Aino Virus Research Articles

In this issue

In this issue

Cleavage of Akabane virus S RNA in the brain of infected ruminants

Since 2002 there has been a rise in arthrogryposis/hydranencephaly incidence in Israel, caused by Akabane viruses (AKAV) and possibly by Aino viruses. In response to the outbreak, serological, molecular-diagnostic and research tools were developed. AKAV sequences were detected by real-time RT-PCR in the brain tissue of 2 out of 20 tested calves and lambs that suffered from hydranencephaly. When the S segments from the two infected calves were characterized, it was concluded that the S genome were cleaved. In order to localize the cleavage site, the 3' segment of the S genome was cloned, sequenced, and found to be 430 bases long, which indicates a cleavage site between nucleotides 430 and 431 of the S segment in the antigenome. This cleavage site was found to be specific and not a result of degradation processes. Analysis of the S segment RNA secondary structure revealed that the cleavage site was located on a loop structure. Furthermore, flunking the cleavage site there are stretches of 7 or 8 bases long that were part of a stem with low free energy, which could stabilize the loop, making it accessible to an, as yet, uncharacterized cleavage mechanism.

Serosurveillance for Japanese encephalitis, Akabane, and Aino viruses for Thoroughbred horses in Korea

Recent global warming trends may have a significant impact on vector-borne viral diseases, possibly affecting vector population dynamics and disease transmission. This study measured levels of hemagglutination-inhibition (HI) antibodies against Japanese encephalitis virus (JEV) and neutralizing antibodies against Akabane virus (AKAV) and Aino virus (AINV) for Thoroughbred horses in Korea. Blood samples were collected from 989 racehorses in several provinces, between October 2005 and March 2007. Sera were tested using either an HI assay or a virus neutralization test. Approximately half (49.7%; 492/989) of the horses tested were antibody-positive for JEV. The HI titer against JEV was significantly correlated with racehorse age (p < 0.05). Horses with an HI antibody titer of 1:160 or higher accounted for 3.9% of the animals tested, indicating that vectors transmitting arthropod-borne viruses bit relatively few horses. In contrast, 3.8% (19/497) and 19.5% (97/497) of horse sera collected in March 2007 were positive against AKAV and AINV, respectively. The presence of antibodies against AKAV and AINV may indicate the multiplication of AKAV and AINV in these horses.

Serosurveillance of Viral Diseases in Korean Native Goats (Capra hircus)

A total of 804 goat sera were collected from 144 goat farms in five regions of South Korea during a period between 2005 and 2006 and screened for the antibodies of viral pathogens in ruminants. The individual seropositive rates for each virus were 13.7% (110/804) for bovine herpesvirus-1 (BHV-1), 9.5% (76/804) for bovine parainfluenza type-3 virus (PI-3V), 5.5% (44/804) for Akabane virus (AKAV), 13.3% (107/804) for Aino virus (AINV), 2.0% (16/804) for Chuzan virus (CHUV) and 1.0% (8/804) for bovine coronavirus (BCoV). Compared with other areas, Chungcheong Province showed higher seropositive rates of 13.6% for PI-3V, 22.3% for AKAV and 28.2% for AINV. The results indicate that among the six viral diseases, BHV-1 infection is quite prevalent, while BCoV infection is less prevalent on domestic goat farms in Gyeongsang and Jeonla Provinces.

Molecular epidemiological analyses of the teratogenic Aino virus based on the sequences of a small RNA segment

The sequences of a small RNA segment of Aino virus isolates were analyzed to define the molecular epidemiology and genetic relationships to other species in the genus Orthobunyavirus in the family Bunyaviridae. The nucleotide and amino acid sequences of the segment were highly conserved among strains isolated from 1964 to 2002 in Japan. These Japanese isolates were segregated into two distinct lineages, one containing the prototype strain JaNAr28 isolated in 1964 and the other containing strains isolated after 1986, by phylogenetic analysis based on the nucleocapsid gene sequences. Japanese strains isolated after 1986 were rather more closely related to Kaikalur virus isolated in India in 1971 than to strain JaNAr28. On the other hand, an Australian strain, B7974, was closely related to Peaton virus. The B7974 strain might have been generated by inter-serotype genetic reassortment between Aino and Peaton viruses in Australia during their evolution. However, recent Aino virus strains isolated in Japan appear to be genetically stable.

Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea

Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.

Highly effective inhibition of Akabane virus replication by siRNA genes

Since 2002 there has been a rise in arthrogryposis/hydranencephaly (AGH) incidence in Israel, caused by Akabane (AKA) and, possibly, Aino viruses. To test the ability to control the disease, three siRNA genes targeted to the S genome segment were designed and prepared in the form of siRNA cassettes. For the design all published S segment were aligned and two conserved target sequences with 100% homology were chosen. A third conserved target that was found exhibited only one base change found in the two Australian isolates and was also designed and tested. It was demonstrated that cells transfected with single siRNA genes showed 99% inhibition, as measured by real-time RT-PCR, virus titration and immunofluoroscence. When cells were transfected with all three genes together the inhibition levels were increased and reached almost 100%.

Dictionary of Parasitology

The authors' intent in writing this dictionary is to provide a concise, clear, up-to-date, accurate use of terms to be used when communicating scientific information in the field of parasitology. This exhaustive text, with more than 11,500 entries, is at first read simply an alphabetized collection of names of organisms and terms associated with the science of parasitology. Upon closer reading, however, one spends more and more time going page by page either refreshing forgotten terminology, or learning new meaning for a particular term or disease. For the student of words, both newcomers to the field or seasoned hands, this book will provide useful information. Some concerns exist, such as continued use of outdated names, e.g., Dipetalonema for a number of filarial infections that have been correctly placed in the genus Mansonella for >20 years. There are also some gaps, such as the absence of an important genus of microsporidia, Enterocytozoon, but overall, readers will be able to find definitions for common and eclectic terms. The dictionary covers a wider range of terms than parasitology; some virology terms, such as Aino virus, are included, as are some far afield terms, including hundredweight, hydrogen half-cell, and zwitterions. These additions add to the level of interest as the reader leafs from page to page looking for familiar friends and making new acquaintances. Zwitterions, in case you have forgotten, are ions that carry both a positive and negative charge.

Apoptosis in Vero cells infected with Akabane, Aino and Chuzan virus

Akabane, Aino and Chuzan virus are arthropod-borne (arbo) viruses mainly associated with reproductive failures in cattle. We investigated apoptosis in Vero cells (C-1586) infected with Akabane, Aino and Chuzan virus. The fragmentation of chromosomal DNA was simultaneously detected with the progress of cytopathic effect from 48 hr to 72 hr post infection, depending on viruses. Although the treatment of cycloheximide blocked apoptosis in Vero cells infected with three viruses, actinomycin D did not prevent DNA oligomerization, thus indicating that de novo viral protein synthesis is critical for viral apoptosis. In addition, the activation of caspase-3 was also detected in Vero cells by indirect fluorescent assay. From the present results, it is of future interest whether apoptotic characteristics of these viruses are related to pathogenecity in vivo.

74 ANATOMICAL ABNORMALITIES IN CALVES PRODUCED BY NUCLEAR TRANSFER

Two limitations currently restrict the acceptability and adoption of bovine cloning as a commercial reality. The first of these is its low rate of efficiency and the second is that some of the cloned calves are not healthy. Abnormalities in the placenta are thought to contribute to many of the losses in early gestation; however, less is known of the pathology of clone deaths in the perinatal period. To date, the majority of perinatal deaths have been attributed to the “large offspring syndrome” characterized by increased birth weight and a range of morphological abnormalities thought to be associated with in vitro culture and manipulation. This report describes multi-systemic abnormalities in aborted, stillborn, and neonatal genetically modified and unmodified cloned calves weighing less than 60 kg at birth and aged between 6 months gestation and 3 weeks postnatal, generated in various experiments. Three of 14 genetically modified cloned calves had cystic renal dysplasia and osteopetrosis. All three and a fourth had irregular nodular, fibrotic livers with biliary abnormalities. Another two had marked flexion of the fetlock joints. Eleven calves derived from an unmodified cloned cell line by nuclear transfer had nodular, fibrotic livers with biliary anomalies, 9 of 11 had cystic renal dysplasia and cardiomegaly, two had osteopetrosis, and two had contracted tendons. In addition, three calves had polymicrocerebral gyri, two had retinal dysplasia, and one had an aortic aneurysm. Only one calf from a second unmodified cloned cell line produced by nuclear transfer had no significant congenital abnormalities. All calves were negative for bovine virus diarrhea virus (BVDV) by competitive-antigen ELISA, and by virus isolation and no BVDV antibodies were detected by AGID assay. Furthermore, all cell lines and media used were negative for BVDV by virus isolation. Two calves were tested and found to be negative for Akabane virus and Aino virus. There are very few reports of the pathological abnormalities of cloned animals. Similar multi-systemic abnormalities have not been found in non-cloned calves, but several analogous conditions occur in humans, including Simpson-Golabi-Behmel and Zellwegers syndromes. Further ultrastructure studies and genetic analysis are needed to investigate the mechanisms of these multi-systemic disorders, which may ultimately elucidate mechanisms for improved reprogramming and increase the efficiency of generating cloned animals with somatic cells by nuclear transfer.

Isolation of Bovine Arboviruses fromCulicoidesBiting Midges (Diptera: Ceratopogonidae) in Southern Japan: 1985–2002

In 1985--2002, surveillance for bovine arboviruses was conducted in Kagoshima, located in the most southern part of the main islands of Japan and known to be an area where bovine arboviral diseases have frequently been epidemic. Culicoides biting midges were collected in a cowshed by light traps. A total of 456,300 Culicoides biting midges representing 13 species were collected, and a portion of each pool of midges were tested for virus isolation. Overall, 85 isolates of six different viruses were obtained from the collected midges. The isolated viruses included two Orthobunyaviruses, Akabane and Aino viruses; three Orbiviruses, Chuzan, D'Aguliar, and Ibaraki viruses; and one unclassified virus, a bunyavirus-like virus. The viruses were most frequently isolated from Culicoides oxystoma Kieffer (85.9% of 85 isolates). Isolations of all viruses except for the bunyavirus-like virus were made from this species. Our data indicated that C. oxystoma is a potential vector for bovine arboviruses in southern Japan.

Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction

Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan, Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyavirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6–10 3.1 median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens.

Detection and quantitation of Akabane and Aino viruses by multiplex real-time reverse-transcriptase PCR

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye—VIC ® for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect ∼3–30 copies of viral S genome.

岡山県におけるアイノウイルスの流行とその病原性

A serological survey of the Aino virus (AINOV) conducted from August to October 2002 in Okayama Pre-fecture showed seroconversion to the virus in 79.6% of the sentinel calves and 90% of the farms examined. AINOV infection from early August 2002 to late March 2003 caused 29 cases of 77 abnormalities. Six fetuses were aborted, 10 calves were stillborn, 4 calves were premature, and 9 calves had congenital defects. Viruses were isolated from blood obtained from 5 sentinel calves in late August 2002 and from the cerebrum of an aborted fetus in early October 2002. Reverse transcription polymerase chain reaction and immunohistochemical analysis resulted in the diagnosis of an animal aged 24 months as AINOV infected. Abortions were more numerous and congenital abnormalities occurred at an earlier state of AINOV infection in this epidemic than in earlier ones. This is the first report to describe AINOV as a cause of encephalitis in naturally infected cattle.

Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.

Serological and genetic characterization of newly isolated Peaton virus in Japan. Brief report.

The viruses were isolated from the blood of sentinel cattle and Culicoides biting midges in the Kyushu district, southwestern Japan, in 1999 and identified by neutralization tests as Peaton (PEA) viruses. Before this study, PEA virus had been isolated in Australia only. The nucleotide identity of the nucleocapsid (N) protein encoded by the S segment ranged from 91.1 to 91.6% between the Australian and Japanese strains. A phylogenetic analysis of the N protein sequence revealed that the PEA virus strains are closely related to Aino (AIN) virus and suggested reassortment events for PEA and AIN viruses.

A pathologic study on ocular disorders in calves in southern Kyushu, Japan.

Of 822 calves, ranging in age between one day and six months necropsied between 1996 and 1998 at Miyazaki University, histological examination showed that 25 (3.0%) had ocular lesions. These ocular lesions consisted of suppurative inflammation (13 cases), cataract (seven cases), and retinal atrophy (five cases). Inflammatory changes were classified as suppurative keratitis (one case), keratitis and uveitis (ten cases), and uveitis and retinitis (two cases). Cataract was subclassified into three categories; cortical (three cases), nuclear (one case), and mature (three cases). These lesions were characterized by degenerative changes in the lens fibers and the appearance of eosinophilic globules known as Morganian globules. In the most severely affected case, there was capsular rupture of the lens, resulting in severe infiltration by eosinophils and histiocytes of the whole anterior chamber. Almost all the calves with retinal atrophy had been suffering from severe hydranencephaly and three had significantly raised levels of neutralization antibodies for the Akabane and/or Aino viruses. This study indicates that congenital arbovirus infections may predispose calves to ocular diseases, especially retinal atrophy.

岡山県におけるアカバネおよびアイノウイルスの流行と異常産発生

岡山県におけるアカバネおよびアイノウイルスの流行と異常産発生

Detection and differentiation of Aino and Akabane Simbu serogroup bunyaviruses by nested polymerase chain reaction.

Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR were developed to detect and differentiate Aino (AINO) and Akabane (AKA) virus S RNA. Two pairs of AINO- and AKA-specific primers for nested PCR were synthesized and examined for their capacity to amplify PCR products using 7 Simbu serogroup viruses isolated in Japan and Australia. RT- and nested PCR using AKA-specific primers amplified cDNA from Tinaroo virus RNA as well as homologous RNA. Nested PCR products were differentiated by Hph I and Bst EII digestion. Peaton (PEA) virus S cDNA was also amplified using AINO- specific primers. The nested PCR products of PEA virus were not digested by 3 restriction enzymes (Ava II, Eco RI and Hae II), whereras those of AINO virus were digested as expected. Using this technique, AINO and AKA viruses were detected at concentrations as low as 10(-3) plaque-forming units (PFU) and 10(-5) PFU, respectively, in a supernatant of virus-infected cells. It was possible to detect AINO and AKA genome from various tissues of experimentally infected mice, and also the AKA nested PCR products from serum samples from sentinel cattle naturally infected with AKA virus. The present nested PCR appears a simple, rapid and valuable method for diagnosing AINO and AKA infection.

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.