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Human parechoviruses (HPeVs) are newly recognized single-stranded RNA viruses that were formerly classified in the Enterovirus genus1). Among 16 HPeV serotypes, HPeV-3 infection occurs most frequently among infants below the age of 3 months2). Since HPeV-3 was first isolated in Japan in 19991), the HPeV serotype has been increasingly identified as an important pathogen of sepsis-like illness and central nervous system infections in neonates and young infants3). Life-threatening illnesses such as hemophagocytic lymphohistiocytosis have been reported in neonatal HPeV-3 infection4). However, the major clinical features displayed by patients with HPeV-3 infection are also common in those suffering from severe infectious diseases caused by other pathogens5). Thus, the diagnosis of HPeV-3 infection is difficult based only on clinical signs.
Recent studies have reported several clinical findings that are characteristic of HPeV-3 infection4678). Clinical features such as palmar-plantar erythema and hyperferritinemia might be diagnostic indicators of an HPeV3 infection in febrile neonates and young infants67). This report describes 2 young infants with an HPeV3 infection who presented with a prolonged fever, palmar-plantar erythema, and hyperferritinemia (>500 ng/mL). These cases may enhance our understanding of the unique features of HPeV-3 infection in young infants.
A 42-day-old male neonate was admitted to Gyeongsang National University of Hospital due to high fever and irritability. He was born at full-term gestational age at a weight of 3,200 g, and he was thriving until this hospital visit. Localized symptoms were not detected, and the results of a physical examination were unremarkable. His healthy older sister was reported to have had a recent febrile respiratory infection. The patient's initial vital signs were as follows: blood pressure 80/50 mmHg, heart rate 168 beats/min, respiratory rate 38 breaths/min, and body temperature 38.8℃. The laboratory findings at admission were as follows: hemoglobin, 9.0 g/dL; white blood cell (WBC) count, 1,990/mm3; absolute neutrophil count, 670/mm3; platelet count, 390×103/mm3; aspartate aminotransferase (AST), 42 U/L (range, 22–63 U/L); alanine aminotransferase (ALT), 23 U/L (range, 12–45 U/L); γ-glutamyl transferase (γ-GT), 36 U/L (range, 12–123); creatine kinase (CK), 147 U/L (range, 5–130 U/L); lactate dehydrogenase (LDH), 283 U/L (range, 170–580 U/L); ferritin, 385 ng/mL (range, 0–400 ng/mL); protein, 5.5 g/dL (range, 4.6–7.4 g/dL); albumin, 4.0 g/dL (range, 1.9–5.0 g/dL); and C-reactive protein (CRP), 0.5 mg/L (range, <7.9 mg/L). No cerebrospinal fluid (CSF) pleocytosis or pyuria was observed. Cefotaxime and ampicillin/sulbactam were administered.
No bacteria were found in blood, CSF, or urine samples. HPeV-3 was detected in CSF and serum samples by reverse transcription polymerase chain reaction (PCR) as described in our previous study9). CSF PCR tests were negative for herpes, enterovirus, cytomegalovirus, Epstein-Barr virus, and HPeV1. In addition, we found no respiratory viruses such as adenovirus, coronavirus, parainfluenza virus, rhinovirus, respiratory syncytial virus, influenza virus, bocavirus, and metapneumovirus. High fever and irritability persisted. At day 5 of admission, an erythematous rash and swelling were observed on the patient's hands and feet. The laboratory findings were as follows: hemoglobin, 8.7 g/dL; WBC count, 3,930/mm3; absolute neutrophil count, 260/mm3; platelet count, 160×103/mm3; protein, 4.4 g/dL; albumin, 2.9 g/dL; AST, 658 U/L; ALT, 162 U/L; γ-GT, 147 U/L; CK, 321 U/L; LDH, 1,324 U/L; ferritin, 2,581 ng/dL; and CRP, 0.3 mg/dL. Intravenous immunoglobulin (IVIG) was administrated because severe systemic inflammatory responses were considered in the patient. After IVIG treatment, the patient's fever subsided gradually and the erythematous rash disappeared. The patient was discharged on day 8 of admission.
The caliciviruses (family Caliciviridae) are non-enveloped, positive sense, single-stranded RNA viruses with diameters ranging from 27 to 40 nm. Caliciviruses cause a wide range of significant diseases in human and animals. At present, there are five recognized genera, i.e., Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus with several additional candidate genera or species proposed and under evaluation by the International Committee on Taxonomy of Viruses (ICTV) [1, 2] (http://www.caliciviridae.com/unclassified/unclassified.htm). In the Vesivirus genus, Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) are two species currently approved by ICTV. Several canine caliciviruses (CaCV) isolates have been identified and shown to be phylogenetically related to vesiviruses with features distinct from both VESV and FCV in phylogeny, serology and cell culture specificities. CaCV is a probable species in the Vesivirus genus, as stated by ICTV. It is still unclassified to date and the evidence presented herein should facilitate the classification and acceptance of CaCV as a species of vesivirus.
Many viruses found in human and other animal species can also infect dogs asymptomatically or cause respiratory, digestive, neurologic and genital diseases with mild to severe symptoms. In response to the use of dogs in military services and laboratory studies, etiological studies of canine diseases were conducted in 1963–1978 at the Walter Reed Army Institute of Research (WRAIR) [3, 4]. In addition to several known canine viral pathogens [5, 6], four unidentified viruses were recovered in Walter Reed Canine Cells (WRCC) producing similar cytopathic effects (CPE). The isolates were not recognized by available human and dog reference virus antisera. Studies of their physicochemical properties and electron microscope observations identified the isolates as likely caliciviruses. Our recent whole genome sequencing of these canine isolates clearly identified them as vesiviruses and elucidated their genetic relationships to the other members of the Caliciviridae family. We herein report the viral isolation and characterization results, which were made in 1963–1978 canine diseases etiological study but were not published, and additional genomics analysis supporting the serological diversity of CaCV strongly suggesting that these isolates and similar CaCV are a unique species within Vesivirus genus [7–9].
ZIKV, an emerging flavivirus, shares common clinical symptoms with DENV and chikungunya virus (CHIKV). The outbreaks caused by these viruses present a large number of diagnostic challenges. The clinical manifestations of ZIKV involve similar clinical symptoms to DENV and CHIKV, which include fever, exanthema, conjunctivitis, retro-orbital headache, and arthralgia (Cardoso et al., 2015). The diagnosis of viral infection has specific management implications for medical personnel. The identification of DENV requires a routine follow-up to examine thrombocytes along with hematocrit, whereas for CHIKV, chronic arthralgia should be assessed due to its high prevalence. In the case of ZIKV, a detailed diagnosis of sexual and maternal-fetal transmission should be performed to confirm the risk of congenital microcephaly in newborn babies (Fauci and Morens, 2016). A variety of arboviral infections (arthropod-borne; DENV is the most common arboviral infection) may have similar clinical presentations; therefore, their circulation may be under-reported if specific diagnostic tools have not been implemented. However, there are several drawbacks in ZIKV diagnosis due to the lack of availability of diagnostic tools and the frequent cross-reactivity of antibodies between flaviviruses, which have resulted in several limitations in the use of serology (Musso et al., 2015). Commonly, no routine testing of virus cultures is performed, and an antigenic detection test is lacking at present (Musso et al., 2015; Saiz et al., 2016).
The symptoms of ZIKV infection usually tend to be mild, and the initial symptoms can escape notice, reducing the opportunity to collect a sample. Although the viremic period has not been completely defined, viral RNA has been detected in serum after the onset of symptoms up to day 10. In addition, RNA particles of ZIKV have been detected in urine over an extended period in the acute phase, leading to the possibility of considering an alternative sample type. Evidence suggests that serum samples should be taken during the first 5 days after the onset of symptoms supported in some more detailed studies (Musso et al., 2015). Symptoms of microcephaly associated with ZIKV during the development of newborns in the uterus have been reported (Oduyebo et al., 2016). For the diagnosis of infant microcephaly, a complete analysis of head circumference is requested (Kallen, 2014), as the diagnostic parameters for severe microcephaly include a head circumference more than 3 standard deviations below the mean (Von der Hagen et al., 2014). Testing should be performed in pregnant women with positive or inconclusive results from ZIKV testing. If diagnostic parameters confirm possibility of congenital ZIKV infection in an infant, further clinical evaluation should be performed in follow-up. Fever is a common presenting symptom in patients testing positive for arboviruses due to their association with multiple illnesses; hence, it is suggested to eliminate differential diagnoses (Kelser, 2016). Patients with DENV and ZIKV present with temperatures >40°C and <38.5°C, respectively. ZIKV is usually self-limiting, with symptoms lasting 2 to 7 days. Jaundice is a distinguishing clinical presentation of yellow fever virus and can aid in identifying patients with ZIKV virus. The presence of nausea, vomiting, and bleeding may be helpful in identifying DENV. Any of the above symptoms in an individual who has been exposed to ZIKV indicates the possibility of ZIKV infection, and immediate serum testing should therefore be performed (Centers for Disease Control and Prevention [CDC], 2015a,b).
Recent scientific and clinical evidence has indicated that the virus is found during upper and lower respiratory tract infections, causing symptoms and signs that do not differ greatly from the symptoms described for the 'old' viruses HCoV-229E and HCoV-OC43. Other systems involvement is still controversial.
Table 1 shows that patients diagnosed with the virus have presented with mild symptoms, indicating upper respiratory tract infection such as fever, cough and rhinorrhoea. On the other hand, the disease is also known to cause significant more alarming lower respiratory tract infection. One of the most alarming symptoms is bronchiolitis, an inflammation of the membranes lining the bronchioles. This symptom was reported by several research groups [25, 50, 64], and although a population-based study in China did not report an association of HCoV-NL63 with bronchiolitis, it is still believed to be one of the presenting symptoms. Several research groups have linked HCoV-NL63 to croup [26, 53]. Croup children present with pharangitis, sore throat and hoarseness of voice, and are considered for hospitalization. Of the rare findings, a group has reported the association between HCoV-NL63 and Kawasaki disease, a form of childhood vasculitis that is presented as fever, polymorphic exanthema, oropharyngeal erythema and bilateral conjuctivitis. However, others fail to report on this association [66, 67].
It is noteworthy to say that the report of symptoms in young children, who represent the majority of patients, is based mainly on parental observations, where other possible subjective signs and symptoms fail to be recognized by the parents. Moreover, most of the studies were conducted on patients reporting to hospitals suffering from acute respiratory tract infection. To date, there are only few population-based studies and the question arises whether larger numbers of such studies might reveal the involvement of other body systems.
Zika virus (ZIKV), the causative agent of the infectious disease Zika fever, is a positive-sense RNA virus that belongs to the family Flaviviridae, genus Flavivirus, and is similar to Dengue virus (DENV), yellow fever virus, Japanese encephalitis virus, and West Nile virus (Sikka et al., 2016). ZIKV was first isolated from Rhesus macaques in Uganda in 1947. Previously, only sporadic cases of negligible concern associated with human ZIKV infection were reported (Hayes, 2009). Now, ZIKV infections have become epidemic throughout the world (Charrel et al., 2016).
In the north-eastern states of Brazil, the public health authorities recently confirmed autochthonous transmission of ZIKV with the first known reported case of ZIKV infection in mainland South America (Campos et al., 2015; Zanluca et al., 2015), followed by 26 countries, including countries in the European Union and the outermost regions of the Americas, such as Barbados, Bolivia, Brazil, Colombia, Costa Rica, Curacao, Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala, Guyana, Haiti, Honduras, Jamaica, Martinique, Mexico, Nicaragua, Panama, Paraguay, Puerto Rico, Saint Martin, Suriname, the US Virgin Island, and Venezuela (Pan American Health Organization [PAHO], 2016; World Health Organization [WHO], 2016). An increased frequency of ZIKV infection among world travelers has been reported in European countries, including Austria, Denmark, Finland, France, Germany, Ireland, Italy, Portugal, the Netherlands, Spain, Sweden, Switzerland, and the UK (European Centre for Disease Prevention [ECDC], 2016).
The virion of ZIKV consists of an approximately 11 kb positive-sense RNA with a single capsid and two membrane-associated envelope proteins (M and E) (Leyssen et al., 2000; Daep et al., 2014; Charrel et al., 2016). Recent outbreaks of ZIKV infections have become fatal on a daily basis in the Americas, where this obscure viral candidate has been placed at the forefront of global healthcare. The reported occurrences of ZIKV infections are thought to be transmitted mainly by the mosquito species Aedes aegypti and Aedes albopictus. Infections have now dramatically increased in highly populated areas of South, Central, and North America due to the increased frequency of the international travel from Zika-infected areas (Bogoch et al., 2016). Considering the calamity of ZIKV infection, there is an urgent need to develop rapid detection methods for ZIKV along with DENV, which shares common clinical symptoms with ZIKV. The purpose of this review is to provide a complete update of the various analytical methods for virus detection, such as molecular, immunological, sensor-based and other detection assays, along with the advantages and limitations of these strategies. Furthermore, we suggest innovative hypothetical approaches for the development of liposome-based rapid detection assays for ZIKV detection, which will provide new insight to medical professionals for controlling this widespread epidemic virus candidate.
Acute respiratory infections (ARIs) are the most common infections in humans of all ages. Children and infants are one of the most vulnerable groups of the population, and ARIs are the most common cause of children's hospitalization worldwide. Although bacteria, fungi, and parasites can cause ARIs, respiratory viruses cause the majority of infections. Most respiratory virus infections in early childhood are confined to the upper respiratory tract. About one-third of infants develop lower respiratory tract infection (LRTI). The most common causative viral agents of ARIs in children, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza viruses (Flu), and adenoviruses (AdV), were the subject of intensive research for years; therefore, clinical characteristics and regional epidemiological features of those ARIs in Croatia are well known [3–6]. However, the list of respiratory viruses is growing due to the rapid advance of laboratory diagnostic methods. In the last ten years, newly discovered viruses have been identified including human bocavirus (HBoV), coronaviruses NL63 (HCoV-NL63) and HKU1 (HCoV-HKU1), new enterovirus (HEV), parechovirus (HPeV), and rhinovirus (HRV) strains. Additionally, despite the fact that some of the respiratory viruses have been well known for a long time, particularly parainfluenza type 4 (PIV-4), the technically demanding cultivation methods and unavailability of commercial tests made it difficult to diagnose PIV-4's infection [8, 9]. Infections caused by some of the newly discovered viruses (i.e., HBoV, HCoV-NL63, and HCoV-HKU1) as well as those difficult to cultivate (PIV-4) have not been recorded in the country yet. There are few recent studies from the region providing valuable but still insufficient data regarding regional epidemiology of infections caused by the abovementioned viruses [10, 11]. Furthermore, the issue of multiple respiratory virus detection, which occurred because of high sensitivity of molecular methods, complicates the interpretation of laboratory diagnosis. The aim of this study was to determine the viral etiology for sixteen viruses tested by multiplex PCR method among children with ARI admitted to the hospital in Zagreb region in two respiratory seasons, in order to demonstrate the need for molecular diagnostics introduced in routine practice. Also, we aimed to investigate the characteristics of infections with single and multiple virus detection, especially regarding the type of virus involved and severity of infection.
ZIKV infection is transmitted mainly by Aedes aegypti mosquitoes, sexual contact, or blood transfusion. It is typically a mild, asymptomatic disease in the general population.
The disease is a self-limiting febrile illness lasting 4–7 days. Infection can be followed by neurological consequences including Guillain-Barre syndromes and microcephaly or other congenital neurological syndromes after vertical transmission from an infected mother to her fetus during pregnancy.
Noroviruses are the most common cause of foodborne disease and acute nonbacterial gastroenteritis worldwide.
Its prevalence was 2% in adults and up to 22% among pediatric transplant recipients with diarrhea, requiring hospitalization in 55% and ICU admission in 27%. Recurrence rate 29%.
Risk factors: second HSCT, intestinal GVHD, children.
Norovirus can cause severe, prolonged disease complicated by enteritis, fever, recurrent hospitalizations for dehydration, chronic diarrhea, acute renal failure, weight loss, malnutrition, pneumatosis intestinalis, peritonitis, secondary bacteremia, and death.
Hand, foot, and mouth disease (HFMD) is an infectious disease that usually affects infants and young children under 5 years of age worldwide. HFMD typically causes self-limiting illness, but development of severe cardiopulmonary and neurologic complications have also been reported [1, 2]. The clinical manifestations are typically ulcerations in the oral cavity, buccal mucosa (enanthema) and tongue with peripherally distributed cutaneous lesions and vesicular rash (exanthema) on the palms of hands and soles of feet. Other parts of the limbs including knees, elbows and buttocks may also be affected. Transmission occurs via person-to-person through direct contact with respiratory secretion, saliva, fluid from blisters, and feces from infected individuals. A number of enteroviruses belonging to the family Picornaviridae cause HFMD, although human enterovirus 71 (EV71) and coxsackievirus (CV) type A16 are two of the most important enteroviruses implicated in many large-scale outbreaks in Asian-Pacific countries including Japan, Taiwan, Malaysia, Singapore, and China [2–4]. Additional enterovirus species including CV-A6, CV-A10 and CV-A4 also cause HFMD [5–9]. Clinical symptoms resulting from CV-A16 as well as other enteroviruses are usually relatively mild and indistinguishable with low incidence of severe complications. In contrast, serious complications such as encephalitis, myocarditis, and poliomyelitis-like illness were observed when EV71 were reported as the causative pathogen [10–12].
HFMD has been continuously present and remains a major cause of morbidity and mortality of young children particularly in Asia. Typically, HFMD exhibits cyclical pattern of outbreaks every two to three years. Factors underlying the prevalence of HFMD remain controversial. Data from countries with long history of HFMD outbreaks suggest that dissemination is associated with socio-economic status, population ethnicity, regional climate [14–16], and attendance in school or care centers of school-age children. The magnitude of HFMD outbreaks appears to fluctuate [3, 18, 19]. HFMD in tropical climate countries, such as Malaysia, Singapore and Thailand, typically showed years-round activity with no discrete epidemic periods although peaks during the rainy and winter seasons were also detected depending on season, year, and geographic regions [20, 21]. At present, no specific treatment for HFMD exists. Vaccines or antiviral drug against EV71 are currently being developed in Taiwan, China and Singapore but are not yet commercially available [22–24]. Prompted by geographically widespread outbreaks, careful monitoring of the spatial and temporal epidemiology is considered to be of great importance to control the spread of HFMD according to the different regional characteristics. As reported by the Bureau of Epidemiology, Ministry of Public Health of Thailand, HFMD has shown an upward trend in the last five to six years. In June 2012, the largest recorded outbreak of HFMD occurred throughout the country. This outbreak affected more than 39,000 individuals, including three deaths, over a period of four to five months with hot spots in Chiang Rai and Mae Hong Son provinces. In our previous study, we monitored HFMD activity in Thailand between 2008 and 2012. Our results revealed that the HFMD epidemic in 2012 was significantly different from previous ones in Thailand including the size of the epidemic and the viruses detected [26, 27]. During the 2012 epidemic, beside a high prevalence of EV71 and CV-A16, multiple EV types such as CV-A6 were also detected. Even though the standardized 5' untranslated region (5'UTR) pan-enterovirus PCR and viral capsid protein 1 (VP1) gene typing PCR assays were used, approximately one third of the suspected cases, mostly young children, were negative for enterovirus by these assays. These findings raised questions regarding the sensitivity of the current assay in identifying causative viruses other than EV71 and CV-A16 that may be present below the limit of detection by conventional PCR. In recent years, metagenomic has become an important strategy for virus discovery in human and animal diseases [28–30]. This technique, based on recognition of sequence similarities following non-specific nucleic acid amplification, circumvents some of the limitations of virus isolation, serology, and the amplification of only known conserved genomic regions. To evaluate circulating enterovirus and previously uncharacterized viruses associated with HFMD, we describe here the virus community (virome) in fecal samples negative by RT-PCR for EV71 and CV-A16/A6 obtained from 29 pediatric patients with HFMD during the outbreak in Thailand in 2012.
A total of 596 of 600 included patients, were discharged as cured after a mean of five days of hospitalization. Two patients died while in hospital; one of meningitis presenting clinically with meningismus and deep coma, one of pneumonia (clinically suspected pulmonary tuberculosis complicated by bacterial pneumonia). One child, diagnosed with Staphylococcus aureus bacteremia, was referred to another hospital after the development of Guillain-Barré syndrome. Another child was taken from the hospital, by his parents, against medical advice (Fig. 1).
Overall, in our study population, neither pre-existing conditions, such as malnutrition or sickle cell anemia nor concomitance of multiple diagnoses were associated with a negative outcome.
A total of 134 children admitted to Children's Hospital Zagreb during two winter seasons (January to March) in 2010 and 2015 with symptoms of ARI and suspected for viral etiology (normal or slightly elevated inflammatory markers, i.e., white cell count) were included in the study. Patients were categorized into three groups according to age (<1, 1–3, and ≥4 years of age) and two groups according to the localization of infection in those with upper respiratory tract infection (URTI) and lower respiratory tract infection (LRTI). URTI was defined by symptoms of the common cold, coryza, cough, and hoarseness often accompanied with fever. Clinical syndromes of respiratory catarrh, rhinitis, and/or pharyngitis are included in URTI category. LRTI was defined according to the clinical symptoms of tachypnea, wheeze, severe cough, breathlessness, and respiratory distress accompanied by LRTI signs such as nasal flaring, jugular, intercostal, and thoracic indrawings, rarely cyanosis, and, on auscultation of the chest, wheeze, crackles, crepitations, and inspiratory rhonchi or generally reduced breath sounds. Clinical syndromes of bronchitis, bronchiolitis, and pneumonia were included in LRTI category. To avoid unnecessary X-ray exposure, chest radiographs were taken only for some of the patients to exclude or confirm bacterial pneumonia. Severe disease and acute respiratory distress syndrome (ARDS) were defined with need for oxygen supplementation and/or mechanical ventilation. The patients' underlying conditions data were collected retrospectively from medical charts. The most common underlying diseases were asthma, anamnestic recurrent wheezing episodes, neurological disorders, prematurity, and anemia. Written consent was obtained from the children's parents or caretakers. The study was approved by the Ethic Committee of the Teaching Institute of Public Health “Dr. Andrija Stampar.”
Nasopharyngeal and pharyngeal flocked swabs from each patient were collected, combined, and placed in viral transport medium (UTM™, Copan, Italy). Specimens accompanied with demographic data and clinical diagnosis were immediately transported to the Molecular Microbiology Laboratory at the Public Health Institute where they were stored at −80°C until tested. Nasopharyngeal and pharyngeal swabs, blood cultures, and serum for serology were searched to exclude bacterial infection. Patients with samples positive on bacteriology testing were subsequently excluded from the study.
Causes of fever in African pediatric populations are more diverse than previously thought. A landmark study conducted in Tanzania showed that due to a change in epidemiology, a broad spectrum of pathogens replaced P. falciparum malaria as the most common cause of disease in children in this area1. However, a few years later, P. falciparum malaria, is still seen to be the main cause of febrile illnesses in Ghana, West Africa2. When unaware, these differences in epidemiology might lead to misdiagnosis as well as inefficient treatment by the medical personnel. The process of medical diagnosis includes the joint interpretation of symptoms, clinical signs and laboratory findings. Careful selection and prioritization of a diagnostic setup are informed by a priori knowledge of the seasonal, local and worldwide frequency and distribution of a given disease3,4.
Our study describes the distribution of infections, co-infections, and co-morbidities in children hospitalized for febrile illnesses at the Albert Schweitzer Hospital (HAS) in Lambaréné, Gabon, as an example for a hospital in a semiurban Central African region. In addition, we present the current spectrum of pathogens causing severe disease identified by Lambaréné Organ Dysfunction Score (LODS) in these children.
Since the coincidental isolation of Seneca Valley virus (SVV), recently termed Senecavirus A (SV-A), as a cell culture media contaminant in 2002, a number of serologically similar viruses were identified and grouped to the classification of Senecavirus. The primary sequence analysis of the conserved polypeptide regions (P1, 2C, 3C and 3D) of the first isolate (SVV-001) showed that the virus is most closely related to cardioviruses in the family of Picornaviridae. The single-stranded RNA genome of SV-A displays the secondary structural features of an internal ribosome entry site (IRES) that resembles the IRES element of classical swine fever virus (CSFV) of the family Flaviviridae, giving rise to the possibility that genetic exchange may have occurred between members of Picornaviridae and Flaviviridae during persistent co-infection in pigs. Importantly, SV-A is a natural oncolytic agent, with the ability to selectively replicate in; and kill human tumor cells of neuroendocrine origin, thus, the virus is being advanced as a tool for potential therapeutic intervention of cancer.
Swine are considered to be the natural hosts of SV-A and all known SV-A sequenced isolates have been obtained from pigs. Previously, by regression analysis of partial genome sequences, it was suggested that different isolates of SV-A had a common ancestor and were assumed to have been introduced into the US pig populations (http://www.europic.org.uk/Europic2006/posters/Knowles.svv.01.pdf). Virus isolated in cell culture from tissue specimens of a diseased pig presenting vesicular lesions on the snout and feet in 2005, was identified by the National Veterinary Services Laboratories’ (NVSL) Foreign Animal Disease Diagnostic Laboratory (FADDL) as SV-A using a broad pan-viral microarray (unpublished data). More recently, this vesicular disease syndrome, with as yet unidentified etiology, has been termed swine idiopathic vesicular disease (SIVD) [5, 6]. Despite the isolation of SV-A in cell culture, FADDL has been unsuccessful at reproducing clinical signs by experimental inoculation of pigs with live virus. Negative observations were also made by other laboratories who conducted animal inoculations with multiple SV-A isolates. Singh et al (2012) proposed SV-A as the causative agent of SIVD from a detailed clinical, diagnostic and histopathological study on a Chester White boar suffering from anorexia, lethargy, lameness and vesicular lesions. However, association of SV-A with SIVD, or as the sole causative agent, is speculative at this time since the virus has also been isolated from pigs lacking clinical disease. SIVD has been reported in pigs in the continents of North America and Australia [6, 9–11]. Although SIVD itself does not pose an economic concern, veterinary diagnosis from clinical signs is complicated since similar vesicular lesions can be formed due to common viral infections such as parvovirus, enterovirus, toxins in food supply, or burns [12–16]. Additionally, SIVD clinically resembles high consequence transboundary animal diseases (TADs) such as foot and mouth disease (FMD), swine vesicular disease (SVD), vesicular stomatitis (VS), and vesicular exanthema of swine (VES). A few laboratory methods have been developed for detection of SV-A including a virus serum antibody neutralizing test and a competitive enzyme linked immunosorbant assay (cELISA), which are not widely available [7, 17]. The principal aim of this study was to develop a specific real-time RT-PCR (RT-qPCR) assay for fast, sensitive, and quantitative detection of SV-A RNA in vesicular diagnostic tissues.
Due to its high specificity and sensitivity, reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid tests are the preferred methods for diagnosis of coronavirus infections, such as SARS-CoV. The majority of nucleic acid amplification tests is designed with the ORF1a/1b, which is genetically stable in coronaviruses, and the nucleocapsid (N) or spike (S) genes (Table 1) [25, 26, 28, 30, 45, 46, 64, 67, 71-73]. Based on in vitro coronavirus expression studies, the N gene has the theoretical advantage of being more abundant in infected cells and therefore of higher sensitivity for PCR assays, but this has not been clearly proven in clinical studies. RT-PCR of nasopharyngeal samples, both frozen and fresh, is the most popular choice for detection of HCoV-NL63 (Table 1) and viral culture is frequently used for confirmation of infection [67, 73].
Seneca Valley virus (SVV) is a single-stranded, positive-sense RNA virus belonging to the species Senecavirus A in the genus Senecavirus in the family Picornaviridae [1, 2]. Although the species name Senecavirus A has been used in some publications as the virus name with an acronym of SVA, in fact the virus name is Seneca Valley virus. The SVV genome (approximately 7.3 kb) contains a single open reading frame (ORF) flanked by a long 5′ untranslated region (UTR; 668 nucleotides) and a short 3′ UTR (68 nucleotides) followed by a poly(A) tail. The polyprotein translated from the single ORF is predicted to be post-translationally processed into four structural proteins (VP4, VP2, VP3, and VP1) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D).
SVV was initially incidentally identified as a contaminant in PER.C6 cell cultures in 2002. From 1988 to 2001, a number of virus isolates were sporadically recovered from pigs in various U.S. states but with no detailed description of the clinical symptoms. Sequence analysis of these retrospective virus isolates suggested that these viruses were the same as SVV. Thereafter, SVV was sporadically identified in pigs with swine idiopathic vesicular disease in Canada in 2007 and in the U.S. in 2010, but not much attention was drawn to this virus. At the end of 2014 and the beginning of 2015, multiple outbreaks of vesicular disease in weaned and adult pigs as well as increasing mortality rates of neonatal piglets (1–4 days of age) were reported in Brazil [6–8]. SVV was consistently detected from the pigs with vesicular lesions while other vesicular viral pathogens were not detected. Starting from July 2015, SVV was consistently detected from increasing swine idiopathic vesicular disease cases observed in exhibition, commercial finisher, and breeding swine herds in the U.S.. Foreign animal disease investigations indicated that other vesicular viral pathogens, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV), were negative in these cases. Subsequently, SVV detection was reported by other laboratories in the U.S. [11–16], China [17–21], Canada, Thailand, and Colombia. Vesicular lesions were induced in pigs following experimental inoculation with the contemporary U.S. isolates of SVV [25, 26], confirming that SVV is a vesicular viral pathogen. In one experimental infection study, a historical SVV isolate (SVV-001) did not cause overt clinical diseases in the inoculated pigs but it established infection in pigs and induced an immune response. Since the vesicular lesions caused by SVV infection are clinically indistinguishable from those caused by other vesicular disease viruses (e.g., FMDV, SVDV, VSV, and VESV), differential diagnosis is mandatory. RT-PCR is a sensitive and fast method commonly used to differentiate vesicular viral pathogens.
A number of SVV specific gel-based (conventional) RT-PCR, nested RT-PCR, real-time RT-PCR (rRT-PCR), reverse transcription droplet digital PCR, and loop-mediated isothermal amplification assays have been described in the literatures although not all of them have been fully validated [6, 28–35]. Compared to the conventional RT-PCR assays, rRT-PCR is generally more sensitive and suitable for high throughput testing with shorter turnaround time. It is noteworthy that conduction of rRT-PCR assays requires trained technicians and expensive instruments; rRT-PCR assays are mainly performed in the laboratory rather than for on-site applications.
In recent years, a fluorescent hydrolysis probe-based insulated isothermal PCR (iiPCR) technology has been described. The iiPCR and RT-iiPCR can be used for the detection of DNA and RNA molecules. The principle of the iiPCR is to amplify the DNA/RNA by cycling the reaction components through different temperature gradients (denaturation, annealing, and extension) in a capillary tube on a simple Nucleic Acid Analyzer [36, 37]. The iiPCR technology and a commercially available field-deployable device (POCKIT™ combo system), which includes a taco™ mini Automatic Nucleic Acid Extraction System and a POCKIT™ Nucleic Acid Analyzer (GeneReach USA, Lexington, MA, USA), allow automatic detection and interpretation of PCR results within 1–1.5 h. It has been shown that iiPCR or RT-iiPCR assays have excellent sensitivity and specificity for the detection of various targets, including swine pathogens, such as classical swine fever virus (CSFV), FMDV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV) [38–41], and various pathogens in shrimp, dogs, cats, poultry, ruminants, and horses [42–52].
In the present study, a SVV rRT-PCR targeting the conserved 5′ UTR and a SVV RT-iiPCR targeting the conserved 3D gene region were developed and validated for the detection of SVV RNA.
Respiratory symptoms are frequently observed in children with Kawasaki disease (KD) during the acute phase. The association rate of KD with antecedent respiratory illness has been reported to be 56 to 83%1,2). Laboratory evaluation for detection of respiratory viruses is occasionally performed in febrile children with respiratory symptoms before confirmation of the diagnosis of KD. Recently, molecular diagnostic tests, such as real-time polymerase chain reaction (PCR), have been more frequently used as a laboratory method, because molecular diagnostic tests for the detection of all epidemic respiratory tract infection-related viruses allow a thorough etiological assessment and better management of children with respiratory tract infection3). However, we propose that identification of respiratory virus during the diagnostic evaluation of febrile children with respiratory symptoms cannot be used as exclusive evidence against the diagnosis of KD. In one study without control subjects, the identification rate of respiratory viruses through multiplex reverse transcriptase (RT)-PCR in children with KD was reported to be 22%4).
According to a recent retrospective study, patients with KD who harbor respiratory viruses have a higher frequency of coronary artery dilatation and are more often diagnosed with incomplete presentation of the disease5). If this is correct, identification of respiratory viruses should be considered to be a risk factor in children with KD.
We planned this study to survey the detection rate of respiratory viruses in children with KD through RT-PCR using a prospective case-control study design and to investigate the clinical impact of the existence of respiratory viruses during the acute phase of KD.
In our patient, we examined the antibody titers of viruses that cause an acute eruptive disease, such as Coxsackievirus (types A2–A5, B1–B4), adenovirus (types 2–4), echovirus (types 1, 3, 5, 7), herpes simplex virus, Epstein–Barr virus, cytomegalovirus, human immunodeficiency virus, and human parvovirus. We analyzed serum samples taken on days 5, 13, and 32 of illness for antibody titers of Coxsackievirus, adenovirus, and echovirus. The day 5 serum sample was taken from the previous hospital. The antibody titer of only Coxsackievirus A4 was increased fourfold on day 13 as compared to day 5 and was decreased on day 32. This strongly suggests that the Coxsackievirus infection was present when KD occurred. Coxsackievirus A4 is a member of the Picornaviridae family of viruses in the Enterovirus genus. It can be a cause of herpangina and myocarditis. Rigante et al reported the cases of Kawasaki syndrome and concurrent Coxsackievirus B3 infection.13 However, to the best of our knowledge, there have been no reports of KD cases and concurrent Coxsackievirus A4 infection thus far. There are various types of adenovirus and Coxsackievirus, and tests for proving these common viral infections (eg, serological antibody, polymerase chain reaction) are performed in few cases. Therefore, such coinfections remain undiagnosed and are thus likely unreported.
Vesiviruses are small (~35-nm), nonenveloped, single-stranded RNA viruses belonging to the family Caliciviridae, which is currently divided into five genera: Vesivirus, Lagovirus, Nebovirus, Sapovirus, and Norovirus. The vesivirus RNA genome is organized into three major open reading frames (ORFs) (1). The nonstructural proteins are encoded within a large polyprotein in ORF1 beginning at the 5′ end of the genome and are released by proteolytic cleavage during replication. The ORF2 sequence encoding a capsid precursor protein is located toward the 3′ end of the genome and overlaps ORF3, which encodes a basic minor structural protein, VP2. The capsid precursor and VP2 proteins are expressed from an abundant subgenomic RNA in infected cells (2, 3). Maturation of the vesivirus major capsid protein VP1 involves proteolytic cleavage of the capsid precursor protein between the capsid leader sequence (LC) and VP1 by the same viral proteinase that mediates processing of the ORF1 polyprotein (4). The assembly of infectious calicivirus particles requires 180 monomers of the VP1 (5). Calicivirus VP1 can be structurally subdivided into two domains, the N-terminal shell (S) domain involved in the assembly of the icosahedral scaffold of the virion and the C-terminal protruding (P) domain that forms arch-like structures on the virion surface (5). The sequence variability of the latter domain defines the antigenic diversity of caliciviruses and is thought to be responsible for the marked differences in tropism among caliciviruses. Accordingly, the P domain contains virus neutralization epitopes and amino acid residues involved in attachment of the virus to cells (6, 7).
Vesiviruses infect a broad range of animal hosts and are associated with various chronic and acute illnesses (8). They cluster into three phylogenetically distinct groups. One group consists of feline calicivirus (FCV) strains, and a second includes canine calicivirus (CaCV) and related strains. The third and largest group (known as the “marine” vesiviruses) includes viruses closely related to the vesicular exanthema of swine virus (VESV), first associated with a foot-and-mouth disease-like syndrome in pigs in the United States in the 1930s (9). The transmission of a marine vesivirus, San Miguel sea lion virus (SMSV), to pigs has been observed experimentally, but with variable results (10, 11), and the frequency of interspecies transmission in nature remains unclear.
The zoonotic potential of these viruses is not known. It has been reported that marine vesiviruses can infect several primate species. VESV-related viruses have been isolated from pygmy chimpanzees, douc and silver leaf langurs, spider monkeys, and lowland gorillas (12–14). The only recorded case of vesivirus isolation from a human patient resulted from the apparent accidental infection of a laboratory worker (15). The researcher, working with CsCl-purified SMSV-5 virions, developed an influenza-like fever and vesicular lesions on all four extremities. The virus was isolated from one of the vesicles, and sequencing of the polymerase region showed a close relationship to the SMSV-5 strain studied in the laboratory (15). The virus was recovered in Vero cells and designated SMSV-5 Homosapien-1 or Hom-1. Here, we report the genetic characterization of this virus and show the ability of Hom-1 to replicate in several human cell lines of hepatic and intestinal origin. Using virus-like particles (VLPs), high-throughput screening of an expression library of human plasma membrane proteins (hPMPs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mutagenesis, we show that the Hom-1 vesivirus can interact with hJAM1 to enter cells and establish a productive infection.
Dog throat, rectal and penile swabs were collected, placed in 2–5 ml of veal infusion broth transport media (Difco Laboratories Inc., Detroit, MI) and frozen at < -60 °C until processed for virus isolation and identification. Blood specimens were collected from each dog and dogs in each cohort at the time and 14–28 days later.
Here, we describe a case of adult-onset KD which revealed to be concurrently infected by Coxsackievirus A4. Adult-onset KD is rare, and this is a very rare case of KD and concurrent Coxsackievirus A4 infection.
KD most commonly develops in infants. The annual incidence is 67 cases per 100,000 children in Japan and 5.6 cases per 100,000 children in the USA. Children under 5 years of age constitute 88.5% of reported cases.2,3 KD occurs predominantly in children while rarely in adolescents and adults.4 The oldest reported case was that of a 68-year-old Caucasian man from France in 2005.9 The diagnostic criteria for KD as defined by the Centers for Disease Control and Prevention include unexplained fever lasting 5 days or more and at least four out of the five following criteria: 1) polymorphic exanthema; 2) changes in the peripheral extremities, that is, erythema and/or indurative edema of the palms and soles (acute phase) or desquamation around the finger tips (convalescent phase); 3) bilateral non-exudative conjunctival injection; 4) changes in the oropharynx, that is, injected or fissured lips, strawberry tongue, and injected pharynx; and 5) acute nonsuppurative cervical lymphadenopathy (>1.5 cm in diameter). Patients with fewer than four of these clinical signs can be diagnosed as having atypical KD if coronary artery abnormalities are present.8 Additionally, it is necessary to exclude the possibility of other diseases that cause fever and rash (eg, toxic shock syndrome, streptococcal scarlet fever, measles, other viral infections, Stevens–Johnson syndrome, or a drug reaction).
Desquamation is typically found during the convalescent phase. However, in patients who develop KD in adulthood, the time of onset of desquamation is varied. Some of these patients have been reported to develop desquamation during the acute phase of KD with fever.9,10 This may be associated with the fact that multiple factors cause KD and that the mechanism of KD differs between adults and children. In our patient, desquamation appeared relatively early and then improved. Although the side effects of cefcapene-pivoxil and loxoprofen should be considered in the differential diagnosis, both drugs were used without adverse effects in this patient before this episode. Item (2) in the diagnostic criteria was satisfied even if desquamation was excluded. Therefore, desquamation does not affect the diagnosis.
Viral infections pose a significant global health burden, especially in the developing world where most infectious disease deaths occur in children and are commonly due to preventable or treatable agents. Effective diagnostic and surveillance tools are crucial for reducing disability-adjusted-life-years (DALYs) due to infectious agents and for bolstering elimination and treatment programs. Previously unrecognized and novel pathogens continually emerge due to globalization, climate change, and environmental encroachment, and pose important diagnostic challenges,.
Dengue virus (DENV) infection is the most common arthropod-borne viral disease of humans, with an estimated 50–100 million clinical infections occurring annually worldwide. DENV infection manifests clinically as dengue fever or the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The increased spread of dengue virus and its mosquito vectors in many subtropical regions over the past several decades, especially in Latin America and Asia, highlights the need for additional methods of dengue virus surveillance. Diagnosing dengue relies on detecting viral nucleic acid or antigens in the blood or confirming the presence of anti-DENV IgM and IgG antibodies and therefore traditionally depends on RT-PCR, ELISA, and viral cell culture methods–. Dengue diagnostics are of crucial importance due to its broad spectrum of clinical presentations, global emergence and spread, unique disease epidemiology, and possible clinical relation to other as-yet unknown tropical febrile pathogens.
Traditional viral detection methods, such as serology, virus isolation, and PCR, are optimized for the detection of known agents. However, novel and highly divergent viruses are not easily detected by approaches that rely on a priori sequence, antigen, or cell tropism knowledge. PCR-based assays that employ degenerate primers may successfully target conserved regions within related virus groups, but unlike bacteria, viruses lack universally conserved genetic regions, such as ribosomal RNA, that can be exploited to amplify all viruses.
Metagenomic analysis enables more systemic detection of both known and novel viral pathogens– and is approached through a variety of microarray and sequencing strategies,. The Virochip is a pan-viral microarray platform that has been previously utilized in the detection and discovery of viruses from both human and animal samples–. Deep sequencing and shotgun sequencing of human clinical samples has been used for viral detection–, novel virus discovery–, and divergent virus genome recovery. Viral metagenomic approaches have also been employed as a diagnostic supplement to pathogen detection as part of public health monitoring systems, but have been limited to shotgun sequencing of viral-enriched libraries and have yet to utilize deep sequencing data. Currently available sequencing platforms can generate millions to billions of sequencing reads per run, far exceeding large-scale shotgun sequencing. Deep sequencing of clinical samples, in which hundreds of thousands to millions of sequencing reads are generated per sample, can be incorporated into stepwise virus detection pipelines. Database searches using Basic Local Alignment Search Tool (BLAST) and other alignment tools can be used to identify sequences in samples that correspond to known and novel viruses, including those present at low concentrations or deriving from viruses that may be too divergent to be detected with PCR or microarray methods. Deep sequencing represents an unbiased, highly sensitive method for identifying viral nucleic acid in clinical samples.
This study describes the use of the Virochip microarray and deep sequencing for the direct viral diagnosis of serum from cases of acute pediatric febrile illness in a tropical urban setting. Patient clinical data and serum samples were collected between 2005 and 2009 as part of an ongoing pediatric dengue study in Managua, Nicaragua. Virochip and deep sequencing were performed on positive control samples and on 123 dengue virus-negative serum samples. Using these methods, viruses were detected in 45 of 123 (37%) previously negative samples. Sequences derived from known and apparently divergent viruses. The viruses identified in some of the cases are known to induce symptoms consistent with those observed, though the definitive causative agent of these infections remains to be determined.
Those with PCP present with symptoms of subtle onset dyspnoea, a low-grade temperature and a non-productive cough, and when examined, the chest is clear on auscultation. However, this may rapidly change with the onset of hypoxia requiring admission to a critical care unit. Imaging of the chest with X-ray reveals bilateral perihilar interstitial infiltrates that become increasingly homogenous and diffuse as the disease progresses. Computed tomography (CT) scans show extensive ground-glass attenuation or cystic lesions (Thomas and Limper 2004).
Patients have a 50% mortality associated with the development of PCP; prompt diagnosis and treatment are warranted with adherence to prophylactic cover. Due to the difficulties of culturing samples, the diagnosis of PCP is made through microscopic examination of sputum or bronchoalveolar fluid or by polymerase chain reaction (PCR).
Swimming pools have been implicated in the transmission of infections. The risk of infection has mainly been linked to fecal contamination of the water, generally due to feces released by bathers or to contaminated source water. Failure in disinfection has been recorded as the main cause of many of the outbreaks associated with swimming pools.
The majority of reported swimming pool-related outbreaks have been caused by enteric viruses. Sinclair and collaborators reported that 48% of viral outbreaks occur in swimming pools, 40% in lakes or ponds, and the remaining 12% in fountains, hot springs, and rivers (4% each).
Viruses cannot replicate outside their host’s tissues and cannot multiply in the environment. Therefore, the presence of viruses in a swimming pool is the result of direct contamination by bathers, who may shed viruses through unintentional fecal release, or through the release of body fluids such as saliva, mucus, or vomitus. Evidence suggests that skin may also be a potential source of pathogenic viruses.