H. Ushijima** * Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand ** Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan
Astroviruses are members of the large and growing family Astroviridae. The viruses can infect both humans and a wide variety of mammals and avian species, including lambs, sheep, calves, pigs, dogs, cats, deer, mice, minks, bats, cheetahs, sea lions, dolphins, rats, rabbits, chickens, ducks, turkeys, and pigeons. In humans, astroviruses cause acute gastroenteritis and mainly affect children under 2 years old with general prevalence rates of up to 10%. Using advanced diagnostic assays, recent epidemiological studies have highlighted the impact of astrovirus-associated gastroenteritis, with additional novel astroviruses MLB and VA being discovered in human stool samples. Continued surveillance studies and the molecular characterization of the viral genome will permit the identification of new strains and potential zoonotic transmission of astroviruses in different host species.
Astroviruses belong to the family Astroviridae, which are small, nonenveloped, single-stranded, positive-sense RNA viruses. The genomes range from 6.1 to 7.9 kb in size and contain three open reading frames (ORFs). The ORF1a and ORF1b encode nonstructural proteins, whereas ORF2 encodes the capsid protein precursor (Mendez and Arias, 2013) (see Chapter 4.1). Although astroviruses have been known to be major causative agents for gastroenteritis in humans worldwide, there is relatively little information on their association with gastroenteritis diseases in other animal species. Surveillance investigations conducted previously in several countries worldwide indicated that human astroviruses are involved in 2–30% of diarrhea in children (De Benedictis et al., 2011).
Astroviruses have been found to be morphologically unique and typically appear as a star-like structures by electron microscopy, with the icosahedral capsid particles measuring approximately 28–30 nm in diameter (Appleton and Higgins, 1975; Madeley and Cosgrove, 1975). The term “astron” (greek) refers to the appearance of the icosahedral capsid which is similar to a star shape. The Astroviridae family contains several important human and animal pathogens and is further differentiated into two genera: Mamastrovirus (MAstV) and Avastrovirus (AAstV). MAstV and AAstV have been known to infect many mammalian and avian species, respectively. Astroviruses identified thus far have been classified on the basis of their host tropism and viral genomic variation by the International Committee on Taxonomy of Viruses (ICTV). Currently, 19 species within the Mamastrovirus genus (MAstV1-19) and 3 species within Avastrovirus (AAstV1-3) have been officially approved (Fig. 4.3.1). Recently, several novel astroviruses from different animal host species have been discovered, mostly by metagenomic pyrosequencing.
The prototype astrovirus species was originally isolated from humans, called the classic human astrovirus (HAstV) and now belongs to MAstV1 species. The classic human astroviruses are genetically highly diversed and have been classified into eight genotypes (HAstV1-HAstV8). Beside MAstV1, three additional astrovirus species, MAstV6, MAstV8, and MAstV9 have been recently identified in human stool samples. The MAstV6 or MLB1 was detected during an outbreak of diarrhea in Australia (Finkbeiner et al., 2008). MAstV8 is comprised of the human astrovirus strains VA2, VA4, and HMOAstV-A, while MAstV9 includes the VA1, VA3, HMOAstV-B, and HMOAstV-C strains (Finkbeiner et al., 2009a,b; Jiang et al., 2013; Kapoor et al., 2009). Therefore, it is evident that there are at least three clades or genetic clusters of astroviruses cocirculating in humans: MAstV1 (classic human astrovirus: HAstV), MAstV6 (MLB clade), and MAstV8 together with MAstV9 (VA clade). In addition to humans, a wide range of animals has been found to be infected by different astrovirus species, such as cats, pigs, dogs, dolphin, mink, and sheep, infected by MAstV2, 3, 5, 7, 10, and 13, respectively. Moreover, California sea lions have been discovered to be infected by MAstV4 and 11, while several bat species are reported to be positive for MAstV12 and MAstV14–19 (Bosch et al., 2014; De Benedictis et al., 2011). The avian astroviruses are clustered exclusively within the Avastrovirus genus (AAstV); most recently a mammalian-like astrovirus has been reported in birds in 2011 (Pankovics et al., 2015).
3. Detection and diagnosis
By electron microscopy, the astrovirus particle is a small, round virus with a distinctive 5–6 pointed star-like structure (Appleton and Higgins, 1975; Madeley and Cosgrove, 1975). In 1981, the virus was successfully propagated in a primary cell culture, initially using human kidney epithelial cells (HEK) and later a human colon carcinoma (CaCo-2) cell line, which has been found to be more sensitive for the propagation of most human astrovirus strains (Willcocks et al., 1990). In addition, astroviruses could be propagated in several types of adenocarcinoma cell lines (SK-CO-1, T-84, and HT-29) and monkey kidney cell lines (Vero, MA104, and Cos-1) (Brinker et al., 2000). The success of growing human astroviruses in cell cultures was a big step forward in the characterization of these viruses. For the diagnosis of astrovirus infection, several different methods are now available. These include enzyme immunoassay (EIA), an immunochromatography (IC) test, reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, loop-mediated isothermal amplification, and nucleic acid sequence analysis (Jonassen et al., 1995; Khamrin et al., 2010; Wei et al., 2013). Of all of these methods, RT-PCR and nucleic acid sequence analysis are the most widely used methods for the detection and genotype identification of astroviruses. These techniques have replaced the traditional culture assays and immunological tests and become the gold standard for diagnosis of astrovirus infections in both humans and animal species.
4. Molecular epidemiology
4.1. Human Astroviruses
4.1.1. Classic Human Astroviruses (HAstV)
Since the human astrovirus was discovered, epidemiological studies of classic human astroviruses (HAstVs) have been extensively performed and reported from several countries around the world (Bosch et al., 2014; De Benedictis et al., 2011). The viruses have been identified in sporadic nonbacterial diarrhea cases and have been reported to be associated with a wide range of clinical illnesses including diarrhea, vomiting, fever, abdominal pain, bronchiolitis, and otitis (Mendez and Arias, 2013).
The HAstV positivity rates in acute gastroenteritis patients have been reviewed and summarized in Table 4.3.2. Surveillance of HAstV conducted in several countries worldwide indicate that the prevalence of HAstVs is variable from <1–40% of diarrhea cases. The variations of positivity rates are dependent on geographical area, study periods, age groups, and screening methods. However, in most studies the prevalence rate of HAstVs is approximately 10%. The virus is detected at a lower prevalence in comparison with group A rotavirus (RVA) and norovirus GII (NoV GII) (Chaimongkol et al., 2012; Chhabra et al., 2013). HAstVs predominantly infect young children with the highest rate of infection in children under 2 years of age; mixed infections with RVA are frequently observed (Iturriza Gomara et al., 2008). Although different seasonal peaks have been recorded in HAstV studies, the highest rates of infection have been reported for the cool season in the USA, Netherlands, Spain, China, and Japan (Chan-it et al., 2010; Chhabra et al., 2013; Enserink et al., 2015; Fang et al., 2006; Guix et al., 2002).
Human Astrovirus Positivity Rates in Patients With Acute Gastroenteritis (Reviewed From 60 Studies Worldwide)
EIA, Enzyme immunoassay; IEM, immune electron microscopy; RT-PCR, reverse transcription-polymerase chain reaction; RVA, rotavirus of species A; NoV, norovirus; BoV, bocavirus; AdV, adenovirus; H, hospitalized; O, outpatient; B, both hospitalized and outpatient.
Among HAstV circulating in the human population, at least eight genotypes have been described (HAstV1–HAstV8). Based on the genome sequences and phylogenetic analyses, the genotypes of HAstV are tightly correlated with the serotypes (Mendez and Arias, 2013). The HAstV1 has been reported to be the most predominant genotype worldwide in children with diarrhea. However, the detection rate varies by geographical region (Fig. 4.3.2). HAstV1 is a major genotype which represents 30–84% of all HAstV genotypes in each continent. It should be noted that the HAstV1 alone represents over 80% of the strains circulating in Asia, while other genotypes (HAstV2–HAstV8) are found to be uncommon and responsible for only 1–6% of astrovirus infections. Lower detection rates of HAstV1 are observed in North America, South America, and Africa at 30, 43, and 46%, respectively. The HAstV1 together with HAstV2-HAstV4 are found regularly in Europe, North America, and South America, accounting for 89–98% of all astrovirus strains, while HAstV5–HAstV8 are less common and represent only 2–11% of astrovirus infections. In contrast, the distribution of HAstV genotypes in Africa is different from that of other regions since the uncommon genotypes, HAstV8 and HAstV5, are found to be the third and fourth common types after HAstV1 and HAstV3.
Within each HAstV genotype, the strains can be further subdivided into lineages or subtypes. Based on the capsid sequences, the new subtype must have a nucleotide sequence divergence of >7% from the existing lineages (Guix et al., 2002). Thus far, HAstV1 has been subdivided into six lineages (1a–1f), HAstV2 into four lineages (2a–2d), HAstV3 into two lineages (3a–3b), and HAstV4 into three lineages (4a–4c) (Martella et al., 2013). In Japan, molecular characterization of HAstV1 lineages from the year 2006–15 demonstrated that during the first half of the surveillance studies from 2006–09 the detection rate of HAstV1d reached a peak of 96% prevalence, while HAstV1a was detected with a low prevalence of 4%. In contrast, from 2010 to 2015 HAstV1a reemerged with the prevalence of up to 98% as a predominant genotype (Chan-it et al., 2010; Dey et al., 2010; Thongprachum et al., 2015). Moreover, a surveillance study in Vietnam carried out in 2005-2006 revealed that HAstV1d was the most predominated astrovirus subtype (Nguyen et al., 2008). In Italy, during the period 2002–05 all HAstV1 were classified as HAstV1d, while during 2008 and 2009 both HAstV1a and HAstV1d subtypes were found to be equally predominant (De Grazia et al., 2013). These data imply that HAstVs cocirculating in children with diarrhea worldwide are genetically highly diverse pathogens.
4.1.2. MLB Astroviruses
The first MLB astrovirus prototype strain was identified in 2008 using the mass genome sequencing approach (Finkbeiner et al., 2008). The virus was detected from archived stool samples collected in 1999 from a 3-year-old boy with acute gastroenteritis in Melbourne, Australia, and later the virus was named as “MLB1.” The complete nucleotide sequence and genetic organization of MLB1 were characterized and demonstrated that MLB1 was considerably distinct from other known classic human astroviruses (HAstV1–HAstV8) and should be classified as a new astrovirus species in the genus Mamastrovirus (Fig. 4.3.1). One year later, the same research group reported the discovery of MLB2 in stool specimens collected from children with diarrhea in the United States and India (Finkbeiner et al., 2009a). In 2013, MLB3 astrovirus strains were identified in five stool specimens which had been collected in India. Within these, four of the MLB3 positive samples were obtained from patients with diarrhea, while 1 positive sample was from an asymptomatic case (Jiang et al., 2013).
The role of astroviruses of the MLB clade as causative agents of gastroenteritis has not been clearly established. Although most of the positive stool samples for this new clade were found in patients with diarrhea, a cohort study with a control population, carried out in Indian children, found no association of MLB1 with diarrhea (Holtz et al., 2011a). In addition, it was reported that MLB1 astrovirus infections were common in an American population since a greater than 60% seropositivity rate was found in children younger than 1-year old and reached 100% by adulthood (Holtz et al., 2014). However, the potential role of a systemic infection being related to febrile illness has been recently described in patient with MLB2 viremia (Holtz et al., 2011b).
After the discovery of the novel MLB astrovirus clade, these viruses have been reported in acute gastroenteritis pediatric patients in the USA, Nigeria, India, Japan, China, Hong Kong, Bhutan, Russia, Italy, Egypt, and Turkey (Bosch et al., 2014). In Japan, during 2013 and 2014, MLB astroviruses were found in 35 out of 330 (10.6%) fecal specimens collected from children with acute diarrhea. Among these, 2 types of MLB were identified (MLB1: n = 32 and MLB2: n = 3). It is interesting to note that the detection rate of MLB astroviruses was higher than that of classic human astroviruses (5.2%); the peak incidence was found in March and April (Khamrin et al., 2016). Since the first MLB astrovirus was identified in 2008, research into MLB astrovirus molecular biology, pathogenicity, and molecular epidemiology remained scarce.
4.1.3. VA Astroviruses
The VA astrovirus prototype strain was initially isolated during a sporadic outbreak of acute diarrhea in a child care center in Virginia, USA, in 2008. Using the pyrosequencing approach, the viral genome was found to be highly divergent from those of all previously known astroviruses, and initially named as “VA1” after the original place of viral discovery (Finkbeiner et al., 2009b). At the same time, another research group also reported the discovery of a novel astrovirus in fecal specimens collected from Nigeria, Pakistan, and Nepal, and the characterization of their genomes revealed that these viruses were phylogentically closely related to mink and ovine astroviruses. Hence, these viruses are provisionally named Human, Mink, and Ovine-like astrovirus (HMOAstV) and subdivided into the three types of HMOAstV-A, HMOAstV-B, and HMOAstV-C (Kapoor et al., 2009). Later, it was found that HMOAstV-C shared a close genetic relationship with VA1 astrovirus. Subsequently, VA2 and VA3 were identified in a cohort study of children with diarrhea in India and the USA (Finkbeiner et al., 2009a). Most recently, VA4 astrovirus strains were identified in two stool specimens collected from children with diarrhea in Nepal (Jiang et al., 2013). Taken together, VA astroviruses are classified into two phylogenetic clusters as VA1 and VA3, together with HMOAstV-B, HMOAstV-C, and are genetically more closely related with Mamastrovirus 9, while VA2, VA4, and HMOAstV-A are grouped together within Manastrovirus 8 (Fig. 4.3.1) (Finkbeiner et al., 2009a; Jiang et al., 2013; Kapoor et al., 2009).
Since the discovery of VA astrovirus was initially published in 2009, the role of VA astrovirus as a cause of gastroenteritis remains unclear (Finkbeiner et al., 2009a; Jiang et al., 2013; Kapoor et al., 2009). Although VA astrovirus infections are thought to be limited to the gastrointestinal tract, some studies have recently described the association of VA1 astrovirus with neurologic disorders in immunocompromised individuals. The VA1 astrovirus has been reported to cause encephalitis in a 15-year-old boy with agammaglobulinemia (Quan et al., 2010). The VA1 astrovirus can escape from the gastrointestinal tract into the circulatory system and is capable of invading the brain, with viral RNA being detected in brain biopsies and cerebrospinal fluid (CSF) (Naccache et al., 2015). In recent epidemiological studies, the virus has been detected at low prevalence rates in sporadic acute gastroenteritis cases in the USA, India, Pakistan, Nepal, Japan, China, and Nigeria (Bosch et al., 2014; Finkbeiner et al., 2009a; Jiang et al., 2013; Kapoor et al., 2009; Khamrin et al., 2016).
4.2. Animal Astroviruses
4.2.1. Ovine Astroviruses (OAstV)
The OAstV was first identified in 1977 in Scotland in lambs suffering from diarrhea shortly after the description of astroviruses in human. The OAstV was identified by electron microscopy (Snodgrass and Gray, 1977) and was purified and characterized structurally in 1981. Subsequently, the nucleotide sequences of the capsid region (ORF2) and the complete genome sequence were determined in the period from 2001 to 2003, and the virus was named OAstV-1 (Y15937) (Jonassen et al., 2003). Recently another OAstV strain was identified in samples taken from healthy domestic sheep in Hungary (Reuter et al., 2012). Sequence and phylogenetic analyses of ORF1b/ORF2/3′UTR of this isolate showed genetic divergence from OAstV-1, and it was therefore named OAstV-2. So far, only two OAstVs have been identified, one from lamb with diarrhea and the other one from a healthy sheep, suggesting that OAstV may not play a crucial causative role of diarrhea in sheep.
4.2.2. Bovine Astroviruses (BoAstV)
The first BoAstV was reported in England in 1978 in samples taken from calves with acute enteritis (Woode and Bridger, 1978). Several years later, another BoAstV, antigenically related to the first BoAstV, was isolated from a calf with diarrhea in Florida, USA. Although, these BoAstVs were isolated from calves with acute diarrhea, they could not induce diarrhea in calves infected experimentally. It was, therefore concluded that BoAstV is not associated with diarrhea in calves under natural conditions. Most recently, three bovine astroviruses associated with neurologic symptoms, so called BoAstV-Neuro S1 (KF233994), were detected in the brain tissue of cattle with histologically confirmed encephalomyelitis and ganglioneuritis, suggesting the virus to be a potential cause of neurologic disease in cattle (Li et al., 2013). Additionally, another bovine neurotropic astrovirus strain (BoAstV-CH13) was identified in cattle with nonsuppurative encephalitis in Switzerland (Bouzalas et al., 2014). Phylogenetically, BoAstV-CH13 was closely related to BoAstV-Neuro S1. These findings support the notion that infection with BoAstV is a common cause of encephalitis in cattle.
4.2.3. Porcine Astroviruses (PoAstV)
The PoAstV was first detected in piglets with diarrhea in association with calicivirus and rotavirus infections (Bridger, 1980). The virus was isolated later in 1990 in an established cell line derived from porcine embryonic kidney tissue. Molecular characterization of the capsid sequence (ORF2 gene) of this isolate was successfully carried out in 2001 and this PoAstV was named as PoAstV-1 (Jonassen et al., 2001). Later, PoAstVs were reported from several countries around the world, suggesting a wide geographical distribution. To date, five different PoAstV types, PoAstV-1–PoAstV-5, have been identified from different countries, including the Czech Republic, Columbia, Canada, the USA, China, and Hungary (De Benedictis et al., 2011). Although PoAstVs were described as a common virus detected in piglets with diarrhea, they were also detected in apparently asymptomatic healthy pigs (Luo et al., 2011). A prevalence of PoAstV infection in pigs of 62% has been reported recently from the United States (Mor et al., 2012). However, examination of the fecal contents of healthy slaughtered finisher pigs revealed a 79% positivity rate for PoAstV (Luo et al., 2011). Taken together, the clinical significance of PoAstV infection in pigs has not been completely elucidated.
4.2.4. Canine Astroviruses (CaAstV)
The CaAstV was first detected by electron microscopy in stool samples of beaglepuppies with gastroenteritis (Williams, 1980). The presumptive identification made by electron microscopy was confirmed by the genetic analysis of viral RNA extracted from clinical specimens. Recently, CaAstV has been propagated in MDCK cell cultures and virus growth induced a clear cytopathic effects (CPE) (Martella et al., 2011a). To date, CaAstVs have been reported from the USA, the UK, Australia, Italy, Germany, France, Brazil, China, Korea, and Japan (De Benedictis et al., 2011; Caddy and Goodfellow, 2015; Takano et al., 2015).
The prevalence of CaAstV infection associated with gastroenteritis in puppies was reported from Italy at 25%, France at 21%, the United Kingdom at 6%, China at 12%, Korea at 2%, and Japan at 10% (De Benedictis et al., 2011; Caddy and Goodfellow, 2015; Takano et al., 2015). It should be noted that the prevalence of CaAstV infection in puppies in Europe is relatively higher than that in Asia. CaAstV was also detected in asymptomatic puppies at an incidence of 9% (Martella et al., 2011a). The information relating to the clinical significance of CaAstV in dogs is limited and requires further investigation.
4.2.5. Feline Astroviruses (FeAstV)
The FeAstV was first detected using electron microscopy in the feces of domestic kittens with diarrhea (Hoshino et al., 1981). Further investigation demonstrated that FeAstV was commonly found in the stools of cats with and without diarrhea (Rice et al., 1993). So far, FeAstV in the feces of domestic cats has been reported from the USA, Australia, Germany, England, New Zealand, and Italy (De Benedictis et al., 2011). The clinical impact of FeAstV infection in cats seems to be low since kittens experimentally infected with FeAstV showed only pyrexia and mild diarrhea (Harbour et al., 1987).