Herpes Simplex Virus and Human CNS Infections

169

7 Herpes Simplex Virus and Human CNS Infections

Marcela Kúdelová and Július Rajcáni

7.1 IntroductIon

The exact origin of herpes in humans is unknown. Herpes simplex virus (HSV) causes fever blisters or cold sores around the lips or in the genital area. The dis-ease caused by the virus has been known for thousands of years. The characteristic

contents

7.1 Introduction .................................................................................................. 1697.2 Biological Properties .................................................................................... 171

7.2.1 HSV Virion Structure ....................................................................... 1717.2.2 HSV Genome .................................................................................... 173

7.2.2.1 Structure of HSV Genome ................................................. 1737.2.2.2 Classification and Functions of HSV Genes ...................... 174

7.2.3 Expression of HSV Genes ................................................................ 1777.2.4 Replication Cycle of HSV ................................................................. 1807.2.5 Latency ............................................................................................. 180

7.3 Clinical Presentation..................................................................................... 1827.3.1 General Considerations ..................................................................... 1827.3.2 HSV Encephalitis.............................................................................. 1837.3.3 Chronic Neural Disorders and Mental Diseases Associated with

HSV-1 or HSV-2 ............................................................................... 1867.4 Diagnosis of HSV Encephalitis .................................................................... 188

7.4.1 Neuroimaging ................................................................................... 1887.4.2 Detection of HSV DNA in the CSF .................................................. 189

7.5 Pathology and Pathogenesis .......................................................................... 1907.6 Epidemiology ................................................................................................ 1977.7 Prognosis and Treatment .............................................................................. 199

7.7.1 General Considerations ..................................................................... 1997.7.2 Treatment of HSVE .......................................................................... 201

7.8 Conclusions (Future Perspectives) ................................................................202Acknowledgment ...................................................................................................203References ..............................................................................................................203

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170 Neuroviral Infections: General Principles and DNA Viruses

vesicles were described by the ancient Greeks when Hippocrates documented sores that seemed to “to creep or crawl.” The name of the virus comes from the Greek word herpes referring to the lesions caused by the virus. Astruc, physician to King Louis, was the first to describe herpes genitalis and data describing recurrences of the lesions were published by Unna (1883). In 1873, Vidal (1873) first demonstrated that HSV was infectious. Finally, Gruter isolated HSV in 1924 and showed how it could be transmitted from rabbit to rabbit (Gruter 1924). Later on, Burnet and Williams defined that herpes simplex infections seem to persist in the host when the virus remains lifelong latent, but under the stimulus of trauma, fever may be pro-voked and result to visible herpetic lesion (Burnet and Williams 1939). Schneweiss demonstrated that, in fact, two serotypes of HSV existed, HSV type 1 (HSV-1) and HSV type 2 (HSV-2) (Schneweiss 1962).

To date, more than 100 herpesviruses that infect vertebrates are known. Her-pesviruses have been divided into three subfamilies: alpha, beta, and gamma. The eight known herpesviruses of humans can be referred to as human herpesviruses (HHVs) 1 through 8 as designated by the International Committee on Taxonomy of Viruses (Davison et al. 2005; Roizman et al. 1981) (Table 7.1). Older names for HHV-1—HSV-1—and HHV-2—HSV-2—are still in common use and are used below. Alphaherpesviruses have a broad host range infecting a wide variety of cul-tured cells or experimental animals. They spread rapidly in cultured cells with a

tABLe 7.1taxonomy of Medically Important Members of the Herpesvirus Family

Formal name Abbreviation common name Abbreviation

Subfamily AlphaherpesvirinaeGenus Simplexvirus Human herpesvirus HHV-1,2 Herpes simplex

virus 1,2HSV-1,2

Genus Varicellovirus

Human herpesvirus 3 HHV-3 Varicella-zoster virus

VZV

Subfamily BetaherpesvirinaeGenus Cytomegalovirus

Human cytomegalovirus 5

HHV-5 HCMV

Genus Roseolovirus Human herpesvirus 6 HHV-6

Human herpesvirus 7 HHV-7

Subfamily GammaherpesvirinaeGenus Lymphocryptovirus

Human herpesvirus 4 HHV-4 Epstein–Barr virus EBV

Genus Rhadinovirus Human herpesvirus 8 HHV-8 Kaposi’s sarcoma-

associated virus

KSHV

Source: According to Davison, A.J. and Scott, J.E. (1986). The complete DNA sequence of varicella-zoster virus. J Gen Virol, 67, 1759–1816, and www.ictvonline.org.

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171Herpes Simplex Virus and Human CNS Infections

short reproductive cycle and efficiently destroy infected epithelial and fibroblastic cells. In their natural host, latent infections are established usually in sensory neu-rons and lytic infection often occurs in epidermal cells. The human viruses belong to two genera, Simplexvirus (HSV-1, HSV-2) and Varicellovirus (HHV-3—Varicella-zoster virus [VZV]). Classification has been based on biological properties, but as more sequence data become available, these data are also used. Serotypes HSV-1 and HSV-2 share about 50% sequence identity and produce a similar disease, but usu-ally infect different parts of the body. HSV-1 infects the facial area, whereas HSV-2 infects the genital area. Because of the relative ease of experimental manipulation, HSV has been intensively studied as a model for the entire group of herpesviruses.

7.2 BIoLogIcAL ProPertIes

7.2.1 HSV Virion Structure

Virion- and virus-related particles of HSV-1, the prototype of all herpesviruses, have been subjected to the most extensive structural studies. The whole HSV virion was defined as a spherical particle with an average diameter of 186 nm, which extended to 225 nm with spikes. The virion is composed of four elements. An icosadeltahedral capsid surrounding an electron-dense core contains DNA. The capsid is surrounded or embedded by a largely unstructured proteinaceous layer called the tegument. The last element is an outer lipid bilayer membrane envelope exhibiting glycoprotein spikes in its surface. The tegument is composed of virus-encoded proteins, and its thickness can vary, even within a single virion. Herpesvirus morphology visualized by electron microscopy and a schematic model of virion structure is shown in Figures 7.1 and 7.2. The core of a mature herpes virion contains the double-stranded DNA (dsDNA) genome retaining a toroidal form in a liquid crystalline state (Booy et al. 1991). A small fraction of the virion DNA may be circular (Strang and Stow 2005). Viral DNA in the torus is wound around the proteinaceous core. Highly purified

FIgure 7.1 Herpesvirus visualized by electron microscope.

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virions contain the polyamines spermidine and spermine in a nearly constant ratio but no highly basic proteins that would neutralize the negative charges on viral DNA to allow proper folding within the capsid.

The icosadeltahedral capsid is composed of 162 capsomers (12 pentons and 150 hexons) (Newcomb et al. 1993; Schrag et al. 1989). The outer shell of the capsid is composed of viral proteins, the major capsid protein VP5 (UL19), VP26 (UL35), VP19C (UL38), and VP23 (UL18) (Zhou et al. 2000). The pentons are located on the vertices of the capsid and the hexons are found on the faces and edges. A hetero-trimeric complex consisting of two VP23 and one VP19C connects the capsomeres and act as a scaffold for the capsid (Zhou et al. 1999). Hexons also have six copies of VP26, forming a ring around each capsomere (Zhou et al. 1995). The capsid also contains a VP24 (UL26) protease that processes the scaffolding during DNA encap-sidation and a UL16 protein that plays a role in DNA encapsidation. The tegument, which is made up of approximately 20 proteins, is the most diverse structural ele-ment of the virus in terms of polypeptide composition and functions. It functions as a delivery compartment for proteins during the early course of infection and also plays a role in virion assembly. The most notable proteins associated with tegument are the VP16 virion transactivation protein (also called alpha trans-inducing factor [α-TIF], encoded by UL48); the virion host shutoff (VHS) protein (UL41); the VP22 protein (UL49), which was reported to have the ability to bind membranes; and a very large protein (VP1–2), which may play a role in DNA release during viral entry. The fol-lowing are other proteins found in the tegument: UL14, UL17, VP11/12 (UL46), VP13/14 (UL47), US10, and US11. The localization of VP16 and VP22 to nuclear compartments and the cytoplasm suggests that they have multiple roles in replication and egress (Elliott et al. 1995; LaBoissiere and O’Hare 2000; Pomeranz and Blaho 1999). Three tegument proteins (UL11, UL47, and UL49) were identified to package mRNA in herpes simplex virions (Sciortino et al. 2002).

The outermost section of herpesvirus virion is an envelope of modified cellu-lar membranes (Epstein 1962). At least 11 viral glycoproteins have been found in the envelope, with gC (VP8, UL44), gD (VP17 and VP18, US6), gE (VP12.3 and

Spikes

Nucleocapsid

Tegument

DNA

Envelope

FIgure 7.2 Model of herpesvirus virion.

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VP12.6, US8), gG (US4), gH (UL22), gI (US7), gL (UL1), gM (UL11), and gB (VP7 and VP8.5 encoded by UL27) being accepted as essential for HSV entry into cells (Reske et al. 2007). Virion envelopes contain also at least two other proteins (UL20 and US9). The envelope accommodates 600–750 glycoprotein spikes that vary in length, in spacing, and in the angles at which they extrude from the lipid membrane (Grunewald et al. 2003). The glycoproteins involved in virion morphogenesis are currently not well defined. It was assumed that HSV acquires the lipids found in the envelope from its host.

The nucleocapsid is formed in the nucleus. A procapsid is assembled with the for-mation of the capsid shell and the internal scaffolding structure. Then, the procapsid is converted into mature nucleocapsid when the morphogenic internal scaffolding protein is released and replaced by the viral DNA genome, concomitant with a major conformation change of the capsid shell (Newcomb et al. 1999). The mature nucleo-capsid and tegument assemble in the nucleus and bud through the nuclear membrane to obtain an envelope through repeated fusion with and detachment from nuclear membranes and other cellular membranous structures. Eventually, the mature infec-tious virion particles are released into the extracellular space via cellular secretory pathways. The mechanisms by which the extracellular virion obtains its envelope are still not well understood. During this assembly process, different virus-related particles and structures, including the mature nucleocapsids and virions, as well as the intermediate and aberrant products, can be found in the infected cells and the extracellular media.

7.2.2 HSV Genome

7.2.2.1 structure of HsV genomeMost of the packaged genome of HSV is linear and dsDNA. A small fraction of it is circular, and most linear DNA circularizes rapidly in the absence of protein syn-thesis after it enters the nuclei of infected cells (Poffenberger and Roizman 1985). A large-sized HSV genome is approximately 150 kbp long. Data defined by complete sequencing of genome of strains of medically important herpesviruses are shown in Table 7.2. The HSV genome consists of two covalently linked components, desig-nated L (long) and S (short). Each component consists of unique sequences—long (UL ) and short (US)—bracketed by two pairs of inverted repeats. Repeats of the L component are designated a b and aʹ b , whereas those of the S component are aʹ cʹ and c a. The short redundant sequence a at either end of the genome may be repeated 0 or more times (n) or one to many copies (m). The inverted sequences b and c flank-ing UL and US components are also designated b/TRL, bʹ/IRL, cʹ/IRS, and c/TRS (Roizman et al. 2007). The structure of HSV genome can then be represented as

aLanb – UL – bʹaʹmcʹ – US – caS.

While the unique sequences (UL and US) together comprise 128 kbp out of about 150 kbp dsDNA, the repetitive sequences represent 9 and 6 kbp, respectively. The length of the basic redundant a and aʹ sequence unit ranges from 200 to 500 bp. Its structure is highly conserved but consists of a variable number of repeat elements,

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the size of which varies from strain to strain. The L and S components of HSV are found reverted relative to one another, to yield four linear isomers. Populations of unit-length DNA from wild-type, virus-infected cells consist of equimolar concen-trations of the four predicted isomers designated P (prototype), IL (inversion of L component), IS (inversion of S component), and ISL (inversion of both L and S com-ponents). Comparison of the complete DNA sequences of the HSV-1 and HSV-2 genome confirmed the colinearity of their genetic maps. Nevertheless, intertypic as well as intratypic polymorphism of HSV was evidenced. Proteins encoded by her-pesviruses are commonly designated according to its localization in the genome (as, e.g., UL or US) or to its localization in cells or function or property (as infected cell protein [ICP], virion protein [VP], or glycoprotein, e.g., gB).

7.2.2.2 classification and Functions of HsV genesHSV DNA encodes, by current count, about 90 unique transcriptional units. With three known exceptions, each viral transcript encodes a single protein. The first exception is a single transcript for both open reading frame (ORF) P and ORF O proteins, the second one is the UL26 gene encoding two proteins, and the third one is an mRNA containing ORFs of UL1, UL2, and UL3 proteins. There are several pairs of transcripts that are 3ʹ co-terminal; thus, the proteins encoded contain the same part of carboxy-terminal residues (US1.5 and α 22 gene; UL26 and UL26.5; US3 and

tABLe 7.2representative strains of Medically Important HHVs with Full sequenced genome

common name strainAccession

no.size (bp)

g + c (%) reference

Herpes simplex virus type 1

17 X14112 152,261 68.3 McGeoch et al. 1988

Herpes simplex virus type 2

HG52 Z86099 154,746 70.4 Dolan et al. 1998

Varicella-zoster virus Dumas X04370 124,884 46.0 Davison and Scott 1986

Oka vaccine AB097932 125,078 Gomi et al. 2002

Oka parental AB097933 125,125 Gomi et al. 2002

Human cytomegalovirus

Merlin AY446894 235,645 57.5 Dolan et al. 2004

AD169 X17403 229,354 Chee et al. 1990

Human herpesvirus 6 U1102 HHV6A X83413 159,321 42.4 Green et al. 1981

Z29 HHV6B AF157706 162,114 42.8 Dominguez et al. 1999

Human herpesvirus 7 JI U43400 144,861 35.3 Nicholas 1996

RK AF037218 153,080 Megaw et al. 1998

Epstein–Barr virus (B95-8) 4 AJ507799 171,823 59.5 de Jesus et al. 2003

B95-8 V01555 172,281 Baer et al. 1984

Human herpesvirus 8 BC-1 U75698 137,508 53.5 Russo et al. 1996

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US3.5). Some of the expressed ORFs are antisense to each other (γ134.5 and ORF P; UL43 and UL43.5; gB and UL27.5).

Few of the transcripts accumulating in infected cells arise as a consequence of splicing of RNA. Introns were located within coding domains (ICP0 and UL15 of HSV-1 and both genes plus γ134.5 of HSV-2) or in the 5ʹ noncodimg domains (genes encoding ICP22, ICP47). Furthermore, several transcripts appear not to encode pro-teins. Those of the best known are the latency-associated transcripts (LATs) and the OriS mRNA, which is expressed late in the infection and is 3ʹ co-terminal with the mRNA encoding ICP4. The function of the latter is unknown. Thus, the number of single-copy ORFs encoding proteins is most probably 84. Organization of viral genes are described in Table 7.3.

Genes of HSV are classified into at least three general kinetic classes: α or imme-diate early (IE), β or early (E), and γ or late (L). α genes map near the termini of the L and S components. α0 and α4 map within the inverted repeat of the L and S components, respectively, and therefore, each present in two copies per wild-type genome. With few exceptions, β and γ genes are localized in the unique sequences of both the L and S components. The exceptions are γ134.5 and ORF P genes located in the reiterated sequences. The β genes specifying the DNA polymerase and the ICP8 single-stranded DNA (ssDNA) binding protein flank the L component origin of DNA synthesis (OriL). The γ genes specifying membrane glycoproteins D, E, G, I, and J map next to each other within unique sequences of the S component.

Recent kinetic classification to EI, E, and L gene transcripts determined by DNA microarray and the functions of genes identified in the HSV-1 genome is shown in Table 7.4 (Stingley et al. 2000). Comparison of abundance profiles of the tran-scription (transcriptomes) exhibits that the temporal pattern of accumulation of four

tABLe 7.3orFs Localization Within the HsV-1 genome

dnA region orF (notice) number of orFs

aL (terminal sequence) pac1 and pac2 signals 0

an (redundant terminal sequence)

0

b/TRL RL1, RL2, LAT/ORF1,LAT/ORF2, ORFs P/O

6

UL UL1–UL56, UL8.5, UL9.5, UL10.5, UL12.5, UL15.5, UL20.5, UL26.5, UL27.5a, UL43.5, UL49.5

66

bʹ/IRL RL1, RL2, LAT ORF1, LAT, ORF2, P/O ORFs 6

am (redundant terminal sequence)

0

cʹ/IRS IS1 (ICP4) 1

US US1–US12, US1.5, US8.5 14

c/TRS RS1 (ICP4) 1 1

as (terminal sequence) pac1 and pac2 signals 0

a After subtraction of the uncertain UL27.5 and of the LAT/ORF polypeptides.

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tABLe 7.4HsV-1 genes, Kinetic class, and Functions of their transcripts

transcript Kinetic class Function

ICP34.5 L Neurovirulence

ICP0 IE trans activator

RHA6 L 1400 nt 3ʹ end of LAT cap

ORF-X L Low abundance

ORF-Y L Low abundance

UL1/2 L/E gL/uracil DNA glycosylase, fusion/remove uracil from DNA

UL3 L Unknown

UL1X ? Antisense to UL1, low abundance

UL4/5 L/E Unknown/part of helicase–primase complex

UL6/7 L/? Portal protein/encapsidation protein, penton for encapsidation/nuclear egress

UL8/9 E/E Helicase–primase subunit, origin binding

UL10 L gM, gN associated

UL11 L Cytoplasmic egress tegument protein, secondary envelopment

UL12 E Alkaline exonuclease, recombination

UL13 L Protein kinase, phosphorylation, regulation

UL14 L Encapsidation chaperon protein

UL15 L Terminase ATPase subunit 1, spliced-DNA packaging

UL16/17 L/L Unknown/capsid transport in the nucleus, packaging of DNA

UL18/20 L/L VP23 (triplex)/VP5 major capsid protein/membrane associated

UL19/20 L/L Very weak poly(A) site

UL21 L Auxiliary virion maturation function, egress

UL22 L gH, fusion

UL23 E Thymidine kinase

UL24 L Unknown regulated poly(A) site, putative membrane

UL25/26/26.5 L/L Capsid maturation/maturational protease/scaffolding protein

UL27/28 E/L gB, heparan binding, capsid maturation, fusion/terminase DNA binding subunit 2

UL29 E ssDNA binding protein, DNA fork, recombination

UL30 E DNA polymerase

UL31/34 L/L/L/L Nuclear phosphoprotein/capsid maturation/capsid maturation/phosphoprotein

UL35 L Small capsid protein, capsomer tips, capsid transport

UL36 (E)/L ICP1/2 very large tegument protein (two transcripts), uncoating, secondary envelopment

UL37 E Tegument phosphoprotein

UL38 L Efficiency of poly(A) site usage varies with cell type, triplex monomer, capsid structure

UL39/40 E/E Large and small subunits of ribonucleotide reductase

UL41 L Virion-associated host shutoff protein

UL42/UL43.5 E/L Part of helicase–primase complex/tegument

UL43 E(?) Unknown

(continued )

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transcripts (UL4, UL29, UL30, and UL31) differs in infections between HSV-1 and HSV-2 (Aguilar et al. 2006).

7.2.3 expreSSion of HSV GeneS

A schema of the productive herpesvirus infection is shown in Figure 7.3. More than 80 HSV proteins are expressed in a highly regulated cascade fashion. Transcription of viral DNA takes place in the nucleus and all viral proteins are synthesized in the cytoplasm. The first step is attachment to cellular receptor and entry by pH-dependent fusion of the viral membrane with the cell plasma membrane mediated by specific viral glycoproteins on the surface of the virion. Then, tegument proteins

tABLe 7.4 (continued)HsV-1 genes, Kinetic class, and Functions of their transcripts

transcript Kinetic class Function

UL44/45 L/L gC/virion associated

UL46/47 L/L Modulate α-TIF

UL48 L α-TIF

UL49/49.5 E/L Tegument protein/unknown

UL50 E dUTPase, recombination

UL51 L Cytoplasmic egress facilitator 1

UL52/53 E/L Helicase–primase complex/gK

UL54 IE RNA transport/inhibition of splicing

UL55 E? Unknown, pathogenesis

UL56 E? Unknown, pathogenesis

LAT/(Orf O/P) poly(A) site

Reactivation/modulate ICP0, ICP22/modulate ICP4?

ICP4 IE Broad-range trans activator

US1 IE Host range

US2 E? Unknown

US3/4 E/E? Protein kinase/gG

US5/6/7 E/E gJ/gD (entry)/gI (Fc binding)

US8/9 E/E gE (Fc binding)/unknown

US10/11/12 E/E/IE Unknown/RNA binding phosphoprotein/α 47(inhibits MHC-I presentation)

Source: According to Stingley, S.W. et al. (2000). J Virol, 74(21), 9916–9927. General information con-cerning genetic functions can be found in Roizman, B. and Sears, A.E. (1996), Herpes simplex viruses and their replication, in Fields in Virology, 3rd ed., Knipe, D.M. and Howley, P.H. (eds), 2231–2295, Philadelphia, PA: Lippincott Williams & Wilkins; Mocarski, E.S., Jr. (2007), in Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis, Arvin A. et al. (eds), 44–61, Cambridge University Press, New York. Modified according to Schmutzhard, E. (2001), J. Neurol., 248, 496–477.

Note: L, late; IE, immediate early; E, early; kinetic class of transcripts as determined in HeLa cells infected with HSV-1 by DNA microarray.

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are released, causing shutoff of host protein synthesis, and the nucleocapsid is transported to nuclear pores. α-TIF (VP16) is transported to the nucleus where the viral DNA is released and circularizes and enters the nucleus. The host RNA poly-merase II is responsible in producing mRNAs of all viral genes during infection. Transcription of α genes occurs immediately on entry of the DNA into the nucleus in the absence of other de novo synthesized viral proteins. Transcription of these genes is transactivated by α-TIF. α-TIF interacts with a cellular transcription factor called Oct-1, which recognizes octomer sequences. A complex of Oct-1, α-TIF, and cellular factor called C1 binds to the consensus sequence TAATGARAT in the HSV genome (R = A or G). This binding results in transcription of α genes.

Six α genes are transcribed to so-called intracellular (ICP) proteins that are trans-ported to the nucleus, ICP0, ICP4, ICP22, ICP27, ICP47, and US1.5, which have regu-latory roles in viral replication and are required to activate the β genes. ICP0 is not essential for viral replication, but it promotes viral infection and viral gene expres-sion (Hagglund and Roizman 2004). ICP4 represses its own gene by binding to its cognate DNA binding site located across the transcription initiation site (Faber and Wilcox 1986; Watson and Clements 1980). Besides that, ICP4 performs the functions of a transcription activator transactivating HSV genes together with ICP0. ICP27 is a multifunctional regulatory protein (Dai-Ju et al. 2006). It activates expression of β genes and blocks splicing of cellular pre-mRNAs, thereby interfering with host pro-tein synthesis. It shuttles between the nucleus and the cytoplasm at late times. ICP22, not required in cultured cells, was shown to mediate the phosphorylation of RNA polymerase II (Long et al. 1999). ICP47, the only α protein not found to stimulate β genes, blocks presentation of antigens to cytotoxic T cells. HSV infection inhibits host transcription, RNA splicing and transport, and protein synthesis to facilitate transition from cellular to viral gene expression. Expression of β genes requires at least the presence of functional ICP4 but not the onset of viral DNA synthesis. Most

Capsid mRNAGolgimaturation

NucleusCapsids C

Capsid B

Capsid A

Cytoplasm

2 α, β, γpolypeptides

DNA1

VirionEgress

FIgure 7.3 Replication of herpesviruses in permissive cells.

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of the β genes (classified into two groups, β1 and β2) encode proteins required for DNA replication and nucleotide metabolism, which include a DNA polymerase, a primase/helicase, a DNase, both dsDNA and ssDNA binding proteins, thymidine kinase (TK), ribonucleotide reductase, dUTPase, uracil DNA glycolase, and a pro-tein kinase (Honess and Watson 1974).

Synthesis of the β proteins allows start of DNA replication. In that time, chroma-tin is degraded and nucleoli are disaggregated. There are three origins of replication, but only one origin appears to be required. Replication takes place by a rolling circle mechanism to give head-to-tail concatemers. Unit-length DNA is cut from the rolling circle by viral nucleases that recognize specific signals at the ends of the linear DNA. The presence of β proteins and DNA replication lead to transcription of more than 30 γ (late) genes (divided into two groups—γ1 early/late and γ2 late), which mostly encode the structural proteins that form progeny virions (empty capsids) (Godowski and Knipe 1986; Honess and Roizman 1974). The nucleocapsid and the tegument are assembled in the nucleus and bud through the nuclear membrane. Cleavages in capsid proteins occur during assembly, associated with uptake of DNA. Three types of capsids, called A-, B-, and C-capsids, have been identified from infected cell nuclear extracts. All three types of capsids contain VP5, the major capsid protein. The capsomers are linked by triplex structures composed of one molecule of VP19C and two molecules of VP23. A- and B-capsids lack DNA, but B-capsid cavities are filled with VP22a and VP21, the cleaved forms of the scaffolding protein and a viral protease, VP24 (Spencer et al. 1998). The scaffolding protein but not the protease is removed on encapsidation of DNA. C-capsids, which contain viral DNA, can mature into infectious virions by budding through the nuclear membrane. A-capsids are not filled with DNA or scaffolding protein and are believed to be abortive forms that result from failed attempts to package DNA (Roizman and Knipe 2001). Completed particles, after receiving envelope and tegument at the nuclear membrane, mature in Golgi apparatus and then exit by exocytosis. The functions of VP are shown in Table 7.5. Almost all genes of HSV appear to be required for infection of humans, but more than half are not required in cultured cells. During lytic infection, the host cell

tABLe 7.5capsid Proteins of HsV and their Functions

Protein gene Properties and Functions

VP5 UL19 Structural, major capsid protein

VP19C UL38 Structural, capsid composition

VP23 UL18 Structural part of capsid A, function in fragmentation of DNA

VP26 UL35 Structural, located on the outer tips of hexons

VP22a UL26.5 Structural, a scaffolding protein of B capsid

VP21 UL26 Structural, a scaffolding protein of B capsid

VP24 UL26 Structural, serine protease, functions in capsid B and C

UL6 Structural, encapsidation of DNA

UL12.5 Nonstructural, encapsidation of DNA

UL15 Nonstructural, terminase, ends the process of DNA encapsidation

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protein synthesis is shut off by the so-called VHS that inhibits the host very early by mediating the degradation of cellular mRNA. Later synthesis of new viral proteins leads to a more profound inhibition of host expression. Lytic infection is also accom-panied by characteristic fragmentation of the nucleolus and degradation of the host cell chromosomes. The profound inhibition of host macromolecular synthesis results in the death of the host cell.

7.2.4 replication cycle of HSV

In 1997, a model for HSV DNA replication was formulated (Boehmer and Lehman 1997). Once the β proteins are expressed, a number of proteins localize into the nucleus and assemble in DNA replication complexes at prereplicative sites, where viral DNA synthesis initiates on the circular molecule. Then, the UL9 (the origin binding) protein binds to specific elements—origin of replication (either OriL or OriS)—thus beginning to unwind the DNA. Then, it recruits ICP8 (the ssDNA bind-ing protein) to the unwound ssDNA and they both recruit the five other viral repli-cation proteins (helicase–primase complex of three proteins UL5, UL8, and UL52, viral DNA polymerase catalytic subunit UL30, and its processivity factor UL42) to begin the initial round of θ (theta) form replication (Wu et al. 1988). Leading strand synthesis involves the unwinding of the DNA and synthesis of a primer by the HSV helicase–primase complex. Then, replication switches from θ form to rolling cir-cle mode, producing long head-to-tail concatemers of viral DNA by an unknown mechanism (Jacob et al. 1979). Concatemers are cleaved into monomeric molecules during packaging.

7.2.5 latency

After the infection of epithelial tissues at the primary site of infection, HSV infects sensory nerves that serve these tissues, and the nucleocapsid is transported by retro-grade axonal transport to the nucleus in the cell body of the neuron. About 5– 10 copies of viral DNA persist in the nucleus in a circular episomal form associated with nucleosomes. Less than 1% of the neurons within a ganglion appear to be latently infected. During latent infection, lytic gene expression is repressed. Only one viral transcript called LAT is expressed, which yields several RNA species on splicing.

The family of these RNA species, referred to as LATs, map to the inverted repeats flanking the UL sequence. No replicating virus can be detected in the sensory gan-glia during latent infection. In a fraction of neurons harboring latent HSV, the virus is periodically reactivated. Infectious virus is carried by anterograde axonal transport to peripheral tissues, usually to cells at or near the site of initial infection. Reactivation of virus occurs sporadically, in response to stressful stimuli such as fever, exposure to UV light, menstruation, or emotional stress. The frequency of recurrence varies in different people from monthly to less often than once per year. Depending on several factors, including the host immune status, the reactivation may be asymptomatic or lead to a recurrent lesion. Most of the data about latent infec-tion by HSV were gained in studies on animal model systems—mice and rabbits for HSV-1 and guinea pigs for HSV-2.

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During latent infection, the expression of LATs at abundant levels was first detected in latently infected murine ganglia (Stevens et al. 1987). The LATs were subsequently found in latently infected human (Krause et al. 1988) and rabbit (Rock et al. 1987) ganglia. In latently infected neurons, viral genomes acquire the charac-teristics of endless or circular non-methylated DNA (Fraser et al. 1986; Mellerick and Fraser 1987) assembled in nucleosomal chromatin (Deshmane and Fraser 1989). The current model for HSV latent infection involves the silencing of all genes except for the LAT gene by heterochromatin (Stevens et al. 1987). The full-length 8.3-kb transcript of LAT gene expressed from a neuron-specific promoter or enhancer accumulates at low levels in latently infected neurons. Two LATs—2.0- and 1.5-kb introns processed from full-length transcript—are abundant and accumulate in the nucleus. These introns are highly stable (Farrell et al. 1991). The 2.0 kb LAT is the major species detected in neuronal nuclei. Despite reports of proteins encoded by LAT transcripts, most studies have found no evidence for expression of proteins by the LAT (Drolet et al. 1998). Expression of LAT has been shown to reduce viral gene expression and replication in cultured cells. Thus, LATs probably protect the neuron from death. LAT most likely exerts its effect on latent infection by exerting a microRNA (miRNA) effect on ICP protein expression or small interfering RNA (siRNA) silencing of the lytic genes at the chromatin level.

Although the events occurring during the first few hours after entry of viral DNA into dorsal root ganglia (establishment of latency) have not been defined in detail, a key factor is likely to be the absence of expression of α genes. It has been postulated that the host Oct-2 factor represses α gene expression through its interaction with TAATGARATT sites, thereby blocking Oct-1 activation (Lillycrop et al. 1991). The lack of α gene expression is likely explained by, e.g., lack of nuclear forms of host factors or by hormonally regulated repression of viral gene expression (Wilcox and Johnson 1987) or by LAT-mediated repression. The LAT transcript could promote heterochromatin on viral lytic gene promoters through a siRNA silencing mecha-nism acting directly on viral chromatin or indirectly through effects on host genes. Alternatively, the LAT transcripts might exert miRNA or antisense effects on ICP0 expression (Corey et al. 1988; Grey et al. 2008). Latent HSV infection occurs through continued antigenic stimulation of DC and macrophages as well as continued T-cell activation in the ganglionic tissue. Prolonged expression of cytokines and chemo-kines and CD8+ T-cell infiltration (Halford et al. 1996) are found in the latently infected murine ganglia, and prolonged expression of cytokines and chemokines is found in human latently infected ganglia. In addition to inducing the recruitment of immune cells into the ganglia, it leads to perturbation of neuronal host cell gene expression. Numerous cellular genes were shown up- or down-regulated in latently infected murine trigeminal ganglia using the microarray technique (Kramer et al. 2003). More recent data about the relatively high copy number of viral DNA in neurons (20–30 DNA molecules in a single murine neuron; Thompson and Sawtell 2000) and about 11 DNA molecules per human neuron (Weiss et al. 2001) suggest that some leaky viral replication may occur in ganglion cells. In summary, it is likely that several mechanisms (repression of viral lytic gene expression by host cell factors, lack of host factors, inhibition of viral lytic gene expression by viral functions, and inhibition of viral replication by the host immune response) combine to comprise the

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events that lead to latent infection of sensory neurons by HSV. On induction of reac-tivation by in vitro explant, LAT transcript levels decrease (Spivack and Fraser 1988) and the histones associated with the LAT gene become deacetylated. Similarly, lytic genes such as the ICP0 gene become associated with acetylated histones, and lytic gene transcripts accumulate (Chen et al. 2000). It has been postulated that viral gene expression during reactivation follows the α–β–γ paradigm (Devi-Rao et al. 1994).

In humans, latent virus is reactivated after local stimuli such as injury to tissues innervated by neurons harboring latent virus or by systemic stimuli (e.g., physical or emotional stress, hyperthermia, exposure to UV light, menstruation, hormonal imbalance), which may reactivate virus simultaneously in neurons of diverse ganglia (e.g., trigeminal and sacral).

7.3 cLInIcAL PresentAtIon

7.3.1 General conSiderationS

HSV-1 and HSV-2 infect the stratified (squamous) epithelium cells of oral and geni-tal mucosa and of the skin surface. Thereafter, the virus enters various mucosal or nerve endings from which it spreads predominantly by axonal route to correspond-ing neurons (Bergstrom and Lycke 1990; Goodpasture 1929; Nahmias and Roizman 1973; Whitley et al. 2007). As a result of this, and in combination with occasional viremia, both HSVs can cause many fold clinical symptoms (Table 7.6). Some of

tABLe 7.6clinical syndromes and diseases caused by HsV-1 or HsV-2

Acute Primary Infection recurrent disease (reactivation of Latency)a

Oropharyngeal (stomatitis, pharyngitis, tonsillitis, stomatitis)a

Labial herpes, herpetic whitlow (mainly HSV-1), rarely severe and widespread cutaneous or mucocutaneous lesions such as herpes facialis, esophagitis, eczema herpeticum (see below)b

Eczema herpeticum (HSV-1)

Genital lesions vesicular, pustular, or ulcerations (mainly HSV-2)a

Recurrent genital lesions (vesicles, ulcerations) or asymptomatic virus shedding (mainly HSV-2)

Ocular herpes (keratoconjuctivitís, chorioretinitis), usually HSV-1

Corneal ulcer (mainly HSV-1)

Herpes in newborn: generalized cutaneous involvement, meningitis, meningoencephalitis, interstitial pneumonia, hepatoadrenal necrosis, (caused either by HSV-2 as a result of perinatal infection or by HSV-1 in newborns)

Meningitis, encephalitis, or meningoencephalitisc

a The establishment of latency may result from overt disease (less frequently) or from asymptomatic replication at the site of entry (more frequently).

b In immunocompromised patients: esophagitis, pneumonia, hepatitis (also in pregnant women).c Either primary infection of adults (rarely) or reactivation (more frequently).

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them are well-defined clinical entities such as herpetic encephalitis, keratoconjunc-tivitis, and neonatal infections (including encephalitis, hepatitis, suprarenal gland necrosis, and disseminated skin disease). The classical recurrent blister within the hornified skin epithelium may turn into pustule, while ulcerations occur in the oro-pharyngeal or genital area.

Precise description of all herpetic lesions is out of the scope of this chapter, which is devoted solely to CNS involvement. The latter appears either as spontaneous encephalitis with rapid onset in adults or as a part of a generalized perinatal disease of newborns, or due to neonatal HSV-2 infection. In addition to acute or subacute herpetic encephalitis with confirmed HSV etiology, we shall consider the putative role of HSV-1 latency within brain tissue as related to schizophrenia, polar psychotic disorders, and Alzheimer’s disease (AD).

7.3.2 HSV encepHalitiS

Sporadic HSV encephalitis (HSVE) accounts for about 10%–20% of severe virus infections of the CNS, probably representing the most frequent sporadic encepha-litis of nonbacterial etiology (Schmutzhard 2001). The patient suddenly develops high fever and shows signs of meningeal irritation. Soon thereafter, bizarre psy-chotic behavior occurs followed by altered state of consciousness and occasional epileptiform seizures (see Table 7.7 for the most frequent clinical symptoms). The initial change of mental status presented as mild aphasia may progress into confu-sion and delirium within 2–3 days (Khan and Ramsay 2006). Computer tomography (CT) and magnetic resonance imaging (MRI) reveal changes pointing at edema and congestion of the temporal and frontal lobe areas, occasionally including the limbic system, especially the insular cortex and angular gyrus (Kapur et al. 1994; Kennedy 2004). However, rare cases with focal disseminated scattered cortical involvement, clinically manifesting as somnolence and sudden generalized convulsions, were also described (Naito et al. 2007). Typical changes on electroencephalography include focal spikes and slow wave abnormalities followed by epileptiform lateralizing

tABLe 7.7clinical signs in HsVe According to Frequency and time course

early symptoms Percentagesymptoms of developed

disease Percentage

Alterations of consciousness 96% Personality change 80%–90%

CSF pleocytosis 97% Dysphasia 67%–76%

Fever and headache 90% Autonomic dysfunction 58%–80%

Seizures 67% Ataxia 40%

Vomiting 46% Seizures 38%–47%

Hemiparesis 33% Hemiparesis 38%

Memory loss 24% Visual field loss 12%–14%

Source: Modified from Schmutzhard, E., J Neurol, 248, 496–477, 2001.

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paroxysmal discharges (Procházka et al. 1995a, 1995b). The periodic discharges may not develop in the case of a quick onset of specific antiviral therapy, but diffuse slowing is always present. In the cerebrospinal fluid (CSF), mild pleocytosis can be found, mainly due to the increased number of white blood cells (over a hundred per cubic millimeter) represented with a clear predominance of lymphocytes (over 60%). Nevertheless, red blood cells can also be present. In the beginning, protein concen-tration may be normal, but later on, it increases over 100 mg/dl, quickly reaching extremely high levels (Koskiniemi et al. 1984). In contrast, the glucose level is not increased.

Within a week, significant levels of specific IgM as well as IgG antibodies occur in the CSF as detected by enzyme-linked immunosorbent assay (ELISA). These changes are associated with a significantly high IgM antibody index (Klapper et al. 1981), to be calculated from comparing the elevated antibody levels within the CSF with those in the patient’s serum. HSV-1 cannot be isolated from CSF. In older stud-ies, HSV-1 was usually isolated from brain biopsies (Johnson et al. 1972; Nahmias et al. 1982; Procházka et al. 1995a). The morphological examination of brain biop-sies for inclusion bodies and virus-specific antigen might be a less effective diag-nostic tool (Kennedy 2004). The debate on the possible beneficial effect of brain biopsy (to achieve some relief from brain edema) has been finally settled (Fishman 1987; Hanley et al. 1987). It is clear now that brain biopsy is not a suitable procedure for HSVE management. Its diagnostic value is not superior to the detection of HSV DNA by the polymerase chain reaction (PCR) technique (Anderson et al. 1991). Since the application of PCR for detection of HSV DNA in the CSF of patients suffering from HSVE, DNA amplification became the gold standard for noninva-sive diagnostic procedures (Bártová et al. 1987; Purchhammer-Stockl et al. 1990; Rowley et al. 1990).

In adults, HSV-1 replication within brain tissue results in acute inflammation, congestion, and hemorrhages occurring predominantly in the temporal lobe and lim-bic areas. As a rule, these lesions appear asymmetrically (Boos and Esiri 1986). The meninges overlaying the involved areas are congested and thickened due to infiltra-tion with mononuclear cells. The early inflammatory lesions within the cortex are not dramatic, but later on, the perivascular cuffing and formation of nodular infiltrates become prominent. Damaged neurons are surrounded by mononuclear phagocytes (neuronophagia). The vascular lesions (vasculitis and thrombosis of small blood ves-sels) located in the cortex and subcortical white matter cause anoxia resulting in necrosis and hemorrhages. The typical intranuclear inclusion bodies can be seen only in a few neurons and glial cells. More often, the affected neural cells reveal smudgy hemophilic and homogenous nucleoplasm in which the nascent viral capsids can be seen by electron microscopy (Figure 7.4a and b). Thus, the inclusions may not be regarded as a good diagnostic sign. The positive outcome of immunofluorescence or immunohistochemical staining for HSV antigen(s) is more reliable in this respect. As mentioned above, classic histological diagnostic methods (Boos and Kim 1984) may still be useful at retrospective evaluation of older paraffin-embedded material, but not for recent diagnostics, since the brain biopsy technique has already been fully replaced by noninvasive procedures, mainly PCR amplification of HSV DNA in CSF samples.

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The damage to individual neurons and glial cells related to HSV replication is caused by apoptosis triggered by the virus (Athmanatan et al. 2001; Aurelian 2005; deBiasi et al. 2002; Nguyen and Blaho 2007). The apoptotic changes within neurons were clearly found in the necroptic material from older cases, which have been stored in the archive of pathology departments, either by anti-caspase-3 antibody stain-ing or by demonstration of cellular DNA fragmentation (Terminal deoxynucleotidyl transferase-mediated deoxyUridine triphosphate Nick End Labeling, i.e., TUNEL assay). Experimental results showed that apoptotic changes are directly triggered by virus replication in neurons and glial cells already during the early phase of CNS infection. Caspase-3 activation is mediated by transcription of the IE HSV genes (Kraft et al. 2006). To make things more complicated, HSV regulates apoptosis also by an additional polypeptide (protein kinase US3). The US3-deficient HSV-2 virus, when given intranasally, would not spread to adjacent neurons beyond the entry of the olfactory area (Mon et al. 2006). Apoptotic neurons and glial cells affected at very early intervals of encephalitis should be considered as immediate targets of specific antiviral therapy. Taken together, the acyclovir (ACV) therapy must start as soon as possible (provided that prompt diagnosis by PCR is available) to avoid severe sequelae or possible lethal outcome. If ACV therapy starts within 2–3 days after the appearance of first clinical signs, a complete recovery might be expected.

As mentioned above, apoptosis of neurons was seen not only along with the signs of cortical inflammation but also in their absence, in areas with no other signs of destruction, thus before inflammation and neuronal loss occurred. The complex net-work of intracellular signaling and related neurotransmitter release may influence the functional state of yet uninfected surrounding neurons. The inflammatory response, including the later recruitment of killer T cells, provides an additional mechanism of tissue injury. Even the toll-like receptors on the surface of brain microglial cells soon interact with the HSV virions. In response to this, reactive oxygen species and inflam-mation mediators (proinflammatory cytokines and chemokines such as CXCL9/10) are produced in great amounts (Schachtele et al. 2010). This explains why investiga-tors described an increased resistance to HSVE in mice, which were deficient for CXC chemokine receptors (Wickham et al. 2005). Despite increased virus titer in the brain stem of such mice, their survival rate still remained high, indicating that che-mokine receptor–deficient animals are resistant against the development of encepha-litis, otherwise occurring in wild-type mice. The toll-like receptors TLR2 and TLR9 synergistically control the outcome of HSV brain infection through activation of the expression of chemokine receptors (such as TNFα [tumor necrosis factor alpha] and IFNγ [interferon gamma]) and stimulate the nonspecific (innate) adaptive response to infection (Sorensen et al. 2000). Interestingly enough, HSVE has been reported to develop in patients receiving treatment with TNFα inhibitors at rheumatologic disorders (Bradford et al. 2009). Such case reports deserve special attention since immunosuppressive treatment otherwise does not activate HSVE, though it has been postulated to occur owing to reactivation of latent virus (compare Section 7.5).

Before the introduction of ACV treatment (see Section 7.7), HSVE had been a severe disease with high mortality rate (about 70%); normal brain function returns in only fewer than 3% of the survivors during those times (Whitley 1990). As com-pared to other viral encephalitides, the mortality rate of HSVE before introduction

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of specific antiviral therapy was disproportionally high. In addition to differences in the onset of ACV treatment and its dosage and duration, further variables may influence the outcome of the disease, especially as far as the severity of sequelae is concerned. According to Taira et al. (2008), two signs are important for defining the prolonged clinical course, which predicts a worse outcome in adult patients despite the standard AVC therapy. These are a higher coma scale (Glasgow Coma Scale) and an early appearance of significant temporal lesions at CT examination. Interestingly enough, corticosteroid administration in combination with a satisfactory and effi-cient ACV therapy did not contribute to the prolonged course. Corticosteroids along with ACV might have a beneficial effect, since they reduce the extent of meningeal and perivascular mononuclear infiltration. In addition to possible prolonged course of acute encephalitis, the relapsing form of HSVE may occur because of reactivation of persisting HSV DNA within brain tissue (Yamada et al. 2003). As stressed by the latter authors, the frequency of relapsing cases of HSVE may be underestimated. When encountering epilepsy in a patient who recovered from HSVE in the past, the presence of HSV DNA in his CSF by PCR should be immediately tested. If positive, specific antiviral (ACV) therapy should be started without any delay.

7.3.3 cHronic neural diSorderS and mental diSeaSeS aSSociated witH HSV-1 or HSV-2

During the last quarter of the twentieth century, an old interest concerning the putative viral etiology of serious mental disorders such as schizophrenia has been revived. During a 20-year period, from the report of Rimon and Halonen (1969), till the thorough review by Yolken and Torrey (1995), over 30 serological studies were published, evaluating the significance of increased levels of HSV-1 antibodies in the serum of schizophrenic patients. Early papers reported higher neutralizing antibody titers; recent investigators used enzyme immunoassay (EIA). A slight majority of authors claimed that the serum antibody titers to various herpesviruses were higher in schizophrenic patients than the corresponding values from matched controls. Out of 17 positive reports from the abovementioned review, at least 7 mentioned solely HSV-1 as the dominantly positive antigen, while others also found elevated titers to cytomegalovirus (CMV), VZV, and Epstein–Barr virus (EBV) antigens. Nevertheless, conclusions drawn were not convincing, since the number of sera inves-tigated in individual studies varied considerably and because many studies (43%, i.e., 13 out of 30) claimed negative results. The most comprehensive study by Libíková (1983) encompassed 265 schizophrenic and 430 control sera; she found that the mean titers in schizophrenics were higher than those in controls. In addition, Libíková also found HSV-1 antibodies in 42% of CSF samples tested. The latter finding was confirmed in a repeated study encompassing CSF samples from 262 patients, which were tested by both neutralizing antibody and EIA techniques (Bártová et al. 1987). Later studies focused their attention to the detection of HSV DNA in nc. amygdalae specimens obtained by stereotactic surgery from psychotic and aggressive patients (Kúdelová et al. 1988). These results were compared with the examination of vari-ous areas of the brain stem, limbic system, and temporal cortex coming from rela-tively fresh autopsy cases with no signs of neural disease. Interestingly, a similar

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proportion (15%) of control autopsy brains yielded positive dot blot hybridization results as did the brain samples from the aggressive schizophrenic patients (Rajčáni et al. 1991a). In conclusion, elevated serum antibody levels among schizophrenics were not significant, while the DNA hybridization results pointed at the possibility of latent HSV-1 infection in basal and frontolateral areas of the brain as well as in the brain stem of healthy adults (see Section 7.5). In contrast, Carter et al. (1987), using the classical hybridization method, found neither HSV-1 DNA nor other herpesvirus DNA sequences in the postmortem brains of patients with schizophrenia and indi-viduals who committed suicide. A similar conclusion was drawn by Alexander et al. (1992), who detected neither HSV-l- nor VZV-specific DNA sequences by PCR. More recently, Prasad et al. (2007) found significant reduction in the gray matter of 30 schizophrenic patients in the dorsolateral prefrontal cortex (Brodmann area 9) and anterior cingulate cortex (Brodmann area 32) using optimized voxel-based mor-phometry as compared to 40 controls. These findings were in accord with the cere-bral morphological changes otherwise reported in schizophrenia. Interestingly, the morphometric changes within the brain cortex structure were associated with HSV-1 antibody level differences, since the schizophrenic patients in question showed ele-vated HSV-1 antibody ratios. Furthermore, an exonic polymorphism of the MHC-1 class polypeptide sequence B gene (rs1051788) was found associated with reduced gray matter volume as well as with the elevated HSV-1 serum antibody titers in the first episode of schizophrenia (Prasad et al. 2010). Longitudinal follow-up showed significant loss of gray matter in the posterior cingulate gyrus among the HSV-1 seropositive schizophrenia subjects over a period of 1 year, but not in other groups. The prefrontal gray matter areas did not show longitudinal changes. These observa-tions suggested that HSV-1 exposure may be associated with gray matter loss in posterior cingulate gyrus and decline in executive functioning among subjects with schizophrenia (Prasad et al. 2011).

AD is another example of slowly developing chronic brain disorder, in which the presence of putative latent HSV-1 within brain tissue might impair the clini-cal outcome (compare Section 7.5). The temporal and frontal cortex as well as the hippocampus were found to harbor the latent HSV-1 DNA by PCR technique in proportion aged persons with and without senile dementia or AD (Jamieson et al. 1992). More recently, Mori et al. (2004) detected HSV-1 DNA in the frontal lobe of familial as well as sporadic cases of AD and in the temporal lobe of familial forms of AD. It should be mentioned that among their 11 cases, these authors included 2 cases of schizophrenia, in whose brains they did not find HSV DNA sequences. Itzhaki et al. (1997) postulated that limited reactivation of latent HSV-1 within brain tissue (for example, in response to immunosuppression) would cause further dam-age in AD patients. They also pointed at the combined effect of the apolipoprotein ε4 allele along with the presence and moderate reactivation of latent HSV DNA. Such combination might represent a stronger risk factor for AD development than the ApoE4 gene alone (Corder et al. 1993). ApoE, a major component of very low density lipoproteins, is involved in mobilization and redistribution of lipids and cho-lesterol within the membranes of neurons.

One of the known hallmarks of AD pathology is the aggregation and depo-sition of the β-amyloid polypeptide within the brain, which is a cleavage product

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(39–43 amino acids) of the larger amyloid precursor protein. It was shown that HSV-1 gB has a sequence homology to β-amyloid (Cribbs et al. 2000). In addition, HSV infection of neurons may decrease the level of amyloid precursor protein but increase the amounts of its cleavage product, a C-terminal fragment with a molecular weight of 55 kDa (Shipley et al. 2005). The latter finding might be a link between HSV infection in the brain and accelerated development of the AD. The effect of apolipoprotein E has been recently analyzed in ApoE3 and ApoE4 transgenic mice and ApoE knockout mice (Burgos et al. 2006). The ApoE (murine) knockout mice had very low levels of latent HSV DNA in their nervous system as compared to wild-type mice. The ApoE3 (human gene) transgenic animals had lower levels of the HSV DNA in midbrain than the ApoE mice. The ApoE4 (human gene) transgenic mice developed latent infec-tion of the brain in an extent similar to the standard inbred (wild-type) mice. During latent infection of ganglion cells, which undergo slow reactivation (see Section 7.5), the HSV gB molecule may be preferentially expressed on the surface of neurons with an early β-kinetics. A gB 498–505 peptide-specific CD8+ T cell clone was found to block HSV-1 reactivation from latency in ex vivo trigeminal ganglion cultures (Khanna et al. 2003). This phenomenon explains the immune recognition of HSV DNA carrying cells before the onset of fully functioning virus replication process. In addition, it challenges the concept that HSV-1 latency represents a silent infection only (see Section 7.5). The less frequent, but possibly also present persistent form of HSV-1 latency combined with the ApoE4 allele can accelerate the development of AD. The essential role of HSV-1 as risk factor for development of AD was recently confirmed by the analysis of serological results (Letenneur et al. 2008). After controlling for age, gender, ApoE4, and the baseline Mini-Mental State Examination, the hazard ratio of developing AD was strongly associated with an elevated HSV antibody status of IgM class indicating the onset of productive virus replication (compare Section 7.5).

7.4 dIAgnosIs oF HsV encePHALItIs

7.4.1 neuroimaGinG

In HSVE, an acute necrotizing inflammation soon develops at characteristic local-izations (temporal, insular, orbitofrontal, and cingular regions). Therefore, MRI becomes an early and helpful diagnostic tool. At the above-mentioned typical sites, hyperintense abnormalities are seen on T2-weighted images by MRI examination. Occasionally, signal characteristics of hemorrhages may be also noticed as witnessed by hypointense areas on T1-weighted images (Demerel et al. 1992; Tien et al. 1993). The meningeal thickening may be demonstrated after administration of gadolinium. Fluid-attenuated inversion recovery sequences clearly demonstrate the definition of temporal lobe abnormalities as compared to standard (control) T1- and T2-weighted images (White et al. 1995). Asymmetric (unilateral) involvement of frontal lobe (cor-tex and underlying white matter with perisylvian distribution) may develop as well (Taylor et al. 2005). Rarely, the MRI shows single disseminated lesions at atypical sites such as cerebellum, occipital lobes, and parietal cortex. For conformation of the MRI diagnosis, noninvasive detection of viral DNA in the CSF is strongly recom-mended rather than brain biopsy (compare Section 7.3.2).

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7.4.2 detection of HSV dna in tHe cSf

The detection of the HSV DNA within CSF looks back over 20-years experience. In an extensive earlier study, the CSF from 257 patients with suspected HSVE was ana-lyzed by PCR as compared with the outcome of other noninvasive diagnostic meth-ods such as the CSF versus blood antibody ratio (see the next section). HSV DNA amplification by PCR has confirmed the diagnosis of HSVE not only in 9 serologi-cally proven cases but also in 14 additional cases, which were suspected of having HSVE but could not be definitively proven when only the serological approach was used (Purchhammer-Stockl et al. 1993). Thus, PCR from the very beginning seemed superior to serological examinations, which did not fulfill the main demand of the forthcoming therapeutic era marked by introduction of ACV therapy. The new and efficient lifesaving drug management strategy called for quick and precise diagnosis. At the time of the introduction of the PCR technique for detection of HSV DNA in CSF, different laboratories amplified different HSV genes. We recommended a highly specific primer set, which flanks the main immunogenic area of the gD gene (Kúdelová et al. 1995), while the majority of laboratories preferred the amplification of an HSV-specific gB gene fragment. Some investigators suggested examining the pellet from the CSF (i.e., its DNA extract); the identity of the amplified fragment could be confirmed by a simple dot blot hybridization method (Guffond et al. 1994) or by restriction enzyme digestion.

Several questions have arisen when the PCR technique has been introduced for routine monitoring for the efficiency of ACV therapy. The main task was how to compare the levels of HSV DNA in subsequent CSF samples and how to avoid false-negative results coming from the presence of Taq-polymerase inhibitors. The first quantification of HSV DNA levels used internal calibration controls prepared in tis-sue culture (Revello et al. 1997). The HSV DNA copy number in the CSF was later on estimated by using an improved internal in vitro prepared control, which could not be mistaken for the fragment coming from naturally present virus (Domimges et al. 1998). For this purpose, the HSV-1 specific calibration standard (a segment of the gB gene) was manipulated to contain a 25 bp deletion. The amount of HSV DNA copies detected by this technique ranged from <25 up to 25,000 copies per microliter of CSF. Soon thereafter, the real-time (RT)-PCR method was intro-duced. In contrast to classic PCR reactions, this method has the advantage of using a LightCycler instrument, which allows high-speed thermal cycling and continuous monitoring of the DNA amplification cycles (Kessler et al. 2000). Using appropriate standards for calibration, about 2–5 × 103 copies of HSV DNA per milliliter could be detected, i.e., 2.5–6.3 genome equivalents per run. The most frequently used detec-tion kits for LightCycler RT-PCR machines amplified the DNA polymerase gene. The LightCycler tests based on either the gB or the DNA polymerase primers were found consistent with each other (Altuglu et al. 2006).

The attempts to compare the efficiency of HSV antigen presence by ELISA with the sensitivity of the genome detection clearly showed the superiority of the PCR technique. The increased sensitivity and accuracy of the RT-PCR and the introduc-tion of automated instruments for DNA detection revealed the smashing advantages of this new technology also for routine diagnosis in samples other than CSF, where

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classic virus isolation prevailed (Espi et al. 2000). The RT-PCR technique has been recommended also in comparison with the highly sensitive but time-consuming nested PCR. For concurrent detection of a variety of herpesvirus genomes, including HSV-1, HSV-2, CMV, VZV, EBV, and HHV-6 within the same CSF sample, a her-pesvirus DNA family common primer set was designed. This approach has turned out useful for example in association with materials from human immunodeficiency virus (HIV)–infected patients (Drago et al. 2004). For the latter purpose, a consen-sus fragment can be amplified first in the screening test to not miss the sequence of any of the above-mentioned herpesvirus DNAs (Calvario et al. 2002). Elaborating the consensus test was of great importance for assessing the expanding spectrum of acute or subacute herpesvirus infections of the nervous system, including some chronic disorders with putative herpesvirus etiology (Kleinschmidt et al. 2001).

For evaluation of RT-PCR in the differential diagnosis of patients with acute meningitis, meningoencephalitis, and other neurological disorders, a recent study of 146 patients (Gaeta et al. 2009) showed the following. The CSF of HIV-infected patients revealed the presence of HHV-6 and EBV DNAs. The DNA of any of the six above-mentioned HHVs (HHV-7 and HHV-8 were not included in the described study, compare Table 7.1) was detected in 33.5% of the CSF samples submitted. The diagnosis of meningoencephalitis was confirmed in 39 patients, in which VZV and HHV-1 were most frequently present. The HHV-6, HSV-2, and EBV DNAs were less frequently detected. In addition, a few patients with polyradiculoneuritis or Guillian–Barré syndrome were positive for CMV, VZV, or HHV-6. Coinfection of two herpesviruses was rarely found. If so, it occurred in combinations such as HSV-1 and HSV-2, EBV and HSV-2, and VZV and HSV-2. In an older study (Studahi et al. 2000), in 69 (10.4%) out of 662 patients with suspected viral neuroinfections, one of the six above-mentioned herpesviruses was found in their CSF. The majority of posi-tive patients (87%) were noncompromised. These findings have raised the abandoned question of searching for the presence of local (intrathecal) antibodies within CSF, in order to specify the diagnosis. As concluded by Plentz et al. (2008), the detection of low levels of any herpesvirus DNA in the CSF in the absence of other than acute or subacute meningoencephalitis symptoms needs critical and careful evaluation. This is especially true if the DNA copy number turns out low. The classical serology at least due to emphasis on noninvasive diagnostic tools is still of actual interest, espe-cially in cases that mimic HSVE (Whitley et al. 1989).

7.5 PAtHoLogy And PAtHogenesIs

The histopathological changes in HSVE were already described under Section 7.3.2. Briefly, an asymmetric (rarely diffuse) meningoencephalitis develops along with hemorrhagic and partially necrotic lesions. The meningeal infiltrate and perivascular cuffings consist of mononuclear cells, mainly lymphocytes, while the pericapillary nodules consist mainly of mononuclear phagocytes and activated microglia (Figure 7.4e). Neurons of brain cortex, astroglia, oligodendroglia of white matter, the walls of small vessels, and microglial cells are soon affected.

In the beginning, the neuronal changes are triggered by the virus (apoptosis and cytopathic changes including intranuclear Cowdry type 1 inclusions). Later on, the

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(a) (b)

(c) (d)

(e) (f )

FIgure 7.4 Experimental HSV-1 neuroinfection in Balb/c mice. (a) The sensory neuron and its satellite as seen by electron microscopic examination. The large pale “empty” nucleus of the pseudounipolar neuron and the elongated nucleus of the satellite cell show chromatin accumulated at their nuclear membranes. The interior of the nuclei contains viral capsids. (b) At larger magnification, the envelopment of capsids can be seen on the inner lamella of nuclear membrane of the satellite cell; the nuclear membrane shows invaginations in the direc-tion of the nucleoplasm. (c) The HSV antigen can be seen in neurons and satellite cells of a sensory (spinal) ganglion after intracutaneous inoculation of HSV-1 (alkaline phosphatase labeling, monospecific anti-HSV serum). (d) Balb/c mouse was inoculated into scarified cor-nea with HSV-1 (SC16 strain); after 3 months, the ganglion was removed, minced, and kept in culture for 7 days. A small ganglion fragment was examined by day 4 in culture. A pseu-dounipolar neuron, in which the synthesis of viral antigens started owing to reactivation, can be seen stained by immunofluorescence method. (e) Experimental HSVE in mice as seen in hematoxylin– eosin-stained section. The meninges are infiltrated with lymphocytes, and peri-vascular cuffings around small vessels can be seen in the temporal cortex. (f) Positive staining of pyramidal neurons for HSV-1 antigen and of smaller glial cells in the cortical gray matter.

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neurons undergo destruction by neuronophagy and because of the extensive inflam-matory (cytotoxic T cell) response. The enhanced permeability of the blood–brain barrier leads to edema and the development of temporal lobe mass detected by CT examination. By immunofluorescence or immunohistochemical (labeled antibody) staining, the HSV antigen can be found not only in neurons but also in glial and satellite cells (Figure 7.4f).

The pathogenesis of sporadic HSVE encephalitis is not quite clear despite exten-sive studies of acute as well as latent HSV-1 or HSV-2 infections in various animal models. It has been hypothesized that HSV-1, which regularly spreads into the tri-geminal ganglion after orofacial primary infection, may rarely reach the second neu-ron within the brain stem or the olfactory and limbic system (Kúdelová and Rajčáni 1990). The virus may spread along the tentorial nerves that innervate the basal meninges (Davies and Johnson 1979). Since HSV-1 antibodies in the serum reach high levels relatively early after the outbreak of the HSVE symptoms, the cases of sporadic encephalitis in adults are expected to occur because of reactivation of latent virus rather than because of primary infection. According to an older theory, HSVE was believed to develop in those seronegative individuals, who escaped the other-wise common primary infection (see Section 7.6). If the main source of reactivation of latent virusanticipates axonal and a forthcoming transsynaptic spread to intercon-nected neurons. In contrast, the primary infection hypothesis assumes viruses spread via the bloodstream, either within the infected carrier cells or along the olfactory nerves (Figure 7.5).

�e trigeminal route

SkinLip

Reversedneuralspread

Trigeminal ganglion

�e olfactory routeOlfactory bulb

Cribriform lamina

Neurons ofolfactory mucosa

Rare spreadto brain stem

Neurons and satellite cells

FIgure 7.5 Pathogenesis of primary HSV infection, latency, and reactivation. The tri-geminal route has been well established as well as the reversed virus movement (HSV “round trip”). The spread from pseudounipolar neurons of trigeminal ganglion to the brain stem is less frequent. The alternative route along olfactory nerves is hypothetical but explains the distribution of HSV in encephalitis cases (Dingwell et al. 1994; Rajčáni et al. 1991b).

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Which factors play an essential role under conditions of various experimental situations depends on the type and age of animals (newborn versus adults; mice, guinea pigs, and rabbits), on the inoculation site (peripheral or intracerebal), on the dose of inoculated virus, and, last but not least, on the properties of the virus strain administered. Bergstrom and Lycke (1990) elaborated an in vitro model of a two-chamber system for neuron cultures to test the spread of various HSV isolates from a chamber containing nerve endings to another chamber containing neuron bodies. They showed that neuronal uptake of virions at nerve endings and their spread along axons and virus replications within the bodies of the neural cells occurred more efficiently with HSV-1 isolates coming from encephalitis cases than with those of dermal origin. The complex property of HSV isolates and strains that ensure their entry into the nervous system was termed neurovirulence (or neuroinvasiveness). It most probably depends on the efficiency of virion attachment and entry to nerve end-ings (Lycke et al. 1984; Vidal 1873).

The uptake of HSV-1 by nonneural cells, which are also present within dissoci-ated nerve cultures, was quicker than their entry into nerve endings (Rajčáni and Scott 1972). Nevertheless, the bodies of neurons were found fully resistant to virion penetration. Similar differences among the HSV-1 isolates, as had been found in neural cultures, were also seen at in vivo experiments. If the given virus strains and isolates were compared for their pathogenicity after peripheral versus intracerebal inoculations to Balb/c mice, the resulting neuroinvasivity indices could be calculated (Dix et al. 1983). In an earlier study, Rajčáni and Szantó (1973) described differences in the pathogenic behavior as well as in the lethal doses after peripheral inoculation as compared to direct intracerebral injection in between several high-passaged ver-sus low-passaged virus strains. The neuroinvasiveness was expected to be a function of genes governing virulence, i.e., encoding proteins important for virus adsorp-tion, penetration, and replication (Rajčáni 1992). A classical gene, the absence of which impairs the efficiency of HSV replication in neural tissue, is TK (reviewed by Tenser 1991). The TK-negative mutants are hampered in acute replication within neurons (owing to defective viral DNA synthesis) but establish latency within sen-sory ganglia as measured by the presence of LATs (explained below). Nevertheless, the TK-negative mutants are impaired in their ability to reactivate since they do not replicate well in neurons anyway (Kramer et al. 1998).

The genes determining neurovirulence as indicated by neural uptake of the cor-responding virus strain do not always correlate with genes that are important for virus entry to nonneural cells. For example, the syn3gB region, anticipated to play some role in cell fusion, does not determine neurovirulence (Rajčáni et al. 1996). In contrast, the gE/gI envelop glycoproteins, which are not essential for in vitro replica-tion, have an important role at the entry of HSV to nerve endings.

In addition, the gD envelope gene, which is inevitable for adsorption to surface protein receptors, may be also important for the neural uptake; i.e., it may influ-ence neurovirulence (Rajčáni et al. 1994). The latter assumption was confirmed by the finding that mice lacking the nectin receptor(s), the so-called herpesvirus entry mediator, are resistant to HSVE (Taylor et al. 2005; Kopp et al 2009). Finally, an important factor for HSV neurovirulence is the protein ICP34.5, which may con-tribute to virion production and to cell-to-cell spread. In contrast to the wild-type

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virus, the ICP34.5 deletion mutants are neuroattenuated, though they can still rep-licate in the brain ependyma cells (Kesari et al. 1998). At in vitro experiments, the protein ICP34.5 redirects the interferon action, which is mediated by the increased activity of cellular RNA-dependent phosphokinase PKR pathway (Roizman and Knipe 2001).

As already mentioned, the route of HSV-1 and HSV-2 spread in experimental animal models depends on virus strain, virus dose, inoculation site determining the route of virus spread, and, last but not least, the status of immune competence of the infected individual. In suckling mice, HSV-1 is used to spread via the bloodstream to brain tissue so that the role of mononuclear phagocytes appeared essential for limit-ing and regulating virus dissemination (Johnson 1964a, b). In our hands, the spread along nerves predominated regardless of whether outbred suckling mice or 21-day-old mice infected by various inoculation routes are used (Rajčáni et al. 1969, 1970). However, in highly susceptible mice, the involvement of Schwann cells seemed extensive, as detected by immunofluorescence after various inoculation routes or by electron microscopy of the trigeminal nerve after virus inoculation to the cornea (Rajčáni and Conen 1972). Under these conditions, the acute neuritis during virus spread to regional sensory ganglia caused ganglionitis associated with the replica-tion of the virus in pseudounipolar neurons and their satellite cells (Figure 7.4a–c). In contrast to suckling and young mice, in adults, no clear-cut neuritis could be seen. Mice that underwent inoculation in the skin, scarified cornea, or ear pinna did not develop encephalitis but had latent infection, which was established in the regional sensory ganglia (either paravertebral, trigeminal, or cervical) corresponding to the regions supplied by given sensory nerves (Stevens et al. 1972). Alternatively, HSV latency has been demonstrated in the rabbit model after virus inoculation in scarified cornea (Rajčáni et al. 1977; Stevens et al. 1972). The explantation technique (keeping ganglion fragments in culture) has been used for detection of latent HSV. According to biological definition, the latent virus does not replicate at the time of ganglion removal but starts producing infectious virus when kept for several days in culture (Figure 7.4d). In animal models for the establishment of latency, no involvement of Schwann cells could be seen during virus transmission along the corresponding nerves. Thus, in such models, the HSV-1 and HSV-2 had to spread via the intraax-onal route since another mechanism was not possible (Kristensson et al. 1971). The presence of virions within axons during intraaxonal transport was first demonstrated by Hill et al. (1972), who examined the peripheral nerves after intracutaneous virus administration, viewing them in longitudinal sections.

As a result of intraaxonal (quick) neural spread, the HSV-1 and HSV-2 reach the regional sensory ganglion and immediately establish latency. In contrast to situa-tions where the virus replicates in the bodies of pseudounipolar neurons, the virus may become latent without initiating productive replication cycle. According to our experience, the HSV-1 DNA reaches the trigeminal ganglion of adult DBA-2 mice after corneal inoculation by 16 h, moving across a distance of about 16 mm, i.e., at least 1 mm/h. The second question concerns the molecular basis of latency. Several questions are of interest in this respect. The first problem concerns the molecular basis of the quick axonal spread. This is mediated by dynein, a cellular transporter protein, which moves along neurotubules and associates with the viral capsid protein

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VP19. The viral particles (capsids) are already uncoated during the axonal transport; i.e., they are devoid of their envelope, which had been removed during entry. As found by Efstathiou et al. (1986), the latent genome resides in the nuclei of neurons in the form of a circularized nonintegrated plasmid-like (ring-shaped) structure, which survives and lives for an extremely long time (possibly lifelong); thus, a given neural cell harbors the viral DNA during its lifetime.

The circularized genome is silent, which means that the IE transcription is blocked. It is probable that the tegument protein VP16, which is needed for initia-tion of IE (alpha) transcription, may not reach the neuron along with the quick DNA transport (Kristie and Roizman 1988). Nevertheless, the relative lack of the VP16 tegument protein is probably not the single reason why latency is so frequently estab-lished from the very beginning (Sears et al. 1991). The low level of viral transcription initiation factor VP16 within neurons may explain why HSV replication starts rarely and in a very few neurons only. The IE polypeptide (molecular weight, 175 kDa), designated VP175 or ICP4, was found in small amounts in the murine trigeminal ganglia (Green et al. 1981). Also, according to our findings, the latently infected ganglia removed from rabbits with established latent infection produced the ICP4 IE protein mRNA at low amounts (Režuchová et al. 2003). In accord with this, the syn-thesis of the IE ICP0 (110 kDa) transactivator protein, which is absent during latency, is always the sign of reactivation (Preston and Efstathiou 2007). In the absence of detectable virus production, the term spontaneous molecular activation of IE tran-scription has been suggested to indicate the transition to a dynamic form of latent infection (Feldman et al. 2002). The reactivation process can be hampered by two different mechanisms. One depends on viral genome products interacting with cellu-lar cofactors of transcription within neurons (Preston 2000, Rajčáni and Ďurmanová 2000). The second mechanism is the outer immune control. For example, if a por-tion of explanted ganglion fragments removed from rabbits with established latent infection was cultured in the presence of rabbit immune serum, the reactivation rate was lower than in the absence of such serum (Rajčáni et al. 1977). If reactivated, the virus returns to peripheral skin or mucosa (Figure 7.5), where it may (but need not) find favorable conditions for further replication. If the virus replicates at periph-eral mucosa or skin tissues, either recurrent virus shedding or blisters occur. A nice example for creating favorable conditions of virus replication at peripheral skin was described in the ear model (Hill and Blyth 1976; Hill et al. 1975). These investiga-tors found that prostaglandins produced after skin trauma or UV light irradiation would enhance the replication of recurrent HSV-1. As known from the experience, when transsection of one root of the trigeminal nerve in patients with severe trigemi-nal neuralgia was a therapeutic measure, herpetic blisters often occurred after such surgery (Paine 1964). As shown by Walz et al. (1974), neurectomy of the trigeminal nerve root reactivated the latent HSV harbored within ganglion cells. The round trip of reactivated virus usually ends with infection of additional neurons in the regional sensory ganglion, since at least some additional neurons get infected and start har-boring the latent genome.

A frequently discussed issue regarding the interaction between viral DNA and host cells (neurons, glial cells) during latency is the presence of LATs. The LATs com-prise a family of transcripts (at least mRNA molecules of different length) encoded

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by the long repeat region of the HSV genome. During the productive replication cycle, a few LAT molecules are transcribed, which belong to the 2.0 kb LAT spe-cies (Fraser et al. 1992). A highly probable hypothesis says that LATs are functional RNAs (not mRNAs), which do not translate proteins. Even though the various LAT species were shown to encode at least two proteins, which have been translated in vitro, the synthesis of these polypeptides was never demonstrated in vivo. The 2.0 kb LATs overlap (at least in part) the IE ICP0 mRNA, since they are transcribed from the opposite (complementary) viral DNA strand. Possibly, they function as antisense regulators of the ICP0 mRNA (transactivator protein) expression. The long 8.0 kb LAT precursor molecule also overlaps the ICP4 gene sequence. Under conditions when very few VP16/α-TIF molecules are at disposal, and when the usually acting cellular cofactors of IE HSV transcription such as HCF-1 and Oct-1 are not avail-able, the HSV genome remains silenced. Furthermore, other cofactors such as Oct-2 and Brn-3 present in neurons cannot associate with the viral VP16/α-TIF (Lilliecrop et al. 1992, 1994). Taken together, the HSV replication cycle may be blocked from the very beginning owing to the lack of IE transcription and the absence of IE trans-activation proteins. The factors latency maintenance and reactivation are summa-rized in Table 7.8 (modified from Valyi-Nagy et al. 2007).

tABLe 7.8Molecular Mechanisms of Latency Maintenance and reactivation

Latency continues, no Infectious Virus Is Produced

HsV reactivates, Infectious Virus Production Begins

The transcription activation cofactor HCF-1 is sequestered within the cytoplasm, out of the reach of the circularized HSV genome within the nucleus.

The block of IE transcription can be overcome by HCF-1 translocation or increased Oct-1 production, owing to their increased expression.

The Oct-1 cellular cofactor of transcription is present in low levels only.

The available cellular cofactors of transcription (Brn-2, Oct-2) do not interact with VP16/α-TIF (viral transcription factor).

No satisfactory amounts of VP16/α-TIF are present.

The ICP4 transactivator protein may be produced in some neurons, which slowly promotes infectious virus production.

The HSV genome is silenced by histone deacetylase (HDAC), which leads to chromatin condensation.a

LAT prevents apoptosis.b

LATs function as molecules antisense to the IE ICP0 transactivation protein mRNA.

The ICP0, if produced in small amounts, overcomes the block of IE transcription starting the activation cascade of the β and γ genes (see Section 7.3.2).

a Roizman et al. (2005).b Perng et al. (2000).

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The use of LAT-deficient HSV mutants in various experiments has shown that LATs are not inevitable for establishment of latency, but rather they are related to reactivation. However, their function is even more complex. As already mentioned, in sensory neurons, either latency is established or the productive virus replication starts. If LATs are produced in abundance, they switch the regulation process toward nonproductive latency.

The LAT-deficient (or null) mutants would destroy a large amount of neurons within the infected sensory ganglion during the acute phase of infection (Thomson and Sawtel 2001). As a result of this, the LAT-null mutant would reactivate less frequently, since the number of neurons harboring the viral genome is low. The reduced reactivation of LAT-negative mutants does not necessarily imply that LAT is directly involved in the molecular mechanism of reactivation. The antiapopto-sis activity of LAT has been independently confirmed in tissue culture and in the mouse model of ocular HSV-1 (Ahmed et al. 2002). This further suggests that the antiapoptosis activity mediated by LAT is important for latency reactivation (Perng et al. 2000, 2002).

Extensive studies describing various aspects of LAT in maintenance and reac-tivation of the latent genome still within regional sensory ganglia do not explain the pathogenesis of HSVE. As already mentioned, HSV spreads along nerves also beyond the first pseudounipolar neuron and may establish latent infection within the brain stem and possibly also in the olfactory brain cortex and related struc-tures of the limbic system (amygdala, hippocampus) as shown in a rabbit model (Kúdelová and Rajčáni 1990) as well as in human CNS samples by in situ hybrid-ization (Rajčáni et al. 1991a). The significance of these preliminary findings was later on widely accepted (Wang et al. 1997) and, therefore, further analyzed. Interestingly, if the specimens from the amygdala removed during stereotactic sur-gery were cultured, no virus could be rescued as it was the case with the trigemi-nal ganglion explants. It was assumed that if a second HSV-1 strain would reach the brain in which latency had been previously established with another HSV-1 strain, a recombination effect might occur between the two virus DNAs, creat-ing a neurovirulent recombinant that would cause the sudden onset of encephali-tis. However, neurons harboring the latent virus and expressing LAT are protected from the superinfecting virus (Mador et al. 2002). Furthermore, superinfection of animals with established latency using another replication-defective (ICP6 minus) HSV-1 mutant did not reactivate the latent virus (Wang et al. 1997). In conclusion, despite extensive experimental research, the main questions—Which routes do the HSV-1 spread into CNS take? Why does the virus reactivate suddenly?—are still not fully understood.

7.6 ePIdeMIoLogy

HSVs are among the most ubiquitous of human infections. Worldwide, about 90% of people have one or both types of HSV. The presence of antibody is thought to persist after infection in humans. HSV-1 is one of the more prevalent viruses, with 65% and about 50% of persons having antibodies to HSV-1 in the United States and Europe, respectively (Xu et al. 2002). In the developing world, HSV-1 is almost

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universal, and usually acquired in early childhood (Whitley et al. 1998). Rates of HSV-1 infections are similar for men and women. Primary infection (mostly oral) often occurs early in life, but may be delayed in some fraction of the population. It is usually inapparent or produces only minor illness. Because of its mode of trans-mission (almost exclusively during sexual activity), HSV-2 is less common, with a 20%–30% range for most European countries and for the United States (Enders et al. 1998; Malkin et al. 2002; Varela et al. 2001) and more that 50% prevalence in the general populations in Africa (Weiss et al. 2001).

In communities with high prevalence of infection, demographic rather than behavioral factors (a number of lifetime patrners) reflect HSV-2 risk more accu-rately (Sucato et al. 2001). However, both factors play a role in infection rates. In the United States, among people who report near 50 lifetime partners, the risk of infection exceeds 35% for white women, 60% for African American women, 40% for white men, and about 50% for African American men (Fleming et al. 1997). In almost all studies, and in all populations, having HSV-2 infection increases the risk of HIV acquisition (Freeman et al. 2006; Wald and Link 2002). Primary infection with HSV is usually inapparent or produces only minor illness. However, because of the neurotropism of the virus, primary infection or reactivation leads to serious illness of neonates and, occasionally, of adults, mainly those whose immune func-tion is compromised by suppressive therapy for organ transplant or by infection with HIV.

HSV infection of neonates is almost always symptomatic and frequently fatal. The frequency of neonatal herpes varies by region and is estimated to occur from 1 in 3200 (in the United States) to 1 in 15,000 pregnancies (Brown et al. 1997; Gutierrez et al. 1999; Kimberlin 2007). The majority of cases are caused by HSV-2. The risk of transmission is increased with primary maternal infection during the third trimester. As reviewed by Kimberlin (2007), 85% of neonatal HSV cases occur owing to peripartum transmission, whereas 10% occur via postnatal transmission and only 5% are due to transmission in utero. Infection in utero, during delivery, or shortly after birth usually leads to a disseminated infection often accompanied by encephalitis. The pathogenesis of CNS involvement in neonatal HSV infections differs depending on whether or not the infection is disseminated. Of all infants with neonatal HSV infections, approximately 30% have CNS disease, while about 25% have disseminated disease that also covers, in more than half the cases, CNS invol-vement (James et al. 2009; Whitley et al. 1988). HSV DNA detection in CSF and serum is highly sensitive for the diagnosis of neonatal HSV infections but does not replace virus isolation and antigen detection. Early detection of HSV in the neonate is mandatory; however, prevention by Cesarean section or prenatal therapy of the mother is the best option.

Herpes encephalitis in adults is the most common cause of sporadic encephali-tis, with an estimated frequency of 2–4/1,000,000 persons per year (Schmutzhard 2001). About 50 cases of herpes encephalitis occur yearly in the United States, and this incidence may be underestimated (Strauss and Strauss 2002). The age distribution appears bimodal, with a smaller peak among youth and a larger peak among the elderly. Encephalitis can develop both during primary infection (usu-ally among younger people) and during reactivation of HSV in CNS (usually among

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older people). HSVE is the most devastating disease if untreated; it either is usually fatal or leaves patients with serious neurological damage. The usual agent is HSV-1, although HSV-2 meningoencephalitis has also been described in immunosuppressed patients (Gateley et al. 1990). Detection of HSV DNA in CSF by molecular methods has become the gold standard of diagnosis of HSVE, avoiding, in particular, invasive brain biopsy.

Very recently, the reactivation of HSV in the CNS has proved to be a causa-tive agent of Mollaret’s meningitis with different prognoses as described for HSVE (Tyler 2004). Most often, HSV-2 is implicated, although HSV-1 has also been reported (Yamamoto et al. 1991). Women are at higher risk than men. The initial epi-sode often develops during acquisition of genital HSV-2, with subsequent recurrent episodes. Thus, the epidemiology of Mollaret’s meningitis parallels that of genital herpes. However, not infrequently, meningitis is the presenting complaint, and the association with HSV-2 is not always recognized. Spinal fluid findings are consis-tent with “aseptic meningitis,” with positive results of PCR diagnostics searching the presence of HSV DNA (also see Section 7.4.2). Patients usually respond well to antiviral therapy.

Sensitive molecular approaches have revealed that HSV DNA can be found in many tissues previously not recognized as an infection site. Several studies provided the evidence for a link between HSV-1 exposure and some of the cerebral morpholo-gical changes often reported in schizophrenia. HSV-1-associated differences in brain structure were not detected among healthy subjects (Preston 2000). Herpesvirus DNA has been identified in the brains of schizophrenia patients, and elevated anti-body levels to herpesviruses were found in some but not all studies (reviewed by Yolken and Torrey 1995). On the basis of the high correlation between HSV-1 in brains of AD and Parkinson’s disease sufferers, HSV was suggested as a significant etiological factor of these neurological diseases (Hemling et al. 2003; Pyles 2001; Wozniak et al. 2009). More recent findings described that infection with herpes-viruses is more frequent in the brain and CSF of multiple sclerosis (MS) patients than in patients with other neurological disease (Mancuso et al. 2007; Sanders et al. 1996). However, it seems that HHV-6, EBV, and VZV rather than HSV may play a role in the pathogenesis of MS. Furthermore, HSV could also be a contributory cause of arteritis leading to atherosclerosis, an idea supported by some animal stud-ies (Álvarez-Lafuente et al. 2008; Haahr and Höllsberg 2006). In all cases where HSV is associated with chronic inflammatory lesions, experimental verification of a causative role is lacking.

7.7 PrognosIs And treAtMent

7.7.1 General conSiderationS

HSV-1 and HSV-2 infections can be treated with several highly efficient drugs (Table 7.9), most important of which are ACV and its derivatives (Ehrlich and Mills 1985). ACV (9-[(2-hydroxyethoxy)methyl]guanine) is a guanosine analogue that becomes phosphorylated in HSV-infected cells by the virus-coded TK. This ensures the virus-dependent accumulation of the active drug (ACV triphosphate) just in cells

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replicating the virus (Figure 7.6). The essential step of ACV activation by the viral TK is its conversion into ACV monophosphate, while additional phosphorylation can be made by the cellular enzymes. This may be of importance for inhibition of viral DNA synthesis in neurons, which do not possess their own TK. In such cells, the whole activation process (ATP triphosphate synthesis) is virus dependent. The activated ATP triphosphate not only acts as a competitive inhibitor at viral DNA replication, but also causes chain termination when incorporated into the nascent viral DNA strand. Additional drugs occasionally recommended for the therapy of HSV act as inhibitors of the viral DNA polymerase complex. They can be useful in cases of ACV resistance, which occasionally develops owing to mutations in the viral TK molecule.

tABLe 7.9Most Frequently used Inhibitors of Alphaherpesvirus replication

drugcommercial designation

recommended therapeutic use dosea

ACV AcyclostadGalmedAcycloviralHerpesinTelviranVirolexZovirax

Generalized herpes of newborn

EncephalitisMucocutaneous herpes, ocular herpes

Varicella, pneumoniaHerpes zoster

15–25 mg/kg daily i.v. for 14–21 days

30 mg/kg daily i.v. for 10–21 days200 mg at 6 h intervals orally for 7 days

locally (ointment or drops)10 mg/kg daily i.v. for 7–10 days200–400 mg 5 times daily orally for 10 days

Valaciclovir Valtrex Mucocutaneous herpesLong-term therapy of latency

Herpes zoster

500 mg 1–2 times daily for 5 days, orally

200 mg daily, oral therapy500 mg daily orally for 5–7 days

Famciclovir Famvir Mucucutaneous herpesLong-term therapy of latency

Herpes zoster

Long-term peroral therapy with 250 mg or 500 mg daily for 5–7 days

Phosphonoformate FoscavirFoscarnet

Mucocutaneous herpesHerpes zoster

Only locally, 3–5 times daily (ointment); at resistance to ACV 90 mg/kg i.v. by 12 h intervals for 14 days

Bromovinyl deoxyuridín (brivudin)

HelpinZovudex

Herpes zosterImmune deficiency statesResistance to ACV

125 mg 4 times daily orally for 7–14 days

Jododeoxyuridín StoxilIdoxuridine

Keratoconjunctivitis Ocular drops

a The given examples of drug dose are not a guide for individual case therapy.

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7.7.2 treatment of HSVe

Since introduction of AVC therapy and the HSV DNA monitoring in CSF by PCR, the prior highly lethal prognosis of HSVE has considerably improved. Nevertheless, the long-term outcome in many ACV-treated patients may not be optimal. Up to 30% of cases of HSVE are still lethal, while the great majority of the rest (about 70%) show sequelae of various extent (McGrath et al. 1997). The survivors regain independence for simple daily activities, but they show symptoms such as memory impairment (69% of survivors), behavioral and personality abnormalities (45% of survivors), and epileptic seizures (24% of survivors). Only a low percentage (5%–10%) of patients recovers into full health. The favorable outcome depends on the early onset of ACV therapy, which should be started immediately, when any one of the typical diagnostic signs of HSVE occurs. ACV is a nontoxic drug causing no

First phosphorylation

ACV

ACVMP

ACVTP

Additionalphosphorylations

HSV DNA strandBase

Base

H2NH2N

H2N

H2N

OO

O

OO

O

O O OOOOO

OH

OH

HN

O

O

O

OO O O

O

OO

OO

OO

O

O

O

HNHN

HN

HO

N

N

N N

N

N

N

NN

N

N

N

P

P

P

P

P

P

×Chain termination by ACVTP

FIgure 7.6 ACV action: the first phosphorylation, essential for ACV activation, is pro-vided by the virus-coded TK.

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harm even when later laboratory results would not confirm the diagnosis. If so, the ACV therapy can be terminated after a shorter regime.

Repeated exacerbation of encephalitic symptoms may occur in some patients either with or without reappearance of the positive PCR reaction in their CSF. In such case, repeated ACV therapy is absolutely indicated. Such relapses probably occur because of the lower total ACV dose administered during initial therapy; this might be either because of shorter duration of the therapy (10 versus 14 days) or because of a lower daily dose (25 mg/kg versus 30 mg/kg). Reactivation of HSV in the CNS of adults is associated with two distinct syndromes—HSVE and Mollaret’s meningitis, which have different prognoses. Untreated HSVE has >70% fatality rate (Whitley and Lakeman 1995). The first episode of Mollaret’s meningitis (often deve-loped during acquisition of genital HSV-2) as well as subsequent recurrent episodes usually respond well to antiviral therapy.

Most cases of neonatal herpes appear to occur after delivery in women who acquired genital HSV-1 or HSV-2 infections or when the virus was transmitted to the newborn via infected secretions during labor (Arvin et al. 1982; Brown et al. 2003). Newborns with clinical evidence of disseminated disease, often including skin lesions, may be premature and have a poor prognosis. Therefore, management of newly recognized genital herpes during pregnancy should include consideration of administration of ACV locally to women toward the end of pregnancy. Scheduled Cesarean delivery appears to be a protective as well as a prophylactic antiviral ther-apy of the newborn (Prober et al. 1992; Sheffield et al. 2003). Clinically, neonatal herpes has been divided into three syndromes: disease present only on skin, eye, and mouth (about 42%); disseminated disease (about 23%); and CNS disease (about 35%). The first form of infection has the best prognosis, with negligible mortality and up to 70% of treated infants having normal course (Kimberlin et al. 2001; Whitley et al. 1988). But some results show that infants without any evidence of CNS involvement may subsequently present with neurologic deficits. Disseminated HSV infection in neonate disease has the highest mortality, reaching about 60% even with therapy. Normal development of survivors is noted in about 60%. CNS disease is associated with low mortality but the highest morbidity. Prompt antiviral therapy is associated with decreased morbidity and mortality, but the prognosis remains grave for most children. Newborns with this form of neonatal herpes have about a 50% chance of normal development. HSV-2 appears more neuroinvasive in newborns than HSV-1 and, as such, has a worse prognosis (Corey et al. 1988). In babies and small children developing HSVE independently of their birth (in age of several months), the correct diagnosis may be difficult, since the cortical functions are inapparent. Therefore, in case of sudden seizures the examination of CSF for HSV DNA is reasonable. (Frenkel 2005; Kimura et al. 1991). Long-term valaciclovir therapy by oral route can be applied after the acute course in order to avoid relapses (Lim et al. 2009).

7.8 concLusIons (Future PersPectIVes)

During the past decades, research on HSV-1 and HSV-2 infections has brought great progress. The genomes of several HSV strains (and isolates) have been sequenced, the transcription of their genes was mapped, and the function of many nonstructural

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as well as structural HSV-coded proteins (and glycoproteins) has been thoroughly studied. The pathogenesis of HSV infection was elucidated at several levels, starting from distribution of viral antigens or viral particles in various cells and tissues, to the recent molecular studies describing the spread of viral DNA and the presence of their transcripts in experimental animals. Nevertheless, our knowledge on precise mechanisms of HSV infection of CNS is still incomplete. Both HSVs can use vary-ing strategies for acute spread versus long-term persistence within the peripheral and central nervous systems. Both viruses may spread via the bloodstream in the neonates or immunocompromised subjects, but in healthy adults, they rather move along nerves. When entering nerve endings, as a rule, they use the quick axonal transmission to establish nonproductive latency in neurons. On the other hand, non-productive latency within some neurons is labile enough to provide reactivation and productive replication in a few ganglion cells, a phenomenon that may cause recur-rent disease. While HSV can, at very high frequency, establish latency in sensory neurons located outside the brain, it rarely continues to spread to the second neuron located within the CNS itself. However, the occasional presence of HSV DNA within brain tissue has been demonstrated by several investigators. Because of the contro-versial experimental findings, an exact mechanism explaining the sudden outbreak of herpes encephalitis, which is a rare but life-threatening disease, is still not fully understood. On the basis of a thorough knowledge of HSV replication in cell culture, the drug ACV was soon developed. ACV is possibly the best antiviral drug, till now introduced into practical treatment. Even though it is highly efficient for treatment of lesions outside the nervous system, it fails to prevent latency and seems less efficient in nearly one-third of encephalitis cases, which may be still lethal. One reason for this unsatisfactory statistic may be the lower availability of ACV within brain tissue as compared to its blood levels. At least in the beginning, when many affected neu-rons undergo apoptosis, the availability of active ACV triphosphate seems essential. However, because of the lack of endogenous TK within neurons, the conversion of ACV monophosphate into active triphosphate may suffer some delay. The early onset of ACV therapy in a sufficiently high dose and the repeated monitoring of the HSV DNA within the CSF are important factors favoring a better recovery from HSVE.

AcKnoWLedgMent

This work was supported by the joint agency (the Slovak Ministry of Education and the Slovak Academy of Sciences [VEGA]) grants #2/0126/10 and #2/0091/13, and the Operational Programme Research and Development Diagnostics of socially impor-tant disorders in Slovakia, based on modern biotechnologies, ITMS:26240220058, cofunded by the European Regional Development Fund.

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Herpes Simplex Virus and Human CNS Infections

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