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Citation Information : Postępy Mikrobiologii - Advancements of Microbiology. Volume 58, Issue 3, Pages 301-315, DOI: https://doi.org/10.21307/PM-2019.58.3.301
License : (CC-BY-NC-ND 4.0)
Received Date : May-2019 / Accepted: June-2019 / Published Online: 05-October-2019
Parechoviruses are small, non-enveloped, icosahedral-shaped capsid viruses belonging to the
1. Introduction. 2. Classification, structure and replication. 3. Cellular receptors and HPeV variability. 4. Course of infection 5. HPeV types in the world 6. Diagnosis 7. Pathogenesis 8. SummaryTranslated
1. Wprowadzenie. 2. Klasyfikacja, budowa i replikacja. 3. Receptory komórkowe i zmienność HPeV. 4. Przebieg zakażenia 5. Typy HPeV na świecie 6. Diagnostyka 7. Patogeneza 8. Podsumowanie
In the United States, in 1956, viruses from the faeces of infants with diarrhoea were isolated, they proliferated in the monkey kidney cell cultures; however, did not concurrently infect monkeys or suckling mice. It was also noted that the viruses were neutralised by the human gamma globulin . Based on these properties, they were classified as enteroviruses (EV), and more specifically as the ECHO (Enteric Cytopathogenic Human Orphan) viruses and were assigned consecutive numbers 22 and 23 (E22, E23). The first strains, named Harris and Williamson (E22 and E23, respectively), differed significantly from other enteroviruses in their growth in the cell cultures, despite meeting the criteria for belonging to the ECHO viruses . Molecular studies have also showed significant homology of both strains, while at the same time having little similarity to the sequences of known enteroviruses. In 1996, detailed studies on the biological properties and recognition of the nucleotide sequence of the viral genome led to the creation of a new genus in the Picornaviridae family – Parechovirus, which initially included two types, HPeV-1 and HPeV-2 [36, 44, 68, 83].
Infections caused by parechoviruses are common in people all over the world and, like enterovirus infections, are mostly asymptomatic. If the infection occurs, it usually manifests itself through mild symptoms in the digestive and respiratory systems and is almost exclusively limited to the population of young children of up to 5 years of age. Human parechoviruses (HPeV) are also etiological agents of severe diseases, which require hospitalisation, especially in the cases of the central nervous system (CNS) infection. There is also an increasing amount of information on the possible participation of parechoviruses in the development of many different diseases, not only neurological ones. In recent years, numerous independent research groups have reported that parechoviruses are the second factor, after enteroviruses, causing sepsis in infants and viral meningitis in children.
In connection with these findings, there is a growing need to introduce rapid diagnosis differentiating enterovirus and parechovirus infections, especially in relation to severe CNS infection in the youngest patients. It also seems necessary to develop strategies to prevent the spread of the HPeV infections and to expand the therapeutic possibilities. Intensive virological and epidemiological studies will allow this to take place. Similarly to what is done in the case of diseases with enteroviral etiology, certain countries have established parechovirus surveillance. The HPeV infection monitoring program operates, among others, in the USA, the UK, the Netherlands [1, 33, 46], and in Australia where HPeV was included in the monitoring system used for new pathogens causing severe diseases .
The aim of this study is to present parechoviruses as pathogens of growing clinical significance and to assess the state of knowledge about the key issues from the diagnostics, epidemiology and treatment points of view.
The Parechovirus genus belongs to the Picornaviridae family and includes four virus species (Parechovirus A-D), which are mammalian pathogens. The human parechoviruses belong exclusively to the A species (Parechovirus A), which currently consists of 19 types, from HPeV-1 to HPeV-19 .
They are small (30 nm), non-enveloped, icosahedral-shaped viruses, whose genetic material is a positive-sense single-stranded RNA (+ssRNA), composed of 7.3 thousand nucleotides. A single open reading frame encodes 3 structural proteins (P1 region) and 7 non-structural proteins (P2 and P3 regions) (Fig. 1). Untranslated regions (UTR) involved in the replication process are found at the ends of the RNA strands. The viral VPg protein is covalently bound to the end of the 5’ strand, and the 3’ end is terminated by the poly(A) sequence. In the 5’UTR region, an IRES (Internal Ribosomal Entry Site) structure is located, which initiates the translation of the viral polyprotein in a manner independent of the cap structure. Similarly to cardio-, afto- and hepatoviruses, parechoviruses have a type II IRES, whereas type I is present in entero- and rhinoviruses. The characteristically folded 5’UTR region also participates in the replication of the viral RNA, and structures such as SLS (stem – loop structure), including SL-A and SL-B, as well as the structure of the pk-C pseudoknot type, are involved in this process. The 3’UTR end, with a single SLS structure, also participates in the viral replication process , similarly to another hairpin structure – CRE (cis-acting replication element), located in the region coding the VP0 capsid protein [4, 36, 75, 83].
Viral RNA functions as mRNA; therefore, translation is possible following the entry of the virus into the cell. The translation is probably initiated by the AUG codon, similarly to cardio- and aftoviruses [36, 83]. The resulting polyprotein is proteolytically cleaved by the 3C viral protease, whereas in contrast to other picornaviruses, the 2A protein has no proteolytic properties [36, 83]. In the first stage, three precursor peptides are formed (P1, P2, P3), the P1 peptide is subsequently cleaved into three structural proteins: VP0, VP1 and VP3, and P2 and P3 peptides are cut into seven non-structural proteins (Fig. 1), which include: RNA-dependent RNA polymerase 3Dpol (RdRp), VPg protein (3B), NTPase/ helicase (2C), 3Cpro protease [36, 44, 75, 83].
The spatial structure of the parechovirus capsid is similar to other viruses in the Picornaviridae family [36, 42] . The VP1, VP3 and VP0 structural proteins form a protomer – a basic building unit of the capsid, and the entire capsid consists of 60 protomers assembled into 12 pentamers. During maturation of the virion, VP0 protein is not cleaved into VP2 and VP4, as is the case with enteroviruses; therefore, there is no VP4 internal protein in the capsid of parechoviruses (Fig. 2). Also the depression in the protomer, named canyon, is not as deep as in enteroviruses [44, 83, 89]. Comparably to other picornaviruses, parechoviruses’ capsid lacks envelope, which determines the stability of the virus inside the host organism and in the environment. The capsid proteins are responsible for binding to the receptor on the surface of susceptible cells. The most immunogenic parechovirus protein is VP1 and its nucleotide sequence is the most variable .
Viral RNA replication involves viral proteins, which form a replication complex associated with the endo-plasmic reticulum (ER) membranes: 2A, 2C, 3A and 3B, with the participation of the SLS and CRE structures, and the viral 3D polymerase. The viral RNA replication process starts with the uridylation of the VPg protein by the viral polymerase on the poly(A) matrix in the presence of the CRE structure, resulting in the formation of VPgpU (pU). This in turn initiates the synthesis of the negative stranded RNA, which is complementary to the genomic RNA, resulting in double-stranded replication forms (dsRNA). The (–) ssRNA strand becomes a template strand, on which numerous copies of genomic RNA are produced [10, 11, 42, 75, 77, 83, 89].
Polyprotein is synthesised directly on the genomic RNA matrix, and the viral proteins are produced by its cleavage. By inhibiting cellular processes, these proteins lead to the lysis of the cell and the release of the resulting virus progeny. The synthesis of the host proteins is not switched off in the cells infected with parechoviruses, as it is the case with enteroviruses. The mechanism of maturation and assembly of virions is not fully understood [10, 42, 44, 83].
Following the binding of the virus to the receptor, structural changes of the capsid and the cell membrane occur and, as a result, the genetic material of the virus is released into the cytoplasm. The form of tropism of different types of HPeV depends on the receptor that a given type uses to enter the cell. Some types of parenchoviruses (HPeV-1, 2, 4, 5, 6) contain the so-called RGD motif composed of amino acids: arginine, glycine and aspartate at the C-terminus of the VP1 protein. This structure is also responsible for the binding of some enteroviruses (Coxsackie A-9 and E9) to the cellular surface receptors known as integrins [4, 27, 36, 44, 83]. The remaining types of parechoviruses do not have this motif and are thought to bind to another cellular receptor (RGD – independent) or to interact with integrins in another way [3, 31, 45, 49, 54, 72]. HPeV-1, 4 and 5 strains were found, which were exceptionally missing the RGD motif [9, 65].
Parechoviruses are characterised by high genetic variability, which is manifested by the numerous genotypes. Based on the analysis of the gene encoding the VP1 structural protein, currently 19 types of human parechovirus are classified . It has been assumed that sequences belonging to the same genotype are characterised by a nucleotide similarity of min. 77% and amino acids similarity of min. 87%, while for different types it is below 73% and 81%, respectively . The HPeV genome is constantly changing, the rate of the parechovirus mutation is estimated at 2.2–2.8 × 10-3 substitutions per site per year . It has been reported that the rate of the HPeV-3 mutation in the most variable region coding VP3/VP1 is estimated at 2.83 × 10-3 substitutions per site per year, which means one nucleotide change per 400 per year, and the HPeV-1 mutation rate is at least twice as large .
The reasons for the HPeV variability include numerous mutations, selection pressure of the host immune system and frequent recombination. Recombination occurs when a cell is simultaneously infected by two closely related viruses – belonging to the same type (intratype recombination) or two different types of HPeV (intertype recombination). Recombination contributes to the creation of new variants of the virus, and thus to the increase in the pace of evolution, it leads to the acquisition of genetic differences conditioning the change in virulence. European HPeV-1 isolates recombine on average every 1–3 years . HPeV-3 rarely comes into contact with other parechoviruses in the same cell, hence the limited recombination and lower genetic diversity in comparison to HPeV-1 [10, 40, 98].
The evidence for the participation of recombination in the HPeV evolution process may be the sequence similarity within the 5’UTR region with the Cardiovirus and Aphthovirus . Two regions are indicated in HPeV: 5’UTR/P1 and P1/P2, in which recombination occurs frequently . Also based on the analysis of the VP3/VP1 and 3Dpol regions, the phenomenon of recombination has been observed in many HPeV types [4, 5, 6]. Sequence similarity was also found in the P2 and P3 regions in HPeV-1 and HPeV-7 , as well as in HPeV-3 and HPeV-4 . The possibility of recombination between HPeV-4 and types 1, 2, 3 and 15 [8, 65], as well as between HPeV-1 and types 3 and 6 has been demonstrated . HPeV-1 strains with sequences similar to types 6 and 7 in the non-coding region  and strain HPeV-5, which is a recombinant resulting from recombination of three types 1, 3 and 4 , were isolated. The highest recombination rate was demonstrated in the HPeV-1B strains [22, 23].
Canadian studies on HPeV-1 showed that the isolates from 1985–2004 differed significantly from the prototype sequence (available at http://www.picornaviridae.com) . A large similarity to the Harris prototype strain (1956) were showed by the strains isolated in Bolivia in 2002–2003 and in India in 2006–2010 [65, 70], as well as most strains detected in China in the years 2012–2013 [21, 22]. Based on the VP1/VP3 region sequence analysis, genotype 1 was divided into two clades 1A (Harris-like), whose representative is the prototype strain and 1B . It appeared that clad 1B belongs to the majority of presently isolated HPeV-1, including the Canadian isolates [8, 9, 20, 23, 98].
HPeV-2 is closely related to type 1. It has 87.9% amino acid identity with HPeV-1; the highest values of nucleotide sequence divergence were observed in the regions of the capsid proteins, and the highest identity in the 3B region . For the type 3 prototype sequence, nucleotide and predicted amino acid similarities were 77.6% and 86.8% with type 1 and 77.2% and 84.7% with type 2, respectively . Type 4 showed the highest similarity of the nucleotide sequence with type 2 (72.2%) , and type 5 with type 4 . Type 6 is the most similar to type 1, while the similarities of the nucleotide and amino acid sequences to other known HPeV types are 76.7–79.5% and 85.9–90.7%, respectively . The type 7 prototype strain is most related to type 3 and its similarity to the other prototype strains was determined at 75.6–80.8% for the nucleotide sequence and 84.8–89.1% for the amino acid sequence . Type 8 has the highest amino acid sequence identity with type 7 (76.3%) and its close relationship to types 1 and 6 has also been shown . The type 10 prototype sequence was most closely related to type 3, showing 69.1% homology in the nucleotide sequence and 82.8% in the amino acid sequence . Type 11 showed the highest nucleotide and amino acid similarity with type 10 (64.7% and 73.6%, respectively) . Analyses revealed close identity of HPeV-12 prototype sequence with type 10 (80% in the nucleotide sequence and 80.5% in the amino acid sequence) , while for type 17, the similarity of the nucleotide sequences with the remaining prototype strains was determined at 79–82% . HPeV-17 from Thailand showed the highest similarity with type 3 (74–78%) .
Human is the host for human parechoviruses; however, there is a high probability that some mammals may act as reservoirs for these pathogens. The support for this theory comes from the detection of HPeV in the faeces of macaques (Macaca sp.) in China  and domestic pigs (Sus scrofa domestica) in Bolivia .
Parechoviruses spread mainly through the faecaloral route, less frequently respiratory routes [42, 43, 75]. In utero transmission of HPeV is also suspected, which is evidenced by the occurrence of infections in newborns in the first two days of life . Viruses enter the host organism through the mouth and pass into the lymphatic vessels of the upper respiratory tract and/or the stomach, and then into the intestines. They multiply in the epithelial cells of the oropharyngeal mucosa, as well as in the intestinal mucosa in the so-called Peyer’s patches [25, 27]. The infection at this stage is often asymptomatic [42, 45, 75, 79]. Viremia occur in some of the infected people and, as a result, viruses multiply in numerous organs (liver, heart, lungs), penetrate the skin, mucous membranes and further lymph nodes, and sometimes into the nervous system, which is associated with the systemic symptoms [27, 43]. To reach the central nervous system, the virus uses the retrograde axonal transport or crosses the blood-brain barrier . A hypothesis exists, according to which the parechoviral CNS inflammation is not a result of a direct infection, but is a consequence of the infection of meningeal vascular smooth muscle cells, resulting in hemorrhage and disturbances in vascular flow in the brain .
Parechovirus infections are usually manifested by mild symptoms in the digestive and respiratory systems. The most frequently mentioned symptoms include: diarrhea, fever, vomiting, wheezing and tachypnea, apnea, cough and coryza [42, 72, 75, 83, 95, 97]. Studies done in Sweden demonstrated that the percentage of intestinal symptoms was twice as high as the percentage of respiratory symptoms in the course of the parechovirus infection [40, 41].
Diseases of the nervous system are more severe in their course, in which case patients absolutely require hospitalisation . In the case of the CNS infections, fever, loss of appetite and irritability (“red, hot and angry”) , neurological symptoms (seizures, paralysis, generalized hypotonia, decreased tendon reflexes, peripheral neuropathy), respiratory and gastrointestinal symptoms, rash [14, 32, 43, 48, 75, 93] and a syndrome similar to sepsis, the so-called “Sepsis-like viral illness”  are most commonly observed. CNS infection of the parechovirus etiology is associated with many disease entities (Tab. I): meningitis [13, 34, 51, 73, 93, 95], encephalitis [18, 24, 29, 51, 59, 61, 74], encephalomyelitis , acute flaccid paralysis (AFP) [3, 34, 54, 65, 81, 93], transient paralysis , neonatal sepsis [8, 14, 16, 18, 47, 53, 73, 84, 92, 95], Guillain-Barré syndrome [55, 71] and Reye’s syndrome [43, 79, 93]. A case of a five-year-old girl from Germany is known for the occurence of acute disseminated encephalomyelitis (ADEM) in the two weeks after the parechoviral respiratory tract infection, as until recently enteroviruses were considered a potential factor for this disease . Severe cases of neurological infections with parechoviruses may end in death [35, 78].
Multifocal changes occurring in the CNS as a result of parechovirus infection are polymorphous and involve mainly the white matter of the brain, where the periventricular and subcortical leukomalacia, as well as gliosis and the formation of cavities in the neural tissue may occur. The consequences of the past disease often include: epilepsy, hypotonia, visual impairment, cerebral palsy, as well as developmental delay [10, 32, 43, 75, 79]. The parenchoviral infection of the CNS manifests itself in a similar manner to the bacterial one; however, without causing an increase in the parameters of the inflammation in the cerebrospinal fluid (CSF) and blood .
Sepsis in the course of the HPeV infection (“sepsis-like viral illness”) was defined by Wolthers et al. as a fever or hypothermia with symptoms of circulatory or respiratory disorders in the form of tachycardia and bradycardia, as well as low blood pressure and decreased saturation; however, it can also proceed with a broad spectrum of respiratory, intestinal (jaundice) and life-threatening neurological symptoms [10, 43, 97]. In addition, sepsis of HPeV etiology may be accompanied by a maculo-papular or erythematous rash on the hands and feet [32, 92, 97].
The following diseases are associated with parechovirus infections (Tab. I): necrotizing enterocolitis [12, 56], pneumonia , otitis media [14, 30, 87], myocarditis and cardiomyopathies [58, 59, 96], conjunctivitis and uveitis [14, 28], haemolytic-uremic syndrome (HUS) , lymphadenitis [7, 21, 93], myositis and myalgia [60, 82, 93], hemophagocytic lymphohistiocytosis , hemorrhagic hepatitis syndrome (hepatitis with coagulopathy) , TORCH syndrome (congenital infection syndrome, TORCH is an acronym meaning: T – toxoplasmosis, O – others, R – rubella, C – cytomegaly, H – Herpes) . There have been reports of sudden infant death syndrome associated with the HPeV infection . Kolehmainen did not exclude HPeV as a risk factor for the development of gender-related type 1 diabetes .
It has been noticed that certain types of the virus preferentially cause more of the certain disease entities. Type 1 is associated with intestinal infections more than type 3 [90, 95]. CNS diseases are mainly caused by types 1, 3 and 6 [48, 75], but in the cases of paralysis, the presence of types 2, 4, 5, 7, 9 and 12 was also detected. The types 1, 5 and 6 are correlated with Reye’s syndrome [43, 79, 93]. In the respiratory infections, the most frequently detected types are HPeV-1 and HPeV-6 [23, 41]. Newly discovered types usually cause mild respiratory and gastrointestinal symptoms .
Symptoms of the parechovirus infections are nonspecific; therefore, they are often assigned the enteroviral etiology due to the similarity of the clinical manifestations [52, 68, 75]. The distinction is possible only after the use of diagnostic methods . According to the Dutch research, in comparison to enteroviruses, parechoviruses are associated with a lower incidence of meningitis and a higher incidence of intestinal and respiratory infections .
Parechoviruses multiply in the host cells even after the symptoms disappear. They are isolated from swabs from the upper respiratory tract up to 3 weeks after the onset of symptoms  and from faeces for a period of several weeks to 5 months (average 51 days) [32, 84, 86]. The viruses achieve high titres in the faeces , and the long shedding translates into a wide HPeV transmission. They enter the environment through the waste and they have repeatedly been detected in surface waters  and in wastewater .
Infections caused by HPeV are noted worldwide, but the occurrence of the individual types is limited to certain geographical regions and time intervals (Tab. II).
Infections caused by type 1 occur all over the world, they are most often asymptomatic or in the form of mild gastrointestinal symptoms and respiratory diseases [8, 41, 95, 98] accompanied by influenza-like illness . This type is responsible for the development of pneumonia and bronchitis (China, Japan) [93, 98]. It has been isolated from the cases of gastrointestinal and respiratory infections in Japan , South Korea , the UK  and Croatia . Moreover, it has been detected in patients with gastrointestinal infections in many distant places of the world (the Netherlands, Hungary, Germany, Thailand, China, India, Brazil, Sri Lanka, Ghana) [5, 8, 21–23, 25, 31, 37, 49, 70, 72, 74, 76, 95], including the necrotising enterocolitis (Israel, Croatia) [12, 56]. It has also been detected in the acute infections of the central nervous system e.g., in encephalitis and meningitis (Finland, Hungary, Jamaica) [34, 51, 74], encephalomyelitis (Persian Gulf) , acute flaccid paralysis (Pakistan, Bolivia, Jamaica) [34, 54, 65], epilepsy (Germany)  and sepsis (the Netherlands, France) [8, 47]. Some infections were associated with the Guillain-Barré syndrome , Reye’s syndrome , uveitis , myocarditis and cardiomyopathy [58, 96], otitis media , HUS syndrome .
Type 2 is rarely detected, and is most often associated with mild gastrointestinal tract and respiratory system infections [6, 68, 90]. It was identified in people with AFP in Bolivia , uveitis in the Netherlands , it was also isolated from the cases of diarrhea and gastroenteritis in Ghana, India and Thailand [23, 37, 70], as well as from healthy children in Norway .
Type 3 is a commonly detected genotype globally and regarded second only to HPeV1 [10, 46]. It was first isolated in Japan in 1999 from the faeces of a one-year-old child with transient paralysis . It was detected in Canada in newborns with sepsis in 2001 . It was also detected in Europe (Bulgaria, Denmark, the Netherlands, the UK, France, Italy, Spain, Austria, Germany) and other parts of the world (USA, Australia, Taiwan, Japan) in patients with sepsis and CNS infections [1, 8, 9, 13, 14, 16, 18, 19, 35, 40, 46–48, 53, 61, 73, 84, 90–93, 95]. Type 3 has been associated with cases of AFP in Pakistan  and Bolivia . It was also isolated from patients with gastrointestinal infections (Sri Lanka, China, India, New Zealand, Thailand) [21–23, 62, 70, 72] and respiratory tract infections (Taiwan, the UK, Japan) [19, 41, 93]. It is considered as a risk factor for the sudden infant death syndrome (SIDS) . In Japan, the correlation between the type 3 infection and hemophagocytic lymphohistiocytosis (HLH)  was observed, and in the USA with hepatitis with coagulopathy . HPeV-3 has been detected in patients with otitis media and conjunctivitis , uveitis (the Netherlands) , myositis, myalgia and myocarditis (USA, Japan) [59, 60, 82, 93], but also in healthy children in Norway , as well as patients with fever in Spain  and in Sweden .
The type 4 parechovirus has been circulating since the seventies of the last century ; however, it was only described in 2006 in the Netherlands after its detection in the faeces of a neonate with fever . In the case of infection with this type, a wide spectrum of symptoms is observed, from influenza-like illness (Thailand) , diarrhea and gastroenteritis (Thailand, India, Ghana, South Korea, China, Hungary, Sri Lanka) [21–23, 37, 38, 49, 70, 72, 74], to AFP and sepsis (Bolivia, France) [47, 65]. HPeV-4 infection was associated with the TORCH syndrome  and lymphadenitis (Japan) [7, 21, 93].
The HPeV-5 prototype strain was isolated in the USA in 1986 from the faeces of a 2-year-old child with a fever, categorising it on the basis of serological research to EV [4, 68]. Presence of type 5 was indicated in patients with AFP in Pakistan  and Reye’s syndrome in the USA . It was detected in the cases of gastrointestinal infections in Thailand, India, Ghana, Brazil, China and Sri Lanka [21–23, 31, 37, 70, 72, 85]. Symptomatically similar cases were reported in Europe, including the Netherlands [9, 30], Spain , Bulgaria  and Denmark .
HPeV-6 was isolated for the first time from the cerebrospinal fluid of a one-year-old child with Reye’s syndrome in Japan in 2000 . After HPeV-1, it is the second type, most commonly causing respiratory infections (the UK) . It was detected in patients with influenza-like illness (Thailand)  and with otitis media (the Netherlands) . In the course of the infection, gastroenteritis, diarrhea [5, 21–23, 70, 93] and rash are primarily observed . However, HPeV-6 may also result in CNS infections (the Netherlands) , including acute flaccid paralysis (Pakistan, Japan) [54, 93] and Guillain-Barré syndrome (Italy) .
The most often detected types are HPeV-1 to HPeV-6 and they are, therefore, considered to be the most important in the diagnosis of clinical cases, the remaining genotypes are rarely isolated.
HPeV-7 was detected in 2009 in the faeces of a healthy Pakistani child, who came into contact with a person affected with AFP , similar cases of association with AFP were observed in Bolivia . HPeV-7 was also isolated from patients with diarrhea in Ghana , in the USA  and in India .
HPeV-8 was found in the faeces of children with gastroenteritis in Brazil in 2009 . HPeV-8 was first isolated in 2011 in Europe from a nearly one-year-old child with encephalitis in Bulgaria . The eighth type was also detected in the faeces of children with gastroenteritis in Ghana , India  and China .
Reference strains of relatively recently discovered types, from HPeV-9 to HPeV-19 were described by: Nix (2013), Böttcher (2017), Benschop (2008) and Graul (2017) . Type 9 was detected in patients with AFP in Bolivia  and patients with diarrhea in Ghana . Type 10 was isolated from patients with influenzalike illness in Thailand , from patients with AFP in Bolivia , and patients with enteritis and diarrhea (Bulgaria, Pakistan, India, Sri Lanka) [2, 49, 61, 70, 72]. Similarly, type 11 was detected in patients with gastroenteritis in Sri Lanka [49, 72] and in India .
The discovery of another HPeV type was reported in 2012. HPeV-12 was detected in Pakistan in an 18 month-old-child with gastroenteritis and AFP , and was subsequently also isolated from a child with AFP contact in Bolivia . Types 13 and 15 were detected in patients with gastroenteritis in Pakistan  and India . HPeV-14 was identified in the material obtained from patients with diarrhoea and gastroenteritis in the Netherlands, China, Ghana, India and Thailand [9, 21–23, 25, 37, 70], as well as patients with fever in Pakistan . HPeV-16 was detected in patients with enteritis in India . Similarly, type 17 was identified in patients with enteritis and diarrhea in Thailand  and Ghana , as well as in a healthy child in Côte d’Ivoire . HPeV-18 was detected in the faeces of patients with diarrhoea in Ghana .
HPeV-1 is the most frequently detected type of parechovirus in the world, which is confirmed by numerous European and Asian studies. The second type of parechovirus in terms of detection frequency is HPeV-3, followed by HPeV-4, 5 and 6 [5, 6, 8–10, 22, 30, 45, 46, 86, 93]. There are exceptions when type 3 is identified with a similar or higher frequency to type 1, [30, 93] e.g. in Amsterdam . In Denmark, HPeV-3 was detected five times more often than HPeV-1 . In the USA in the years 1983–2005, the dominant type was HPeV-2 , while in the years 2009–2013 HPeV-3 .
Parechovirus infections are reported worldwide. The observed differences in the circulation of particular types are of geographical nature, also the seasonality of cases is related to geographical latitude. In Ghana, for instance, a country in the equatorial climate zone, no differences in the incidence rates depending on the season of the year were observed . Cases of diseases of parechovirus etiology coincide with the enterovirus pattern in a moderate climate (summer and autumn to early winter) [6, 21–23, 35, 41, 43, 75, 79, 83, 86]. Parechoviruses are detected with the highest frequency in the autumn and winter [6, 8–10, 72, 90, 93, 95], less frequently in the spring [52, 83] or in the summer [33, 52]. In Europe, HPeV-3 was detected in the spring [18, 90], in the summer [8, 10, 18, 46, 90, 95] and in the autumn [18, 92, 95]. Circulation of more than one HPeV genotype is often observed in a specific region [2, 5, 45, 72, 75, 86].
Parechoviruses, which cause CNS infections appear every 2–3 years . It is noted that HPeV-3 exhibits a two-year cycle in Europe, starting in 1988 [6, 8, 9, 40, 42, 46, 90, 91, 97]; however, this is not confirmed by some European and Japanese studies [45, 73, 92, 95]. In Denmark, type 3 was detected every year during the investigated period (2009–2012), which suggests endemic circulation of the virus . In contrast, in North America and Australia, it appears in odd years, mainly in the summer months [10, 16, 95]. In Asia, “intermittent” circulation of type 3, characterised by some enteroviruses (E9 and E30), was observed [10, 93].
Diagnosis of parechoviral infections, similarly to enterovirus infections, is based on the isolation of viruses in the cultures of sensitive cells and/or on the detection of the virus genom (RNA) in the investigated material using molecular biology methods. Diagnosis based on clinical symptoms alone is not possible . In the diagnosis of HPeV, various types of clinical materials are used: faeces, cerebrospinal fluid (CSF), serum, plasma, throat and nasopharyngeal swabs, tissue biopsies, bronchoalveolar lavages (BALF) . In the case of CNS infections, the best diagnostic material is CSF, in which the HPeV detection clearly confirms the etiology of the infection. In the case of neuroinfections, identification of the virus in the faeces and/or throat swab is very helpful in the diagnosis, but does not clearly identify the cause of the disease [5, 9, 63, 86]. In the gastrointestinal and respiratory infections, the appropriate materials are faeces and throat swab; however, the presence of the virus in these materials does not definitively confirm the etiology of the infection . Faeces and swab are the best diagnostic materials, regardless of the nature of clinical symptoms, because large amounts of the virus are shed by the infected people in the faeces and respiratory secretions for a long time after recovery [10, 41]. Type 1 is most frequently detected in the faeces and type 3 in CSF, exceptionally in China, HPeV-1 was detected in 90% of CSF samples from patients with the parechovirus CNS infection [46, 79].
Parechoviruses are isolated in cell lines sensitive to infection, among which the epithelial cell line of African green monkey (Chlorocebus aethiops) kidney epithelial cells – Vero is optimal; it allows multiplication of many diagnostically relevant HPeV types (including 1, 3, 4, 6) [6, 10, 14, 20, 45, 93, 98]. Other cell lines of importance in the diagnosis of HPeV are: HT29 (human colon carcinoma cells) [6, 10], A549 (human lung adenocarcinoma cells) [6, 20], tMK (Cynomolgus monkey kidney cells) [6, 30], LLCMK2 (Rhezus monkey kidney epithelial cell) , T84 (human colon carcinoma cells) , DBTRG-5MG (human glioblastoma cells) , and commonly used in the diagnosis of infections towards EV: RD (human rhabdomyosarcoma cells)  and L20B (mouse fibroblasts with the polio virus receptor) .
As in the case of enteroviruses, no cell line allows isolation of all HPeV types. Moreover, different types of parechovirus have varying ability to multiply in particular cell lines . Therefore, as in the case of EV diagnostics, several different cell lines are considered, although this does not guarantee isolation of all HPeV types present in the tested material.
Observation of the infected cell cultures lasts from a few to several days , and the cytopathic effect (CPE) indicating the presence of the virus in the material is similar to that caused by EV [6, 75]. Obtained isolates are then serotyped (serological methods) or genotyped (molecular methods) to identify the type of virus. Serological methods are rarely used due to the limited availability of polyclonal sera (available only for HPeV-1 and HPeV-2) [4, 10, 42, 79]. The traditional method of virus isolation in cell cultures is used in epidemiological studies, as it provides data on the types circulating in the population, and also allows monitoring of the dominant clinical and environmental types . This method has numerous limitations; oftentimes observation for several days greatly prolongs the waiting time for the result, which is why HPeV isolation in cell cultures is of no value from the rapid diagnostics point of view (CNS infection, sepsis). Low isolation sensitivity in cell cultures associated with low viral titer in some types of clinical material (e.g. CSF) or the inability of certain types of virus to multiply in some cell lines may result in false negatives . Isolation of types 7 to 19 is often difficult . Today, the isolation method in cell lines is used to multiply viruses from selected PCR positive materials, which are then assigned a type. Serological methods are used in the “late diagnosis” and are based on the detection of antibodies appearing in response to a past infection [10, 14].
Increasingly, molecular biology methods are used to diagnose HPeV infections. These approaches are based on the detection of viral RNA in the examined clinical material. The highest sensitivity is obtained by examining faeces , but in the case of neuroinfections, it is necessary to study CSF in order to confirm the etiology of the infection. Molecular biology methods also allow identification of new types. Due to the fact that the genetic material of parechovirus is RNA, diagnostic molecular methods are based on reverse transcription PCR (RT-PCR). Most often, the highly conserved 5’UTR region is amplified. The PCR technique is currently the gold standard in the diagnosis of parechoviral infections enabling the identification of all HPeV types [6, 27, 42]. It requires different primers than the reaction identifying EV, since the sequences of the 5’UTR regions of both types are very different . The first tests based on RT-PCR detected only types 1 and 2. With time, the PCR methods for subsequent types were determined [7, 27, 40, 41, 63]. Numerous studies show that the molecular diagnostics of HPeV is faster, simpler and more sensitive than isolation in cell lines .
Currently, different types of RT-PCR are used for the diagnostic purposes. Multiplex-PCR techniques with high specificity and sensitivity have been used in diagnostics differentiating e.g. the HPeV/EV infection. Reactions of this type can identify up to several pathogens in one sample, allow for quick diagnostics of CNS and sepsis infections, which can contribute to shortening of the hospitalisation time and rapid implementation of treatment . The multiplex reaction works well in the diagnosis of material with low virus titre (e.g. CSF) and allows for detection of the most common types of HPeV (1–6) [7, 10]. Real-time RT-PCR reduces the risk of non-specific reactions compared to typical RT-PCR [7, 63]. It is successfully used for rapid diagnostics, while simultaneously detecting EV and HPeV in clinical material [18, 92]. Many HPeV detection reactions have been established and their efficiency is adjusted based on the tested material e.g., stool, CSF, blood or swab [9, 86]. Real-time RT-PCR is a method even a thousand times more sensitive than isolation in cell lines [5, 63]. In turn, nested PCR relies on a two-stage amplification of the VP3/VP1 region and allows to reduce the risk of amplification of a non-specific fragment of the genetic material. It is a method suitable for the diagnosis of CNS infections, which improved the HPeV typing from CSF [40, 92]. It is also useful in molecular epidemiology in the study of geographical distribution of genotypes and for the identification of new HPeV types [40, 41].
The new diagnostic method, VIDISCA, is based on the use of cDNA-AFLP, so the polymorphism of the length of amplified cDNA fragments and allows faster detection of RNA or DNA of viruses amplified in cell lines. It is used to identify types when the classical PCR technique fails (e.g. HPeV-5) [30, 81].
HPeV typing is also carried out using molecular methods. Genotyping uses a PCR technique based on the amplification and sequencing of the highly variable region encoding the VP1 protein [6, 40, 43, 64, 75]. In recent years, the direct genotyping method has become popular, allowing the identification of the type directly from the clinical material, which avoids the difficult isolation of the virus in cell lines, e.g. CSF. Conducted from faeces, it allows HPeV detection with even greater sensitivity [6, 9, 10, 40].
Age is the main risk factor for parechovirus infection and the development of severe disease. Children aged from 6 months to 5 years are particularly at risk. Among people infected with parechovirus, it is young children that constitute the majority (approx. 90%), and a significant proportion of them is less than a year old [1, 35, 83]. It is mainly boys who become ill, although this relationship is usually not statistically significant. Among the cases of parechovirus 1 and 2 infections registered in the USA in the years 1970–2005, children below 1 year constituted 73% and 68% of the total number respectively, while children up to 5 years 95% and 88%, respectively [10, 43, 75]. Studies on the level of anti-HPeV antibodies in Norway showed that parechovirus infection had 43% of children up to 1 year of age, 86% up to 2 years of age, and 94% of children up to 3 years of age . It is estimated that over 90% of children under 2 years old were infected with at least one type of HPeV [27, 43]. In China, among patients with gastrointestinal infection of the parechovirus etiology, children up to 2 years of age constituted 97.7% .
Infants are particularly vulnerable to parechovirus infections because they have an immature immune system . In the case of children up to 6 months of age, protection is provided by maternal antibodies, the level of which falls rapidly after 6 months of life . The number of people with immunity and HPeV antibodies increases with age. Low levels of antibodies in young children translate into high incidence. Seroprevalence studies in Finland from 1998 and 2007 showed that as many as 95% of newborns had HPeV antibodies [42, 83], whereas only 20–30% of children aged 2–12 months, while seroprevalence significantly increases in the age range of 1–2 years (89%), reaching 98% in children up to 3 years of age [42, 45, 79, 86]. In a Norwegian study, children up to 1 year old had twice as many anti-HPeV-1 antibodies (43%) as children from Finland. Similar results were obtained in the remaining age groups . It is estimated that 92–99% of the adult population have the anti-HPeV antibodies [10, 42, 83, 95].
The issue of protection against type 3 is different from protection against other types of HPeV. Only 15% of children up to 1 year of age have the anti-HPeV antibodies. This number increases to 45% for children in the age range of 2–3 years, to 85% in children aged 4–6 and to 90% in adolescents. In the second and third decades of life, the percentage of people with antibodies falls and interestingly, among women of childbearing age in Japan (20–39 years), lower levels of anti-HPeV-3 antibodies (57–74%) have been demonstrated compared to the standard group . In Finland and the Netherlands, respectively, only 10% and 13% of the subjects had antibodies against type 3 [13, 79, 95]. Low level of type 3 antibodies in adults may result in low level of maternal antibodies, and thus ineffective protection of infants in the early months of life. This may be a possible explanation for the serious course of infection with type 3 compared to HPeV-1 [10, 32, 42, 43, 95].
There is a clear differentiation in the age of children infected with individual types of HPeV. The average age of children infected with type 1 is approx. 6 months, while for type 3 the average is 1–2 months [8, 35, 42, 46, 90]. Severe parechoviral infections (CNS, respiratory), often caused by type 3, are almost exclusively limited to infants up to 3 months of age [9, 10, 13, 40, 41, 47, 73, 75, 84, 91, 92]. In 2016 in the UK, among children with severe parechovirus infections, 92% were infants up to 3 months old, of which as much as 43% were newborns . In Switzerland, 66% of children infected with HPeV were under 3 months . In Spain, types 3 and 5 were isolated in newborn infants with fever without a source (FWS), sepsis and encephalitis .
In comparison to enteroviruses, parechoviruses are rarely detected in older children and adults and usually constitute only a small percentage of the studied subjects (a few percent) [10, 23]. Exceptionally, in 2008 and 2014 in Japan, myositis and myalgia in adults with HPeV etiology were observed [32, 60]. A similar case was a pregnant woman with myalgia and muscle weakness in whom HPeV-3 was detected . In the Netherlands, parechoviruses have been detected in adults with uveitis (28), whereas in Thailand in adults with gastroenteritis . Other known cases include myocarditis in a 16-year-old girl in the USA and a 26-year-old man in Australia [50, 59]. An incidence of meningoencephalitis in an adult has also been reported in Australia .
Coinfections may affect the course of diseases with parechovirus etiology. In the respiratory tract infections, up to two-thirds of the patients exhibited HPeV coinfection with at least one respiratory virus, e.g. adenovirus and RS virus (RSV, respiratory syncytial virus) [32, 41]. In Sri Lanka, 67% of gastroenteritis cases were a result of a mixed infection with HPeV and viruses resulting in diarrhea (rotavirus, norovirus and adenovirus) . Similarly, in China, in half of the people with gastroenteritis, in addition to parechoviruses, other diarrheal viruses (rotaviruses, noroviruses, adenoviruses, astroviruses, sapoviruses) were identified, among which the most common pathogen were rotaviruses [21, 22]. Coinfections with noroviruses and enteroviruses have been observed in diarrhea cases in Thailand  and Ghana . In addition, in the Netherlands mixed infections with HPeV and EV accounted for between 1% and 3% of the studied cases [6, 9].
In a Norwegian study, 11% of children were diagnosed with HPeV, regardless of symptoms [42, 86]. A similar result (11.6%) was obtained in Germany among children with pathological symptoms, which did not significantly differ from the result in the group of healthy children. Interestingly, one of the highest viral titers was marked in the material from a healthy child. In a German study no significant association between HPeV and gastroenteritis was observed . No correlation between viral load and disease severity was found either . The above observations suggest that the course of the disease depends on additional factors.
The role of TLR7 and TLR8 receptors in the pathogenesis of encephalitis and damage to white matter in response to the HPeV infection has been suggested [10, 27, 32]. TLRs are transmembrane proteins, which when bound to specific antigens, induce the secretion of pro-inflammatory cytokines. They play the role of so-called sensors (host sensors) for HPeV. Following the binding of viral RNA by TLR, the immune response is initiated, and the pro-inflammatory cytokines and reactive oxygen species are released, leading to cell death [10, 27, 32]. The presence of a large number of TLR8 receptors in a developing nervous system may affect a serious course of infection, associated with neuronal apoptosis and irreversible changes in the brain.
HPeV-3 is considered to be the most pathogenic type of parenchovirus . The reasons for this may be the differences in viral biology (lack of RGD), involving the use of a different receptor than the other types [9, 10, 32, 40–43]. It was noticed that HPeV-3 proliferates significantly more efficiently in neural cells than HPeV-1, which may indicate greater neurotropism and explain the serious course of infections with this type [9, 27, 32]. It is the most frequently detected parechovirus in CNS infections and sepsis in newborns, and is associated with meningitis, sepsis, fever and rash more than type 1 [27, 90, 95]. It also causes more severe disease course in younger children [8, 9, 47, 48, 84, 92, 95]. It is also the second cause of viral meningitis, encephalitis and newborn sepsis after enteroviruses, and is responsible for 3 to 17% of cases of meningitis and encephalitis [10, 27, 75, 97]. Other types sporadically cause severe forms of the disease . After an infection, a reinfection with another type almost always occurs within the following three years .
Patients with parechovirus infections receive symptomatic treatment, and a more severe course of the disease requires hospitalisation and monitoring of vital signs. There is no effective antiviral treatment. Patients with myocarditis have been successfully treated with intravenous immunoglobulin (IVIG), which is commonly used on patients with enterovirus infections. Immunoglobulin reduces inflammation by affecting a network of cytokines involved in the inflammatory reaction in response to an infection [32, 43, 59, 89, 96]. Some researchers find it unlikely that immunoglobulin therapy would be effective in the cases of severe disease caused by HPeV-3 due to low seroprevalence. Higher IVIG efficacy was observed in HPeV-1 infections than in other types [10, 27, 79]. So far, the effectiveness of drugs used to treat enterovirus infections has not been confirmed. This includes pleconaril (blocks the receptor binding site of the capsid), rupintrivir and SG85 (3C protease inhibitors), as well as helicase/ATPase 2C inhibitors (e.g. GuHCl and HBB), which is probably due to differences in the capsid structure and genetic differences between EV and HPeV. Nonetheless, the most promising drugs appear to be preparations from the group of capsid inhibitors and 3C protease inhibitors, as well as substances targeting the host factors used by the virus during replication, as in the case of anti-HCV therapy [32, 89]. Monoclonal antibodies and ribavirin (nucleotide analogue) may turn out to be a promising form of therapy [10, 32, 43, 89].
Parechoviruses are pathogens, which commonly occur throughout the world. Most often, they cause harmless infections of the gastrointestinal and respiratory tracts in young children. However, some infected people develop acute symptoms and severe forms of disease. The key factor in the development of severe stages of the disease is age, which makes young children particularly susceptible to parechovirus infections.
Numerous reports describe the connection of parechoviruses with many disease entities. Also the observed spectrum of symptoms is broad and unspecific, and the fact that the course of the parechovirus and enterovirus infections is similar, makes the differential diagnosis a big challenge, especially in the area of standardisation of diagnostic methods. Many research groups are observing the trend of parechoviruses rising to a second place after enteroviruses in the ranking of etiological factors of viral meningitis and sepsis, especially in newborns and infants. In spite of this, only few laboratories carry out diagnostics for parechovirus infections, which is performed mainly in reference laboratories.
Currently, 19 genotypes of HPeV are known, which differ in biological properties, the range of geographical occurrence, pathogenicity, as well as in the frequency of detection. Although six decades have passed since the discovery of the first parechovirus, our knowledge of these variable pathogens is still incomplete. An increasing number of research groups, including European ones, are aware of the need to conduct more extensive research into the biology of parechovirus and to supervise the incidence of HPeV infections. Monitoring the circulation of genotypes in the population is of particular importance, as well as collecting data from clinical history. The current knowledge on parechovirus shows the need for further virological and epidemiological studies in order to understand and assess the threat of these small viruses.