorabies virus (PRV) genome consists of two components, long (U(L)) and short (U(S)) regions. The U(S) region is the only one capable of inverting itself relative to the U(L) region during productive infection, generating two equimolecular isomeric forms of viral DNA. Here we describe a recombinant virus (gIp2) generated by genetic recombination between pseudorabies viral isomers. This recombination event was observed in the parental virus gIS8, which was obtained by insertion of the alpha4-TK herpes simplex virus type 1 (HSV1) gene. The growth of gIS8 virus in the presence of 5-bromodeoxyuridine (BrdU) yielded gIp2. This was generated by nonhomologous recombination either between the two viral genomic isomers of gIS8, P and I(U/S), or between the same P isomer using nonhomologous and homologous recombination, with loss of the HSV1 sequences and duplication of the PRV US3-encoded protein kinase gene. Virus gIp2 is negative for TK, gI, gE, 11K and 28K and shows an in vitro replication capacity in neuronal cells approximately 22 times lower than that of parental virus gIS8, and similar to that of the Bartha vaccine virus strain in monkey kidney and human neuronal cells.     

Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector

Summary Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and theE. coli -galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, -gal plaque assays, and by immunoprecipitation experiments on extracts from³H-mannose-labelled cells. The recombinant IBRV expressing -gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.     

Deleting valine-125 and cysteine-126 in glycoprotein gI of pseudorabies virus strain NIA-3 decreases plaque size and reduces virulence in mice

Summary We investigated the function of antigenic domains on gI in virulence and immunogenicity. Three PRV gI mutants were constructed by deleting nucleotides coding for the following amino acids: valine-125 and cysteine-126, located in a discontinuous antigenic domain (M 303); glycine-59 and aspartic acid-60 located in a continuous antigenic domain (M 304); and arginine-67 and alanine-68, located in a discontinuous antigenic domain (M 305). Mismatch primers in the polymerase chain reaction were used to introduce the deletions. Anti-gI monoclonal antibodies were used in an immunoperoxidase monolayer assay to distinguish PRV gI mutants from wild-type PRV. The gI mutant viruses were tested for their growth in vitro and for their virulence in mice. The growth properties of PRV gI mutant virus M 303 were comparable to the growth properties of a PRV gI-negative mutant (M 301): both mutants produced small plaques in various cells, and when grown on swine kidney cells and chicken embryo fibroblasts, their growth was disadvantaged compared to wild-type PRV. However, in embryonal Balb/c mouse cells expressing gI, gI mutant viruses and wild-type PRV produced plaques of the same size, confirming that the mutations in gI are responsible for the small plaque phenotype. The growth properties of PRV gI mutant viruses M 304 and M 305 were comparable to the growth properties of wild-type PRV. When the mean time to death was used as the criterion, the gI mutant viruses M 301 and M 303 were significantly less virulent in mice than wild-type PRV. Four other, independently obtained, PRV mutants all carrying the valine-125 and cysteine-126 deletion (M 308, M 309, M 310 and M 311 respectively) exhibit the same phenotype. Our results show that deleting valine-125 and cysteine-126 in gI decreases plaque size and reduces virulence in mice to the same degree as deleting the gI protein.     

A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells

Summary. In alphaherpesviruses, glycoprotein I (gI) and glycoprotein E (gE) form a heterodimer that functions in cell-to-cell spread of the virus. Generally, alphaherpesvirus mutants that lack these glycoproteins are replication competent in cell culture but show a reduced capacity for cell-to-cell spread and hence smaller plaque sizes. Infectious laryngotracheitis virus (ILTV), or Gallid herpesvirus 1, is an alphaherpesvirus that causes respiratory disease in chickens. The roles of gI and gE in ILTV have not been investigated previously. In this study, a glycoprotein I and glycoprotein E deletion mutant of ILTV (gI/gE-ve ILTV) was generated by replacing the region of the ILTV genome coding for the adjacent gI and gE genes with the gene for enhanced green fluorescent protein (eGFP). This gI/E-ve ILTV was readily propagated in cell culture in the presence of wildtype ILTV (wt ILTV). However, in the absence of wt ILTV the propagation of gI/gE-ve ILTV was severely impaired. Infection of permissive cell cultures with gI/gE-ve ILTV failed to produce plaques but single infected cells could be identified by fluorescence microscopy. This suggests that gI/gE has a more significant role in the cell-to-cell spread of ILTV in vitro than in many other alphaherpesviruses.     

Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB,gC, gD,and gE

Summary We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.     

Genetic variability of sexual behavior in a natural population ofDrosophila melanogaster

Although many experiments on laboratory stocks ofDrosophila have suggested that mate choice is a major feature of sexual selection in this organism, few attempts have been made to measure its extent in wild populations. In this study, a crossing design was used to obtain a set of 13 genetically identical independent lines representative of genotypes from an African population ofDrosophila melanogaster. They were tested for variation in sexual behavior using dyadic tests. Significant variation in orientation and vibration latencies was found for males, and in mating speed and copulation duration for both sexes. No evidence of assortative mating, either positive or negative, was found. The absence of a correlation in mating speed between males and females sharing the same genotype leads us to doubt the applicability of the notion of male eagerness and female reluctancy inDrosophila and the importance of vigor as a factor in mating speed. The absence of mate choice in natural populations ofDrosophila seems to us the most likely hypothesis on the basis of both theory and empirical evidence.     

Biosynthesis and interaction of glycoproteins E and I of canine herpesvirus.

In cells infected with canine herpesvirus (CHV), the mature form of glycoprotein E (gE) had a molecular weight of 94 kDa, and that of glycoprotein I (gI) had a broad range of molecular weights of 55-62 kDa. gE and gI formed a complex like gE and gI of other alphaherpesviruses. When cells were infected with the gI minus mutant of CHV (gI/Z), the mature form of the 94 kDa gE was not formed, but a 76 kDa gE polypeptide was found. Similarly, no mature gI was formed in cells infected with the gE minus mutant of CHV (gE/Z), but a 40 kDa gI polypeptide was formed. When cells were coinfected with gE/Z and gI/Z, the molecular masses of gE and gI were increased from 76 to 94 kDa and from 40 to 55-62 kDa, respectively. We constructed vaccinia virus recombinants which expressed CHV gE or CHV gI. Only when cells were coinfected with both the vaccinia recombinant which expressed gE and the vaccinia recombinant which expressed gI, gE and gI were processed into their mature forms. Our results suggest that the presence of both gE and gI is necessary for efficient processing of the precursors of gE and gI to their mature forms.     

用减毒型亲神经性跨神经元传递的假狂犬病毒追踪神经通路

用减毒型亲神经性跨神经元传递的假狂犬病毒追踪神经通路杨明（ＹａｎｇＭｉｎｇ）ＬＷＥｎｇｕｉｓｔＪＰＣａｒｄＲＲＭｉｓｅｌｉｓ（美国宾夕法尼亚州大学动物生物学系，美国普林斯顿大学分子生物学系，美国匹茨伯格大学神经科学系）假狂犬病病毒（Ｐｓｅｕｄｏｒａｂ...     

The extracellular part of glycoprotein E of bovine herpesvirus 1 is sufficient for complex formation with glycoprotein I but not for cell-to-cell spread

Summary.  Glycoproteins gE and gI of bovine herpesvirus 1 (BHV-1) are type I transmembrane proteins that can form a complex that is involved in cell-to-cell spread mechanisms. The extracellular domains of both proteins have cysteine-rich regions that are also found in the homologous proteins of other alphaherpesviruses. The extracellular domain of gE has two conserved cysteine-rich regions: C1 and C2. The other conserved regions in gE are located between C2 and transmembrane region and in the cytoplasmic domain of gE. We studied the complex formation between gE and gI using a series of truncated gE proteins and a full length form and a secreted form of gI. All proteins were expressed in recombinant baculoviruses. To analyse the complex formation between these polypeptides we used monoclonal antibodies (MAbs 67 and 75) that specifically react with the gE/gI complex and not with separately expressed glycoproteins gE and gI alone. This analysis showed that the BHV-1 gE/gI complex can be formed in insect cells after a co-infection with baculoviruses expressing gE and gI in their full length form. When secreted forms of gE and gI were expressed after co-infection, the gE/gI complex was still formed and could also be detected in the tissue culture medium. This gE/gI complex was also formed after mixing the tissue culture media of insect cells expressing the secreted form or gE or gI separately. The smallest part of gE that still formed a complex is encoded by the first 246 residues of gE. This extracellular domain contains only the C1 region, showing that the C2 region is not essential for gE/gI complex formation. Shorter forms of gE encoding the C1 region did not form a detectable complex. We also found that the formation of gE/gI complex is not sufficient for normal cell-to-cell spread of BHV-1. A recombinant BHV-1 gE TM-virus, expressing a truncated glycoprotein E from which the transmembrane and cytoplasmic domain were removed, forms plaques as small as a gE null mutant. Accepted July 26, 1999/Received March 5, 1999     

Phagocytosis and Chemiluminescence Response of Granulocytes to Monodisperse Latex Particles of Varying Sizes and Surface Coats

The response of human granulocytes to polystyrene latex beads of diameter 0.1–7 m was measured by luminol-dependent chemiluminescence. In all instances, the response to beads of 3–7 m was definitely higher than with smaller beads. In protein-free medium, the chemiluminescence response was slow compared to that of opsonized zymosan, and the highest response was only 9% of the response to opsonized zymosan. Scanning electron microscopy showed that granulocytes in suspension bound the particles, occasionally by extending rope-like protrusions. When the beads were coated with albumin, the chemiluminescence diminished to about 1/3 of that seen with uncoated beads; however, preincubating the beads in serum led to a large increase with beads of 1.1 m (to 25% of the maximal response to opsonized zymosan) and 3.19 m (to 42%), but with the smallest beads, no increase was noted. "Priming" of the cells with tumor necrosis factor- caused a further increase with serum-coated beads. When uncoated beads of 1.1 m were tested with "primed" cells, there was an increase of 6 times in the chemiluminescence compared to un-"primed" cells.     

Vasopressin studies in the rat

Summary In previous experiments single i.v. injections of 0.2–10 U ADH were made in alcohol anesthetized rats and the amount of extra water reabsorbed during the antidiuretic phase (urine deficit) was measured. The dose response curve resembled a saturation curve with a fairly linear rise up to 1–1.5 U. When 4–6 U were injected the urine deficit was not appreciably greater while 75% of the ADH injected appeared in the urine during antidiuresis. It was concluded that during a single injection of ADH 1–1.5 U were bound almost instantaneously at receptors of the tubular wall and inactivated during the slow process of water reabsorption, while the excess ADH was excreted in the urine. It was estimated that 1 U ADH was needed for the reabsorption of approximately 5 cm³ water. The time required for this process is short at a high rate of reabsorption and vice versa.In the present investigation the single i.v. injections were repeated with 20 U. The higher dose permitted the separate determination of ADA in 3 consecutive samples of the urine collected during the antidiuretic phase. The result fully confirmed the working hypothesis e.g.:1. The antidiuretic activity (ADA) obtained with 20 U Pitressin was not greater but even (though not significantly) smaller than that obtained previously with 5 U Pitressin or 1 U Tonephin.2. 95±15% of the ADA injected appeared in the urine. This means that the difference between the 20 U injected and the 18.5–19 U appearing in the urine after deduction of the 1–1.5 U ADH supposedly bound at tubular pore sites was too small to be detected with our bioassay.3. Under the assumption that 1 U Pitressin was used up for the reabsorption of approximately 4 cm³ water a vasopressin-water-equivalent in the order of 1 mole vasopressin for 10⁸ mole water reabsorbed, could be calculated.4. The amount of vasopressin excreted by the kidney follows an exponential function with a half life of 5 min.5. The vasopressin clearance is approximately 1.0 cm³/min · rat and lies within the range of inulin clearance (1.2 cm³/min · rat). It is suggested that elimination of excess vasopressin proceeds by a simple filtration process.6. Calculating on a weight basis the ADH-requirement of the 200 times heavier human kidneys leads to the value 200–300 U. Using a vasopressin-water-equivalent of 4–5 cm³ water per 1–1.5 U (action time 50 min) it can be predicted that the human kidney must lose approximately 261 water per day under the condition of a complete lack of vasopressin. This agreement with the actual observations in diabetes insipidus patients supports the belief that some of the concepts worked out in the alcohol anesthetized rat are valid under circumstances other than the strict conditions of this preparation.

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Zusammenfassung In früheren Experimenten wurden Einzelinjektionen von 0,2–10 E ADH in Alkohol narkotisierte Ratten gemacht und die Wassermenge bestimmt, die während der antidiuretischen Phase rückresorbiert wurde. Die Dosis-Antwort-Kurve hatte den Charakter einer Sättigungskurve mit einem praktisch linearen Anstieg bis 1–1,5 E. Der antidiuretische Effekt war bei einer Injektion von 4–6 E nicht deutlich größer als bei der kleineren Dosis, gleichzeitig fanden sich 75% der injizierten ADH-Menge im Urin der antidiuretischen Phase. Es wurde geschlossen, daß während einer Einzelinjektion von ADH 1–1,5 E fast augenblicklich von Receptoren der Tubuluswand gebunden und dann während des langssamen Prozesses der Wasserresorption inaktiviert werden, während das überschüssige ADH im Urin ausgeschieden wird. Schätzungsweise war 1 E ADH erforderlich, um 5 cm³ Wasser rückzuresorbieren. Die für den Resorptionsvorgang erforderliche Zeit war relativ kurz bei hoher Resorptionsrate und umgekehrt.In den vorliegenden Untersuchungen wurden die Einzelinjektionen mit 20 E wiederholt. Die höhere Dosis erlaubte, die antidiuretische Aktivität (ADA) in drei aufeinanderfolgenden Urinportionen der antidiuretischen Phase getrennt zu bestimmen. Das Resultat bestätigte die Arbeitshypothese.1. Die mit 20 E Pitressin erzielte ADA von 4 cm³ war nicht größer, sondern (nicht signifikant) kleiner als diejenige, die in den früheren Versuchen mit 5 E Pitressin oder 1 E Tonephin gefunden wurden.2. 95±15% der injizierten ADA erschienen im Urin. Das heißt, die Differenz zwischen den 20 injizierten E und den 18,5–19 E, die nach Abzug der vermutlich an der Tubuluswand gebundenen 1–1,5 E ausgeschieden wurden, war zu klein, um mit unserem Bioassay entdeckt zu werden.3. Unter der Annahme, daß 1 E Pitressin verbraucht wurde für die Resorption von ungefähr 4 cm³ Wasser, kann ein Vasopressin-Wasser-Äquivalent von ungefähr 1 Mol Vasopressin für 10⁸ Mol Wasser errechnet werden.4. Die durch die Niere ausgeschiedene Vasopressin-Menge folgt einer e-Funktion mit einer Halbwertszeit von 5 min.5. Die Vasopressin-Clearance beträgt ungefähr 1,0 cm³/min · Ratte und liegt somit wenig unter der Inulin-Clearance (1,2 cm³/min · Ratte). Diese Tatsache legt die Vermutung nahe, daß das überschüssige Vasopressin durch einfache Filtration ausgeschieden wird.6. Wenn man unter Berücksichtigung der unterschiedlichen Nierengewichte den ADH-Bedarf der 200mal schwereren Menschenniere schätzt, so kommt man auf 200–300 E. Setzt man ein Vasopressin-Wasser-Äquivalent von 4–5 cm³ pro 1–1,5 E ADH bei einer Wirkzeit von 50 min in Rechnung, so sollte die menschliche Niere ca. 261 Wasser pro Tag bei völligem Fehlen von ADH verlieren. Die Übereinstimmung mit dem wirklichen Wert könnte dafür sprechen, daß ein Teil der Resultate, die an der Alkohol-narkotisierten Ratte gewonnen wurden, nicht nur unter den strikten Bedingungen dieses Präparates gelten.

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This work was supported by Contract AF 61 (052)-947 of the USAF School of Aerospace Medicine, European Office of Aerospace Research (OAR), U.S. Air Force and the Deutsche Forschungsgemeinschaft.     

Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types

Summary The effect of specific pseudorabies virus (PRV) antibodies on the enlargement of plaques produced by PRV were studied in monolayers of different cell types. The plaque size was used as parameter for the efficacy of the cell-associated spread (CAS) of PRV. First, the effect of anti-PRV hyperimmune serum on the plaque growth was examined in monolayers of the continuous cell lines ST, SK-6 and MDCK and monolayers of the primary cultures of porcine fibroblasts, endothelial cells and endometrial cells. A tenfold increase in the serum concentration did reduce the plaque size with 50% in both SK-cells and fibroblasts and with 40, 28 and 16% in MDCK, endothelial and endometrial cells, respectively. In ST cells, no change in size was observed with increasing antibody concentrations. Secondly, the effects of monoclonal antibodies (mAbs) directed against PRV glycoproteins gB, gC, gD and gE and polyclonal antibodies against gC were evaluated in SK-6 cells. MAbs against gB, gD and gE were able to reduce the CAS with a cumulative effect between mAbs against gD and either mAbs against gB or mAbs against gE. Monoclonal and polyclonal antibodies against gC did not change the plaque size.     

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains

Summary.  Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex. Received September 15, 1999/Accepted December 16, 1999     

Meningoencephalitis in Mice Infected with an Equine Herpesvirus 1 Strain KyA Recombinant Expressing Glycoprotein I and Glycoprotein E

One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.     

Identification of two plaque variants of Guaroa virus

Summary The prototype CoH35211 strain of Guaroa virus contained 2 different plaque populations when French or Mann agarose were used for solidifying the overlay medium. The pl1L (large) and p12S (small) plaque variants were similar with respect to pathogenicity for suckling mice (SM), titers achieved in SM brains after intracranial inoculation and time of appearance of plaques under agarose overlay. The prototype CoH35211 strain and the 2 plaque variants were indistinguishable by complement-fixation and plaque-neutralization tests. However, the 2 plaque variants differed with respect to plaque size and sensitivity to the inhibitory effects of sulfated agar polysaccharides. The only different serological relationship was a one-way cross-reaction between p12S antibody and California encephalitis and Tahyna viruses by plaque-neutralization test.     

Varicella-Zoster Virus Glycoproteins E and I Expressed in Insect Cells Form a Heterodimer That Requires the N-Terminal Domain of Glycoprotein I

Varicella-zoster virus (VZV) glycoproteins E and I (gE and gI), which are major components of the virion envelope, form a noncovalently linked complex. To understand their properties and functions, we expressed and purified soluble forms of gE and gI in the baculovirus system. Extracellular domains of gE and gI were cloned into baculoviruses, using either native or insect-derived signal peptides. Each recombinant virus yielded soluble protein in culture medium although a higher level of secretion was achieved with insect-derived signal peptides in recombinant gE baculoviruses. A soluble gE–gI complex was formed by co-infecting insect cells with recombinant gE and gI baculoviruses and detected by immunoprecipitation followed by Western blotting analyses. By gel filtration and cross-linking studies, we showed that the VZV gE–gI complex expressed in insect cells is a heterodimer. Interestingly, two recombinant gI proteins in which signal peptides were replaced with insect-derived signal peptides did not associate with gE. Amino-terminal sequencing and site-specific mutational studies showed that the replacement of only the signal peptides did not prevent complex formation but alterations in the processed amino-terminus of gI abrogated its ability to complex with gE. These findings indicate that the mature amino-terminus of gI is required for gE–gI complex formation by the external domains of VZV gE and gI.