Hirohito Ogawa

Prevalence of swine Torque teno virus genogroups 1 and 2 in Japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex

Torque teno virus (TTV) was first isolated from a human hepatitis patient in 1997. TTV was also identified in several animals, including pigs, cattle, sheep, cats and dogs. In this study, we analysed the prevalence of swine TTV genogroups 1 (TTV1) and 2 (TTV2) in Japanese swine populations with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease by using a nested polymerase chain reaction method. Of 153 serum samples from 16 different herds in Japan, TTV1 was detected in 46 samples (30%), TTV2 in 47 samples (31%) and both in 15 samples (10%). There was no significant difference in the detection rate among geographical regions. The overall prevalence rate of TTV genogroups was significantly lower in < or = 30-day-old pigs (11%) compared to that in older age groups (54-82%). These results suggest that swine TTV may be widespread in post-weaning pigs and could play aetiological roles in pig diseases in Japan. This is the first report on the prevalence of swine TTV in Japan.

Characterization of the envelope glycoprotein of a novel filovirus, lloviu virus.

ABSTRACT Lloviu virus (LLOV), a novel filovirus detected in bats, is phylogenetically distinct from viruses in the genera Ebolavirus and Marburgvirus in the family Filoviridae. While filoviruses are known to cause severe hemorrhagic fever in humans and/or nonhuman primates, LLOV is biologically uncharacterized, since infectious LLOV has never been isolated. To examine the properties of LLOV, we characterized its envelope glycoprotein (GP), which likely plays a key role in viral tropism and pathogenicity. We first found that LLOV GP principally has the same primary structure as the other filovirus GPs. Similar to the other filoviruses, virus-like particles (VLPs) produced by transient expression of LLOV GP, matrix protein, and nucleoprotein in 293T cells had densely arrayed GP spikes on a filamentous particle. Mouse antiserum to LLOV VLP was barely cross-reactive to viruses of the other genera, indicating that LLOV is serologically distinct from the other known filoviruses. For functional study of LLOV GP, we utilized a vesicular stomatitis virus (VSV) pseudotype system and found that LLOV GP requires low endosomal pH and cathepsin L, and that human C-type lectins act as attachment factors for LLOV entry into cells. Interestingly, LLOV GP-pseudotyped VSV infected particular bat cell lines more efficiently than viruses bearing other filovirus GPs. These results suggest that LLOV GP mediates cellular entry in a manner similar to that of the other filoviruses while showing preferential tropism for some bat cells.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections.

Multiplex PCR and multiplex RT-PCR were developed to identify nine viruses in pigs with multiple infections. These viruses are: porcine circovirus type 2 (PCV2), suid herpesvirus 1, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus, porcine rotavirus A (PoRV-A), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Getah virus. These methods were shown to be high specificity and sensitivity. In the clinical application, a total of 75 field samples were examined by our methods and previously reported methods for PCV2, PRRSV, TGEV, and PEDV. As a result, the detection rates of our multiplex PCR and multiplex RT-PCR were higher than those of the previously reported methods. Furthermore, it was confirmed that 24 PCV2 positive samples were co-infected with other viruses, 11 with PRRSV, 10 with PPV, 2 with PoRV-A, and 1 with TGEV by a combination of multiplex PCR and multiplex RT-PCR. PPV and PoRV-A were newly detected by multiplex PCR and multiplex RT-PCR. These results suggest that the combination of our multiplex PCR and multiplex RT-PCR is useful for rapid and accurate identification of nine major pathogenic viruses in pigs with multiple infections.

Detection of all known filovirus species by reverse transcription-polymerase chain reaction using a primer set specific for the viral nucleoprotein gene

The filoviruses, Marburg virus (MARV) and Ebola virus (EBOV), are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates. Sporadic outbreaks of filovirus infection have occurred in Central Africa and parts of Asia. Identification of the natural reservoir animals that are unknown yet and epidemiological investigations are current challenges to forestall outbreaks of filovirus diseases. The filovirus species identified currently include one in the MARV group and five in the EBOV group, with large genetic variations found among the species. Therefore, it has been difficult to develop a single sensitive assay to detect all filovirus species, which would advance laboratory diagnosis greatly in endemic areas. In this study, a highly sensitive universal RT-PCR assay targeting the nucleoprotein (NP) gene of filoviruses was developed. The genomic RNAs of all known MARV and EBOV species were detected by using an NP-specific primer set. In addition, this RT-PCR procedure was verified further for its application to detect viral RNAs in tissue samples of animals infected experimentally and blood specimens of infected patients. This assay will be a useful method for diagnostics and epidemiological studies of filovirus infections.

Duplex Shuttle PCR for Differential Diagnosis of Budgerigar Fledgling Disease and Psittacine Beak and Feather Disease

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298‐bp fragment of the t/T antigen region of APV genome and a 495‐bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV‐positive, 15 PBFDV‐positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV‐positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.

Genetic Diversity and Epizootiology of Chlamydophila psittaci Prevalent among the Captive and Feral Avian Species Based on VD2 Region of ompA Gene

To study genetic diversity and occurrence of Chlamydophila psittaci, a total of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and captive birds were examined using ompA gene based nested PCR. Three types of chlamydiae: C. psittaci (94.12%), C. abortus (4.41%) and unknown Chlamydophila sp. (1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes‐59, Ciconiiformes‐8 and Passeriformes‐1). Based on nucleotide sequence variations in the VD2 region of ompA gene, all 64 detected C. psittaci strains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and 7.35% samples respectively. A single strain of unknown Chlamydophila sp. was found phylogenetically intermediate between Chlamydophila species infecting avian and mammalian hosts. Among Psittaciformes, 28 out of 81 tested species including 10 species previously unreported were found to be chlamydiae positive. Chlamydiosis was detected among 8.97% sick and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was observed that though various genetically diverse chlamydiae may cause avian chlamydiosis, only a few C. psittaci strains are highly prevalent and frequently associated with clinical/subclinical infections.

Human–animal anthrax outbreak in the Luangwa valley of Zambia in 2011:

There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4–56.0) were from humans, 42.1% (95% CI, 21.1–66.0) were from hippopotamuses and 20.0% (95% CI, 6.61–44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.

Identification of a novel polyomavirus from vervet monkeys in Zambia

To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n = 100 each) of yellow baboons and vervet monkeys (VMs) (n = 50 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broad-spectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48 % nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen.

Molecular epidemiology of paramyxoviruses in frugivorous Eidolon helvum bats in Zambia.

ABSTRACT In this study, we describe the detection of novel paramyxoviruses from the Eidolon helvum species of fruit bats. We extracted RNA from 312 spleen samples from bats captured in Zambia over a period of 4 years (2008–2011). Semi-nested RT-PCR detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. Among the positive samples, seven novel paramyxoviruses were detected. Five viruses were closely related to the genus Henipavirus, while two viruses were related to the unclassified Bat paramyxoviruses from Ghana and Congo Brazzaville. Our study identified novel Henipavirus-related and unrelated viruses using RT-PCR in fruit bats from Kansaka National Park and indicated the presence of similar Bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. The presence of these viruses in fruit bats might pose a public health risk.