First isolation and genomic characterization of bovine parechovirus from faecal samples of cattle in Japan.

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5' untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2-64.5 %, 58.6-59.7 % and 66.3-66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Rapid molecular diagnosis of Parechovirus infection using the reverse transcription loop-mediated isothermal amplification technique.

OBJECTIVES: Human parechovirus (HPeV), especially HPeV A3 (HPeV3), causes sepsis-like diseases and sudden infant death syndrome in neonates and young infants. Development of rapid and easier diagnostic laboratory tests for HPeVs is desired. METHODS: Original inner primers, outer primers, and loop-primers were designed on the 5' untranslated region of HPeV3. HPeV3 ribonucleic acids (RNAs), other viral RNAs, and clinical stool samples were used to confirm whether the designed primers would allow the detection of HPeV3 with the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique. RESULTS: Three combinations of primers were created and it was confirmed that all primer sets allowed the detection of HPeV3 RNAs. The primer sets had cross-reactivity with HPeV type 1 (HPeV1), but all sets showed negative results when applied to coxsackievirus, echovirus, enterovirus, norovirus, and adenovirus genomes. Four of six stool samples, obtained from newborn and infant patients with sepsis-like symptoms, showed positive results with our RT-LAMP technique. CONCLUSIONS: This manuscript is the first description of an RT-LAMP for the diagnosis of HPeVs, allowing a faster, easier, and cheaper diagnosis. This technique is clinically useful for newborns and infants who have sepsis-like symptoms.

Neutralizing Ljungan virus antibodies in children with newly diagnosed type 1 diabetes.

Ljungan virus (LV), a Parechovirus of the Picornavirus family, first isolated from a bank vole at the Ljungan river in Sweden, has been implicated in the risk for autoimmune type 1 diabetes. An assay for neutralizing Ljungan virus antibodies (NLVA) was developed using the original 87-012 LV isolate. The goal was to determine NLVA titres in incident 0-18 years old newly diagnosed type 1 diabetes patients (n=67) and school children controls (n=292) from Jämtland county in Sweden. NLVA were found in 41 of 67 (61 %) patients compared to 127 of 292 (44 %) controls (P=0.009). In the type 1 diabetes patients, NLVA titres were associated with autoantibodies to glutamic acid decarboxylase (GADA) (P=0.023), but not to autoantibodies against insulin (IAA) or islet antigen-2 (IA-2A). The NLVA assay should prove useful for further investigations to determine levels of LV antibodies in patients and future studies to determine a possible role of LV in autoimmune type 1 diabetes.

Severity of parechovirus infections in infants under 3 months of age and comparison with enterovirus infections: A French retrospective study.

While enteroviruses (EV) are a well-recognized cause of aseptic meningitis in children, human parechoviruses (HPeV), especially genotype 3, have been increasingly reported as a frequent cause of sepsis-like illness and meningitis among young infants. The aim of this study was to describe the epidemiological, clinical, and laboratory characteristics of HPeV infections in infants and to compare them with those of well-known EV infections. This monocentric retrospective study was carried out at the pediatric unit of Nantes University Hospital from January 2015 to August 2018. All patients under 18 years of age with diagnosis codes referring to fever, for whom viral infection was suspected and cerebrospinal fluid (CSF) specimens were collected, were included. All CSF specimens were screened by duplex real-time polymerase chain reaction (PCR) assay that allows for the simultaneous detection of EV and HPeV in clinical samples. During the study period, 1373 CSF specimens from patients under 18 were included. A total of 312 CSF samples were positive for HPeV (n=34) or EV (n=278). Among the 34 HPeV-positive patients, 97% (33/34) were under 3 months of age, whereas the rate was 54% (149/278) for EV-positive patients (P<0.001); thus, patients under 3 months of age were defined as the study population for the rest of this work. A review of the medical records was carried out for the positive cases. In this population, the HPeV detection rate was 5.6% versus 25.3% (P<0.001) for EV. All but one of the HPeV samples available for genotyping were HPeV-3. No seasonality was observed for HPeV infections. Length of hospital stay tended to be longer for children infected with HPeV compared with those infected by EV (3 days vs. 2 days, P=0.05). Clinicians reported more severe illness presentations among HPeV-infected infants, with more frequent administration of fluid bolus (P<0.02). Regarding laboratory characteristics, a significant lack of cellular reaction in the CSF (P=0.004) as well as lower C-reactive protein (CRP) levels (P=0.006) and neutrophil counts (P<0.001) were noted for HPeV infections compared with EV infections. Our results confirm the early onset of HPeV infections (more than 95% of patients aged under 3 months). The clinical presentation and laboratory characteristics of the two infections was similar. However, some higher clinical severity criteria and a lack of CSF pleocytosis were regularly observed in patients with HPeV infections. Considering the significant proportion (5.6%; 95% CI, 3.7-7.5) of all CSF samples in our series, HPeV detection should be systematically included in the microbiological diagnosis of febrile children under 3 months of age.

Multicystic Encephalomalacia: The Neuropathology of Systemic Neonatal Parechovirus Infection.

The Neuropathology of Human Parechovirus (HPeV) is not widely described due to the relatively recent discovery of the virus combined with a limited number of autopsy case reports. We report the case of an infant boy born at 38 weeks who, six days after birth, presented with fever and severe neurological dysfunction. Human Parechovirus Type 3 (HPeV3) RNA was detected in his cerebrospinal fluid (CSF) and blood. He died five days after his initial presentation. Neuropathologic examination demonstrated multicystic encephalomalacia (ME). This case report confirms that white matter pathology is dominant in HPeV3 infection. A unique feature, of HPeV encephalomalacia is absence of CSF pleocytosis and minimal inflammation in the meninges. The findings permit comment on the pathogenesis of brain injury by this virus.

The yield of CSF molecular testing in febrile neonates.

We retrospectively examined the yield of a cerebrospinal fluid (CSF) multiplex real-time PCR assay of febrile young infants undergoing a full sepsis work-up. Eighty infants were included in the study: Forty-nine (61%) neonates and 31 (39%) 29-90 day-old patients were included in the study. A viral pathogen was detected in 59% (47/80) of the samples, human enterovirus in 53% (42/80) and Human parechovirus in 6% (5/80). The CSF of nearly half of the subjects with CNS infection was without pleocytosis; all CSF cultures were negative. Multiplex PCR CSF testing enhances the diagnosis of pathogen-specific viral CNS infection among febrile young infants.

Acute heart failure due to dilated cardiomyopathy exacerbated by systemic parechovirus A1 infection in an infant.

Parechovirus A1 (PeV-A1) often causes mild respiratory or gastrointestinal disease. Herein we report a case of acute heart failure due to dilated cardiomyopathy exacerbated by acute PeV-A1 infection in a 10-month-old infant. He was brought to our hospital with acute respiratory distress and compensated shock. Echocardiogram showed a dilated left ventricle and severe mitral regurgitation, consistent with dilated cardiomyopathy. PeV-A1 infection was confirmed by (1) positive PCR test results for PeV-A in multiple anatomical sites, including blood, stool, and throat, (2) the genetic sequence of viral protein, and (3) an increase in paired serum PeV-A1-specific neutralizing antibody titers. A few, scattered case reports in infants and young children also indicate the association between myocarditis and/or dilated cardiomyopathy and PeV-A1 infection. In conclusion, PeV-A1 infection could be associated with exacerbation of myocardial diseases in infants and young children; thus PeV-A1 needs to be evaluated as a viral cause of such a condition.

Year-Round, Routine Testing of Multiple Body Site Specimens for Human Parechovirus in Young Febrile Infants.

OBJECTIVES: To test our hypothesis that routine year-round testing of specimens from multiple body sites and genotyping of detected virus would describe seasonal changes, increase diagnostic yield, and provide a better definition of clinical manifestations of human parechovirus (PeV-A) infections in young febrile infants. STUDY DESIGN: PeV-A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was incorporated in routine evaluation of infants aged ≤60 days hospitalized at Nationwide Children's Hospital for fever and/or suspected sepsis-like syndrome beginning in July 2013. We reviewed electronic medical records of infants who tested positive for PeV-A between July 2013 and September 2016. Genotyping was performed with specific type 3 RT-PCR and sequencing. RESULTS: Of 1475 infants evaluated, 130 (9%) tested positive for PeV-A in 1 or more sites: 100 (77%) in blood, 84 (65%) in a nonsterile site, and 53 (41%) in cerebrospinal fluid (CSF). Five infants (4%) were CSF-only positive, 31 (24%) were blood-only positive, and 20 (15%) were nonsterile site-only positive. PeV-A3 was the most common type (85%) and the only type detected in CSF. Although the majority (79%) of infections were diagnosed between July and December, PeV-A was detected year-round. The median age at detection was 29 days. Fever (96%), fussiness (75%), and lymphopenia (56%) were common. Among infants with PeV-A-positive CSF, 77% had no CSF pleocytosis. The median duration of hospitalization was 41 hours. Four infants had bacterial coinfections diagnosed within 24 hours of admission; 40 infants had viral coinfections. CONCLUSIONS: Although most frequent in summer and fall, PeV-A infections were encountered in every calendar month within the 3-year period of study. More than one-half of patients had PeV-A detected at more than 1 body site. Coinfections were common. PeV-A3 was the most common type identified and the only type detected in the CSF.

Enterovirus and Parechovirus Coinfection in a Sudden Unexpected Infant Death.

Viruses are suspected to play a role in the multifactorial pathogenesis of sudden infant death. We described a sudden and unexpected death in a 5-month-old boy, with detection of both enterovirus and parechovirus RNA in the blood. This is the first report of a dual viraemia of enterovirus and parechovirus and its potential association with a sudden unexpected infant death. Extensive sampling and testing especially using molecular methods currently available is needed to better understanding the "hypothetical" link between viral infections and sudden infant death.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Prevalence and molecular characterization of parechovirus A in children with acute gastroenteritis in Shenzhen, 2016-2018.

Parechovirus A (PeV-A), which causes a wide variety of diseases, is prevalent among young children. However, little is currently known about PeV-A infections in children with acute gastroenteritis in mainland China. In this study, we investigated the molecular epidemiology of acute gastroenteritis in Shenzhen, southern China, with an emphasis on PeV-A infections. A total of 1220 stool specimens from 1220 outpatient children under 5 years old with acute gastroenteritis were collected from January 2016 to December 2018. Viral RNA was detected by a real-time RT-PCR and PCR method. The PeV-A isolates were genotyped by sequencing the VP3/VP1 region. Of 1220 specimens, 148 (12.1%) were positive for PeV-A. The predominant genotype was PeV-A 1B (68.9%), followed by PeV-A 4 (12.2%), PeV-A 14 (6.1%), PeV-A 1A (5.4%), PeV-A 6 (2.7%), PeV-A 3 (2.7%) and PeV-A 5 (2.0%). It was found that 68.2% of PeV-A infections occurred in the summer and rainy months (June to September) in southern China. The majority of PeV-A-positive patients (97.3%) were younger than 24 months old. PeV-A coinfection with norovirus, rotavirus, astrovirus and adenovirus was found in thirty specimens (30/148, 20.3%), five specimens (5/148, 3.4%), five specimens (5/148, 3.4%), and two specimens (2/148, 1.4%), respectively. Coinfections with more than one other enteric virus were not observed in any of the PeV-A-positive specimens. Phylogenetic analysis revealed that the PeV-A isolates from Shenzhen were closely related to each other and to strains circulating in China, suggesting endemic circulation of PeV-A in China. The results of this study indicate that PeV-A is one of important pathogens of acute gastroenteritis in young children and that coinfection is a possible mode of PeV-A infection. PeV-A associated with acute gastroenteritis exhibited high genotypic diversity in Shenzhen, southern China.

Identification and molecular characterization of the first complete genome sequence of Human Parechovirus type 15.

Using a metagenomics approach, we have determined the first full-length genome sequence of a human parechovirus type 15 (HPeV15) strain, isolated from a child with acute flaccid paralysis and co-infected with EV-A71. HPeV15 is a rarely reported type. To date, no full-length genome sequence of HPeV15 is available in the GenBank database, where only limited VP1 sequences of this virus are available. Pairwise comparisons of the complete VP1 nucleotide and deduced amino acid sequences revealed that the study strain belongs to type 15 as it displayed 79.6% nucleotide and 93.4% amino acid identity with the HPeV15 prototype strain. Comparative analysis of available genomic regions and phylogenetic analysis using the P2 and P3 coding regions revealed low nucleotide identity to HPeV reference genomes. Phylogenetic and similarity plot analyses showed that genomic recombination events might have occurred in the UTRs and nonstructural region during HPeV15 evolution. The study strain has high similarity features with different variants of HPeV3 suggesting intertypic recombination. Our data contributes to the scarce data available on HPeVs in Africa and provides valuable information for future studies that aim to understand the evolutionary history, molecular epidemiology or biological and pathogenic properties of HPeV15.

Parechovirus-A3 encephalitis presenting with focal seizure mimicking herpes simplex virus infection.

BACKGROUND: Febrile neonates and young infants presenting with seizure require immediate evaluation and treatment. Herein we experienced two young infants with parechovirus-A3 (PeV-A3) encephalitis, initially presented with focal seizure suspecting herpes simplex virus (HSV) encephalitis. CASES: We have experienced 2 infantile cases, initially presented with focal seizure. At presentation, HSV encephalitis was strongly suspected and empiric acyclovir therapy was started; however, serum and/or cerebrospinal fluid (CSF) PCR for HSV were negative. Instead, serum and/or CSF PCR for parechovirus-A was positive. PeV-A3 infection was confirmed by genetic sequence analyses. Both cases required multiple anticonvulsant therapy and intensive care for intractable seizure. Diffusion-weighted imaging of brain magnetic resonance imaging (MRI) showed distinct findings; high-intensity lesions in the gray matter of parietal and occipital lobes in Case 1, and bilateral decreased diffusion of the deep white matter and corpus callosum in Case 2. We have followed two cases more than four years; Case 1 developed epilepsy, has been on an anticonvulsant to control her seizure. Case 2 has significant neurodevelopmental delay, unable to stand or communicate with language. CONCLUSIONS: PeV-A3 encephalitis needs to be in differential diagnosis when neonates and young infants present with focal seizure, mimicking HSV encephalitis. Special attention may be necessary in patients with PeV-A3 encephalitis given it could present with intractable seizure with high morbidity in a long-term.

Parechovirus A prevalence in adults in The Netherlands.

Human parechoviruses (HPeV) of the species Parechovirus A are highly prevalent disease-causing pathogens in children worldwide. HPeVs are capable of causing severe disease in adults as well, but the prevalence in adults may be much lower. The aim of our present study was to determine the prevalence of HPeV in clinical samples from adults sent in for diagnostic procedures in a tertiary hospital in the Netherlands. From a total of 10,645 samples obtained from 6175 patients, 20 samples from 11 patients (0.18%) tested positive for HPeV by RT-PCR. Two patients were positive for HPeV-1, two for HPeV-3, and one for HPeV-6. Six HPeVs could not be typed. Eight of the 11 HPeV-positive patients were immunocompromised. Due to comorbidity, we were unable to attribute the patients' clinical symptoms to the HPeV infection. The HPeV prevalence in adults found in this study is low compared to HPeV prevalence in children. This may be largely explained by the high seropositivity rates in adults, although there could be other mechanisms involved.

Association of Picornavirus Infections With Acute Otitis Media in a Prospective Birth Cohort Study.

BACKGROUND: Human rhinoviruses (HRVs), human enteroviruses (HEVs) and human parechoviruses (HPeVs) have been linked to acute otitis media (AOM). We evaluated this association in a prospective birth cohort setting. METHODS: A total of 324 healthy infants were followed up from birth to age 3 years. Nasal swab samples were collected at age 3, 6, 12, 18, 24, and 36 months and screened for HRV and HEV using real-time reverse-transcription quantitative polymerase chain reaction. Stool samples were collected monthly and analyzed for HRV, HEV, and HPeV. AOM episodes diagnosed by physicians were reported by parents in a diary. The association of viruses with AOM was analyzed using generalized estimation equations, and their relative contributions using population-attributable risk percentages. RESULTS: A clear association was found between AOM episodes and simultaneous detection of HEV (adjusted odds ratio for the detection of virus in stools, 2.04; 95% confidence interval, 1.06-3.91) and HRV (1.54; 1.04-2.30). HPeV showed a similar, yet nonsignificant trend (adjusted odds ratio, 1.44; 95% confidence interval, .81-2.56). HRV and HEV showed higher population-attributable risk percentages (25% and 20%) than HPeV (11%). CONCLUSIONS: HEVs and HRVs may contribute to the development of AOM in a relatively large proportion of cases.

Repeated viral meningitis in a newborn.

Human enteroviruses (EV) are the most common cause of viral meningitis in children. Human parechoviruses (HPeV) are increasingly being recognized as a cause of central nervous system (CNS) infections and sepsis-like disease in children. Both viruses belong to Picornaviridae family. The clinical picture in EV and HPeV infections is usually nonspecific. Therefore, molecular detection of both viruses is needed for etiological diagnosis. In this case report, we describe and discuss clinical and laboratory findings of two consecutive episodes of viral meningitis caused by EV and HPeV, respectively, occurring in the first month of a newborn's life.

Complete genome sequences of the first parechoviruses A associated with sporadic pediatric acute gastroenteritis in Russia.

Parechovirus (the Picornaviridae family), recently classified as Parechovirus A and formerly known as Human parechovirus (HPeV), can cause a wide range of human diseases. Over the past decade, several studies have reported HPeV epidemiology in different regions; however, information from Russia is limited. A total of 632 stool samples collected in Novosibirsk, Russia during January-March 2012 were screened for HPeV by RT-PCR. The study cohort comprised 572 patients with acute gastroenteritis and 60 healthy children. Seven of 572 (1.2%) gastroenteritis cases were HPeV-positive, including one co-infection with rotavirus and astrovirus. All positive patients were ≤1 year old, and five of them were younger than 3 months. None of the healthy controls provided an HPeV-positive sample. Six HPeV isolates were classified as HPeV-1 and one as HPeV-5 using phylogenetic analysis. Two complete genome sequences of HPeV-1 and one of HPeV-5 were determined and analyzed. Phylogenetic analysis showed that the studied Russian strains are probably recombinants. P1 region sequences of two Russian HPeV-1 strains clustered with rare contemporary HPeV-1A strains, whereas their P3 regions were phylogenetically closer to the archival Harris strain. The Russian HPeV-5 strain formed a common cluster with other HPeV-5 strains only for the P1 region, while the P3 region grouped with the German HPeV-2 strain. In the Russian HPeV-5 strain, the lack of the arginine-glycine-aspartic acid (RGD) motif at the C-terminus of VP1 was observed. This is the first complete genome characterization of the Russian HPeV strains detected in sporadic cases of pediatric acute gastroenteritis.

Novel scoring system for differentiating parechovirus-A3 and enterovirus infection in neonates and young infants.

BACKGROUND: Parechovirus-A3 (PeV-A3) and the enteroviruses (EVs) are the most common viral pathogens responsible for sepsis and meningoencephalitis in neonates and young infants; however, differences in the clinical presentations of two infections are not well described. OBJECTIVES: To describe the clinical presentations of PeV-A3- and EVs-related diseases and develop a novel scoring system to differentiate two diseases. STUDY DESIGN: This prospective study used real-time PCR and genetic sequencing to evaluate viral etiologies of febrile neonates and infants <4 months with suspected sepsis or meningoencephalitis in Niigata area, Japan, in 2014-2016. The clinical manifestations of PeV-A3- and EVs-infected patients were compared, and a novel scoring system was developed after identifying the most distinguishable clinical findings, followed by the external cohort validation. RESULTS: In 210 patients evaluated, we identified 56 PeV-A3-infected (27%) and 43 EVs-infected (20%) patients. The following clinical manifestations were significant in PeV-A3-infected patients, as compared with EVs-infected patients; a higher body temperature (38.9°C vs. 38.5°C, P < .01) and heart rate (181/min vs. 168/min, P = .01), cold extremities (72% vs. 34%, P < .01) and skin mottling (65% vs. 23%, P < .01), lower white blood cell count (5,200/µL vs. 8,900/µL, P < .01) and incidence of cerebrospinal fluid (CSF) pleocytosis (2% vs. 63%, P < .01). Using some of these significant findings, the scoring system successfully distinguished the diseases (accuracy: 86% and 83% for the derivative and external validation cohorts, respectively). CONCLUSIONS: We found significant clinical manifestations in PeV-A3-infected patients compared to EVs-infected patients. The scoring system may be helpful to distinguish two infections, especially at onset of outbreak.