New Advances in Viral and Microorganism Disinfectants.

Nanomaterials are used to develop simpler, cheaper, and faster methods for disease diagnosis [...].

Antiviral agents and disinfectants for foot­and­mouth disease (Review).

Fluorouracil, 5-azacytidine, 6-azauridine, ribavirin, favipiravir (T-705) and its derivative (T-1105) exhibit anti-foot-and-mouth disease virus (FMDV) effects. In particular, T-1105 exhibits promising results when administered to guinea pigs orally, and pigs in their feed. FMDV is excreted in the early stages of infection in aerosols and oral or nasal droplets from animals. T-1105 along with the FMDV vaccine can be used to combat foot-and-mouth disease (FMD) epidemics. Several studies have shown that sodium hypochlorous solutions are widely used to inactivate viruses, including FMDV. However, these solutions must be stored under cool and dark conditions to maintain their virucidal effects. Interestingly, a study indicated that the virucidal activity of a calcium bicarbonate solution with a mesoscopic structure (CAC-717) did not decrease after storage at room temperature for at least four years outside direct sunlight. Numerous lessons acquired from the 2010 FMD outbreak in Japan are relevant for the control of COVID-19. However, the widespread use of chlorite can cause environmental issues. Chlorite can be combined with nitrogen to produce chloramine or N-nitrosodimethylamine, which plays a role in carcinogenesis. Therefore, risk assessments should be conducted in aquatic environments. Moreover, there is a need to develop nonchlorine disinfectants that can be used during epidemics, including FMD. The approach of 'One Health' should be shared between the public health and veterinary fields to improve the management of viral outbreaks, including those due to FMD.

Universal Virucidal Activity of Calcium Bicarbonate Mesoscopic Crystals That Provides an Effective and Biosafe Disinfectant.

We investigated the virucidal effects in solution of a new type of disinfectant, calcium bicarbonate mesoscopic crystals, designated CAC-717, against various types of virus. CAC-717 in solution is alkaline (pH 12.4) and has a self-electromotive force that generates pulsed electrical fields. Upon application to human skin, the pH of the solution becomes 8.4. CAC-717 contains no harmful chemicals and is thus non-irritating and harmless to humans and animals. Its virucidal effects were tested against six types of animal virus: enveloped double-strand (ds)-DNA viruses, non-enveloped ds-DNA viruses, non-enveloped single strand (ss)-DNA viruses, enveloped ss-RNA viruses, non-enveloped ss-RNA viruses, and non-enveloped ds-RNA viruses. The treatment resulted in a reduction in viral titer of at least 3.00 log10 to 6.38 log10. Fetal bovine serum was added as a representative organic substance. When its concentration was ≥20%, the virucidal effect of CAC-717 was reduced. Real-time PCR revealed that CAC-717 did not reduce the quantity of genomic DNA of most of the DNA viruses, but it greatly reduced that of the genomic RNA of most of the RNA viruses. CAC-717 may therefore be a useful biosafe disinfectant for use against a broad range of viruses.

Virucidal Effect of the Mesoscopic Structure of CAC-717 on Severe Acute Respiratory Syndrome Coronavirus-2.

Here, the virucidal effect of calcium bicarbonate with a mesoscopic structure (CAC-717) on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was determined. Assays showed that CAC-717 had a strong virucidal effect on all examined SARS-CoV-2 isolates, including variant strains. The viral infectivity decreased within 15 s, and the virucidal activity of CAC-717 at a 1:49 ratio was similar to that of ethanol disinfectant. CAC-717 neutralization eliminated this virucidal effect, indicating that the alkaline condition of CAC-717 is important for virus inactivation and is an indicator of its mesoscopic structure and virucidal activity. The virucidal effect was observed in the presence of organic matter (bovine serum albumin). CAC-717 is a non-invasive and non-flammable substance with a low environmental burden, and its usefulness as a novel disinfectant has been confirmed.

Period of excretion of equine herpesvirus 3 (EHV-3) from a stallion before showing clinical signs of equine coital exanthema and the effect of acyclovir treatment on the duration of EHV-3 excretion.

In 2017, two Thoroughbred stallions, A and B in Farms A and B, respectively, in Hokkaido in Japan showed clinical signs of equine coital exanthema (ECE). In 2020, stallion C in Farm B showed clinical signs of ECE. Eighteen mares were mated within five days before stallion A developed ECE. Ten mares that mated within 3 days before onset showed clinical signs of ECE on the external genitalia. Equine herpesvirus 3 (EHV-3) was isolated from vaginal swabs from three mares that mated within 2 days before onset. Swabs from 12 mares that mated within 4 days before onset were real-time PCR (rPCR)-positive and nine of those mares had an increased EHV-3 antibody titer. The three stallions were administered valaciclovir orally and topical acyclovir ointment was applied. Treatment started on the next day after onset in stallion A and on the day of onset in stallions B and C. EHV-3 was firstly isolated from penis swabs of stallions A and B before treatment and from penis swabs of stallion C 2 days after treatment. EHV-3 was not isolated after 8, 5 and 8 days from onset in stallions A, B and C, respectively. However, swabs were rPCR-positive for at least 12, 9 and 15 days after onset of stallions A, B and C, respectively. EHV-3 was excreted from the stallions at least within 4 days before the onset of ECE, and acyclovir treatment resulted in the termination of excretion within 8 days after onset.

Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation.

Instrument cost is a major problem for the transduction of DNA fragments and proteins into cells. Water-in-oil droplet electroporation (droplet-EP) was recently invented as a low-cost and effective method for the transfection of plasmids into cultured human cells. We here applied droplet-EP to livestock animal cells. Although it is difficult to transfect plasmids into bovine fibroblasts using conventional lipofection methods, droplet-EP enabled us to introduce an enhanced green fluorescent protein (EGFP)-expressing plasmid into bovine earlobe fibroblasts. The optimal transfection condition was 3.0 kV, which allowed 19.1% of the cells to be transfected. For swine earlobe fibroblasts, the maximum transfection efficacy was 14.0% at 4.0 kV. After transfection with droplet-EP, 69.1% of bovine and 76.5% of swine cells were viable. Furthermore, droplet-EP successfully transduced Escherichia coli recombinant EGFP into frozen-thawed bovine sperm at 1.5 kV. Flow cytometry analysis revealed that 71.5% of spermatozoa exhibited green fluorescence after transfection. Overall, droplet-EP is suitable for the transfection of plasmids and proteins into cultured livestock animal cells.

Isolation of an Equine Foamy Virus and Sero-Epidemiology of the Viral Infection in Horses in Japan.

An equine foamy virus (EFV) was isolated for the first time in Japan from peripheral blood mononuclear cells of a broodmare that showed wobbler syndrome after surgery for intestinal volvulus and the isolate was designated as EFVeca_LM. Complete nucleotide sequences of EFVeca_LM were determined. Nucleotide sequence analysis of the long terminal repeat (LTR) region, gag, pol, env, tas, and bel2 genes revealed that EFVeca_LM and the EFV reference strain had 97.2% to 99.1% identities. For a sero-epidemiological survey, indirect immunofluorescent antibody tests were carried out using EFVeca_LM-infected cells as an antigen against 166 sera of horses in five farms collected in 2001 to 2002 and 293 sera of horses in eight farms collected in 2014 to 2016 in Hokkaido, Japan. All of the farms had EFV antibody-positive horses, and average positive rates were 24.6% in sera obtained in 2001 to 2002 and 25.6% in sera obtained in 2014 to 2016 from broodmare farms. The positive rate in a stallion farm (Farm A) in 2002 was 10.7%, and the positive rates in two stallion farms, Farms A and B, in 2015 were 40.9% and 13.3%, respectively. The results suggested that EFV infection is maintained widely in horses in Japan.

Isolation and molecular characterization of a variant of Chinese gC-genotype II pseudorabies virus from a hunting dog infected by biting a wild boar in Japan and its pathogenicity in a mouse model.

We isolated a variant of Chinese pseudorabies virus from a hunting dog with symptoms similar to Aujeszky's disease and designated the isolate MY-1 strain. The dog developed symptoms 6 days after hunting and biting a wild boar and died the day after onset. The Bam HI restriction profile of MY-1 DNA was different from those of the Japanese reference strain Yamagata-S81 and two vaccine strains, Bartha and Begonia, and resembled Bam HI-RFLP (restriction fragment length polymorphism) type IV. Complete nucleotide sequences were determined, and phylogenetic analyses revealed that MY-1 belonged to the same cluster of old Chinese strains and variant strains isolated recently in China, but most of the open reading frames of MY-1 were located on a different branch from those of these Chinese strains. Based on a gC phylogenetic analysis, MY-1 belonged to gC-genotype II composed of those Chinese strains. In mice, the 50% lethal dose (LD50) of MY-1 (103.0 TCID50) was almost the same as those of Yamagata-S81 and Bartha. The LD50 value of Begonia was 10≥4.5 TCID50. The mean survival periods of mice after infection with 104 TCID50 of MY-1, Yamagata-S81 and Bartha were 3.9 days, 2.3 days, and 8.0 days, respectively. The results suggested that the variant of Chinese PRV with slightly weaker pathogenicity than that of wild virulent viruses might be maintained in wild boars in Japan. Furthermore, we would like to propose that old Chinese strains, recent Chinese variant strains, and MY-1 should be grouped as an Asian type PRV.

Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions.

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

Evaluation of calcium hydrogen carbonate mesoscopic crystals as a disinfectant for influenza A viruses.

In this study, the virucidal effect of a novel electrically charged disinfectant CAC-717 was investigated. CAC-717 is produced by applying an electric field to mineral water containing calcium hydrogen carbonate to generate mesoscopic crystals. Virus titration analysis showed a >3 log reduction of influenza A viruses after treatment with CAC-717 for 1 min in room temperature, while infectivity was undetectable after 15 min treatment. Adding bovine serum albumin to CAC-717 solution did not affect the disinfectant effect. Although CAC-717 is an alkaline solution (pH=12.39), upon contact with human tissue, its pH becomes almost physiological (pH 8.84) after accelerated electric discharge, which enables its use against influenza viruses. Therefore, CAC-717 may be used as a preventative measure against influenza A viruses and for biosecurity in the environment.

Occurrence of equine coital exanthema (ECE) in stallions in Japan and effectiveness of treatment with valacyclovir for ECE.

Equine coital exanthema (ECE) has been reported in many countries, but equine herpesvirus 3 (EHV-3) has been isolated only once in Japan. In 2015, symptoms of ECE were found, and EHV-3 was isolated in two stallions. Valacyclovir, an anti-herpesvirus agent, was administered orally. The stallions rested from mating for more than two weeks, causing enormous financial losses because of their high fees. This is the first study in which valacyclovir was administered for ECE. Though valacyclovir treatment did not shorten the duration of healing, the affected area did not expand after administration of valacyclovir. Valacyclovir therefore seems to be effective for suppression of EHV-3 infection. Further investigation about the administration protocol might be required.

Isolation of equine herpesvirus 3 (EHV-3) from equine coital exanthema of two stallions and sero-epidemiology of EHV-3 infection in Japan.

In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms.

The full genome sequences of 8 equine herpesvirus type 4 isolates from horses in Japan.

Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.

Isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (Hypsipetes amaurotis) in Japan.

Five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified in the genus Orthoreovirus. Their genomes each consist of 10 dsRNA segments. A novel orthoreovirus was isolated from the haemorrhagic intestine of a dead brown-eared bulbul (Hypsipetes amaurotis) in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed non-enveloped capsids of ~70 nm diameter, which were characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and σC, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV, but was located on a phylogenetic branch different from that of ARV and NBV. The virus had the closest phylogenetic relationship to two reovirus strains: SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The 10 RNA segments had a 3' pentanucleotide sequence (UCAUC-3') conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal heptasequence (5'-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggested that the novel virus might form a new species with the two strains in the genus Orthoreovirus.

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins.

Identification and functional characterization of novel feline cytochrome P450 2A.

1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

Genomic reassortants of pandemic A (H1N1) 2009 virus and endemic porcine H1 and H3 viruses in swine in Japan.

From 2010 to 2013 in Japan, we isolated 11 swine influenza viruses (SIVs) from pigs showing respiratory symptoms. Sequence and phylogenetic analyses showed that 6 H1N1 viruses originated from the pandemic (H1N1) 2009 (pdm 09) virus and the other 5 viruses were reassortants between SIVs and pdm 09 viruses, representing 4 genotypes. Two H1N2 viruses contained H1 and N2 genes originated from Japanese H1N2 SIV together with internal genes of pdm 09 viruses. Additionally, 1 H1N2 virus contained a further NP gene originating from Japanese H1N2 SIV. One H1N1 virus contained only the H1 gene originating from Japanese H1 SIV in a pdm 09 virus background. One H3N2 virus contained H3 and N2 genes originating from Japanese H3N2 SIV together with internal genes of pdm 09 virus. The results indicate that pdm 09 viruses are distributed widely in the Japanese swine population and that several reassortments with Japanese SIVs have occurred.

Changes in acute-phase proteins and cytokines in serum and milk whey from dairy cows with naturally occurring peracute mastitis caused by Klebsiella pneumoniae and the relationship to clinical outcome.

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1ß in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 µg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.

Virological and serological studies of porcine respiratory coronavirus infection on a Japanese farm.

We detected transmissible gastroenteritis virus (TGEV) antibodies in pig farms in Tochigi prefecture, although the farms had no past record of TGEV vaccination or TGE. Among the farms, Farm A showed a high antibody incidence. We could not confirm if either TGEV or porcine respiratory coronavirus (PRCV) induced the antibodies, since conventional tests failed to discriminate PRCV from TGEV. Therefore, we conducted virological and serological examinations of this farm for 4 years to establish the etiology - TGEV or PRCV. Although no TGEV was detected, PRCVs were isolated from the nasal samples of pigs. Using a commercial ELISA kit, it was found that the antibodies detected in pigs of all the raising stages and sows were raised against PRCV but not TGEV. The phylogenetic analysis of the nucleotide sequences of the isolates showed that they were closely related to each other, and formed a separate cluster apart from the U.S.A. and European strains. In Cesarean-derived, colostrums-deprived piglets inoculated with a PRCV isolate, no clinical signs were seen, and the viruses were mainly isolated from the nasal samples. Moreover, viral genes were detected from the nasal sample of the contact pig. The result suggested that PRCV infection was located in the nasal cavity of pigs, and horizontal transmission easily occurs. From these results, PRCVs with different origins from the exotic PRCVs might be prevalent in pig farms in Japan.