Hikaru Fujii

Reston Ebolavirus antibodies in bats, the Philippines.

To the Editor: Filoviruses cause highly lethal hemorrhagic fever in humans and nonhuman primates, except for Reston Ebolavirus (REBOV), which causes severe hemorrhagic fever in macaques (1,2). REBOV epizootics among cynomolgus macaques occurred in 1989, 1990, 1992, and 1996 (2) and among swine in 2008 (3). African fruit bats have been suggested to be natural reservoirs for Zaire Ebolavirus and Marburg virus (4–6). However, the natural reservoir of REBOV in the Philippines is unknown. Thus, we determined the prevalence of REBOV antibody–positive bats in the Philippines. Permission for this study was obtained from the Department of Environment and Natural Resources, the Philippines, before collecting bat specimens. Serum specimens from 141 wild-caught bats were collected at several locations during 2008–2009. The bat species tested are summarized in the Table. Captured bats were humanely killed and various tissues were obtained. Carcasses were then provided to the Department of Environment and Natural Resources for issuance of a transport permit. Table REBOV-specific IgG in Rousettus amplexicaudatus bats and other bats, the Philippines* We used immunoglobulin (Ig) G ELISAs with recombinant nucleoprotein (NP) and glycoprotein (GP) of REBOV (7) to determine REBOV antibody prevalence. REBOV NP and GP were expressed and purified from Tn5 cells infected with recombinant baculoviruses AcResNP and AcResGPDTM, which express NP and the ectodomain of GP with the histidine tag at its C-terminus. We also used histidine-tagged recombinant Crimean-Congo hemorrhagic fever virus NP as a negative control antigen in the IgG ELISA to confirm specificity of reactivity. In IgG ELISAs for bat specimens, positive results were detected by using rabbit anti-bat IgG and horseradish peroxidase–conjugated anti-rabbit IgG. Anti-bat (Rousettus aegyptiacus) rabbit IgG strongly cross-reacts with IgGs of other bat species, including insectivorous bats (8). Bat serum samples were 4-fold serially diluted (1:100–1:6,400) and tested by using IgG ELISAs. Results of IgG ELISAs were the sum of optical densities at serum dilutions of 1:100, 1:400, 1:1,600, and 1:6,400. Cutoff values (0.82 for both IgG ELISAs) were determined by using serum specimens from REBOV antibody–negative bats. Among 16 serum samples from R. amplexicaudatus bats, 5 (31%) captured at either the forest of Diliman (14°38′N, 121°2′E) or the forest of Quezon (14°10′N, 121°50′E) had positive results in the IgG ELISA for REBOV NP, and 5 (31%) captured at the forest of Quezon had positive results in the IgG ELISA for REBOV GP. The REBOV NP antibody–positive bats serum samples were confirmed to be NP antibody positive in the IgG ELISA by using glutathione-S-transferase–tagged partial REBOV NP antigen (9). Three samples had positive results in both IgG ELISAs (Table). Serum samples from other bat species had negative results in IgG ELISAs. All bat serum samples were also tested by indirect immunofluorescence assays (IFAs) that used HeLa cells expressing NP and GP (10). In the IFAs, 2 samples from R. amplexicaudatus bats captured at the forest of Diliman and the forest of Quezon had high titers (1,280 and 640, respectively) of NP-specific antibodies, and 1 sample from an R. amplexicaudatus bat captured at the forest of Quezon had a positive result in the GP-specific IFA (titer 20). All IFA-positive samples were also positive in the IgG ELISA (Table). The forest of Diliman is ≈30 km from the monkey facility and the Bulacan farm where REBOV infections in monkeys and swine, respectively, were detected. The forest of Quezon is ≈60 km from the monkey facility. Samples from other bat species had negative results in IFAs. We also performed heminested reverse transcription PCR specific for the REBOV NP gene with spleen specimens from all 16 R. amplexicaudatus bats but failed to detect any REBOV-specific amplicons. REBOV-specific antibodies were detected only in R. amplexicaudatus bats, a common species of fruit bat, in the Philippines. In Africa, R. aegyptiacus bats, which are genetically similar to R. amplexicaudatus bats, have been shown to be naturally infected with Zaire Ebolavirus and Marburg virus. Thus, R. amplexicaudatus bats are a possible natural reservoir of REBOV. However, only 16 specimens of R. amplexicaudatus bats were available in this study, and it will be necessary to investigate more specimens of this species to detect the REBOV genome or antigens to conclude the bat is a natural reservoir for REBOV. We have shown that R. amplexicaudatus bats are putatively infected with REBOV or closely related viruses in the Philippines. Antibody-positive bats were captured at the sites near the study areas, where REBOV infections in cynomolgus monkeys and swine have been identified. Thus, bats are a possible natural reservoir of REBOV. Further analysis to demonstrate the REBOV genome in bats is necessary to conclude that the bat is a reservoir of REBOV.

Bat Coronaviruses and Experimental Infection of Bats, the Philippines

Virus-infected fruit bats showed no signs of clinical infection.

Roles of p53 in Herpes Simplex Virus 1 Replication

ABSTRACT p53 is a critical factor in the cellular response to a broad range of stress factors through its ability to regulate various cellular pathways. In this study, tandem affinity purification of transiently expressed herpes simplex virus 1 (HSV-1) regulatory protein ICP22 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP22 interacted with p53 in HSV-1-infected cells. In p53−/− cells, replication of wild-type HSV-1 was reduced compared to that in parental p53+/+ cells, indicating that p53 had a positive effect on HSV-1 replication. In contrast, the levels of viral replication of an ICP22-null mutant virus were similar in both p53−/− and p53+/+ cells. At 2 h postinfection, the level of expression of ICP27, an essential viral regulatory protein, in p53−/− cells infected with wild-type HSV-1 or the ICP22-null mutant virus was lower than in p53+/+ cells. In contrast, at 18 h postinfection, the level of expression of ICP0, a critical viral regulatory protein, in p53−/− cells infected with the ICP22-null mutant virus was higher than in p53+/+ cells, although the levels of ICP0 expression in p53−/− and p53+/+ cells infected with wild-type HSV-1 were almost identical. These results suggested that p53 overall promoted HSV-1 replication and that p53 played both positive and negative roles in HSV-1 replication: upregulating ICP27 expression very early in infection and downregulating ICP0 expression later in infection, which was antagonized by ICP22.

Detection of a new bat gammaherpesvirus in the Philippines.

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3′ part of the gB gene and the 5′ part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

ABSTRACT Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.

Functional analysis of Rousettus aegyptiacus "signal transducer and activator of transcription 1" (STAT1).

Abstract Bats are now known as the source of several diseases in humans, but few studies regarding immune responses and factors associated with bats have so far been reported. In this study, we focused on STAT1, one of the critical components in interferon (IFN)-signaling and antiviral activity, which is often targeted by viral proteins to reduce antiviral activity and increase viral replication. We found that Rousettus aegyptiacus STAT1 (bat STAT1) is phosphorylatable and translocates to the nucleus when stimulated with human IFN-α (hIFN-α). Furthermore, phosphorylation of bat STAT1 and inhibition of nuclear translocation was observed in IFN-stimulated cells infected with the HEP-Flury strain of rabies virus, in the same manner as in other mammals. Additionally, quantitative real-time RT-PCR revealed that bat STAT1 mRNA was highly expressed in the liver, while low in muscle and spleen.

The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation

ABSTRACT The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.

Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.