Enterovirus B types cause severe infection in infants aged 0–3 months

Enterovirus (EV) are small non-enveloped RNA viruses belonging to the family Picornaviridae, with genomes approximately 7500 nucleotides in length. Currently, more than 100 EV types, assigned to four species, have been found to infect humans, namely enterovirus A (EV-A), EV-B, EV-C, and EV-D, based on genetic divergence [1, 2]. EV are distributed worldwide and have a seasonal incidence patterns in temperate regions during summer and fall and occur year-round in the tropics [3]. EV infection in children manifest as a spectrum of clinical disorders, including non-specific febrile illness, hand-foot-mouth disease (HFMD), meningitis, viral encephalitis, myocarditis, sepsis, and pulmonary edema. The severity and mortality of EV infection are generally inversely related to age, especially in newborns within the first few days of life [4, 5].

In recent decades, several EV-A-associated HFMD outbreaks, particularly those caused by EV-A71, coxsackievirus (CV) A6, CVA10, and CVA16 types, involving millions of children under 5 years of age, have been described in detail [6‐9]. Unlike EV-A infections, EV-B infections (echovirus (E) 1–7, E9, E11–21, E24–27, E29–33, CVB1–6, and CVA9) are commonly identified in children less than 1 year old and are characterized by serious disease, with high mortality rate [10, 11]. The circulation of EV-B types has been associated with several EV outbreaks in Asia, Europe, and the USA in recent years [12‐14]. However, the incidence of infant infections and circulating types varies markedly across countries and regions.

There is growing interest in the understanding of epidemiology of EV in infants, particularly due to their association with severe disorders. Awareness of the clinical features associated with severe conditions in infants, recognition of the risk factors, and monitoring of the infection types might help pediatricians diagnose severe cases promptly and treat them appropriately to reduce mortality. In this study, we described the epidemiological, clinical, and genetic characteristics of EV infections in a cohort of children admitted to a tertiary care center in Guangzhou, China. Furthermore, with the clinical and molecular epidemiological patterns of EV infection, we aimed to explore the clinical spectrum and relationship of individual EV types with the risk factors of severe infection in infants.

This study showed that seasonal variations in EV infections with respect to age were evident. Most infants aged 0–3 months had a seasonal pattern of infections. They presented during summer, with a peak from April to July, which was apparently different from the epidemic features in older children, and consistent with previous studies [4, 11, 18]. Our current study also showed that the circulating EV types, affecting each age range, differed substantially. The most common EV detected in children older than 3 months was EV-A, whereas EV-B, represented by the E11 type, was significantly more frequent in younger infants. In the USA and Europe, EV-B is the one most commonly reported in neonates [12, 13, 19, 20].

EV infection in neonates can present with clinical symptoms, ranging from asymptomatic, non-specific febrile illness to severe, life-threatening disease, and it is difficult to distinguish from non-EV infection based on the clinical signs only. In this study, fever was the most common symptom in both infants aged 0–3 months and older children. However, the other clinical symptoms, including rashes, tachycardia, coughing, vomiting, startle, and convulsions, frequently observed in children with HFMD, were not typical for infants, as suggested in earlier retrospective studies [13, 21]. Rashes are often suggested as a diagnostic basis for viral infection. However, only 6.0–27.3% of cases of infants with EV infection have been reported with rashes [4, 13, 22]. In this study, only 8% (4/50) of the infants developed cutaneous rashes. As an important indication of host inflammation in viral infection, less frequent rashes make the early clinical diagnosis of EV infection difficult for neonatologists.

Severe illnesses due to EV are commonly seen in neonates; mortality is exceptionally high when meningitis, myocarditis, and hepatitis occur [4, 13, 23]. More than three-quarters of neonates were diagnosed with severe infection in this study, particularly in the first two weeks of life. Meningitis, sepsis, pneumonia, myocarditis, and/or hepatitis are the most common clinical presentations. In contrast, only 35.4% of children older than 3 months showed severe infection. Meningitis is often associated with age-specific pleocytosis and/or neurological symptoms. Previous studies had shown that EVs can invade the central nervous system and disrupt the blood–brain barrier, resulting in more than 75% of meningitis cases, some of which are life-threatening [7, 13, 24]. In our study, out of 36 patients with meningitis, 28 (77.8%) were infants aged 0–3 months. It could be due to the immature brain of neonates and their imperfect blood–brain barrier. A higher detection rate of 76.7% (23 of 30) for EVs in CSF samples available from 30 neonates further substantiated this finding.

Pneumonia is the most common comorbidity in lower respiratory tract-EV-B infection, and can be rapidly progressive, leading to severe pneumonia [25]. In this study, more than one-third (44/115) cases were accompanied by pneumonia. EV-B types were the predominant pathogens in patients with pneumonia. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection associated with a wide range of pathogens, and pediatricians frequently encounter it in young infants [13, 19, 26]. Hepatitis is often associated with EV-B infections, and mortality is especially high when it occurs concomitantly with myocarditis [27]. In this study, a total of 8 cases (6 infants and 2 children) developed symptoms of hepatitis during hospitalization, and all were associated with EV-B infection.

Understanding the risk factors and monitoring the parameters associated with severe infection may lead to effective prophylaxis and prompt aggressive treatment to reduce morbidity and mortality. Infection with EV-B and younger age are considered the major risk factors for developing severe infections [5]. A retrospective study on 2356 infants with known EV types during 1983–2003 reported that CVB1–5 and E11 have an increased risk of neonatal infection, with CVB4 being associated with the highest mortality rate of 40% [28]. In our study, we found that EV-B infection and age less than 3 months increased the risk of EV infection 4.260-fold and 6.474-fold, respectively. Moreover, of the two deceased children, one was positive for CVB3 and the other was for E18, which illustrated that EV-B infection is a major risk factor. Notably, the mother of the 7-month-old child was also positive for E18 as per stool sample test, with 100% similarity in VP1 gene compared to that of the children, but showed no obvious clinical symptom. Previous studies had shown that transmission of EVs from mother to infant is relatively common, occurring in 30–50% of cases [23, 29, 30].

Xiao-Qing Lv et al. had previously reported that an abnormal platelet count could be an independent predictor of severe EV infection [31]. Our results also showed that if the platelet count was abnormal, the risk of severe infection was increased 2.745-fold. Furthermore, abnormal CSF test result, such as pleocytosis, constituted a significant risk factor for developing severe EV infection, as observed by previous investigators [32], and elevated protein levels and pleocytosis in CSF increased the risk of severe infection 13.913-fold and 9.481-fold, respectively. Moreover, we elucidated that the positive result of EV in CSF was an independent predictor of severe infection, and the OR increased to 12.071.

Genomic recombination is a major driving force in the evolution, diversification, and shaping of genetic architecture of EVs [33], and time-correlated recombination events of EV-B are more frequent than those of other human EV species [34]. However, until recently, only limited complete genome sequences of E11 strains were available in the public database, with most of them coming from non-clinical isolates. In this study, we obtained 5 full-length E11 genomes, analyzed their phylogenetic characteristics, and found homologous recombination events to have occurred with E6 strains in China in 2018. Multiple phylogenetic studies presented previously provided evidence that RNA recombination of EVs only occurred throughout the entire non-structural region, and recombination sites were mainly located in region P2 [33‐36]; notably, the recombination site was in the junction between 2B and 2C, as per our study. These observations suggested that non-structural proteins may be functionally interchangeable with other variants within EVs. Furthermore, in the infected host, effective recombination events are critical for RNA viruses to overcome tissue-type specific antiviral selection, establish robust infection and virulence, and adapt rapidly to dynamic selective environments [37, 38]. However, the changes in phenotypic characteristics of E11 recombination, including their fitness and pathogenicity, need to be investigated further.

In summary, EV affects infants aged 0–3 months differently and more severely than in older children. Clinical manifestations in infants with EV mainly included meningitis, sepsis, pneumonia, and even death. EV-B types were the most common in neonatal EV infection, and recombination events were observed in the P2 and P3 regions of predominant type E11 with E6 from China. In addition, we identified independent predictors of severe EV infection, including EV-B infection, age less than 3 months, elevated ALT level, abnormal platelet count, and abnormal CSF characteristics. Taken together, EV-B infections should be routinely considered in neonates with meningitis, sepsis, and pneumonia, with or without a rash, particularly during EV season.