Enterovirus 70 (EV70) and an antigenic variant of coxsackievirus A24 (CA24v) are the two major pathogenic agents inducing acute hemorrhagic conjunctivitis (AHC). The ocular symptoms caused by EV70 and CA24v are clinically indistinguishable and constitute the AHC syndrome. Therefore, AHC should be used as the term representing an enteroviral eye disease characterized by the AHC syndrome. AHC in this chapter includes conditions caused not only by EV70 and CA24v but also by echoviruses 7 and 11 whose epidemic occurred in Sweden in 1977; AHC caused by adenovirus type 11 is excluded.

EPIDEMIOLOGY

Explosive epidemics of AHC were initially reported in West Africa and Indonesia in 1969 with subsequent pandemic spread.³ A causative agent of AHC was first recovered by Kono and coworkers⁴ from conjunctival scrapings of patients with AHC. The isolate was identified as a new type of enterovirus when the first outbreak of AHC in Japan occurred, during the worldwide pandemic of 1969 to 1972. The first pandemic of AHC was caused by EV70 in Africa, Southeast Asia (including Singapore and Hong Kong), Japan, and India in 1969 to 1971, with several million cases. In 1970, a year before EV70 was found, Yin-Murphy⁵ reported a large outbreak of epidemic conjunctivitis in Singapore, where she isolated a new enterovirus from the conjunctival scrapings of patients. The virus was identified as the causative agent of the disease, which was called Singapore epidemic conjunctivitis by Lin and Yin-Murphy.⁶ The virus was later renamed CA24v.⁷

Epidemics of this disease recurred during the last quarter of the 20th century. Both viruses cause the same disease, according to viral isolation, serologic investigation, and the reverse transcription-polymerase chain reaction (RT-PCR) method. AHC spread throughout Southeast Asia and first occurred outside Asia in American Samoa in 1986, where nearly half the population was affected.⁸ After a sporadic outbreak in French Polynesia in 1982, EV70 was introduced into the Western hemisphere, including the United States.⁹ CA24v was responsible for the epidemics of 1975¹⁰ and 1985.^11,12

There is a noticeable difference in the geographic distribution of these two viruses. EV70 has widely spread to various parts of the world from its original focus in West Africa, and it has so far caused two major pandemics in 1969 to 1972 and 1980 to 1982. AHC caused by CA24v first appeared in Singapore and Malaysia in 1970, and since then, AHC epidemics have been intermittently observed only in Southeast and East Asia and India.

CLINICAL CHARACTERISTICS

The onset is sudden and alarming. After beginning with one eye, the other often becomes inflamed in a matter of hours. Most patients develop signs in the second eye within 1 to 3 days (Fig. 1). Pain accompanies the other symptoms.

The most common symptoms are a profuse and predominantly watery discharge, a foreign body sensation, burning, photophobia, swelling of the eyelids, and pain in the upper eyelid that is aggravated on bending over. Although patients often complained of coryza, fewer than 5% showed systemic manifestations, such as fever or malaise. Ocular symptoms are often usually severe enough, however, to disrupt routine activities for several days.

Signs of AHC caused by EV70 and CA24v are similar, and it is impossible to determine the causative virus from clinical characteristics. Edematous swelling of the lid occurs in 72% to 100% of patients, although severity varies. Swelling usually subsides within 3 or 4 days. Preauricular lymph nodes become enlarged and palpable in 65% of patients, and approximately 80% are tender.¹³ The upper conjunctivae become inflamed from a mild to a severe degree, with injection, infiltration, and follicle formation. In moderately inflamed eyes, the fornix is reddened and cloudy, obscuring blood vessels.

The most characteristic finding is marked hemorrhagic involvement of the bulbar conjunctival and subconjunctival layers. Punctate hemorrhages often appear within a few hours of onset of symptoms and rapidly coalesce to form larger hemorrhagic patches (Fig. 2). These patches tend to form ridges concentric with the corneoscleral limbus and occasionally become confluent (Fig. 3). Although 40% of patients have with unilateral involvement, more than 90% have bilateral disease when examined the next day.

Corneal complications are generally rare. The incidence of epithelial keratitis ranges from 0% to 30% (Fig. 4). Extraocular symptoms and signs are usually mild and resolve without sequelae.

Most symptoms resolve by the fourth day of illness, as do most physical findings. The most persistent signs are conjunctival hemorrhages and follicles, which may aid physicians in making the diagnosis of AHC when patients present during the convalescent phase of disease.

VIRAL MORPHOLOGY AND PHYSICOCHEMICAL PROPERTIES

The virions of EV70 and CA24v are spherical and have diameters of 30 ± 1 nm, showing a typical enterovirus morphology. Virions of these viruses have a buoyant density of 1.34 g/cm³ in CsCl₂, and their sedimentation coefficient is 160S in sucrose.^14,15 The virion consists of a single-stranded RNA genome and four structural proteins, similar to other enteroviruses.¹⁶

The sedimentation coefficient of the genome RNA is 34S, and its molecular weight is 2.5 × 10⁶ daltons.^14,15 The single-stranded genomic RNA has positive-strand sense, and it codes in a single open reading frame for the four capsid proteins and additionally for functional proteins with, for example, RNA polymerase and protease activities. The genome RNA contains 7,391 nucleotides.

The capsid consists of 60 protomers, each of which contains four nonglycosylated virus proteins. The capsid proteins are virus protein (VP) 1 (molecular weight, 35 kilodaltons), VP2 (28 kilodaltons), VP3 (27 kilodaltons), and VP4 (9 kilodaltons). VP4 is myristoylated at its N-terminus. Structure analysis by x-ray crystallography and biochemical accessibility studies have shown that the capsid proteins VP1, VP2, and VP3 are at the capsid surface, whereas VP4 is covered inside the capsid shell and has contact to the viral RNA (Fig. 5). VP1, VP2, and VP3 together make up a pseudoequivalent packing arrangement in the capsid. VP1 and VP3 form a depression or canyon (approximately 2.5 nm deep and up to 3 nm wide) that is oriented around the fivefold symmetry axis of the capsid. It is proposed that the canyon is the recognition site for the virus-specific receptor; this canyon hypothesis has also been described for the related picornavirus, human rhinovirus 14.¹⁷

BIOLOGICAL PROPERTIES

Portal of Entry

For most enteroviruses, the primary site of infection is the alimentary tract, where the viruses replicate and produce systemic infection through viremia. For EV70 and CA24v, however, the primary infection site seems to be the conjunctiva, and there has been no definite evidence to suggest active replication of these viruses in the gastrointestinal tract. In the occasional case, these viruses are recovered from the feces of patients with AHC. Although one cannot completely exclude the possibility that EV70 and CA24v replicated in the intestine, in such cases, it is more reasonable to assume that these viruses grew in the conjunctiva, passed from the nasopharynx into the alimentary tract, and were then incidentally recovered from feces. This assumption is also supported by in vitro findings that EV70 replicates better at 32°C to 34°C than at 37°C and usually does not replicate at 39°C,^18,19 suggesting that the virus cannot readily grow in the alimentary tract. This temperature-sensitive growth of EV70 suggests that these viruses have acquired human pathogenicity as a result of their adaptation to the lower temperature of the ocular surface through evolution. However, in contrast, as for CA24v, no difference was observed in the isolation rate in HeLa cell culture at 33°C and those incubated at 37°C.^20,21 Studies to investigate molecular events leading to a temperature-sensitive defect in EV70 replication showed that viral RNA synthesis was blocked at the nonpermissive temperature (39°C).^22,23 The temperature-sensitive defect of EV70 replication is most likely the result of lack of uridylation of VPg at the restricted temperature.²⁴

Virus-specific Receptors

Most human enteroviruses, with a few exceptions, grow only in primary or established cell cultures derived from human and monkey tissues. EV70 is one of those that replicate in nonprimate cell cultures. Replication of EV70 occurs in nonprimate cell cultures at 33°C, and this virus can adsorb and replicate in cells from the mouse (L), hamster (BHK21), rabbit (RK13), pig (IB-RS-2), and cow (BK1).²⁵ In contrast, a CA24v can replicate and cause cytopathic effect only in primate cell cultures,²⁶ like poliovirus and many other enterovi-ruses. Although a specific viral receptor of CA24v has not been determined, an additional membrane protein, decay accelerating factor (CD55), is the receptor for echoviruses 6, 7, 12, and 21,²⁷ some of which are considered to cause AHC, and for EV70.²⁸

Viral Reproduction

The early phase of the enterovirus reproduction cycle can be divided into adsorption, penetration, and uncoating. Viral entry into the host cell (penetration) occurs by way of receptor-mediated endocytosis followed by a pH-dependent release of viral RNA from the virus capsid. The internal capsid protein VP4 is released from the virus, and the N-terminus of the capsid VP1 becomes accessible. After the virus has been uncoated, synthesis of viral protein and RNA are initiated by using the viral parental positive-strand RNA as a template. One reproduction cycle of enterovirus lasts 6 to 8 hours, at which time as many as 10,000 virus particles have been synthesized in a single cell.

ENTEROVIRUS 70

To establish the etiologic diagnosis of AHC caused by EV70, viral isolation and serologic tests are required. The selection of sensitive batches of cells or of sensitive sublines is important. To effectively isolate EV70, more sensitive cell types should be selected among primary cell cultures (e.g., human embryonic kidney, human embryonic lung, or monkey kidney cells) and diploid (e.g., human embryonic lung fibroblast or WI-38) or heteroploid (e.g., HeLa or HEp-2) cells. Suitable specimens should be inoculated onto at least two different cell types sensitive to EV70 propagation.

Although commonly used diagnostic tests for enterovirus infection are based on virus isolation in cell culture, followed by identification of serotypes with neutralizing antisera, or on serologic tests, no EV70 strain has been isolated by the cell culture method since 1988.²⁹ Changes in the characteristics of the virus may explain this phenomenon. Takeda and coworkers³⁰ have shown with oligonucleotide fingerprinting that EV70 isolates from cases of AHC in 1981 were genetically distinct from isolates observed during the 1970-1971 pandemic. Immunofluorescence,³¹ enzyme-linked immunosorbent assay,³² and electron microscopy³³ have been applied to detect epidemic EV70 that could not be isolated by cell culture.

Recently, the sequences of the full-length genome of EV70 standard strain were determined.³⁴ Uchio and coworkers³⁵ have previously reported that out of culture-negative specimens from conjunctival swabs of patients among the population of an AHC epidemic in Okinawa, Japan in 1994, several specimens were positive by RT-PCR (Fig. 6). Three sets of primers (VP1, VP2, and VP3 regions) were designed based on the nucleotide sequence of the standard strain of EV70, J670/7120. That no PCR products obtained from specimens from patients with symptoms of more than 3 days' duration gave positive results indicates that the virus might be present in the eye for only a brief duration, and timely collection of specimens may be important.³⁶ This method has been applied for phylogenetic analysis of EV70 using specimens from patients among a community outbreak of AHC in Israel.³⁷

COXSACKIEVIRUS 24

Identification of CA24v can be carried out by the conventional neutralization test in test tubes or more economically in microtitration plates.³³ The micrometabolic inhibition test in microtitration plates is also reliable and convenient.³⁸ The CA24v specific antiserum should be used for identification by the neutralization test because the virus is known to be antigenically distinct from the prototype (Joseph strain) and other variants of CA24v.⁷

TYPING

Although the neutralization test is generally reliable for enterovirus typing, it is labor-intensive and time-consuming and may fail to identify the serotype of clinical isolates because of antigenic drift, recombination, or the presence of virus mixtures in the specimen being tested. The technique of viral protein fingerprinting has recently been used for the typing and characterization of clinical enterovirus isolates.³⁹ The method is specific and 97% accurate, but the radiolabeled proteins generate radioactive waste, and the method requires the use of specialized instrumentation for data acquisition and analysis. Obserste and coworkers⁴⁰ have developed a molecular typing system based on RT-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The serotype of an unknown may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototypes strains of all 66 human enterovirus serotypes. The antigenic and molecular typing results agreed for all isolates. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunologic reagents, such as recent isolates of EV70.

DISINFECTANTS

Enterovirus 70 is readily killed by various germicides, such as phenol, cresol, formaldehyde, and sublimate, under the conditions by which other enteroviruses are not or are only partially inactivated. However, neither EV70 nor any of the other enteroviruses were inactivated by the common preservatives: benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, and hexachlorophen at levels of practical use.⁴¹

Iodine is widely used in eye clinics. The solution containing free iodine at the concentration of 0.0025% to 0.01% iodine is generally used for disinfection of hands and instruments. Kono and Yoshii⁴² studied the virucidal activity of the Pliacid-Nutra flow system containing iodine and found it effective for EV70. Previous investigation showed that among six enteroviruses (polio 1, echo 7, coxsackie A1, coxsackie B5, EV70, and EV71), EV70 was most sensitive to methanol, ethanol, isopropanol, and n-propanol.⁴³

ANTIVIRAL AGENTS

Antiviral chemotherapy cannot yet be used for the treatment of enterovirus infection of humans. Some antiviral compounds have been proven to be potent only in vitro. Guanidine and some benzimidazoles specifically inhibit the viral RNA polymerization. Hydrophobic compounds intercalating in the viral capsid protein VP1 stabilize the virus, resulting in inhibition of viral uncoating.⁴⁴ Arildone is a broad-spectrum antiviral agent that has been shown to selectively inhibit replication of picornaviruses. The mechanism of action of arildone is consistent with inhibition of the uncoating process.⁴⁵ Arildone inhibited the infectivity of AHC viruses, EV70, and CA24v in tissue culture. Arildone did not inhibit interferon production or interferon activity.

ENTEROVIRUS 70

Although it was reported that EV70 isolates grew well in the primary cultured epithelial cells derived from mouse, rabbit, and monkey conjunctival and corneal tissue, no laboratory animals have so far been found to develop noticeable conjunctivitis.²⁶ Unlike coxsackievirus, enterovirus isolates are nonpathogenic to suckling mice and adult mice.⁴ However, EV70 showed a distinct neurovirulence when inoculated into the central nervous system of cynomolgus monkeys.⁴⁶ Monkeys inoculated with EV70 simultaneously in the spinal cord and thalamus developed paraplegia or monoplegia of the lower limbs. This finding gave virologic evidence to the later recognized clinicoepidemiologic observation that EV70 infection is occasionally complicated by poliolike motor paralysis, which usually appears several weeks after the onset of conjunctivitis.⁴⁷

COXSACKIEVIRUS 24

Although suckling mice are the most popular isolation system for the coxsackievirus A group, they are not sensitive as tissue cell cultures for the isolation of CA24v. The pathogenicity of the virus for mice can be enhanced by passing the virus through the intracerebral route into consecutive batches of suckling mice.⁴⁸ Higgins and coworkers¹⁵ observed stunting of growth and paralysis in some newborn mice inoculated subcutaneously and intracerebrally with the Singapore and Hong Kong strains of CA24v. They also observed that the Hong Kong strain produced a higher morbidity and mortality rate than did the Singapore strain and that the virus was found in the brain and torso of paralyzed mice. Loss of striation and fragmentation of fibers in the voluntary muscles occur with infiltration of mononuclear cells, polymorphonuclear cells, and eosinophils typical of coxsackievirus A infection.⁴⁹

The phylogenetic tree of EV70 indicates that the virus originated from one focus around 1967 and was transmitted among humans since then.⁵⁰ West Africa is considered to be the most likely place where EV70 was born. A hot and humid coastal monsoonal climate and a dense population are favorable factors for the spread of EV70. The phylogenetic tree of EV70 showed an evolutionary pattern in divergent fashion; the virus branched into many lineages during the pandemic period that were transmitted independently of one another, resulting in the appearance of vastly polymorphic viruses in the nucleotide sequence.

According to the evolutionary tree of CA24v, the strains during the early epidemic in Singapore and Hong Kong were closely related.⁵¹ In contrast, all late isolates from various areas including Singapore, Pakistan, Taiwan, and Japan were distinct from the strains of early epidemics. The tree indicated that all strains isolated in 1985 and 1986 were the descendants of the virus that prevailed between 1978 and 1980. After branching, the viruses transmitted independently in certain areas, and after 1985, one of them spread to Pakistan, one to Singapore, and another to Taiwan and Japan. The tree indicated that all isolates were derived from a common ancestor that appeared around 1965 leading to the hypothesis that AHC due to CA24v originated from a Java focus. It is conceivable that one of the strains of CA24v had undergone nucleotide substitutions and pathogenic change and came to preferentially grow in human conjunctiva. Through this new pathogenicity, the virus has spread and caused major epidemics of AHC in humans.

From these reports, the two enteroviruses causing the AHC syndrome, EV70 and CA24v, are entirely different, not only in antigenic properties but also in viral genomes. This was shown by cross-neutralization tests, complement-fixation tests using antisera against purified viral antigens, and RNA-RNA hybridization experiments in which ³²P-labeled ssRNA (-) extracted from infected cells was hybridized with ssRNA (+) of homologous or heterologous virions. EV70 is considered as a new distinct type of human enterovirus, and its origin remains unknown. The first emergence of this virus has been estimated to be around 1967 by molecular epidemiology using oligonucleotide mapping.⁵² Conversely, EH24/70, the representative strain of CA24v, shares nucleotide sequence with the prototype strain of Joseph and other variant strains of coxsackievirus A24 to various degrees. Thus, these two closely related virus seem to arose, one from Africa and the other from Asia, at approximately the same time.

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