Antifungal activity of aminopyrrolnitrin against Trichophyton verrucosum in a guinea pig model of dermatophytosis.

BACKGROUND: Dermatophytosis is a common and major public health concern worldwide. Despite the increasing availability of antifungal drugs, relapses and untreated cases of dermatophyte infections are reported. Therefore, novel antifungal agents are required. Aminopyrrolnitrin (APRN) shows promise for dermatophytosis treatment because of its antifungal activity. OBJECTIVES: This study aimed to assess the antifungal properties of APRN against Trichophyton verrucosum (T. verrucosum), in both laboratory settings and a guinea pig model. METHODS: The minimum inhibitory concentrations (MICs) of APRN and enilconazole against T. verrucosum were determined according to the CLSI M38 method. The skins of 16 male guinea pigs were infected with 1.0 × 108 conidia of T. verrucosum and the animals were grouped into sets of four: negative control group (NC) received normal saline; positive control group (PC) received 2 µg/mL of enilconazole; and APRN4 and APRN8 received 4 and 8 µg/mL of APRN, respectively. Clinical, mycological and histological efficacies were measured after 10 days. RESULTS: The MIC90 of APRN and enilconazole against T. verrucosum was 4 and 2 µg/mL, respectively. The clinical scores of PC, APRN4, and APRN8 were significantly lower than those of NC. Clinical and mycological efficacies were higher for APRN8, APRN4 and PC. No fungi were observed in the skin tissues of APRN4 and APRN8, while fungi were observed in 50% of the PC. CONCLUSION: APRN showed antifungal activity against T. verrucosum in vitro and in vivo and is a potential candidate for the treatment of dermatophytosis.

Distribution and genotypic analysis of Enterocytozoon bieneusi from wild boars in Korea.

Enterocytozoon bieneusi, an important microsporidian fungus, causes chronic diarrhea in humans and animals worldwide. Out of the 502 fecal samples from wild boars, 13 were positive for the E. bieneusi internal transcribed spacer region, with a prevalence of 2.6%. Six E. bieneusi genotypes, D, EbpC, and four novel KWB1-KWB4, were identified with zoonotic potential. Genotypes D (subgroup 1a) and EbpC (subgroup 1d) were first reported in Korean swine and Korea, respectively; KWB1-KWB4 (subgroup 1e) were most prevalent in this study. Because zoonotic genotypes have been identified, E. bieneusi transmission through wild boars must be closely monitored for proper prevention and treatment, despite their low prevalence. LAY SUMMARY: Enterocytozoon bieneusi is an important microsporidian fungus. Its sequences from wild boars were identified with zoonotic potential. Genotypes D and EbpC were first reported in Korean swine and Korea, respectively. E. bieneusi should be closely monitored to properly prevent and treat animals.

Intergenic recombination in feline calicivirus associated with a hemorrhagic-like disease in the Republic of Korea.

Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats. In this study, the complete genome sequence of FCV 14Q315, which was detected from a dead domestic cat with a hemorrhagic-like disease, was analyzed to identify the genetic characteristics. The FCV 14Q315 genome was 7,684 bp. Phylogenetic analyses based on the ORF1, ORF2, and ORF3 sequences indicated that FCV 14Q315 is more closely related to FCV 15D022 than to other FCV strains. ORF1 of FCV 14Q315 shared high sequence similarity with ORF1 of FCVs 15D022 and UTCVM-H1. We further evaluated genetic recombination in ORF1 of FCV 14Q315 and detected intergenic recombination between p30 and the ORF1/ORF2 junction with high significance. Particularly, the non-recombination region in ORF1 of FCV 14Q315 showed high sequence similarity with FCVs GX2019, CH-JL2, and 15D022. The recombination region in ORF1 of FCV 14Q315 showed the highest similarity with FCV UTCVM-H1, which is associated with a hemorrhagic-like disease. The results suggest that the UTCVM-H1-like FCV was introduced into the Republic of Korea and presumably recombined with Korean FCVs by occasional mixed infections. In addition, the Korean FCV strains were located in several phylogenetic clusters with marked genetic diversity in the ORF2 region. These results imply that Korean FCVs possess high genetic diversity owing to mutations and recombination. Furthermore, it is possible that certain FCVs caused cyclical infections in the Korean cat population based on a phylogenetic analysis of FCVs isolated at different time points. Keywords: calicivirus; virulent systemic feline calicivirus; recombination; hemorrhagic-like disease.

Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

Bats have been widely known as natural reservoir hosts of zoonotic diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused by coronaviruses (CoVs). In the present study, we investigated the whole genomic sequence of a SARS-like bat CoV (16BO133) and found it to be 29,075 nt in length with a 40.9% G+C content. Phylogenetic analysis using amino acid sequences of the ORF 1ab and the spike gene showed that the bat coronavirus strain 16BO133 was grouped with the Beta-CoV lineage B and was closely related to the JTMC15 strain isolated from Rhinolophus ferrumequinum in China. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2). Interestingly, 16BO133 showed complete elimination of ORF8 regions induced by a frame shift of the stop codon in ORF7b. The lowest amino acid identity of 16BO133 was identified at the spike region among various ORFs. The spike region of 16BO133 showed 84.7% and 75.2% amino acid identity with Rf1 (SARS-like bat CoV) and Tor2 (human SARS CoV), respectively. In addition, the S gene of 16BO133 was found to contain the amino acid substitution of two critical residues (N479S and T487 V) associated with human infection. In conclusion, we firstly carried out whole genome characterization of the SARS-like bat coronavirus discovered in the Republic of Korea; however, it presumably has no human infectivity. However, continuous surveillance and genomic characterization of coronaviruses from bats are necessary due to potential risks of human infection induced by genetic mutation.

Experimental infection of Korean native goats (Capra aegagrus hircus) with bovine viral diarrhea virus 1b.

BACKGROUND: Bovine viral diarrhea virus (BVDV) infects various ungulates and causes reproductive failure in infected goats. BVDV has been detected among goats in the Republic of Korea, but the route of transmission remains unclear. Here, we aimed to investigate whether BVDV-1b circulating among Korean cattle can be transmitted to Korean native goats (Capra aegagrus hircus) and characterize the outcomes of BVDV infection in these goats. RESULTS: Four goats were inoculated intranasally with the Korean noncytopathic (ncp) BVDV-1b strain. Two goats exhibited clinical signs of illness, including coughing and nasal discharge. Nasal swabs and blood were collected to screen for viral RNA and BVDV antibodies. Using the 5'-untranslated region (UTR), viral RNA was detected in the nasal swabs of two goats (Goat 1 and 3) on 12 day post-inoculation (dpi) and in the blood sample of one goat (Goat 1) on 7 and 19 dpi. Using the N-terminal protease (Npro) region, viral RNA was detected in the blood sample of Goat 1 on 7 and 12 dpi. Antibodies to BVDV were detected in Goats 1 and 3 on 16-21 dpi using enzyme-linked immunosorbent assay. Sequence analysis of the virus from nasal swabs and blood samples, which was detected via RT-PCR, using the 5'-UTR and Npro regions led to the identification of the strain as ncp BVDV-1b and revealed changes in the nucleotide sequence of these goats. CONCLUSIONS: Our results indicate that changes in the nucleotide sequence are associated with the establishment of BVDV infection in Korean native goats; these changes may be owing to a process required for the establishment of infection in a new host reservoir. Broadly, these findings highlight the importance of BVDV surveillance in ungulates other than cattle.

Molecular detection of Enterocytozoon bieneusi from bats in South Korea.

Enterocytozoon bieneusi, which has recently been re-classified as a fungus, was identified in 5.2% (3/58) bat intestinal tissues and 1.9% (4/210) bat feces collected in South Korea. The positive cases were classified into six genotypes including four novel genotypes, KBAT1-KBAT4, based on sequence analysis of the E. bieneusi internal transcribed spacer (ITS) region. In addition, a novel genotype, KBAT3, belonged to group 1, which is considered having zoonotic potential by phylogenetic analysis of the E. bieneusi ITS region. This study expands our knowledge of the host range of E. bieneusi.

Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

Bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (SARS), coronavirus, and other coronaviruses found in mammals. However, little research has been conducted on bat coronaviruses in South Korea. In this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in South Korea. RT-PCR and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. Coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family Rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family Vespertiliodae. To determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the RNA-dependent RNA polymerase (RdRp) gene were analyzed phylogenetically. Thirteen sequences belonging to SARS-like betacoronaviruses showed the highest nucleotide identity (97.1-99.7%) with Bat-CoV-JTMC15 reported in China. The other five sequences were most similar to MERS-like betacoronaviruses. Four nucleotide sequences displayed the highest identity (94.1-95.1%) with Bat-CoV-HKU5 from Hong Kong. The one sequence from a carcass showed the highest nucleotide identity (99%) with Bat-CoV-SC2013 from China. These results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs.

Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

Genetic diversity and phylogenetic analysis of newly discovered bat astroviruses in Korea.

Bats have been identified as a natural reservoir for several potentially zoonotic viruses. Recently, astroviruses have been reported in bats in many countries, but not Korea. We collected 363 bat samples from thirteen species at twenty-nine sites in Korea across 2016 and tested them for astrovirus. The detection of the RNA-dependent RNA polymerase (RdRp) gene in bat astroviruses was confirmed in thirty-four bats across four bat species in Korea: twenty-five from Miniopterus fuliginosusi, one from Myotis macrodactylus, four from M. petax, and four from Rhinolophus ferrumequinum. The highest detection rates for astrovirus were found in Sunchang (61.5%, 8/13 bats), and in the samples collected in April (63.2%, 12/19 bats). The amino acid identity of astroviral sequences identified from bat samples was ≥ 46.6%. More specifically, the amino acid identity within multiple clones from individual bats was ≥ 50.8%. Additionally, the phylogenetic topology between astroviruses from different bat families showed a close relationship. Furthermore, phylogenetic analysis of the partial ORF2 sequence of bat astroviruses was found to have a maximum similarity of 73.3-74.8% with available bat astrovirus sequences. These results indicate potential multiple-infection by several bat astrovirus species in individual bats, or hyperpolymorphism in the astrovirus strains, as well as the transmission of astroviruses across bat families; furthermore, our phylogenetic analysis of the partial ORF2 implied that a novel astrovirus may exist. However, the wide diversity of astroviral sequences appeared to have no significant correlation with bat species or the spatiotemporal distribution of Korean bat astroviruses.

Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-do, Korea.

Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccination before the season in which the Culicoides are active is probably best to prevent arbovirus infections.

Identification of Two novel reassortant avian influenza a (H5N6) viruses in whooper swans in Korea, 2016.

BACKGROUND: On November 20, 2016 two novel strains of H5N6 highly pathogenic avian influenza virus (HPAIVs) were isolated from three whooper swans (Cygnus cygnus) at Gangjin Bay in South Jeolla province, South Korea. Identification of HPAIVs in wild birds is significant as there is a potential risk of transmission of these viruses to poultry and humans. RESULTS: Phylogenetic analysis revealed that Gangjin H5N6 viruses classified into Asian H5 clade 2.3.4.4 lineage and were distinguishable from H5N8 and H5N1 HPAIVs previously isolated in Korea. With the exception of the polymerase acidic (PA) gene, the viruses were most closely related to A/duck/Guangdong/01.01SZSGXJK005-Y/2016 (H5N6) (98.90 ~ 99.74%). The PA genes of the two novel Gangjin H5N6 viruses were most closely related to AIV isolates previously characterized from Korea, A/hooded crane/Korea/1176/2016 (H1N1) (99.16%) and A/environment/Korea/W133/2006 (H7N7) (98.65%). The lack of more recent viruses to A/environment/Korea/W133/2006 (H7N7) indicates the need for analysis of recent wild bird AIVs isolated in Korea because they might provide further clues as to the origin of these novel reassortant H5N6 viruses. CONCLUSIONS: Although research on the origins and epidemiology of these infections is ongoing, the most likely route of infection for the whooper swans was through direct or indirect contact with reassortant viruses shed by migratory wild birds in Korea. As H5N6 HPAIVs can potentially be transmitted to poultry and humans, continuous monitoring of AIVs among wild birds will help to mitigate this risk.

Novel reassortant clade 2.3.4.4 avian influenza A (H5N8) virus in a grey heron in South Korea in 2017.

We report the identification of a novel reassortant clade 2.3.4.4 H5N8 virus from a dead grey heron in Korea in 2017. Outbreaks of clade 2.3.4.4 H5 HPAIVs have been reported worldwide, and they have evolved into multiple genotypes among wild birds. Phylogenetic analysis indicated that this virus likely originated from Qinghai Lake and Western Siberia and further evolved through reassortment with Eurasian LPAI during the 2016 fall migration of wild birds. Enhanced surveillance and comparative genetic analysis will help to monitor the further evolution and dissemination of clade 2.3.4.4 HPAIVs.

Experimental infection of cows with newly isolated Akabane virus strain (AKAV-7) causing encephalomyelitis.

Akabane virus (AKAV), an arthropod-transmitted bunyavirus, is a major cause of congenital abnormalities and encephalomyelitis in ruminants. In 2010, there was a major outbreak of encephalomyelitis in Korea and fifteen AKAV strains, including AKAV-7, were isolated from cows. To identify the neuropathogenicity of AKAV-7, we performed experimental infection of cows. Six-month-old female Korean Holstein dairy cattle were inoculated with AKAV-7 by various routes, including intracerebral (IC), intrasubarachnoid space (IS), subcutaneous (SC) and intravenous (IV); a separate group was vaccinated before intravenous infection. Five of the six cows in the IC group and two of the six cows in the IS group showed clinical signs such as locomotor ataxia and paralysis of the hind limbs. Three of six cows died after IC infection 9-12 days post infection (dpi). Histopathologic changes such as nonsuppurative encephalomyelitis were confirmed in various parts of the central nervous system in the IC, IS and SC groups. Early onset of neutralizing antibodies in the serum and lower viral mRNA levels in the peripheral blood mononuclear cells (PBMCs) and various tissues in the vaccinated group was noticeable compared to the unvaccinated group (IV group). We suggest that the AKAV vaccine currently used in Korea may be partially effective for protection against AKAV-7 in cows.

Molecular characteristics of a novel strain of canine minute virus associated with hepatitis in a dog.

A 5-year-old female Yorkshire terrier dog died a few days following hernia and ovariohysterectomy surgeries. Necropsy performed on the dog revealed that the surgeries were not the cause of death; however, degenerative viral hepatitis, showing intranuclear inclusion bodies in hepatic cells, was observed in histopathologic examination. Several diagnostic methods were used to screen for the cause of disease, and minute virus of canines (MVC) was detected in all parenchymal organs, including the liver. Other pathogens that may cause degenerative viral hepatitis were not found. Infection with MVC was confirmed by in situ hybridization, which revealed the presence of MVC nucleic acid in the liver tissue of the dog. Through sequencing and phylogenetic analysis of the nearly complete genome sequence, the strain was found to be distinct from other previously reported MVC strains. These results indicate that this novel MVC strain might be related to degenerative viral hepatitis in dogs.

Experimental infection of mice with bovine viral diarrhea virus.

The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.). None of the infected mice exhibited any clinical signs of illness; however, the tissues harvested after BVDV challenge showed significant histopathological changes. Blood samples from five mice that were injected IP and one mouse that was inoculated IN were positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was used to assess the presence of viral antigen in the organs of mice infected with three BVDV strains. In IP-injected mice, BVDV antigen was detected in the spleen (5/6), mesenteric lymph nodes (4/6), lymphatic tissue of the lung (3/6), lung (1/6), and stomach (1/6) of the infected mice; however, it was not detected in the liver (0/6) or kidney (0/6). In IN-inoculated mice, BVDV antigen was detected in the lung and mesenteric lymph nodes of one BVDV-infected mouse but was not detected in other tissues. The results of this study suggest that the spleen is the most reliable tissue for BVDV antigen detection using IHC in the IP-injected group. Our study demonstrates that mice can be infected by BVDV. This is the first report of BVDV infection in mice.

Genetic characterization of porcine epidemic diarrhea virus in Korea from 1998 to 2013.

The number of porcine epidemic diarrhea (PED) cases has increased over the past 20 years in Korea, with a major outbreak in 2013. A total of 27 Korean strains from 1998 to 2013 were analyzed (excluding the noncoding regions) and divided into two groups for comparison of the spike (S), ORF3, envelope (E), membrane (M), and nucleocapsid (N) genes with those of reference strains, vaccine strains, and previously identified strains based on phylogenetic analysis. Analysis of the selection patterns of PEDV isolated in Korea indicated positive selection of nine nonsynonymous sites in the S and N proteins and negative selection at 97 sites for all of the proteins. Interestingly, eight nonsynonymous mutations in S showed no significant pattern change over the 15-year period, and one of eight mutation sites was found only in IC05TK, GN05DJ, and KNU0802 in the epidemic years 2005 and 2008. These eight mutations were also present during the epidemic years in China. Furthermore, of the signs of positive selection in the S protein, the conservative substitutions were more frequent than radical substitutions in PEDVs, suggesting that the evolution of Korean strains has been slow. Serological cross-reactivity was detected between three field PEDVs and two vaccine strains, with different serum neutralization titers. In conclusion, although Korean PEDVs have been evolving slowly, their diverse antigenicity and genetics imply that multilateral efforts to prevent future PED outbreaks are required.

Genetic characteristics of the complete feline kobuvirus genome.

We sequenced the complete genome of a feline kobuvirus and determined relationships with other kobuviruses. This kobuvirus has an 8,269-nucleotide-long RNA genome, excluding the poly(A) tail. The genome contains a 7,311-bp open reading frame (ORF) encoding a putative polyprotein precursor of 2,437 amino acids, a 717-bp 5'-untranslated region (UTR), and a 241-bp 3'-UTR. The L protein sequence was found to be the most variable region in the feline kobuvirus genome. Interestingly, the 5'-UTR B and C stem-loops were conserved as observed with other kobuviruses; however, a secondary structure corresponding to stem-loop A was not found in the full length 5'-UTR sequence. Phylogenetic tree analysis showed that kobuviruses can be divided into 3 main groups. The feline kobuvirus belongs to the Aichivirus A species containing Aichivirus, mouse kobuvirus, and canine kobuvirus.

Canine kobuvirus infections in Korean dogs.

To investigate canine kobuvirus (CaKoV) infection, fecal samples (n = 59) were collected from dogs with or without diarrhea (n = 21 and 38, respectively) in the Republic of Korea (ROK) in 2012. CaKoV infection was detected in four diarrheic samples (19.0 %) and five non-diarrheic samples (13.2 %). All CaKoV-positive dogs with diarrhea were found to be infected in mixed infections with canine distemper virus and canine parvovirus or canine adenovirus. There was no significant difference in the prevalence of CaKoV in dogs with and without diarrhea. By phylogenetic analysis based on partial 3D genes and complete genome sequences, the Korean isolates were found to be closely related to each other regardless of whether they were associated with diarrhea, and to the canine kobuviruses identified in the USA and UK. This study supports the conclusion that CaKoVs from different countries are not restricted geographically and belong to a single lineage.

Phylogenetic analysis of bovine astrovirus in Korean cattle.

Bovine astrovirus (BAstV) belongs to a genetically divergent lineage within the genus Mamastrovirus. The present study showed that BAstV was associated with the gastroenteric tracts of cattle in nine positive fecal samples from 115 cattle, whereas no positive samples were found in the brain tissues of 14 downer cattle. Interestingly, the positive diarrheal samples were obtained mainly from calves aged 14 days-3 months. Bayesian inference tree analysis of the partial ORF1ab and capsid (ORF2) gene sequences of BAstVs identified four divergent groups. Eleven BAstVs, four porcine astroviruses, and two deer astroviruses (DAstVs; CcAstV-1 and -2) belonged to group 1; group 2 contained two BAstVs (BAstK08-51 and BAstK10-96) with another two in group 3 (BAstK08-2 and BAstK08-53); and group 4 comprised the BAstV-NeuroS1 strain derived from a cattle brain tissue sample and an ovine astrovirus. The same divergent groups were obtained when the pairwise alignments were produced using both amino acid and nucleotide sequences. The Korean BAstVs isolated from infected cattle had a nationwide distribution and they belonged to groups 1, 2, and 3.

Serological characteristics of affected cattle during an outbreak of bovine enzootic encephalomyelitis caused by Akabane virus.

During an outbreak of bovine enzootic encephalomyelitis caused by the Akabane virus (AKAV) in 2010, 210 serum samples were collected from the affected cattle, and serological investigations for the AKAV were performed using a serum neutralization test (SNT) and an enzyme-linked immunosorbent assay (ELISA). The seropositive rates for SNT and ELISA were 90.0 and 85.2 %, respectively. The titers of SNT (log2) against the AKAV were higher than 4.0 in the highly affected cattle (80.0 %). This finding indicates that most affected cattle were infected with the AKAV and that strong immune responses against this virus were elicited in affected cattle. The strong immune response to the AKAV in cattle may provide insight into the occurrence of bovine encephalomyelitis caused by the AKAV.