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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.

House dust mite is a common cause of atopic dermatitis (AD) both in humans and dogs. Detection of serum IgE to allergens is commonly used to diagnose allergic diseases. However, false-positive reactions due to cross-reactivity and non-specific reactivity may lead to misdiagnosis. We compared human and canine IgE reactivities to mite component allergens. Canine IgE-reactive components of Dermatophagoides farinae and Tyrophagus putrescentiae were identified by tandem mass spectrometry. Recombinant proteins were produced and IgE reactivities to component allergens were assessed by ELISA and inhibition assays using sera from AD patients and dogs. Canine IgE-reactive proteins (Der f 1, Der f 11, Tyr p 4, Tyr p 8, Tyr p 11, Tyr p 28) were identified by proteome analysis. Most patients were sensitized to Der f 1 (93.3%) and Der f 2 (86.7%). Dogs showed high sensitization to Der f 2 (94.1%) and Der f 18 (84.6%). Both patients and dogs showed low IgE binding frequency to Tyr p 8, 43.3% and 4%, respectively. The ELISA inhibition study indicated that canine IgE reactivity to T. putrescentiae is mostly due to non-specific reaction and cross-reaction with D. farinae. Different IgE sensitization patterns were shown between allergic humans and dogs with AD, especially to Der f 18, for the first time in Korea. Furthermore, non-specific canine IgE reactivity to storage mite indicates the possibility of misdiagnoses. Standardizations focused on the major canine allergen content of extracts should be developed. This will allow precision diagnosis and individuated treatments for each patient and atopic dog., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Diarrhea is one of the most common clinical symptoms in cats and can be caused by infectious pathogens and investigation of the prevalence, co-infection and seasonality of enteropathogens are not well-established in diarrheic cats., Results: Fecal samples of 1620 diarrheic cats were collected and enteropathogens were detected using real-time PCR. We retrospectively investigated the clinical features, total/seasonal prevalence, and infection patterns of enteropathogens. The positive infection rate was 82.59%. Bacterial, viral, and protozoal infections accounted for 49.3, 37.57, and 13.13% of cases, respectively. Feline enteric coronavirus (FECV) was the most common pathogen (29.37%), followed by Clostridium (C.) perfringens, Campylobacter (C.) coli, feline parvovirus, and Tritrichomonas foetus. The seasonality of enteropathogens was observed with peaks as follows: bacterial infections peaked in October, viral infections peaked in November, and protozoal infections peaked in August. Viral and protozoal infections showed differences in prevalence according to patient age. In the infection patterns, the ratios of single infections, mixed infections, and co-infections were 35.72, 9.87, and 54.41%, respectively. FECV was predominant in single infections. The most common patterns of multiple infections were C. perfringens and C. coli in mixed infections and C. perfringens and FECV in co-infections., Conclusions: Infection patterns differed according to the enteropathogen species, seasonality, and age distribution in cats. The results of this study might be helpful to understand in clinical characteristics of feline infectious diarrhea. In addition, continued monitoring of feline enteropathogens is required., (© 2021. The Author(s).)

Canine mammary gland tumour (cMGT) is the most common tumour in intact female dogs. Surgery is the only effective treatment for cMGT, and dogs with metastasis at the time of diagnosis or those diagnosed at an advanced stage have poorer prognosis. Thus, novel diagnostic biomarkers and therapeutic targets are needed. Neurokinin-1 receptor (NK-1 receptor) is involved in cancer progression and has been detected in various malignant tumours including breast cancer in humans. Furthermore, NK-1 receptor antagonists inhibit cancer progression. We evaluated NK-1 receptor expression in malignant and benign cMGT compared with that in normal mammary gland tissues and analysed the relationship between the expression of NK-1 receptor and histopathological type or malignancy grade. Specimens from 34 malignant MGT and 35 benign MGT cases were used for immunohistochemistry and scored according to intensity and percentage. Healthy margins from each tumour were used as internal controls. The scores for NK-1 receptor intensity, percentage of positive cells and overall immunohistochemistry were higher in malignant MGT than in benign MGT and normal tissue (p < .000). NK-1 receptor expression was not correlated with either malignancy grade or histopathological type. Expression of the NK-1 receptor in malignant MGT was higher than that in benign MGT and normal tissues. Thus, NK-1 receptor could be considered a novel therapeutic target for cMGT. Further studies using other quantitative tests such as western blotting or PCR and the evaluation of substance P in patient tumour tissue or serum are needed., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging zoonotic virus in East Asia. However, SFTS virus (SFTSV) has not been reported to cause clinical infection in companion dogs to date. We report the case of a 4-year-old companion dog that presented with fever, vomiting, leukocytopenia and thrombocytopenia at a veterinary hospital in the Republic of Korea. It was diagnosed with SFTS, which was confirmed using real-time reverse transcription PCR, sequencing and an indirect immunofluorescence assay, and recovered after supportive care. Further studies are required to investigate SFTSV infection in companion animals, living in close contact with humans, as well as animal-to-human transmission., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

The outbreak of human toxoplasmosis can be attributed to ingestion of food contaminated with Toxoplasma gondii. Toxoplasmosis recently increased in domestic and stray dogs and cats. It prompted studies on the zoonotic infectious diseases transmitted via these animals. Sero- and antigen prevalences of T. gondii in dogs and cats were surveyed using ELISA and PCR, and B1 gene phylogeny was analyzed in this study. Toxoplasmosis antibodies were measured on sera of 403 stray cats, 947 stray dogs, 909 domestic cats, and 2,412 domestic dogs collected at nationwide regions, Korea from 2017 to 2019. In addition, whole blood, feces, and tissue samples were also collected from stray cats (1,392), stray dogs (686), domestic cats (3,040), and domestic dogs (1,974), and T. gondii-specific B1 gene PCR was performed. Antibody prevalence of stray cats, stray dogs, domestic cats, and domestic dogs were 14.1%, 5.6%, 2.3%, and 0.04%, respectively. Antigen prevalence of these animals was 0.5%, 0.2%, 0.1%, and 0.4%, respectively. Stray cats revealed the highest infection rate of toxoplasmosis, followed by stray dogs, domestic cats, and domestic dogs. B1 gene positives were 5 of stray cats, and identified to high/moderate pathogenic Type I/III group. These findings enforce that preventive hygienic measure should be strengthened at One Health level in dogs and cats, domestic and stray, to minimize human toxoplasmosis infections.

A 12-year-old neutered male domestic shorthair cat was presented with a 3-year history of multiple nonpruritic, ulcerated, plaque-like skin lesions but no other clinical signs. A systemic examination revealed mild lymphadenopathy. Histopathologic analysis of the skin lesions revealed multicentric basosquamous carcinoma (BSC). Immunohistochemical analysis, PCR, and sequencing detected Felis catus papillomavirus type 2 (FcaPV-2) within the tumors. As BSC is rare in cats, clinical behavior has not been established. To our knowledge, this is the first case report to demonstrate detection of FcaPV-2 within a BSC in a domestic cat.

A 3-month-old female Maltese puppy was hospitalized with persistent diarrhea in a local veterinary clinic. Blood chemistry and hematology profile were analyzed and fecal smear was examined. Diarrheal stools were examined in a diagnostic laboratory, using multiplex real-time polymerase chain reaction (PCR) against 23 diarrheal pathogens. Sequence analysis was performed using nested PCR amplicon of 18S ribosomal RNA. Coccidian oocysts were identified in the fecal smear. Although multiplex real-time PCR was positive for Cyclospora cayetanensis, the final diagnosis was Cystoisospora ohioensis infection, confirmed by phylogenetic analysis of 18S rRNA. To our knowledge, this the first case report of C. ohioensis in Korea, using microscopic examination and phylogenetic analysis.

Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.

Background: Enteroviruses (EVs) are the leading cause of aseptic meningitis worldwide. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patient care, especially that of neonates and young children., Findings: To establish a sensitive and reliable assay for routine laboratory diagnosis, we compared the sensitivity and specificity of the GeneXpert Enterovirus Assay (GXEA) with that of the reverse transcription polymerase chain reaction (RT-PCR) based assay referred to as real-time one step RT-PCR (RTo-PCR). The sensitivity/specificity produced by GXEA and RTo-PCR were 100%/100% and 65%/100%, respectively., Conclusions: Both methods evaluated in this article can be used for detection of enterovirus in clinical specimens and these nucleic acid amplification methods are useful assays for the diagnosis of enteroviral infection.

Epidemiological and virological studies indicate that noroviruses-contaminated groundwater was the primary source of four acute gastroenteritis outbreaks in South Korea between 2008 and 2012. Furthermore, cabbage kimchi was first identified as the vehicle of transmission between groundwater and infected patients in an outbreak in 2011. The proper treatment of groundwater sources prior to use for drinking or in food preparation is necessary to prevent further outbreaks.

Acute flaccid paralysis (AFP) is described as sudden onset of flaccid paralysis in one or more limbs in children caused by polioviruses (PVs). PV eradication is achieved through intensive immunization and AFP attentive surveillance, according to the World Health Organization. Since 1998, the Korea Centers for Disease Control and Prevention has conducted surveillance system. This is an overview of surveillance in the Republic of Korea during the 10-year period from 2002 to 2011. The surveillance system for wild PV eradication was conducted through reporting and laboratory testing. Cell culture isolates were identified by neutralization tests using standard polyclonal antisera typing. The molecular methods were used for further characterization to improve specificity. For genotyping, semi-nested RT-PCR was used to amplify part of the viral protein 1 gene. Patients below 5 years of age accounted for the largest proportion of cases, and a positive association between age and incidence was found. In the total 285 cases, Guillain-Barré syndrome was the major leading causes of AFP. Non-polio enterovirus was detected in some AFP patients. EV71 was detected in 21 cases and Coxsackievirus (C) A2, CA6, CA9, CB2, CB3, CB4, CB5, and Echovirus (E) 25, E30, Sabin strain polio 2, polio 1 and 3 were also detected in some patients. The present study represents a comprehensive 10-year country-based survey of AFP in the Republic of Korea. This surveillance could provide better understanding of the epidemiologic pattern, and clinical manifestations associated with specific genotypes of AFP in the Republic of Korea., (© 2013 Wiley Periodicals, Inc.)

Enterovirus 71 infection causes hand, foot and mouth disease in children, and can produce diverse neurologic complications. Epidemics occurring in Korea between 2009 and 2012 resulted in the death of some patients. The present study aimed to clarify the correlation between clinical features and MRI findings in patients presenting with acute neurologic manifestations related to enterovirus 71 infection. Based on their clinical features, the patients were classified into four clinical groups: (1) brainstem encephalitis (n=17), characterized by myoclonus, tremor, ataxia, and autonomic dysregulation such as pulmonary hemorrhage; (2) aseptic meningitis (n=2); (3) encephalitis (n=2), characterized by decreased consciousness, seizure, and fever without myoclonus, tremor, ataxia, and autonomic dysregulation; and (4) acute flaccid paralysis (n=1). Thirteen of the 17 patients with brainstem encephalitis showed characteristic lesions in the dorsal brainstem and bilateral cerebellar dentate nuclei on brain MRI, whereas three had no abnormality. One of the two patients with meningitis had a small lesion in the left dorsal pons. Two patients with encephalitis had no apparent MRI abnormality. One patient with acute flaccid paralysis of the right leg had contrast-enhancement of the bilateral ventral nerve roots at the lumbar spine level on MRI. Five of 13 patients with lesions in the bilateral dentate nuclei of the cerebellum exhibited no cerebellar symptoms, while two with no cerebellar lesions developed ataxia. Although most patients presenting with neurologic manifestations of enterovirus 71 infection had characteristic clinical features together with typical MRI findings, the clinical features were not necessarily consistent with MRI findings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

One new flavonol glycoside, 4'-O-methylmyricitrin 3'-O-beta-D-glucopyranoside (1), one new sesquiterpene, reynoudiol (11), as well as the 12 known compounds (2-10, 12-14) quercetin 3-O-methyl ether (2), quercitrin (3), isorhamnetin 3-alpha-L-rhamnopyranoside (4), tamarixetin 3-alpha-L-rhamnopyranoside (5), myricitrin (6), 4'-O-methylmyricitrin (7), isorhamnetin 3-O-beta-D-xylopyranosyl (1-2)-O-beta- D-glucopyranoside (8), isorhamnetin 3-O-beta- D-apiofuranosyl(1-2)-O-beta- D-glucopyranoside (9), (+)-catechin (10), 7-drimene-3,11,12-triol (12), clovane-2 beta,9 alpha-diol (13), and a-cadinol (14), were isolated from the methanol extract of Reynoutria japonica roots. Based on in vitro screening of the anti-influenza activity of the isolated compounds, reynoudiol showed significantly higher activity than that of oseltamivir phosphate at the same concentration, and did not induce any detectable cytopathic effect in MDCK cells. The CC50 of reynoudiol was above 50 micro M and could inhibit influenza virus infection with an IC50 of 0.29 +/- 0.01 microM. The therapeutic index (TI) of reynoudiol against influenza infection was 172.4, and thus, this compound can be potentially used to treat oseltamivir-resistant influenza virus infection.

The epidemiology of enteroviral infection in South Korea during 1999-2011 chronicles nationwide outbreaks and changing detection and subtyping methods used over the 13-year period. Of 14,657 patients whose samples were tested, 4,762 (32.5%) samples were positive for human enterovirus (human EV); as diagnostic methods improved, the rate of positive results increased. A seasonal trend of outbreaks was documented. Genotypes enterovirus 71, echovirus 30, coxsackievirus B5, enterovirus 6, and coxsackievirus B2 were the most common genotypes identified. Accurate test results correlated clinical syndromes to enterovirus genotypes: aseptic meningitis to echovirus 30, enterovirus 6, and coxsackievirus B5; hand, foot and mouth disease to coxsackievirus A16; and hand, foot and mouth disease with neurologic complications to enterovirus 71. There are currently no treatments specific to human EV infections; surveillance of enterovirus infections such as this study provides may assist with evaluating the need to research and develop treatments for infections caused by virulent human EV genotypes.

Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks., (Copyright © 2013 Wiley Periodicals, Inc.)

An epidemiological survey on human norovirus (NoV)-associated gastroenteritis was conducted to clarify the prevalence of NoV infections in children and adults in Korea. Recombinant capsid proteins from three major NoV genotypes (GI-4, GII-3, and GII-4) were expressed using a baculovirus expression system, and the morphology and antigenicity of self-assembled virus-like particles were then confirmed by electron microscopy and Western blotting with a NoV-specific antibody. To determine seroprevalence, an enzyme-linked immunosorbent assay was performed to detect antibodies against virus-like particles antigen in 346 serum specimens collected from persons who visited five public heath care centers for regular physical examination in Jeollanam-do, Korea, between 2005 and 2006. The seroprevalence of immunoglobulin G antibodies against the GI-4, GII-3, and GII-4 NoV genotypes was 84.1%, 76.3%, and 94.5%, respectively. A rapid decrease in seroprevalence occurred after birth, with the lowest levels observed in the <23-month age group, and a steep increase in seroprevalence occurred in early childhood, reaching 60.5% for GI-4, 65.1% for GII-3, and 90.7% for GII-4 at age 2-5 years, and over 80% for all three genotypes in subjects aged 20 years or older. The seroprevalence of different NoV genotypes statistically differed across the age groups (p<0.01).

Prevalence of asymptomatic norovirus infection was investigated in food handlers in South Korea. Among 6,441 subjects, 66 (1.02%) had norovirus infections confirmed by reverse transcription (RT)-PCR (real time and nested). GII-12 and GII-4 were the prevalent genotypes. Our data suggest that infection of asymptomatic food handlers is an important transmission source in norovirus outbreaks.

In 2009, the first outbreak of hand, foot and mouth disease (HFMD) or herpangina (HP) caused by enterovirus 71 occurred in the Republic of Korea. This study inquired into risk factors associated with complications of HFMD or HP. A retrospective medical records review was conducted on HFMD or HP patients for whom etiologic viruses had been verified in 2009. One hundred sixty-eight patients were examined for this investigation. Eighty patients were without complications while 88 were accompanied by complications, and 2 had expired. Enterovirus 71 subgenotype C4a was the most prevalent in number with 67 cases (54.9%). In the univariate analysis, the disease patterns of HFMD rather than HP, fever longer than 4 days, peak body temperature over 39℃, vomiting, headache, neurologic signs, serum glucose over 100 mg/dL, and having an enterovirus 71 as a causative virus were significant risk factors of the complications. After multiple logistic analysis, headache (Odds ratio [OR], 10.75; P < 0.001) and neurologic signs (OR, 42.76; P < 0.001) were found to be the most significant factors. Early detection and proper management of patients with aforementioned risk factors would be necessary in order to attain a better clinical outcome.

This study was performed to analyze epidemiological and molecular characteristics of coxsakievirus (CV) B1 infection associated with severe neonatal illness cases and death in Korea during 2008-2009. Through a nationwide surveillance program, specimens were collected from 104 patients infected with CVB1. The detection of enteroviruses (EVs) from specimens was subjected to a diagnostic real-time polymerase chain reaction (RT-PCR) in the 5'-non-coding region (NCR). A semi-nested PCR was conducted to amplify sequences from the VP1 region and sequence comparison was performed with reference strains registered in Genbank. Male-to-female ratio confirmed approximately 5:4. The major clinical manifestation of patients infected with CVB1 was aseptic meningitis (55.8%). The other clinical symptoms were herpangina or hand-foot-mouth disease (22.1%) and neonatal sepsis (7.7%). The sequences of CVB1 isolates were divided into four genetic clusters (A-D) with at least 15% diversity between the clusters. Almost all the CVB1 isolates in Korea from 2008 to 2009 were in cluster D (except for 2 cases). The homology relationship was also similar between the Korean CVB1 strains and US strain (above 93%). It is possible that Korean CVB1 isolates found during 2008-2009 originated from the US strains found during 2006-2008. The identification of CVB1 in South Korea shows the potential of EVs to cause serious disease in an unpredictable fashion., (Copyright © 2012 Wiley Periodicals, Inc.)

Objectives: This study aims to describe the clinical characteristics of enteroviral meningitis in association with the absence of cerebrospinal fluid (CSF) pleocytosis., Design: This was a retrospective analysis of databases of patients diagnosed with enteroviral meningitis by CSF reverse transcription-PCR testing. Presence of CSF non-pleocytosis at each age group was analysed by use of the two criteria. Clinical variables were compared with regard to the presence of CSF pleocytosis. Multiple logistic regression analysis was used to identify factors that were associated with CSF pleocytosis., Setting: Two hospitals in South Korea, between January 2008 and August 2011., Patients: 390 infants and children with enteroviral meningitis., Interventions: None., Main Outcome Measures: Proportion of enteroviral meningitis without CSF pleocytosis., Results: Among the 390 patients with enteroviral meningitis, 16-18% did not have CSF pleocytosis. In particular, CSF pleocytosis was not present in 68-77% of the neonates with enteroviral meningitis, demonstrating that the proportion of CSF pleocytosis decreased significantly with age (p<0.001). In multivariate models, younger age (adjusted OR 0.981; 95% CI 0.973 to 0.989), lower peripheral white blood cell count (adjusted OR 0.843; 95% CI 0.791 to 0.899), and shorter interval between onset and lumbar puncture (adjusted OR 0.527; 95% CI 0.315 to 0.882) were associated with the absence of CSF pleocytosis in enteroviral meningitis., Conclusions: This study demonstrated high proportion of non-pleocytic enteroviral meningitis in young infants and identified several clinical factors that contributed to the absence of CSF pleocytosis. We suggest that CSF enterovirus PCR testing is likely to detect more cases of enteroviral meningitis, especially in young infants.

Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.

Hepatitis A virus is one of the most prominent causes of fecally transmitted acute hepatitis worldwide. In order to characterize the viral agents causing an outbreak in Korea (comprising North and South Korea) from June 2007 to May 2009, we collected specimens and performed genotyping of the VP1/P2A and VP3/VP1 regions of hepatitis A virus. We then used a multiple-alignment algorithm to compare the nucleotide sequences of the 2 regions with those of reference strains. Hepatitis A virus antibodies were detected in 64 patients from 5 reported outbreaks (North Korea, June 2007 [n = 11]; Jeonnam, April 2008 [n = 15]; Daegu, May 2008 [n = 13]; Seoul, May 2009 [n = 22]; and Incheon, May 2009 [n = 3]). We found 100% homology between strains isolated from the Kaesong Industrial Region and Jeonnam. While those strains were classified as genotype IA strains, strains from Seoul and Incheon were identified as genotype IIIA strains and showed 98.9 to 100% homology. Genotype IIIA was also dominant in Daegu, where strains were 95.7 to 100% homologous. All hepatitis A virus strains isolated from the Kaesong Industrial Region, Jeonnam, Seoul, and Incheon belonged to a single cluster. However, strains from Daegu could be classified into 2 clusters, suggesting that the outbreak had multiple sources. This study indicates that hepatitis A virus strains of 2 different genotypes are currently cocirculating in Korea. Moreover, it documents an increasing prevalence of genotype IIIA strains in the country.

This study aimed to develop a specific and sensitive reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR-ELISA) for detecting hepatitis E virus (HEV). Eight sets of primers and biotinylated probes designed in the ORF2-ORF3 overlapping region of HEV were tested for sensitivity. The ability of nested reverse transcription polymerase chain reaction (RT-PCR) and RT-PCR-ELISA to detect HEV was compared. RT-PCR-ELISA was 10-100 times more sensitive than nested RT-PCR and could detect 0.01 ng/μl HEV in swine stool samples. In terms of specificity, RT-PCR-ELISA did not falsely detect HEV when other viruses such as hepatitis A virus, rotavirus, norovirus genotype I, norovirus genotype II, and Feline calicivirus were present. Therefore, RT-PCR-ELISA appears to be a sensitive and specific method for detecting HEV.

Human astrovirus (HAstV) is a major cause of acute diarrhea among children, resulting in outbreaks of diarrhea and occasionally hospitalization. Improved surveillance and application of sensitive molecular diagnostics have further defined the impact of HAstV infections in children. These studies have shown that HAstV infections are clinically milder (diarrhea, vomiting, fever) than infections with other enteric agents. Among the 8 serotypes of HAstV identified, serotype 1 is the predominant strain worldwide. In addition to serotype 1, the detection rate of HAstV types 2 to 8 has increased by using newly developed assays. HAstV is less common compared with other major gastroenteritis viruses, including norovirus and rotavirus; however, it is a potentially important viral etiological agent with a significant role in acute gastroenteritis. A better understanding of the molecular epidemiology and characteristics of HAstV strains may be valuable to develop specific prevention strategies.

Background: Enteroviruses (EVs) are the leading cause of aseptic meningitis, which is the most frequent central nervous system infection worldwide. We aimed to characterize the EVs involved in an aseptic meningitis outbreak in Korea in 2008. In Korea, Echovirus type 30 (E30) and E6 have been associated with outbreaks and frequent meningitis., Methods: During 2008, through nationwide surveillance, we collected specimens from 758 patients with aseptic meningitis-related clinical manifestations. The detection of EVs from specimens was subjected to a diagnostic real-time RT-PCR in the 5' NCR. A semi-nested polymerase chain reaction (PCR) to amplify sequences from the VP1 region and sequence comparison with reference strains registered in Genbank was performed for the genotype determination., Results: Most patients (98%) in this outbreak were children < 15 years of age. The temporal distribution of the E6 and E30 epidemics showed an obvious seasonal pattern during the short period from June to July. A large majority of the EV-positive patients experienced fever, headache, vomiting, and neck stiffness. Some patients also showed cold symptoms, sore throat, altered mental status, and seizures. We did not observe a higher fatality rate in children with E6 or E30 infection. Most of the patients recovered uneventfully. In most cases, the cerebrospinal fluid (CSF) profile was studied, and generally showed a higher than normal white blood cell count (≥ 5/mm(3)). We detected EVs from 513 patients (67.68%) and identified the EV genotype in 287 patients. E30 (n = 155, 50.4%) and E6 (n = 95, 33.1%) were the predominant genotypes. E9, E1, E7, E16, coxsackievirus A3, 4, 6, coxsackievirus B1, 3, and 10 were also identified. According to phylogenetic analysis, E30 belonged to subgroup 4b, and E6, to the C4 subgroup., Conclusions: Conclusively, aseptic meningitis was the most common manifestation in children with either echovirus 30 or 6 infection. Identification of E6 and E30 as the prominent EVs in the 2008 outbreak in South Korea shows the potential of EVs to cause a serious disease in an unpredictable (fashion. Our findings provide new) insights into the clinical and virological features of the aseptic meningitis outbreak caused by E30 and E6.

Background: Echovirus 30 (Echo30) is one of the most frequently identified human enteroviruses (EVs) causing aseptic meningitis and encephalitis. However the mechanism underlying the pathogenesis of Echo30 infection with significant clinical outcomes is not completely understood. The aim of this investigation is to illustrate molecular pathologic alteration in neuronal cells induced by Echo30 infection using clinical isolate from young patient with neurologic involvement., Methodology/principal Findings: To characterize the neuronal cellular response to Echo30 infection, we performed a proteomic analysis based on two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrophotometric (MS) analysis. We identified significant alteration of several protein expression levels in Echo30-infected SK-N-SH cells. Among these proteins, we focused on an outstanding up-regulation of Triple functional domain (TRIO) in Echo30-infected SK-N-SH cells. Generally, TRIO acts as a key component in the regulation of axon guidance and cell migration. In this study, we determined that TRIO plays a role in the novel pathways in Echo30 induced neuronal cell death., Conclusions/significance: Our finding shows that TRIO plays a critical role in neuronal cell death by Echo30 infection. Echo30 infection activates TRIO-guanine nucleotide exchange factor (GEF) domains (GEFD2) and RhoA signaling in turn. These results suggest that Echo30 infection induced neuronal cell death by activation of the TRIO-RhoA signaling. We expect the regulation of TRIO-RhoA signaling may represent a new therapeutic approach in treating aseptic meningitis and encephalitis induced by Echo30.

Korean red ginseng has been studied various biological activities such as immune, anti-oxidative, anti-microbial, and anticancer activities but antiviral mechanism needs further studies. In this study, we aimed to examine the antiviral effects of Korea red ginseng extract and ginsenosides on norovirus surrogate, including murine norovirus (MNV) and feline calicivirus (FCV). We evaluated the pre-, co-, and post-treatment effects of Korean red ginseng (KRG), ginsenosides Rb1 and Rg1. To measure the antiviral effect and cytotoxicity of KRG extract, and ginsenosides Rb1 and Rg1, we treated Crandell-Reese Feline Kidney for FCV or RAW264.7 cells for MNV with concentrations of 0, 5, 6.7, 10, 20 ug/mL total saponin. There was cytotoxic effect in the highest concentration 20 ug/mL of KRG extract so this concentration was excluded in this study. The FCV titer was significantly reduced to 0.23-0.83 log10 50% tissue culture infectious dose (TCID50)/mL in groups pre-treated with red ginseng extract or ginsenosides. The titer of MNV was significantly reduced to 0.37-1.48 log10 TCID50/mL in groups pre-treated with red ginseng extract or ginsenosides. However, there was no observed antiviral effect in groups co-treated or post-treated with KRG and its constituents. Our data suggest that KRG extract has an antiviral effect against norovirus surrogates. The antiviral mechanisms of KRG and ginsenosides should be addressed in future studies.

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Enterovirus (EV) infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease., Method: To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR) assay with minor groove binder probes and primers modified with complementary locked primer technology (TMC-PCR). We checked the sensitivity of the developed assay by comparing it to a previously published TaqMan probe real-time one-step RT-PCR (TTN-PCR) procedure using enteroviral isolates, Enterovirus Proficiency panels from Quality Control on Molecular Diagnostics (QCMD-2007), and clinical specimens from patients with suspected EV infections., Results: One hundred clinical specimens from 158 suspected viral meningitis cases were determined to be positive by the TMC-PCR assay (63.29%), whereas only 60 were found to be positive by the TTN-PCR assay (37.97%). The positive and negative agreements between the TMC-PCR and TTN-PCR assays were 100% and 59.2%, respectively., Conclusion: This data suggest that the TMC-PCR assay may be suitable for routine diagnostic screening from patient suspected EV infection.

The present study was conducted to survey the prevalence and genotypic distribution of human astrovirus (HAstV) circulating in South Korea. Of 160,027 patients with acute gastroenteritis, 2,057 (1.3%) were positive for HAstV antigen. We determined the genotypes of 187 HAstV strains collected from laboratories across the country. Genetic analysis revealed genotype 1 to be the most prevalent, accounting for 72.19% of the strains, followed by genotypes 8 (9.63%), 6 (6.95%), 4 (6.42%), 2 (3.21%) and 3 (1.60%). Our findings indicate that HAstV is less common but, even so, a potentially important viral agent of gastroenteritis in South Korea, with significant genetic diversity among circulating HAstV strains., (© 2010 University of Sassari. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.)

An outbreak of echovirus 5 (ECV 5) occurred in Korea in 2006, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from a young male patient with aseptic meningitis, we performed sequencing of the Korean ECV 5 strain and compared it with a prototype strain (Noyce). At the nucleotide level, the P1 region (85.3%) had the highest identity value; at the amino acid level, the P3 region (98.0%) had the highest identity value. The two strains shared all cleavage sites, with the exception of the VP1/2A site, which was TY/GA in the Noyce strain but TR/GA in the Korean ECV 5 isolate. In Vero cells infected with the Korean ECV 5 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC₅₀ value >100 μg/mL. Of the five drugs, only amantadine (IC₅₀: 1 ± 0.42 μg/mL, TI: 100) and ribavirin (IC₅₀: 22 ± 1.36 μg/mL, TI: 4.55) had any antiviral activity against the Korean ECV 5 isolate.

A Korean nationwide surveillance on circulating rotavirus strains was conducted from September 2000 to August 2007 aiming to obtain prevaccine data for predicting vaccine effectiveness. The predominant strains among the 2779 strains analyzed varied annually and only approximately 50% had either a G or a P antigen present in both RotaTeq (Merck & Co. Inc., Whitehouse Station, NJ, USA) and Rotarix (GlaxoSmithKline, Brentford, UK)., (© 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Human enteroviruses (HEVs) are a major cause of herpangina, HFMD (hand, foot, and mouth disease), and other neurological diseases in Seoul, Korea., Methods: A total of 56 specimens from hospitalized patients collected from February to December 2009 (37 females and 19 males) in Seoul were tested for HEV from stool, throat swab, and vesicle swab samples taken from patients with herpangina or HFMD using cell culture and RT-PCR in 2009. By the 1D gene, encoding the VP1 capsid protein, seven different HEV genotypes were detected with Coxsackievirus A2, A4, A5, A9, A16 (CA), Coxsackievirus B1 (CB), and Enterovirus 71 (EV71). The most prevalent genotype was CA16 (6, 10.7%), followed by CA2 (4, 7.1%), CA5 (4, 7.1%), EV71 (2, 3.6%), CA4 (1, 1.8%), CA9 (1, 1.8%), and CB1 (1, 1.8%). The 1D gene sequences of two EV71 strains were closely related with one another (98.5% nucleotide similarity) and belonged to the C4 genotype., Conclusions: It is important to continuously survey the genetic characteristics of EV71 and CA16 from patients, which will provide useful data that aids in our understanding of HFMD infections in Seoul, Korea and may contribute to future control.

The prevalence of asymptomatic norovirus (NoV) infection was investigated in children registered for kindergarten in Korea during the winter and summer. Children with no gastrointestinal symptoms, including diarrhea and abdominal pain, during the 2 weeks before and following sample collection were included in this investigation. NoV presence and genetic identification were determined with real-time reverse transcriptase-polymerase chain reaction and conventional nested reverse transcriptase-polymerase chain reaction. The prevalence of NoV in asymptomatic children was 5.5% in the winter and 3.5% in the summer, respectively. GII.4 was the most prevalent NoV genotype, but GII.2 and GI.10 were also found during genetic analysis. This study demonstrates that asymptomatic NoV infection may be an important source of transmission in kindergarten children.

We assessed neurologic sequelae associated with an enterovirus 71 (EV71) outbreak in South Korea during 2009. Four of 94 patients had high signal intensities at brainstem or cerebellum on magnetic resonance imaging. Two patients died of cardiopulmonary collapse; 2 had severe neurologic sequelae. Severity and case-fatality rates may differ by EV71 genotype or subgenotype.

Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Currently, reverse transcription polymerase chain reaction (RT-PCR) is used commonly to detect NoVs in both clinical and environmental samples. However, RT-PCR requires expensive equipment and cannot be performed on site. In this study, a latex agglutination test (LAT) using antibody-labeled latex beads for detecting NoVs was developed. Two kinds of polyclonal antibodies, one generated from synthetic peptides and the other from E. coli-expressed NoV capsid proteins, were used to develop the LAT. Each of these polyclonal antibodies was immobilized on the surface of latex beads and tested for the ability to detect NoVs. Under optimized conditions, our LAT detected GII.4 NoV at concentrations as low as 3.3x10(5) RT-PCR units/ml in stool samples. The detection limit for the LAT was approximately 1.7 103 RT-PCR units. Forty-eight stool samples were tested for NoVs using this LAT. In comparison with an RT-PCR assay, the sensitivity and specificity of the LAT were 35% and 100%, respectively. With further optimization, this LAT used with appropriate antibodies could be applied for convenient detection of NoVs in clinical diagnosis and food monitoring.

Background: Hepatitis A virus (HAV) is a causative agent of acute hepatitis, which is transmitted by person-to-person contact and via the faecal-oral route. Acute HAV infection is usually confirmed by anti-HAV IgM detection. In order to detect anti-HAV IgM in the serum of patients infected with HAV, we developed a rapid assay based on immunochromatography (ICA) and evaluated the sensitivity of this assay by comparing it with a commercial microparticle enzyme immunoassay (MEIA) that is widely used for serological diagnosis., Results: The newly developed ICA showed 100% sensitivity and specificity when used to test 150 anti-HAV IgM-positive sera collected from infected patients and 75 negative sera from healthy subjects. Also, the sensitivity of ICA is about 10 times higher than MEIA used in this study by determining end point to detect independent on infected genotype of HAV. In addition, the ICA was able to detect 1 positive sample from among 50 sera from acute hepatitis patients that had tested negative for anti-HAV IgM using the MEIA., Conclusion: Conclusively, ICA for the detection of anti-HAV IgM will be very effective for rapid assay to apply clinical diagnosis and epidemiological investigation on epidemics due to the simplicity, rapidity and specificity.

This study was conducted to determine the prevalence of Arcobacter species identified or isolated from retail meats in Korea. Multiplex PCR assays for the detection of Arcobacter species were performed for 360 chicken, 100 pork, and 106 beef samples. Arcobacter butzleri and Arcobacter cryaerophilus were detected in 18.9 and 3.3% of chicken samples, respectively. However, Arcobacter species were not found in any of the pork and beef samples. Biochemical testing of isolates selected after enrichment revealed 38 A. butzleri isolates in chicken samples, but no A. cryaerophilus isolates were detected. In this study, A. butzleri was the most prevalent Arcobacter species in chicken meat, and contamination with Arcobacter species in pork and beef may be less prevalent in Korea.

Human enterovirus 71 (EV 71) has caused large-scale outbreaks of hand-foot-and-mouth disease (HFMD), particularly in the Asian-Pacific region. In this study, we report a major outbreak of EV 71 infection in Korea and describe the clinical differences between EV 71 and non-EV 71 enterovirus infections. We prospectively enrolled patients with suspected viral infections during a recent 2-year period through a nationwide surveillance system. We identified 719 patients with suspected HFMD or herpangina using real-time PCR and genotyping based on VP1 sequence analysis. The major pathogen causing HFMD changed substantially from 2008 to 2009, with EV 71 becoming the most common cause of HFMD in Korea in 2009. We successfully identified the enteroviral genotypes for 218 of the 719 patients. Patients with EV 71 infections tended to be younger than those with non-EV 71 enteroviral infections and presented with HFMD and meningoencephalitis. In addition, the occurrence of fever, headache, and neck stiffness was significantly higher in patients with EV 71 infections. Multivariable analysis showed that for patients presenting with HFMD, fever, or a sore throat, each covariate was independently associated with EV 71 infection; the adjusted odds ratios (with 95% confidence intervals in parentheses) for these variables were 31.86 (10.04 to 101.09), 4.76 (1.71 to 13.25), and 0.18 (0.04 to 0.77), respectively. Our results indicate that EV 71 was a major cause of HFMD in Korea during the study period. In addition, we found that clinical symptoms may be helpful in the early identification of patients with EV 71 infections.

Noroviruses are the enteric pathogens most commonly responsible for infectious gastroenteritis and outbreaks of foodborne illness. The GII.4 norovirus, in particular, is responsible for the majority of epidemics. Here, we present data on the distribution of norovirus genotypes in Chungnam, Korea, in 2008, measure genetic variation among GII.4 strains, and compare Korean GII.4 variants with reference strains based on the 237-bp junction of ORF1 and ORF2. We detected 139 different strains, which formed two distinct genetic clusters with significant sequence diversity. One Korean cluster (2008-Korea&#95;a) showed high similarity to the Sakai cluster that appeared in Japan and Europe in 2006. The other cluster (2008-Korea&#95;b) was unique and unrelated to previously reported clusters. Genotype GII.4 was confirmed as the predominant cause of norovirus epidemics in Korea. Foodborne norovirus infections, on the other hand, were generally caused by emerging GII.4 genetic variants similar to those responsible for global epidemics.

Purpose: Hand-foot-mouth disease (HFMD) is a common viral illness in children, which is usually mild and self-limiting. However, in recent epidemics of HFMD in Asia, enterovirus 71 (EV71) has been recognized as a causative agent with severe neurological symptoms with or without cardiopulmonary involvement. HFMD was epidemic in Korea in the spring of 2009. Severe cases with complications including death have been reported. The clinical characteristics in children with neurologic manifestations of EV71 were studied in Ewha Womans University Mokdong Hospital., Methods: Examinations for EV71 were performed from the stools, respiratory secretion or CSF of children who presented neurologic symptoms associated with HFMD by realtime PCR. Clinical and radiologic data of the patients were collected and analyzed., Results: EV71 was isolated from the stool of 16 patients but not from respiratory secretion or CSF. Among the 16 patients, meningitis (n=10) was the most common manifestation, followed by Guillain-Barré syndrome (n=3), meningoencephalitis (n=2), poliomyelitis-like paralytic disease (n=1), and myoclonus (n=1). Gene analysis showed that most of them were caused by EV71 subgenotype C4a, which was prevalent in China in 2008., Conclusion: Because EV71 causes severe complications and death in children, a surveillance system to predict upcoming outbreaks should be established and maintained and adequate public health measures are needed to control disease.

To increase detection sensitivity, we modified primers using complementary locked primer (CLP) technology. The sensitivity of the reverse transcription-PCR (RT-PCR) with CLP-modified primers was 10- to 100-fold higher than that of RT-PCR without these primers. CLP-modified primers can increase sensitivity, providing a widely accessible method for molecular diagnosis.

Background: Enteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections., Methods: Reverse transcription (RT)-PCR ELISA kit for targeting 5' non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them., Results: The detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5'NCR (P=0.016)., Conclusions: Our results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.

A group 2 human coronavirus designated HECV-4408 was isolated from a child with acute diarrhea and is antigenically and genetically more closely related to bovine coronavirus (BCoV) than to human coronavirus OC43 (X. M. Zhang, W. Herbst, K. G. Kousoulas, and J. Storz, J. Med. Virol. 44:152-161, 1994). To determine whether HECV-4408 infects gnotobiotic calves and induces cross-protective immunity against the virulent enteric BCoV DB2 strain, gnotobiotic calves (n = 4) were orally inoculated with HECV-4408 and then challenged with BCoV DB2 at postinoculation day (PID) 21. All calves inoculated with HECV-4408 developed diarrhea at PID 3 to 4 lasting 5 to 9 days. Fecal and nasal virus shedding were first detected by reverse transcription-PCR at PID 3 to 4 and at PID 2 to 4, respectively. After challenge with bovine coronavirus, no diarrhea or virus shedding was detected in calves inoculated with HECV-4408, but a mock-inoculated calf developed diarrhea and fecal and nasal shedding. Fecal immunoglobulin A (IgA) and serum IgG antibodies were first detected at PID 7 and PID 14, respectively. At postchallenge day 7, serum IgG and fecal IgA antibody titers remained the same or increased only twofold compared to prechallenge titers. An additional two gnotobiotic calves were inoculated with HECV-4408 and euthanized at PID 5. Moderate villous atrophy was observed in the small intestines, and viral antigen was detected in villous enterocytes of the small and large intestines by immunohistochemistry. These results support and extend the previous report that HECV-4408 is likely a variant of bovine coronavirus. They confirm its infectivity for calves and complete cross-protection against a bovine coronavirus (DB2 strain) showing 98.2% amino acid identity to HECV-4408 in the S protein.