Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Nectin-2 Acts as a Viral Entry Mediated Molecule That Binds to Human Herpesvirus 6B Glycoprotein B.

Human herpesvirus 6B (HHV-6B) is a T-lymphotropic virus and the etiological agent of exanthem subitum. HHV-6B is present in a latent or persistent form after primary infection and is produced in the salivary glands or transmitted to this organ. Infected individuals continue to secrete the virus in their saliva, which is thus considered a source for virus transmission. HHV-6B primarily propagates in T cells because its entry receptor, CD134, is mainly expressed by activated T cells. The virus then spreads to the host's organs, including the salivary glands, nervous system, and liver. However, CD134 expression is not detected in these organs. Therefore, HHV-6B may be entering cells via a currently unidentified cell surface molecule, but the mechanisms for this have not yet been investigated. In this study, we investigated a CD134-independent virus entry mechanism in the parotid-derived cell line HSY. First, we confirmed viral infection in CD134-membrane unanchored HSY cells. We then determined that nectin cell adhesion molecule 2 (nectin-2) mediated virus entry and that HHV-6B-insensitive T-cells transduced with nectin-2 were transformed into virus-permissive cells. We also found that virus entry was significantly reduced in nectin-2 knockout parotid-derived cells. Furthermore, we showed that HHV-6B glycoprotein B (gB) interacted with the nectin-2 V-set domain. The results suggest that nectin-2 acts as an HHV-6B entry-mediated protein.

Characterization of a Novel Bat Adenovirus Isolated from Straw-Colored Fruit Bat (Eidolon helvum).

Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name "Bat mastadenovirus H". Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission.

Metabolic pathway catalyzed by Vanin-1 pantetheinase plays a suppressive role in influenza virus replication in human alveolar epithelial A549 cells.

Our previous analysis of gene expression profiles in the peripheral blood from patients with influenza A (H1N1) pdm09 pneumonia revealed elevated transcription levels of the vanin-1 (vascular non-inflammatory molecule 1, VNN1) gene, which encodes an epithelial ectoenzyme with pantetheinase activity involved in recycling coenzyme A. Here, to elucidate the role of VNN1 in influenza A virus (IAV) H1N1 infection, we investigated the change of VNN1 expression in the context of IAV infection and the effects of its related substances, i.e., its direct substrate pantetheine and its two metabolites pantothenic acid and cysteamine on the replication of IAV in the human alveolar epithelial carcinoma cell line A549. The messenger RNA expression of VNN1 in A549 cells was significantly increased (by 4.9-fold) after IAV infection under an elevated concentration of pantetheine. Moreover, VNN1 mRNA levels were elevated by > 100-fold in response to pro-inflammatory cytokines, especially TNF-α and IL-1ß. Pantetheine significantly reduced the IAV replication and IAV Matrix 1 (M1) mRNA levels when it was administered prior to and during infection. In addition, cysteamine treatment during IAV infection significantly reduced the viral replication and IAV M1 mRNA levels, whereas pantothenic acid did not. These findings suggest that the metabolic pathway catalyzed by VNN1 pantetheinase plays a suppressive role in IAV infection in the respiratory tract, especially in severe conditions under hypercytokinemia.

Molecular characterization of infectious bursal disease viruses detected in vaccinated commercial broiler flocks in Lusaka, Zambia.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive viral disease of young chickens and remains one of the economically most important diseases threatening the poultry industry worldwide. In this study, 16 and 11 nucleotide sequences of the VP2 hypervariable region (VP2-HVR) and part of VP1, respectively, of IBD virus (IBDV) detected in vaccinated broiler chickens in Lusaka in 2012 were determined. Phylogenetic analysis revealed that these Zambian IBDVs separated into three genotypes of very virulent (VV) IBDVs. Although the majority of these viruses belonged to the African VV type (VV1), which consisted of viruses from West Africa, South Africa and Zambia, one virus belonged to the East African VV type (VV2). Interestingly, a Zambian IBDV belonging to the VV3 genotype (composed of viruses from several continents) clustered with attenuated vaccine strains. Although sequence analysis of VP2-HVR showed that all detected Zambian IBDVs had conserved putative virulence marker amino acids (i.e., 222A, 242I, 256I, 294I and 299S), one virus had two unique amino acid substitutions, N280S and E300A. This study demonstrates the diversity of Zambian IBDVs and documents for the first time the possible involvement of attenuated vaccine strains in the epidemiology of IBD in Zambia. Strict biosecurity of poultry farms, monitoring of live vaccine use in the field, surveillance and characterization of IBDV in poultry and development of a vaccine from local or regional IBDV field strains are recommended for improved IBD control in Zambia.

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

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.

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.

A review of DNA viral infections in psittacine birds.

To date, several DNA viral infections have been reported in psittacine birds. Psittacine beak and feather disease (PBFD) is characterized by symmetric feather dystrophy and loss and development of beak deformities. PBFD is caused by beak and feather virus, which belongs to the Circoviridae, and is the most important infection in psittacine birds worldwide. Avian polyomavirus infection causes acute death, abdominal distention, and feather abnormalities. Pacheco's disease (PD), which is caused by psittacid herpesvirus type 1, is an acute lethal disease without a prodrome. Psittacine adenovirus infections are described as having a clinical progression similar to PD. The clinical changes in psittacine poxvirus-infected birds include serious ocular discharge, rhinitis, and conjunctivitis, followed by the appearance of ulcerations on the medial canthi of the eyes. Internal papillomatosis of parrots (IPP) is a tumor disease characterized by progressive development of papillomas in the oral and cloacal mucosa. IPP has been suggested to caused by papillomavirus or herpesvirus. However, information about these diseases is limited. Here we review the etiology, clinical features, pathology, epidemiology, and diagnosis of these DNA viruses.

A survey of avian polyomavirus (APV) infection in imported and domestic bred psittacine birds in Japan.

Although birds infected with avian polyomavirus (APV) subclinically could be a source of infection, no epidemiological studies of APV in psittacine birds have been reported in Japan. In the present study, we investigated subclinical morbidity rate of APV in imported and domestically bred psittacine birds by polymerase chain reaction (PCR). Of 402 live birds from which blood or feather samples were taken between April, 2003 and March, 2004, 11 (2.7%) were found to be APV positive. The DNA sequences of the APV t/T antigen region were determined for five APV-positive randomly selected samples and were found to be conserved.

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.