The rotavirus genus

~ Pergamon Comp. Immun. Microbiol. infect. Dis. Vol. 17, No. 3/4, pp. 305 320, 1994 Copyright © 1994 Elsevier Science Ltd

0147-9571(94)E0010-Q Printed in Great Britain. All rights reserved 0147-9571/94 $7.00 + 0.00

THE ROTAVIRUS GENUS

GIANFRANCO DONELLI and FABIANA SUPERTI Laboratorio di Ultrastrutture, Istituto Superiore di SanitY, Viale Regina Elena, 299-00161, Roma,

Italy

Abstract--Human rotaviruses, discovered nearly 20 years ago, have been proven to be major cause of paediatric diarrhoeal disease morbidity and mortality. The clinical significance of these viruses stimulated basic studies on their biology, molecular and antigenic properties and epidemiology. General features, clinical relevance, epidemiologic pattern and laboratory diagnosis of human rotavirus infections are here reviewed.

Key words: Rotavirus, diarrhoea.

R~sum~--Les rotaviruses humains, d6couverts depuis vingt ans, sont connus comme la principale cause de morbidit6 et de morbilit6 des maladies diarrheiques en p~diatrie. L'importance clinique de ces viruses a stimul~ des &udes de base sur leur biologie, leurs propriet~s mol+culaires et antig6niques et sur l'6pid~miologie. Dans cette revue sont present6es les caract6ristiques g6nb.rales, l'importance clinique, l'6pid~miologie et le diagnostic de laboratoire des infections par les rotaviruses.

Mots-clefs: Rotavirus, diarrh6e.

INTRODUCTION

Rotav i ruses are a genus be longing to the family Reov i r idae . Firs t identif ied by electron mic roscopy in 1973 [1], h u m a n rotavi ruses are the ma in aet iologic agents o f gas t roenter i t i s in infants and young chi ldren wor ldwide and the ma jo r cause o f infant mor ta l i t y in undeve loped areas.

STRUCTURE AND PHYSICOCHEMICAL PROPERTIES

Rotavi ruses , like o ther members o f the R e o v i r i d a e family, have a double-shel led capsid c o m p o s e d o f an ou te r layer, an inner capsid, and an icosahedra l core (Table 1). Double - and single-shelled par t ic les and core ghosts can be observed by electron mic roscopy (Fig. 1). The double-she l led par t ic les show a dist inct ive m o r p h o l o g y suggestive o f a wheel. The virus name is der ived f rom the Lat in rota, meaning wheel. In tac t part icles, abou t 70 nm in dia, possess a core fo rming a hub, capsomeres rad ia t ing f rom the core as shor t spokes , and a precisely marg ina t ed outer layer forming the r im [2]. The single-shelled part icles , measur ing a b o u t 55 nm in dia, are often descr ibed as rough part icles because they lack the s m o o t h outer layer and their pe r iphery shows pro jec t ing tr imeric subuni ts o f the inner capsid. Cores , a b o u t 37 nm in dia, are less f requent ly observed as ghost part icles in that they usual ly lack genomic R N A [3].

The different par t ic les have dis t inct b iophys ica l and biological proper t ies . Doub le -

CIMID 17-3/4~V 3 0 5

306 GIANFRANCO DONELLI and FABIANA SUPERTI

shelled particles have a density of 1.36 g/cm 3 in CsC1 and a sedimentation coefficient of 520-530S; single-shelled particles have a density of 1.38 g/cm 3 and a sedimentation coefficient of 380-400S; cores have a density of 1.44 g/cm 3 and a sedimentation coefficient of 280S [4-6]. Only double-shelled particles are infectious [4] and treatment with calcium-chelating agents (e.g. EDTA or EGTA), causing the removal of the outer capsid, results in a loss of viral infectivity [7]. Cores can be obtained by caotropic agents, such as sodium thiocyanate [8] or highly concentrated calcium chloride, which degrade the inner capsid of the particles [6].

Rotavirus infectivity is not affected by fluorocarbon extraction or exposure to ether and deoxycholate [7], reflecting the absence of a lipid envelope. Moreover, viral particles are relatively resistant to repeated freeze-thawing, sonication or incubation at 37°C for I h or at room temperature (25°C) for 24h [7,9]. Chloroform treatment reduces infectivity slightly and destroys hemagglutinating activity [7]. Rotavirus infectivity is relatively stable within the pH range 3-9 [7, 10].

Treatment with proteolytic enzymes (trypsin, pancreatin or elastine) results in the enhancement of viral infectivity and is essential for successful cultivation of human rotaviruses [7, 11].

Viral infectivity can be inactivated by disinfectants such as ethanol, phenol, formalin, chlorine, and Lysol [12-14].

ISOLATION AND CULTIVATION

Although the various animal rotaviruses, such as simian rotavirus SA-11, multiply easily in cell cultures, group A human strains have only recently been successfully cultivated in vitro [15-17].

The cultivation of human rotaviruses requires preliminary treatment of the virus with trypsin (10 #g/mi), the presence of the enzyme in the culture medium, the use of rotating cell cultures and incubation at 37°C. Before the establishment of these culture conditions, abortive in vitro infections were often observed, in which, despite the production of viral antigens, infectious particles were not released in the supernatant. Using the cultivation procedures described above, human rotaviruses extracted from stool samples can be

Table 1. General features of rotaviruses

Morphology lcosahedral 65-75 nm particles Two outer capsid shells and a core

Physicochemical properties Resistant to ether (non enveloped) Infectivity stable at pH 3.0 and relatively heat-stable

Nucleic acid 11 dsRNA segments Each segment codes for at least one protein

Proteins Six structural proteins (for group A): VPI, VP2, VP3 (inner core), VP6 (inner capsid), VP4 and VP7 (outer capsid)

Antigenic properties Six VP6-serogroups (A-F) described Two VP6-subgroups recognized within group A Group A serotypes: I1 VP7- and nine VP4-types (G and P, respectively)

Replication Penetration depends on VP4 after specific cleavage by proteases Cytoplasmic replication Inclusion body formation Final assembly occurs within cisternae of ER

The rotavirus genus 307

isolated in MA-104 (simian foetal kidney) cell cultures with an about 70% efficiency. Nevertheless, an efficiency rate of approx. 100% can be achieved in the direct virus isolation from clinical samples when primary cultures of simian kidney cells or a cell line derived from a human colon adenocarcinoma (HT-29) are used [18]. The cultivation of the non-A group rotavirus presents considerable difficulties and, until now, only a group C strain has been isolated in cell cultures.

VIRUS-CELL INTERACTIONS

The identification of the rotavirus protein which mediates viral binding to the host cell has been uncertain. The two virus outer capsid proteins VP7 and VP4 have been considered [19, 20]. The chemical nature of the cellular receptor for rotaviruses has not been completely elucidated. Different glycoconjugates have been shown to bind the virus and to prevent cell attachment [20-26]. The morphogenesis of rotaviruses is unique. Viral particles assemble near the periphery of viroplasm and subsequently bud into the endoplasmic reticulum (ER) cisternae acquiring membrane. Mature non enveloped particles derive from enveloped particles by the loss or transformation of the outer rim and these infective virions are released by lysis from infected cells (Fig. 2).

GENOME

The rotavirus genome contains 11 segments of double strand RNA which range in molecular weight from 2 x 105 to 2.2 x 106 Da [27]. Analysis of genome by polyacrylamide gel electrophoresis (PAGE) is a relatively easy and rapid technique for virus detection and epidemiological studies. In most cases, the electrophoretic pattern is comprised of four high molecular weight dsRNA segments, two middle-sized segments, a distinctive triplet of small segments and two smaller segments (Fig. 3). On the basis of the different electrophoretic mobility of the last two genomic segments (10 and 11), electrophoretic patterns referred to as "long", "short" or "super short" have been identified. However, since distinct RNA patterns can arise by different mechanisms, (shift, drift, rearrange- ments), these profiles cannot be used as the only criteria for classification of a virus strain. Protein products encoded in each of the 11 genome segments is shown in Table 2.

STRUCTURAL PROTEINS

The virion is made up of four major structural proteins: VP4, VP7, VP6 and VP2 [28-29]. VP4 and VP7 are the two surface proteins on the outer capsid. VP4, the product of gene 4, is the viral hemagglutinin [30], forms spike-like structures of about 10 nm [31-33], and is an important determinant of virulence [34]. The cleavage of VP4 into two subunit proteins (VP5* and VP8*) by trypsin is essential for viral penetration into host cells [35-36]. VP7, the product of gene 9, makes up over 90% of the viral surface, is a glycoprotein of the high mannose type and is primarily responsible for determining the viral serotype [37]. VP7 is encoded by genome segment 9 of the SA-11 simian rotavirus, genome segment 8 of the UK bovine virus and genome segment 7 of rhesus rotavirus. The inner capsid of rotavirus is composed of the major structural protein VP6 (the product of gene 6). VP6 makes up 50% of the virion by weight and is primarily responsible for group and subgroup antigenic reactivity [38, 39]. Rotaviruses comprise six distinct groups


Table 2. Rotavirus genome segments and related proteins (Adapted from Estes and Cohen [28] and Estes [3])

Nascent Location Protein polypeptide in virus Main

Segment product (mol. wt) particles functions

1 VPI 125.000 Inner core Slightly basic 2 VP2 102.000 Inner core RNA binding 3 VP3 98.000 Inner core Basic 4 VP4

(VP5* + VP8*) 88.000 Outer capsid

5 NS53 58.600 Nonstructural 6 VP6 44.800 Inner capsid

7 NS34 34.600 Nonstructural 8 NS35 36.600 Nonstructural 9 VP7 34.000 Outer capsid

10 NS20 20.300 Nonstructu ral

11 NS26 21.700 Nonstructural

Hemagglutinin, protease-enhanced infectivity, neutralization antigen, virulence, putative fusion region

Slightly basic Hydrophobic, trimer, group-subgroup

antigen required for transcription Slightly acidic, RNA binding Basic, RNA binding Rough endoplasmic reticulum (RER)

integral membrane protein, neutralization antigen, glycoprotein

RER transmembrane glycoprotein, role in morphogenesis

Slightly basic

(A-F): A, B and C groups are found in both humans and animals, whereas D, E and F groups have been found only in animals to date [3, 28]. The inner shell surrounds the core, which encompasses the genome and is composed predominantly of VP2 (the product of gene 2) as well as minor amounts of VP1 and VP3 [33].

PATHOGENESIS

Rotaviruses infect the mature enterocytes of the upper small intestine. Infection usually spreads from the proximal small bowel to the ileum over a period of 1-2 days. Histopathologic studies have revealed villous shortening, reticular cell enlargement, mitochondrial swelling, lymphocyte infiltration of the villous lamina propria and irregular microvilli. Infection generally lasts about one week; however, the period may be substantially longer in immunocompromised patients [40].

Recent investigations on mice with combined immunodeficiency as well as on immunocompromised children indicate that rotaviruses can also infect the liver under some circumstances [41, 42].

The mechanism of rotavirus-induced diarrhoea is not completely known. The major mechanism appears to be a decreased absorption of salt and water related to selective infection of the absorptive intestinal villous cells, resulting in net fluid secretion. Impaired D-xylose absorption has been observed [43], and some patients have depressed levels of disaccharidases [1] resulting in carbohydrate malabsorption with osmotic diarrhoea.

EPIDEM1OLOGY

Group A rotaviruses have clearly been established as causing severe dehydrating gastroenteritis in young children (under the age of two) in both industrialized and developing countries [44, 45].

Group B rotaviruses have been associated with very large epidemics of diarrhoeal disease primarily in adults in China and have only rarely been identified elsewhere [46].

Fig. 1. Rotavirus particles observed by negative-stain electron microscopy in a stool suspension of child with diarrhoea. Bar represents 100 rim.

Fig. 2. Thin sectioned rotavirus infected MA-104 cells. Viroplasm (V) associated with ER cistcrnae which contain both enveloped (arrow) and non enveloped (arrowhead) viral particles. Bar

represents 200 rim.

I

II

III

IV

5

6

Fig. 3. PO lyacrylan lide gel and (B) a I human stool e segr nents, numbere :d from

elect .rophoresis of rotaviral RNA extr acted xtraq :t and stained by the silver imr lregna the top. can be divided into four siz e-clas

Sl how typical “long” rotavirus pat tern.

from (A) simian 1 virus SA-I I ltion method. Th e I1 RNA

ses (E group I-IV). Botl h lanes

The ro tav i rus genus 313

Group C viruses have been sporadically reported to cause diarrhoeal disease in children in several areas of the world [47] (Table 3).

In temperate climates, group A rotavirus infection may be responsible for Close to half of the hospitalizations of infants during the winter months, whereas in tropical climates, infections tend to occur year around. Transmission of the virus is thought to be primarily by the fecal-oral route, although transmission through food and water has been suggested. Virus specific IgA has been detected in pharingeal secretions of patients with rotavirus gastroenteritis, suggesting the possibility of transmission via a respiratory route or by ingestion of virus aerosols [48]. Moreover, rotavirus infection of the oropharynx and respiratory tract has been recently reported [49].

Infant mortality due to diarrhoeal diseases is high in developing countries, and rotavirus is thought to be of major importance. Chronic disease is not usually found, but may be seen in immunodeficient or immunosuppressed patients [40, 50-52]. Rotavirus infection may also be asymptomatic, especially in newborn children [53-55] and in adults. Outbreaks of rotavirus gastroenteritis among premature infants have been reported [56].

The peak incidence of illness occurs between 6 and 24 months of life, but considerable morbidity is seen throughout the second year. During the first 2 or 3 months of life, infants appear to be relatively resistant to rotaviral illness, most likely due to transplacentai transfer of maternal antibodies [57]. After 3 years of age, children rarely have severe rotaviral illness. Group A rotaviruses are usually not considered common pathogens in adults, but they can cause illness under a variety of circumstances [58]. Nosocomial infections in hospital wards are a known problem and appropriate infection control measures should be undertaken when patients with suspected viral gastroenteritis are admitted [59~50].

The epidemiology of rotavirus infections can be followed by monitoring a variety of serologic markers or by determining the electrophoretic pattern of viral genomic RNA (the electropherotype) in faecal specimens. Several serotypes of human group A rotavirus have been identified, although the great majority of infections appear to be caused by only the 1-4 serotypes [61-63].

IMMUNITY

Seroconversion occurs following infection in both animals and man. Serological surveys carried out in different areas of the world have demonstrated that over 80% of the population has antibodies from the age of about 3 years [64-65]. Immunity may wane among the elderly, rendering them susceptible again to severe disease.

Genetic studies with specific viral reassortants and passive-transfer studies with monoclonal antibodies directed at specific rotaviral proteins have demonstrated that, in general,

Table 3. Clinical relevance and epidemiology characteristics of human rotaviruses

Epidemiologic Clinical Group features symptoms

Major cause of severe dehydrating gastroenteritis in infants and young children worldwide (in winter months in temperate climates) Large epidemics of diarrhoeal disease primarily in adults in China Sporadically reported to cause diarrhoeal disease in children in many areas of the world

Dehydrating diarrhoea for 3-8 days, fever and vomiting frequent

Severe dehydrating diarrhoea for 3-5 days

Dehydrating diarrhoea for 3-8 days, fever and vomiting frequent


Table 4. VP7 (G) antigenic types of group A rotaviruses. (Adapted from Estes and Cohen [28])

G serotype Human (strains)

Host range

Animal (species*)

I KU, Wa, RV-4, K8, D, M37, SI2, Mont

2 DS-I, RV-5, RV-6, $2, HN-126, 1076

3 P, M, RV-3, AU-I lto, Yo, Nemoto, W178

4 VA70, St Thomas-3, Hochi, Hosakawa, 57M

Si, Po, Ca, La, Mu, Eq, Fe

Po

5 Po, Eq 6 Bo 7 Ch, Ty 8 69M, B37 9 Wl-61, AU-32, F45

10 Bo I 1 Po

*Abbreviations: Si, simian; Po, porcine; Bo, bovine; Eq, equine; Fe, feline; La, lapine; Ch, chicken (avian); Ty, turkey (avian); Ca, canine; Mu, murine.

antibodies directed at outer capsid proteins, but not at other rotaviral proteins, can neutralize virus and protect susceptible hosts [29]. Two different neutralization specificities exist on the outer capsid of group A rotaviruses. At least 11 VP7 (G) antigenic types are distinguishable (Table 4) and about nine distinct antigenic (P) "types" of rotavirus VP4 corresponding to separate rotavirus gene 4 groups have been described (Table 5). During primary and secondary infections in humans, IgM, IgG, and IgA immune responses to both surface proteins, VP4 and VP7, have been demonstrated in serum and intestinal secretions [66, 67].

Children under the age of 3 months are quite resistant to rotaviral illness, and this may be related to levels of maternal antibodies. The effects of breast feeding in the prevention of rotavirus infection in humans are not clear. Although some studies have indicated that breast-fed infants have a lower incidence of diarrhoeal disease than formula-fed infants, other surveys have not found a significant influence of breast-feeding on the course of rotaviral illness [68].

Table 5. VP4 (P) antigenic "types" of group A rotaviruses. (Adapted from Estes and Cohen [281)

Host range

P "type" Human (strains) Animal (species*)

Si, Bo Si Si

DS-I, RV-5 Bo

RV-3, M37, 1076 ST3 Po Po

Wa, VA70, KU, P K8

*Abbreviations: Si, simian; Po, porcine; Bo, bovine.


CLINICAL DISEASE

The clinical spectrum of rotavirus infections varies from asymptomatic to severe, with occasional fatal episodes. The commonest clinical findings usually include vomiting (which arise early in the illness), abdominal pain, watery diarrhoea, mild to moderate dehydration with normal serum sodium concentration and mild acidosis [69]. Fever and vomiting frequently precede diarrhoea. Associated clinical findings may include irritability and pharingeal or tympanic membrane erythema. Stools usually do not contain blood or leukocytes. The incubation period is approx. 2 days and the course of the disease is generally 3-8 days. When required, due to severe dehydration, mean hospitalization time is 4 days.

Numerous case reports have associated rotavirus infection with a variety of conditions such as intususception, Reye syndrome, hemolytic uremic syndrome, and Kawasaki syndrome. Evidence for an association with rotavirus has been reported for some cases of neonatal necrotizing enterocolitis [70].

Deaths, rare in developed countries, are frequent in developing countries and typically due to electrolite imbalance.

In adults the infections are usually asymptomatic and accompanied by seroconversion to the virus. Symptomatic infections in adults are usually mild, but severe illness has been reported both in elderly patients [71] and in apparently normal young adults [72]. Infections due to non-group A rotaviruses in China have been reported to cause epidemics of gastroenteritis with severe symptoms in adults [73].

LABORATORY DIAGNOSIS

As clinical manifestations are not sufficiently distinctive, diagnosis requires detection of virus (or viral antigens, or viral nucleic acid) and/or demonstration of a serologic response. The epidemiologic pattern may suggest the diagnosis, but laboratory confirmation is required.

Commercial rotavirus antigen-detection kits are widely available, inexpensive, and allow rapid diagnosis. These include the enzyme immunoassays EIA or ELISA and the latex particles agglutination tests [74-77]. Only small amounts of stools are required for the tests, and samples may be frozen before testing. Since virus shedding is generally maximal during diarrhoea, faecal samples should be obtained as close to illness as possible (within 4 days).

Other methods for virus detection include hybridization of radiolabeled nucleic acid probes to viral RNA [78], or PAGE analysis of viral RNA [77, 79, 80]. Polymerase chain reaction (PCR) techniques have also been developed in the amplification and typing of rotaviral nucleic acid from stool specimens [81, 82]. Direct electron microscopy remains a useful technique for viral detection, in particular for non-group-A rotaviruses, and immune-electron microscopy (IEM) is frequently used as reference method with respect to other assays [83, 84].

Viruses can also be identified by direct isolation from stool samples in cell cultures with the aid of proteolitic enzymes [15-18].

To measure serologic response to infection, there are many techniques such as ELISA, IEM, complement fixation, immunofluorescence, immune-adherence hemagglutination, radio-immunoassay and neutralization tests [85-91]. The complement fixation, initially widely used for the detection of rotavirus antibodies, is efficient for revealing an antibody


response only in patients between 6 and 24 months o f age [85]. An E L I S A IgA has been particularly effective in demonstra t ing serologic responses in young infants who possess passively acquired maternal IgG antibodies, since IgA does not cross the placenta [92]. This assay may also be used to examine stools for coproant ibodies [93].

Virus detection and/or demonst ra t ion o f a serologic response in an individual patient does not necessarily establish an aetiologic association with patient 's illness, especially in newborns and adults who commonly undergo inapparent infection.

PREVENTION, CONTROL AND TREATMENT OF ROTAVIRUS ILLNESS

Extraordinary progress toward the development o f a rotaviral vaccine has been made [94-95] and a number o f trials in developed and less-developed countries have been carried out [96-98]. Current strategies for development o f a rotavirus vaccine range from cell culture cultivation o f human or animal strains to the application o f molecular biology techniques. The most developed candidate strains have been derived from animal rotaviruses (bovine and simian) or are reassortants containing 10 animal rotavirus gene segments and the human rotavirus gene 9 (encoding VP7 in humans) [98-101]. Another approach is based on the use o f "na tura l ly" at tenuated neonatal human strains [98].

Other possible vaccines under study include the use o f synthetic viral proteins (VP7 or VP4) or viral proteins expressed in prokaryot ic or eukaryotic cell systems by cloned rotavirus c D N A [102 104].

Al though passive immunizat ion has shown promising results [105, 106], it is not practical for protect ion against rotavirus illness, with the exception o f immunocompromised individuals, requiring repeated treatments.

Treatment remains mainly symptomatic . The replacement o f fluid and electrolytes lost by vomiting and diarrhoea, preferably with use o f oral rehydrat ion solutions, is highly effective in the great majori ty o f cases [107]. Of course, if the oral rehydrat ion regimen is not able to correct the fluid and electrolyte loss or if the patient is severely dehydrated or in shock, intravenous fluids must be given. Normal and immunodeficient children have been fed with gammaglobul ins [108] or human milk containing rotavirus antibodies [50], respectively, but these therapies are still under study.

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The rotavirus genus

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