Molecular characterization of coxsackievirus B5 from the sputum of pneumonia children patients of Kunming, Southwest China

Enterovirus B is considered to be a common respiratory pathogen in young children and can cause respiratory wheezing disease, including bronchiolitis and exacerbation of asthma [1, 10, 16, 17]. CVB5 belongs to the species Enterovirus B of the Picornaviridae family [5, 9, 23] and the genomic RNA is about 7.5 kb long in length and encodes a large polyprotein, which consists of the structural proteins (VP1-4) and other nonstructural proteins [15]. CVB5 uses DAF as a receptor for virus attachment to cells and it depends on CAR for virus entry and virus replication processes. Aseptic meningitis and viral encephalitis caused by CVB5 occur worldwide, with outbreaks or epidemics reported in Europe, North America, South America and Asia [6]. The first reported clinical symptom of CVB5 was lower respiratory tract infection, and in recent years, the clinical symptoms of CVB5 have included HFMD [24]. CVB5 usually occurs in summer and autumn and the susceptible populations are mainly under 18 years old. To date, the vaccines against enteroviruses are those for poliovirus and EV-A71 [23]. The two vaccines offer no cross-protection against other enteroviruses, and there are no vaccines or specific drugs against CVB5 [4]. Therefore, the study of CVB5 has become an urgent need for respiratory pathogen research and respiratory disease prevention.

We unexpectedly found five cases of CVB5 infection during routine surveillance of rhinoviruses in respiratory tract samples. All five patients were hospitalized with pneumonia symptoms and were not tested for enterovirus and rhinovirus during their hospitalization. But we found CVB5 infection instead of rhinovirus infection in their sputum samples through follow-up experiments. Considering the molecular epidemiological information on respiratory tract samples containing CVB5 is still limited and clinicians lack attention to the detection of enterovirus in patients with respiratory symptoms, we decided to study it. In this study, we obtained five complete genome sequences from the sputum of inpatients in the respiratory department of the Children’s Hospital Affiliated with Kunming Medical University. Meanwhile, nine CVB5 VP1 sequences have been obtained by our team from the feces of patients with HFMD during a large enterovirus infection outbreak in Kunming in 2018 [11, 18]. To explore the molecular characteristics of CVB5 in patients with different symptoms, transmission and evolution of CVB5 in Kunming, we compared these sequences and performed phylogenetic, mutation and recombination analysis, structural analysis and prediction. Our study may benefit the research of the genetic characterization, potential source and evolution of CVB5.

Molecular epidemiological information on respiratory tract samples containing CVB5 is still limited. Under this background, we analyzed the molecular characteristics of CVB5 in patients with different symptoms, transmission and evolution of CVB5 in Kunming.

Phylogenetic and similarity analyses based on the VP1 region showed that the five sputum isolates were highly similar to the fecal isolates. However, there were nucleotide differences between the isolates and sublineage II of E genogroup (8.7–12%), the main strains endemic in Mainland China [18]. These information suggests that the five sputum isolates in 2021 and the nine fecal isolates in 2018 were in the same transmission chain and form an independent branch in genogroup E. The CVB5 isolated from patients with pneumonia sputum samples was not significantly different from the CVB5 we previously isolated in fecal samples from patients with HFMD in the VP1 region, but there were more mutations when comparing with Faulkner. Faulkner was isolated from patients in 1952 [20], nearly 70 years have passed between Faulkner and the CVB5 isolates in this study. The clinical symptoms of CVB5 may have changed due to the increasing differences in genomic sequences caused by the continuous evolution of the virus. In addition, the virus particles were successfully observed under an electron microscope in the harvested positive culture supernatants of KM35, and it will benefit the follow-up research of CVB5.

Amino acid mutations in VP1 were analyzed due to the lower homology of the isolates compared to Faulkner (80.87–80.99%). Four amino acid mutations were substituted at the N-terminus and C-terminus of the five isolates, and four mutations were in loop regions. The loops are located on the surface of the virion and are easily accessible to the host immune system [15]. Mutations at the N-terminal significantly changed the structure and spatial position of the N-terminal of VP1 protein. PROVEAN showed three deleterious mutations and nine neutral mutations. The deleterious mutations may lead to weakened virulence of the CVB5 isolates. Although neutral mutations can affect the binding of proteins, the main research direction is deleterious mutations, and the effect of neutral mutations needs to be further studied [2].

In China, patients with respiratory problems are often not tested for enterovirus to save on medical costs. The detection of enteroviruses in respiratory tract samples has also not received sufficient attention from clinicians. In the respiratory infection detection data from China over the past 11 years (2009–2019) that was released by the China CDC [13], enteroviruses were not monitored. The HFMD surveillance network established in China since 2009 is mainly based on clinical manifestations of diseases, and only several limited pathogens, such as EV-A71, CVA6, CVA10, and CVA16 are monitored [8]. These findings indicate a lack of enterovirus surveillance in patients with respiratory symptoms. CVB5 is the most common type among all coxsackie B viruses [21], but CVB5 has not even been incorporated into disease surveillance systems in China.

It was reported that the selective production of RANTES, IL-8 and MCP-1 by CVB5-infected epithelial cells of the small bronchioles, along with mechanisms of amplification mediated by IFN-γ [19]. This may be the various histologic and inflammatory features of CVB5-induced airway disease. The five patients in our study were all under the age of 10, and their common chief complaint before hospitalization was a prolonged cough. All five patients had almost the same symptoms as other pneumonia patients. Although CVB5 infection was detected in sputum samples from the five patients, they were co-infected with other pathogens. We can’t tell if CVB5 made their symptoms worse, constituted limiting factors in this investigation. In 1960, two children were reported to have died from pneumonia caused by CVB5 infection [3]. This suggests a certain mortality after CVB5 infection and should not be ignored in respiratory symptoms.

In conclusion, a total of 108 sputum samples from children hospitalized with lower respiratory tract infection were collected from the Children’s Hospital Affiliated with Kunming Medical University. We unexpectedly found five cases of CVB5 infection instead of rhinoviruses infection during our routine surveillance of rhinoviruses. This report suggests that enterovirus surveillance should be enhanced in patients with respiratory symptoms and serves as a reference to follow-up studies of molecular epidemiology, virulence, infection, and pathogenicity of CVB5.