Typing VP1 sequence is a suitable method for picornavirus classification (Oberste et al. 1999), but 3CD junction region has been also used for that purpose in AiV (Yamashita et al. 2000; Pham et al. 2007). A good correlation between both classifications has been reported for AiV genotypes (Ambert-Balay et al. 2008).
The first genetic differentiation of AiV genotypes was made by Yamashita et al. (2000). In that study the genetic relation among 17 AiV isolates was predicted by the comparison of 519 bases at the putative junction between the C terminus of 3C and the N terminus of 3D, and two groups were proposed: Group 1 or genotype A and group 2 or genotype B. Since then, 3 distinct genotype categories A, B and C were described (Ambert-Balay et al. 2008; Kitajima and Gerba 2015). Furthermore, several studies suggest some geographical distribution of AiV genotypes (Fig. 5).
Figure 5. Worldwide distribution of human AiV genotypes. Data from AiV related gastroenteritis outbreaks and environmental samples.
Among human population, the presence of genotype A has essentially been reported in Asian countries. Thus, AiV A was detected in samples connected with 12 out of 37 gastroenteritis outbreaks in Japan (Yamashita et al. 2000), most of them associated with oyster consumption, showing prevalences up to 81% (Table 1). In addition, AiV A was detected in stool samples from travelers with diarrhea returning from India, Nepal, Thailand, Indonesia, Singapore and Vietnam, reaching percentage values of almost 100% of genotyped samples (Yamashita and Sakae 2003). Further investigations also reported the same genotype in fecal samples negative for RV, AdV, NoV, Sapovirus and AsV from patients with acute gastroenteritis from Vietnam, Thailand and Bangladesh (Pham et al. 2007).
Country Outbreak Year Source Positive rate (%)a Genotype References Japan 1 1987 Oysters 55.0 A Yamashita et al. (2000) 2 1988 Oysters 71.4 A 3 Oysters 81.8 A 4 1989 Oysters 80.9 A 5 School meal 64.3 A 6 1990 Oysters 50.0 B 7 Oysters 54.5 A 8 1991 Oysters 50.0 A 9 1994 Oysters 14.3 A 10 1997 Oysters 62.5 A 11 1998 Oysters 50.0 A 12 Oysters 33.0 A Germany 1 2006 NSb NS A Oh et al. (2006) France 1 2006 Oysters 50.0 A Ambert-Balay et al. (2008) 2 2006 Oysters 50.0 A 3 Oysters 17.0 A 4 2007 Oysters 100.0 A 5 Oysters 33.0 A 6 2007 Seafoodc 6.0 A aNumbers of fecal specimens positive/Numbers tested.
bNS, not specified.
cShellfish species not specified.
Table 1. Features of the gastroenteritis outbreaks linked to AiV.
In Europe the presence of AiV A was reported for the first time in Germany, from stool samples of patients involved in a gastroenteritis outbreak (Oh et al. 2006). Afterwards the same genotype was found in France, in samples from children and adults involved in gastroenteritis outbreaks and hospitalized for acute illness (Ambert-Balay et al. 2008), in Finland from children under 5 years with gastroenteritis (Kaikkonen et al. 2010) and in a Hungarian sample from a 3 years old girl (Reuter et al. 2009). AiV A was also reported in elderly patients suffering from diarrhea, who were negative for AdV, RV, and calicivirus in Sweden (Jonsson et al. 2012) in Italy (Bergallo et al. 2017) and among different age groups from outpatients in Spain (Rivadulla et al. 2019). AiV A was also reported in Africa. It was detected in stool samples from inpatient and outpatient children collected in Monastir, Tunisia (Sdiri-Loulizi et al. 2009) and, more recently, in young children with watery diarrhea in Ethiopia (Aiemjoy et al. 2019).
AiV B was described at the same time as AiV A in samples from Japan but with less prevalence (about 16% of samples were genotyped as B) (Yamashita et al. 2000). However, places like China, Pakistan, Bangladesh or Malaysia has been reported AiV B from patients with gastroenteritis, in higher percentages than AiV A (reaching percentages of up 100%) (Pham et al. 2007; Yang et al. 2009; Li et al. 2017). On the other hand, in South Korea genotype prevalence is not clearly detected, since both genotypes were detected equally in stool samples from teenagers and adults (Han et al. 2014). In other places as Thailand, alternance of AiV A and AiV B as predominant genotypes along the time was observed (Yamashita et al. 2000; Saikruang et al. 2014; Chuchaona et al. 2017). In Europe, AiV B was also reported. In Finland and Spain AiV B was detected, but at lower prevalence than AiV A (Kaikkonen et al. 2010; Rivadulla et al. 2019). In Germany a genotype drift was detected, and AiV B seems to be more prevalent than in previous surveys (Drexler et al. 2011). AiV B was detected in stool samples from Brazilian children suffering diarrhea (Oh et al. 2006), and in North American children between 15 days and 5 years of age with symptoms of acute gastroenteritis (Chhabra et al. 2013). In Africa, AiV B was detected from one outpatient children in Nigeria (Japhet et al. 2018).
Only one survey reported AiV C from a stool sample of a child, hospitalized for gastroenteritis in France after a trip to Mali (Ambert-Balay 2008; Reuter et al. 2011). Further studies are needed to clarify the real impact of this genotype in human health.
Some investigations of in vivo evolution, based upon nt/aa changes through the complete genome, have been performed for porcine kobuvirus suggesting good adaptation of the virus-host relationship (Reuter et al. 2011). Such research have not been carried out for human AiV yet, but it would be very helpful to understand the virus-species and host-species spectrums.
Since AiV was discovered, various methods have been used to identify it (Table 2). Under electron microscopy, the viral particles have a distinct ultrastructure than other gastroenteritis pathogens as Sapovirus (SaV) or NoV, but there are not easy to distinguish from other small round viruses and could be wrongly be classified as an AsV (Yamashita et al. 1993). Although AiV have not shown cytopathic effect (CPE) in human cell lines as HeLa (Human cervix epitheloid carcinoma), HEL (Human Erythroleukemia Cell Line), RD (Human rhabdomyosarcoma Cell Line) cells or in newborn mice, AiV cause CPE on BSC-1 and Vero (both obtained from kidney of African green monkeys) cells (Yamashita et al. 1993). An enzyme-linked immunosorbent assay (ELISA) was also developed for the detection of AiV antigens in clinical samples using monoclonal antibodies (Yamashita et al. 1993).
Laboratory diagnostic method Advantages Disadvantages Electron microscopy Visualization of viral particles Labour and tedious. Useless for environmental samples Cell culture Variety of sensitive cell lines. Determination of infectivity. Quantitative (TCID50) Labour and tedious. Effect of inhibitors/contaminants ELISAa Sensitivity. Especificity Effect of inhibitors. Limited use for environmental samples LAMPb Sensitivity. Especificity. Rapidity. Isothermal conditions Detection of infective and non-infective particles. Effect of inhibitors RT-PCRc Sensitivity. Especificity. Rapidity Detection of infective and non-infective particles. Effect of inhibitors RT-nested PCR Sensitivity. Especificity. Valid for genotyping coupled with sequencing Detection of infective and non-infective particles. Effect of inhibitors RT-qPCRd Sensitivity. Especificity. Rapidity. Quantification Detection of infective and non-infective particles. Need of standard for quantification. Expensive Digital RT-PCR Sensitivity. Especificity. Rapidity. Absolute quantification Detection of infective and non-infective particles. Hard optimization. Expensive Pyrosequencing Sensitivity. Universal detection Complex sample processing and bioinformatic analysis. Expensive aELISA, enzyme-linked immunosorbent assay.
bLAMP, loop-mediated isothermal amplification.
cRT-PCR, reverse transcription-polymerase chain reaction.
dRT-qPCR, real time quantitative RT-PCR.
Table 2. Laboratory diagnostic methods for AiV.
Reverse transcription-polymerase chain reaction (RT-PCR) is a widely employed method for AiV research. It is a sensitive method applicable for further genetic analysis as genotyping (Yamashita et al. 2000). As mentioned before, the 3CD junction region and VP1 region are suitable for differentiation of AiV genotypes. Nonetheless, no phylogenetic evidence of recombination has been reported between AiV genotypes A and B, suggesting that both genotyping methods are reliable (Lukashev et al. 2012; Kitajima and Gerba 2015). Nested PCR targeting the 3C, VP1, and VP3 regions, as well as multiplex semi-nested PCR on VP0-VP3 region were also developed for genotyping (Oh et al. 2006; Lodder et al. 2013).
Recently, Oshiki et al. (2018) reported a high-throughput detection and genotyping tool for RNA virus, like AiV, using a microfluidic device and next-generation sequencer. In this study the investigators reported detection limits ranging from 100 to 103 copies/μL in cDNA sample, corresponding to 101–104 copies/mL-sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oyster. Simultaneous detection and genotyping techniques are powerful tools for source tracking of human pathogenic viruses.
A loop-mediated isothermal amplification procedure was developed by Lee and coworkers (Lee et al. 2019), for rapid and specific detection of AiV from water samples. The whole protocol can be performed in 2–8 h showing equivalent AiV detection than conventional PCR.
Although conventional culture methods like 50% Tissue culture Infective Dose (TCID50) are used to quantify AiV, methodological advances in molecular biology lead to the development of better technologies. Reverse transcription- quantitative PCR (RT-qPCR) is nowadays the most employed method for AiV detection and quantification. The highly conserved 5'-UTR sequence is a common RT-qPCR target for picornavirus detection (Drexler et al. 2011; Nielsen et al. 2013). However, a RT-qPCR that amplifies the VP0 region, and could quantify and differentiate between genotypes A and B, was developed for determination of viral RNA load in clinical and environmental samples (Kitajima et al. 2013). This RT-qPCR consists in two amplifications: one that uses a universal primer pair that could amplify both genotypes and a universal probe to detect AiV. The second one uses the same primer pair but two different genotype-specific probes. Both methods, VP0 qPCR and 5'-UTR qPCR showed similar efficiency for AiV detection, with the advantage that VP0 qPCR is able to quantify and also differentiate the AiV genotypes.
Although, these techniques have been helpful to clarify the transmission sources of AiV among population, during the recent years, new detection technologies have been developed. Digital RT-PCR (RT-dPCR) is a precise endpoint-sensitive absolute quantification approach, capable of determine the number of target copies without a standard curve. As an example, AiV and eighteen enteric viruses more were targeted with this method and compared with RT-qPCR (Coudray-Meunier et al. 2016), the conclusions were that the limit of AiV detection for RT-dPCR assay was lower (7.8×102 genome copies/μL) than the limit of detection obtained with conventional RT-qPCR (1.0×103 genome copies/μL). This new technology presents many advantages and possibilities for detection of enteric pathogens in environmental and clinical samples. The RT-dPCR divides each reaction mix across thousands of individual PCR reactions, making this method more tolerant to inhibitory substances and also reducing the difficulty of virus quantification (Pinheiro et al. 2012; Rački et al. 2014).
AiV was identified in water in 2010 (Alcalá et al. 2010). Since then, AiV was reported in high percentages in wastewater samples around the world (Table 3). Current treatments applied in wastewater treatment plants (WWTPs), cannot guarantee a total removal of viral pathogens that are continuously discharged to the environment (da Silva et al. 2007).
Countrya Positive Samples Sample type Genotype Copies/L References South Africa 10/12 Raw sewage NS NSb Onosi et al. (2019) Tunisia 10/125 Raw sewage A NS Sdiri-Loulizi et al. (2010) 4/125 Treated sewage A NS 51/102 Raw sewage B NS Ibrahim et al. (2017) US 24/24 Raw sewage A, B 1.2×104–4.0×106 Kitajima et al.(2014, 2018) 24/24 Treated sewage A, B 2.0×103–4.0×105 1/1 Raw sewage NS NS Cantalupo et al. (2011) Iran 7/10 Raw sewage NS 2.1×104–1.9×106 Azhdar et al. (2019) 7/12 Treated sewage NS 4.2×103–6.7×105 Japan 137/207 Raw sewage A NS Yamashita et al. (2014) 12/12 Raw sewage A, B NS Kitajima et al. (2011) 11/12 Treated sewage A NS 12/12 Raw sewage A 1.4×105–2.2×107 Kitajima et al. (2013) 11/12 Treated sewage A Up to 1.8×104 11/12 Raw sewage NS NS Thongprachum et al. (2018) Nepal 1/1 Raw sewage NS NS Ng et al. (2012) 1/1 Raw sewage B NS Haramoto and Kitajima (2017) Thailand 1/1 Raw sewage NS NS Ng et al. (2012) France 61/100 Treated sewage NS Up to 103 Prevost et al. (2015) Italy 6/48 Raw sewage B NS Di Martino et al. (2013) Netherlands 16/16 Raw sewage A, B NS Lodder et al. (2013) Spain 1/1 Raw sewage NS NS Cantalupo et al. (2011) aCountries were ordered by continent and alphabetically within each continent.
bNS, not specified.
Table 3. Worldwide detection of AiV in wastewater.
In Japan AiV was detected in high percentages (from 66.2% to 100%) in raw sewage samples, with viral concentrations ranging from 1.4×105 to 2.2×107 copies/L (Yamashita et al. 2014; Kitajima et al. 2011, 2013). Samples of treated sewage also analyzed showed an AiV prevalence of 91.7% (Kitajima et al. 2011). AiV detection in sewage showed no seasonality, being detected throughout the year (Thongprachum et al. 2018). In other Asian countries, as Nepal, Thailand or Iran, AiV was also reported from untreated sewage samples (Ng et al. 2012; Haramoto and Kitajima 2017; Azhdar et al. 2019). These studies supported the idea of use AiV as a human faecal pollution indicator, due its stability in wastewater and his lower removal percentages during wastewater treatments.
Other surveys detected AiV in Africa, America and Europe. In Africa, AiV was detected in Tunisia but at low prevalences. Thus, Sdiri-Loulizi et al. (2010) detected AiV in 10 out of 125 (8%) samples of influent water samples and 4 out of 125 (3.2%) treated sewage samples. On the other hand, Ibrahim et al. (2017) reported the virus in 51 out of 102 (50%) samples. Moreover, a recent study carried out in South Africa detected AiV in 10 out of 12 pooled sewage samples (Onosi et al. 2019).
In USA, AiV was detected in a sample of untreated wastewater collected from Pennsylvania (Cantalupo et al. 2011). Furthermore, another study carried out in two WWTPs of southern Arizona, detected AiV in all influent and effluent samples, with viral levels of 1.2×104 to 4.0×106 copies/L in influent samples and 2.0×103–4.0×105 copies/L in effluent samples (Kitajima et al. 2014). A clear predominance of AiV B was revelaed in these positive samples (Kitajima et al. 2018).
Finally, in Europe the percentages of detection vary. AiV was observed in sewage samples from France, the Netherlands and Spain with detection rates between 61 and 100% (Cantalupo et al. 2011; Lodder et al. 2013; Prevost et al. 2015). However, other analysis of untreated influent sewage samples collected from four WWTPs in Italy detected AiV in only 12.5% of the samples (Di Martino et al. 2013).
Surface water is infiltrated via spreading basins into aquifers and wells but, due to their small size and their survival capacity, viral pathogens like AiV are not totally removed during this natural filtration (Weiss 2005; Sharma and Amy 2010). Therefore, river water and groundwater are also possible reservoirs for AiV (Table 4).
Countrya Positive Samples Sample type Genotype Copies/L References US 1/2 Reclaimed water NSb NS Rosario et al. (2009) 7/12 Ground waterb NS 1.0×102–1.5×104 Betancourt et al. (2014) Venezuela 5/11 River water B NS Alcalá et al. (2010) Iran 15/28 River water NS 3.4×102–5.9×106 Azhdar et al. (2019) Japan 36/60 River water A + B NS Kitajima et al. (2011) 29/29 River water NS 8.6×102–2.0×104 Hata et al. (2014) 20/52 Surface water NS Up to 104 Hata et al. (2018) Nepal 14/14 River water B 1.2×106–8.3×108 Haramoto and Kitajima (2017) 11/37 Ground water B 5.6×104–2.0×106 1/1 Tap water B 109 France 20/175 River water NS Up to 102 Prevost et al. (2015) Netherlands 12/14 River water A + B NS Lodder et al. (2013) aCountries were ordered by continent and alphabetically within each continent.
bNS, not specified.
Table 4. Worldwide detection of AiV in river, surface and ground waters.
Although AiV had been previously detected in tap water in USA (Rosario et al. 2009), the first study conducted to determine the occurrence and circulation of AiV in river water was carried out in Venezuela. In this study, AiV was detected in 5 out of 11 samples (45%) (Alcalá et al. 2010). Other survey carried out in Japan for a longer period detected AiV in 36 out of 60 samples (60%), demonstrating a higher detection frequency for AiV than for other enteric virus, like NoV or SaV is the same set of river samples (Kitajima et al. 2011). Hata et al. (2014), investigating the effects of rainfall events and water quality on viral occurrence, detected AiV in all the tested samples with relatively higher frequency of detection and concentration (ranging from 1.2×106 to 8.3×108 copies/L) than other enteric viruses. More recently, the same authors detected AiV in 20 out of 50 surface water samples in Japan (Hata et al. 2018).
In Nepal, AiV was detected from river water, groundwater, tap water in a house supplied by tanker water, and from a sewage pipe (Haramoto and Kitajima, 2017). In this study, differences in AiV detection were observed, and a high prevalence of AiV B was reported. The frequence of AiV detection was significantly higher in shallow dug wells, where AiV was found in 10 out of 22 samples (45%) than in shallow tube wells, in which AiV was found in 1 out of 15 samples (7%). In accordance with the study, this could be happened due to the vulnerable structure of dug wells, which are usually made of brick or stone, than tube wells. On the other hand, a recent study from Iran reported the detection of AiV in 50% of the river water samples analyzed (Azhdar et al. 2019).
The first study for AiV detection in groundwater was performed in USA for the assessment of the occurrence and elimination of virus at a full-scale managed aquifer recharge system (Betancourt et al. 2014). In this study, the concentration of AiV was up to 1.52×104 copies/L. Recent environmental studies have demonstrated a high prevalence of AiV in different types of water samples, such as river and groundwater (Kitajima and Gerba 2015).
There are also some surveys that reported AiV from river samples in Europe. In France, 20 out of 175 river water samples were positive for AiV with up to 102 copies/L concentration levels (Prevost et al. 2015). In the Netherlands, AiV was also detected in 12 out of 14 river water samples (Lodder et al. 2013). The importance of that pathogen in this kind of samples is not well understood and more investigation will be needed to evaluate the real impact of AiV. Recently, Bonadonna et al. (2019) reported for the first time the presence and abundance of AiV in marine bathing waters in the Adriatic and Tyrrhenian Seas (Italy).
Bivalve molluscs are associated with viral foodborne disease (Vossen 2001) as they obtain their food filtering small particles suspended in water. Often in these processes, molluscs concentrate and retain pathogens including enteric viruses (Romalde et al. 1994). These viruses are underestimated in molluscan safety controls that are based only on bacterial indicators, becoming this kind of food as a vector for enteric viruses transmission (Polo et al. 2015).
From its first detection, AiV has been suggested as an important etiological agent of gastroenteritis especially in outbreaks associated with contaminated seafood (Table 5) (Yamashita et al. 1991). In Japan, a one-year study carried out between 2005 and 2006, reported AiV A from clam samples, with a prevalence of 73% (Hansman et al. 2008).
Countrya Positive Samples Sample type Genotype Copies/L Reference South Africa 8/12 Mussels NSb NS Onosi et al. (2019) Tunisia 4/60 Shellfish (NS) A NS Sdiri-Loulizi et al. (2010) Japan 19/26 Clams A NS Hansman et al. (2008) France 6/66 Oysters NS NS Le Guyader et al. (2008) Italy 13/108 Mussels NS Up to 102 Fusco et al. (2017) 3/170 Mussels, Oysters, Clams A, B NS Terio et al. (2018) Spain 15/249 Mussels, Clams, Cockles NS NQc— 6.9×103 Rivadulla et al. (2017) aCountries were ordered by continent and alphabetically within each continent.
bNS, not specified.
cNQ, non quantifiable (under the limit of quantification of the method).
Table 5. Worldwide detection of AiV in shellfish.
Other studies reported AiV from shellfish samples worldwide. In Tunisia, Sdiri-Loulizi et al. (2010) reported an AiV prevalence of 4% in shellfish. Aditionally, the phylogenetic analysis revealed several clusters that occurred sequentially in time, pointing out some parallelism in the temporal shifts among environmental and human strains. On the other hand, Onosi et al. (2019) observed prevalence up to 66.6% in mussels from South Africa.
In France, other study detected AiV in oysters that were linked to a gastroenteritis outbreak and the AiV sequences obtained where similar to those from stool samples analyzed in parallel (Le Guyader et al. 2008). More recently, AiV has been detected from shellfish in Spain and Italy (Fusco et al. 2017; Rivadulla et al. 2017; Terio et al. 2018), with prevalence ranging from 1.7 to 12%.