Influence of Tegument Proteins of Pseudorabies Virus on Neuroinvasion and Transneuronal Spread in the Nervous System of Adult Mice after Intranasal Inoculation

ABSTRACT Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6- to 8-week-old CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-ΔUL47-, PrV-ΔUL11-, and PrV-ΔUL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-ΔUL37-infected animals died. Upon closer histological examination, all viruses except PrV-ΔUL37 were able to infect the nasal cavity and propagate to first- and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-ΔUL47, PrV-ΔUL11, and PrV-ΔUL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-ΔUL48 and PrV-ΔUL37 replicated to similar titers in cell culture which were ∼500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-ΔUL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-ΔUL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.

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Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment

Journal of General Virology ◽

10.1099/vir.0.82168-0 ◽

2006 ◽

Vol 87 (12) ◽

pp. 3503-3507 ◽

Cited By ~ 17

Author(s):

Kathrin Michael ◽

Sindy Böttcher ◽

Barbara G. Klupp ◽

Axel Karger ◽

Thomas C. Mettenleiter

Keyword(s):

Protein Interactions ◽

Pseudorabies Virus ◽

Single Gene ◽

Full Length ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Protein Protein Interactions ◽

Tegument Proteins ◽

Terminal Fragment

Proteins of the virion tegument of alphaherpesviruses are involved in protein–protein interactions, which play important roles in virus morphogenesis. Seven single-gene deletion mutants of Pseudorabies virus were analysed for alterations in the overall composition of the virion beyond the loss of the targeted protein. The UL36 protein (pUL36) was present in equal amounts in wild-type virions and mutants lacking pUL21, pUL49, pUL51, pUS3 or pUS8. Virions lacking pUL11 or pUL16 incorporated less full-length pUL36 than wild-type particles, but contained increased amounts of an N-terminal fragment of pUL36 that is present only in traces in wild-type virus and the other mutants.

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The UL7 Gene of Pseudorabies Virus Encodes a Nonessential Structural Protein Which Is Involved in Virion Formation and Egress

Journal of Virology ◽

10.1128/jvi.79.17.11291-11299.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11291-11299 ◽

Cited By ~ 20

Author(s):

Walter Fuchs ◽

Harald Granzow ◽

Robert Klopfleisch ◽

Barbara G. Klupp ◽

Daniela Rosenkranz ◽

...

Keyword(s):

Pseudorabies Virus ◽

Rabbit Antiserum ◽

Wild Type Virus ◽

Rabbit Kidney ◽

Structural Protein ◽

Wild Type ◽

Survival Times ◽

Virion Protein ◽

Infected Cells

ABSTRACT Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-ΔUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-ΔUL7F in UL7-expressing cells. PrV-ΔUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.

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Influence of Pseudorabies Virus Proteins on Neuroinvasion and Neurovirulence in Mice

Journal of Virology ◽

10.1128/jvi.02589-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5571-5576 ◽

Cited By ~ 39

Author(s):

Robert Klopfleisch ◽

Barbara G. Klupp ◽

Walter Fuchs ◽

Martina Kopp ◽

Jens P. Teifke ◽

...

Keyword(s):

Pseudorabies Virus ◽

Past Research ◽

Viral Envelope ◽

Wild Type Virus ◽

Survival Times ◽

Glycoprotein E ◽

Viral Spread ◽

Adult Mice ◽

Kinetics Of

ABSTRACT Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.

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The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

Journal of Virology ◽

10.1128/jvi.73.12.10551-10555.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10551-10555 ◽

Cited By ~ 32

Author(s):

Armin Ensser ◽

André Pfinder ◽

Ingrid Müller-Fleckenstein ◽

Bernhard Fleckenstein

Keyword(s):

Cell Culture ◽

Herpesvirus Saimiri ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Human T Cell ◽

Human T Cells ◽

Small Nuclear Rnas ◽

T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

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Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/1806842 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Zsolt Csabai ◽

Dóra Tombácz ◽

Zoltán Deim ◽

Michael Snyder ◽

Zsolt Boldogkői

Keyword(s):

Single Molecule ◽

Base Composition ◽

Pseudorabies Virus ◽

Point Mutations ◽

Wild Type Virus ◽

Economic Losses ◽

Wild Type ◽

Southeast Europe ◽

Long Read ◽

National Programs

Background. Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.Results. In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.Conclusion. PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

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Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection

Journal of Virology ◽

10.1128/jvi.00352-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6387-6398 ◽

Cited By ~ 27

Author(s):

L. M. Olsen ◽

T. H. Ch'ng ◽

J. P. Card ◽

L. W. Enquist

Keyword(s):

Protein Kinase ◽

Pseudorabies Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Spread Of Infection ◽

Viral Components ◽

Axonal Targeting ◽

Null Mutants

ABSTRACT The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

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Pre-clinical development of cell culture (Vero)-derived H5N1 pandemic vaccines

Biological Chemistry ◽

10.1515/bc.2008.060 ◽

2008 ◽

Vol 389 (5) ◽

Cited By ~ 31

Author(s):

M. Keith Howard ◽

Otfried Kistner ◽

P. Noel Barrett

Keyword(s):

Cell Culture ◽

High Yield ◽

Wild Type Virus ◽

Effective Vaccine ◽

Type Virus ◽

Wild Type ◽

Yield Production ◽

Homologous Virus ◽

Clade 1 ◽

Influenza H5n1

AbstractThe rapid spread of avian influenza (H5N1) and its transmission to humans has raised the possibility of an imminent pandemic and concerns over the ability of standard influenza vaccine production methods to supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains, produced at a variety of manufacturing scales, were demonstrated to be highly immunogenic in animal models without the need for adjuvant. The vaccines induce cross-neutralising antibodies and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from other clades. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high-yield production of a candidate pandemic vaccine.

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UL54-Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture

Journal of Virology ◽

10.1128/jvi.80.2.769-784.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 769-784 ◽

Cited By ~ 17

Author(s):

Jennifer A. Schwartz ◽

Elizabeth E. Brittle ◽

Ashley E. Reynolds ◽

Lynn W. Enquist ◽

Saul J. Silverstein

Keyword(s):

Protein Synthesis ◽

Pseudorabies Virus ◽

Host Protein ◽

Wild Type Virus ◽

Cell Protein ◽

Transcriptional Level ◽

Dependent Manner ◽

Type Virus ◽

Wild Type ◽

In Cells

ABSTRACT The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and λRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJSΔ54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSΔ54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSΔ54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJSΔ54. Interestingly, vJSΔ54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSΔ54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.

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Effects of Hemagglutinin-Neuraminidase Protein Mutations on Cell-Cell Fusion Mediated by Human Parainfluenza Type 2 Virus

Journal of Virology ◽

10.1128/jvi.00460-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8283-8295 ◽

Cited By ~ 13

Author(s):

Masato Tsurudome ◽

Machiko Nishio ◽

Morihiro Ito ◽

Shunsuke Tanahashi ◽

Mitsuo Kawano ◽

...

Keyword(s):

Cell Fusion ◽

Single Cells ◽

Vero Cells ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Amino Acid Mutations ◽

Cell Cell

ABSTRACT The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.

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