Ibaraki Virus Research Articles

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

Partial glycosylation of the Ibaraki virus NS3 protein is sufficient to support virus propagation

Ibaraki virus (IBAV) causes Ibaraki disease. We have previously shown that IBAV NS3 protein is highly glycosylated and that tunicamycin, an inhibitor of N-linked glycosylation, suppressed NS3 glycosylation and viral propagation. Since tunicamycin is known to cause endoplasmic reticulum (ER) stress, we explored the effects of ER stress and NS3 glycosylation on IBAV infection using tunicamycin and thapsigargin. These reagents both induced ER stress and NS3 glycosylation inhibition in a concentration-dependent manner, and as in our previous report, high concentrations of tunicamycin and thapsigargin suppressed IBAV propagation. However, lower concentrations of these reagents produced limited differences in IBAV propagation, despite their ability to suppress NS3 glycosylation and induce ER stress. These findings suggest that a considerable degree of NS3 glycosylation inhibition and ER stress induction does not suppress IBAV propagation. Conversely, lower concentrations of thapsigargin enhanced IBAV propagation, suggesting that moderate ER stress could benefit IBAV.

Ibaraki virus enters host cells by macropinocytosis

Ibaraki virus (IBAV) is the pathogen associated with Ibaraki disease. In a previous study, we suggested that IBAV enters hamster lung (HmLu-1) cells via endocytosis and subsequently escapes into the cytoplasm upon endosomal acidification. However, it is unclear which of the endocytic pathways IBAV utilizes. In this study, we aimed to further elucidate the pathway of IBAV entry into host cells. We found that IBAV replication was not suppressed by inhibitors of clathrin-mediated or caveolin-mediated endocytosis but was markedly suppressed by 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and cytochalasin D, both of which inhibit macropinocytosis. Monensin, which inhibits endosomal acidification, also suppressed IBAV replication. To assess the inhibitory effects of these reagents on endocytosis, dextran and transferrin were used as indicators of macropinocytosis and clathrin-mediated endocytic activity, respectively. Our data confirmed that EIPA and monensin inhibited dextran uptake, and cytochalasin D inhibited the uptake of both. Additionally, we confirmed that endosomal/lysosomal acidification was inhibited by monensin. These results suggest that the macropinocytosis pathway is the major route of IBAV entry and confirm that IBAV infection of HmLu-1 cells is dependent on endosomal acidification.

Endemic and Emerging Arboviruses in Domestic Ruminants in East Asia.

Epizootic congenital abnormalities caused by Akabane, Aino, and Chuzan viruses have damaged the reproduction of domestic ruminants in East Asia for many years. In the past, large outbreaks of febrile illness related to bovine ephemeral fever and Ibaraki viruses severely affected the cattle industry in that region. In recent years, vaccines against these viruses have reduced the occurrence of diseases, although the viruses are still circulating and have occasionally caused sporadic and small-scaled epidemics. Over a long-term monitoring period, many arboviruses other than the above-mentioned viruses have been isolated from cattle and Culicoides biting midges in Japan. Several novel arboviruses that may infect ruminants (e.g., mosquito- and tick-borne arboviruses) were recently reported in mainland China based on extensive surveillance. It is noteworthy that some are suspected of being associated with cattle diseases. Malformed calves exposed to an intrauterine infection with orthobunyaviruses (e.g., Peaton and Shamonda viruses) have been observed. Epizootic hemorrhagic disease virus serotype 6 caused a sudden outbreak of hemorrhagic disease in cattle in Japan. Unfortunately, the pathogenicity of many other viruses in ruminants has been uncertain, although these viruses potentially affect livestock production. As global transportation grows, the risk of an accidental incursion of arboviruses is likely to increase in previously non-endemic areas. Global warming will also certainly affect the distribution and active period of vectors, and thus the range of virus spreads will expand to higher-latitude regions. To prevent anticipated damages to the livestock industry, the monitoring system for arboviral circulation and incursion should be strengthened; moreover, the sharing of information and preventive strategies will be essential in East Asia.

Identification and complete-genome phylogenetic analysis of an epizootic hemorrhagic disease virus serotype 7 strain isolated in China

An epizootic hemorrhagic disease virus (EHDV) strain designated YN09-04 was isolated from sentinel cattle in China. The length of its complete genome was 19,344bp in total, consisting of 10 segments ranging in size from 810bp (S10) to 3942bp (S1). Based on phylogenetic analysis of the S2 sequence, YN09-04 clusters with EHDV serotype 7 (EHDV-7) strains form a distinct, well-supported subgroup, indicating that YN09-04 belongs to EHDV-7. However, the origin of the YN09-04 genome is very complex. The S2 and S6 of YN09-04 cluster with those of Japanese EHDV-7 strains, whereas the S1, S3, S4, S5 and S7 of YN09-04 share high nucleotide sequence identity and a close relationship with those of Japanese Ibaraki viruses, and the S8, S9 and S10 nucleotide sequences of YN09-04 are more similar to those of some Australian EHDV strains than to those of other isolates. These results suggest that the genome of YN09-04 likely originated from a reassortment event between EHDV strains that were similar to the current Japanese and Australian strains and that YN09-04 and some EHDVs from Japan and Australia share the same ancestors. This is the first report of the isolation, identification and complete-genome phylogenetic analysis of an EHDV-7 strain from China.

Apoptosis induced by Ibaraki virus does not affect virus replication and cell death in hamster lung HmLu-1 cells.

Ibaraki virus (IBAV) is an arbovirus that is transmitted by biting midges and causes Ibaraki disease in cattle. IBAV induces apoptosis in several mammalian cell lines, and apoptosis in turn facilitates IBAV replication. In addition, virus-induced apoptosis may contribute to mammalian-specific pathogenicity considering that some arboviruses induce apoptosis in mammalian cells but not in insect cells. In this study, we found that when hamster lung cells (HmLu-1) are used as a virus host, IBAV causes severe cytopathic effects with little induction of apoptosis. Furthermore, pharmacological inhibition of apoptosis did not affect IBAV-induced cytotoxicity. These results indicate the existence of an apoptosis-independent pathway in which IBAV replicates and exerts cytotoxicity in mammalian cells.

Amino acid starvation accelerates replication of Ibaraki virus

Ibaraki virus (IBAV) is a strain of epizootic hemorrhagic disease virus 2 that belongs to the genus Orbivirus of the family Reoviridae. IBAV replication is suppressed by the inhibition of autophagy, and since mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of autophagy, we examined if mTORC1 inhibition by amino acid starvation or mTOR inhibitors (Torin 1 and rapamycin) affects IBAV replication. We found that IBAV replication is significantly enhanced after amino acid starvation of host cells, but not after treatment with mTOR inhibitors, during early stages of viral infection (0–1 hpi). Notably, inhibition of mTORC1 by amino acid starvation was reversible and thus restricted to 0–1 hpi, whereas mTOR inhibitors sustainably suppressed mTORC1 even after the 1-h treatment, suggesting that mTORC1 suppression itself does not affect IBAV replication. To investigate the mechanism of enhanced IBAV replication by amino acid starvation, we examined the endocytic pathway, since IBAV utilizes acidification of endosomes as a trigger for viral replication. Accordingly, we found that amino acid starvation, but not mTOR inhibitors, strongly induced acidification of endosomes/lysosomes and that inhibition of endosomal acidification by bafilomycin A1 effectively blocked enhancement of IBAV replication. Altogether, the inactivation of mTORC1 by amino acid starvation during early stages of infection enhances acidification of endosomes, which in turn enhances IBAV replication.

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.

Monitoring for bovine arboviruses in the most southwestern islands in Japan between 1994 and 2014.

BackgroundIn Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and D’Aguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014.ResultsBlood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D’ Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle (ages 6–12 months: had only been alive for one summer) clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan.ConclusionsWe showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0747-z) contains supplementary material, which is available to authorized users.

The requirement of environmental acidification for Ibaraki virus infection to host cells.

The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. IBAV outer capsid proteins, VP5 and VP2, were removed from virion when purified IBAV was exposed to low pH environment. Further experiment showed that the exposure to low pH buffer facilitated IBAV infection when the cellular endosomal pathway was impaired by bafilomycin A1. Results obtained in this study suggest that acidic environment is essential to initiate IBAV infection.

Epizootic haemorrhagic disease

Summary Epizootic haemorrhagic disease (EHD) is an arthropod-transmitted viral disease of certain wild ungulates, notably North American white-tailed deer and, more rarely, cattle. The disease in white-tailed deer results from vascular injury analogous to that caused by bluetongue virus (BTV), to which EHD virus (EHDV) is closely related. There are seven serotypes of EHDV recognised, and Ibaraki virus, which is the cause of sporadic disease outbreaks in cattle in Asia, is included in EHDV serotype 2. The global distribution and epidemiology of BTV and EHDV infections are also similar, as both viruses occur throughout temperate and tropical regions of the world where they are transmitted by biting Culicoides midges and infect a wide variety of domestic and wild ungulates. However, the global distribution and epidemiology of EHDV infection are less well characterised than they are for BTV. Whereas most natural and experimental EHDV infections (other than Ibaraki virus infection) of livestock are subclinical or asymptomatic, outbreaks of EHD have recently been reported among cattle in the Mediterranean Basin, Reunion Island, South Africa, and the United States. Accurate and convenient laboratory tests are increasingly available for the sensitive and specific serological and virological diagnosis of EHDV infection and confirmation of EHD in animals, but commercial vaccines are available only for prevention of Ibaraki disease and not for protection against other strains and serotypes of EHDV.

Reemergence of Ibaraki disease in southern Japan in 2013

In Japan in 2013, two cattle in the northwestern part of Kagoshima Prefecture developed fever and swallowing difficulty and were suspected of having Ibaraki disease. The epizootic hemorrhagic virus (EHDV) genome was detected from diseased and asymptomatic cattle by reverse transcription-polymerase chain reaction (RT-PCR). High neutralization antibody titers to Ibaraki virus (IBAV) ranging from 1:128 to 1:1,024 were observed in the RT-PCR-positive cattle, and the virus was isolated in one of the IBAV-positive farms. A pairwise alignment and phylogenetic analysis based on the major outer coat protein VP2 encoded in segment 2 revealed a close relationship between the isolated viruses and previous IBAV isolates. The phylogeny of VP2 also suggested that an IBAV variant isolated in 1997 was distinct from IBAV and sorted into a heterogeneous serotype, EHDV serotype 7. The findings revealed the reemergence of Ibaraki disease in Japan after a 26-year absence. Interestingly, the co-circulation of EHDV serotype 1 with IBAV was observed in the affected region, suggesting the potential reassortment between two heterogeneous serotypes in the field. Sentinel surveillance in Kagoshima Prefecture indicated that the incursion of IBAV occurred in October 2013 and that its spread was limited within the small area. Inadequate environmental temperatures for vector transmission in late autumn might have limited the virus spread to a wider region. The reemergence of Ibaraki disease showed us the importance of continuous vaccination to prevent economic losses.

Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV

Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

Seroprevalence of five arboviruses in sentinel cattle as part of nationwide surveillance in South Korea, 2009-2012.

To investigate the possible circulation of arboviruses in South Korea, nationwide surveillance of five arbovirues was conducted in sentinel calves during 2009−2012. We used serum neutralization tests to investigate the presence of antibodies for the Aino virus, Akabane virus, bovine ephemeral fever virus, Chuzan virus and Ibaraki virus. In 2009, 2011 and 2012, the seropositive rates for these five arboviruses were all less than 14.1%. In 2010, however, the seropositive rates for Aino virus and Akabane virus were 33.2% and 40.2%, respectively. High seropositive rates were also associated with a large-scale outbreak of Akabane viral encephalomyelitis in cattle in southern Korea in 2010. Continued seroprevalence surveillance will be useful for monitoring natural arboviral diseases.

The host specific NS3 glycosylation pattern reflects the virulence of Ibaraki virus in different hosts

The non-structural protein NS3 was investigated in Ibaraki virus (IBAV), an epizootic hemorrhagic disease virus, serotype 2. Degree of NS3 glycosylation, cytopathic effect, and virus release efficiency were compared between mammalian and insect cells. The molecular weight of synthesized NS3 was compared in Western blot analysis following the removal of the glycochain by PNGase F treatment and revealed that glycosylation of NS3 occurred only in mammalian cells. Also, it was revealed that the amount of infectious IBAV in the extracellular fraction continued to increase for insect cells even after 60h post infection without disrupting cells. These results suggested that glycosylation of NS3 controls pathogenicity of IBAV in host cells to protect vector insects by altering the release pathway of assembled progeny viruses.

Competitive ELISA for the Detection of Antibodies to Rift Valley Fever Virus in Goats and Cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic Modulation Biosensing for Rapid and Homogeneous Detection of Biological Targets at Low Concentrations

This paper reviews the development of a magnetic modulation biosensing (MMB) system for rapid, simple and sensitive detection of biological targets in homogeneous solution at low concentrations. It relies on condensation and modulation of the fluorescent-labeled probes attached to magnetic beads using an alternating magnetic field gradient. Condensation of the beads from the entire volume increases the signal while modulation separates the signal from the background noise of the non-magnetized solution. We first discuss the motivation and challenges in specific DNA sequences detection as well as current approaches to overcome some of these challenges. We then present the MMB system, DNA detection schemes and magnetic beads manipulation in solution. Rapid detection at sub-picomolar concentrations of fluorescent-labeled probes as well as of coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) without any washing or separation step is also reviewed. Finally, we show preliminary results of protein detection using a 'sandwich'-based assay.

Rapid homogenous detection of the Ibaraki virus NS3 cDNA at picomolar concentrations by magnetic modulation

Magnetic modulation biosensing (MMB) system is experimentally demonstrated for rapid and homogeneous detection of the Ibaraki virus NS3 cDNA. A novel fluorescent resonance energy transfer (FRET)-based probe discriminates the target DNA from the control. When detection is made, the FRET-based probe is cleaved using Taq-polymerase activity and fluorescent light is produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The beads are condensed into the detection area and their movement in and out the orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. 1.9pM of the Ibaraki virus NS3 cDNA was detected in homogeneous solution within 18min without separation or washing steps.

Monitoring of Five Bovine Arboviral Diseases Transmitted by Arthropod Vectors in Korea

A survey was performed in Korea to monitor the prevalence of five bovine arboviruses [Akabane virus, Aino virus, Chuzan virus, bovine ephemeral fever (BEF) virus, and Ibaraki virus] in arthropod vectors, such as Culicoides species. To determine the possible applications of survey data in annual monitoring and warning systems in Korea, we examined the prevalence of bovine arboviruses in arthropod vectors using RT-PCR. To compare the sensitivity and specificity of virus detection, nested PCR was also performed in parallel for all five viruses. Using the RT-PCR, the detection limits were at least up to 10 1.5 , 10 2.8 , 10 2.0 , 10 1.8 , and 10 4.0 TCID??/㎖ for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. When nested PCR was performed using 1 ㎕ of PCR product, the detection limits were increased, to 10 0.05 , 10 1.8 , 10 1.0 , 10 0.008 , and 102.0 TCID??/㎖ for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. Thus, nested PCR increased the sensitivity of the virus detection limit by 1~2 log. We pooled 30~40 mosquitoes in one sample. We collected 113 samples in 2006, 135 samples in 2007, and 100 samples in 2008. Among these samples, Chuzan virus and BEF virus genes were detected at a range between 0.82% and 1.19%, and Akabane virus, Aino virus, and Ibaraki virus genes were detected at less than 0.20%. These data may provide some insight into future epidemiological studies of bovine arboviral diseases in Korea.