Comparison of Antiretroviral Therapies in Pregnant Women Living With Human Immunodeficiency Virus and Hepatitis B Virus: A Randomized Controlled Trial.

OBJECTIVE: To describe the anti-hepatitis B virus (HBV) efficacy, HBeAg serologic changes, HBV perinatal transmission, and safety in pregnant women who are living with human immunodeficiency virus (HIV) and HBV co-infection who were randomized to various antiretroviral therapy (ART) regimens. METHODS: The PROMISE (Promoting Maternal and Infant Survival Everywhere) trial was a multicenter randomized trial for ART-naive pregnant women with HIV infection. Women with HIV and HBV co-infection at 14 or more weeks of gestation were randomized to one of three ART arms: one without HBV treatment (group 1) and two HBV treatment arms with single (group 2) or dual anti-HBV activity (group 3). The primary HBV outcome was HBV viral load antepartum change from baseline (enrollment) to 8 weeks; safety assessments included alanine aminotransferase (ALT) level, aspartate aminotransferase (AST) level, and anemia (hemoglobin less than 10 g/dL). Primary comparison was for the HBV-active treatment arms. Pairwise comparisons applied t test and the Fisher exact tests. RESULTS: Of 3,543 women, 3.9% were HBsAg-positive; 42 were randomized to group 1, 48 to group 2, and 48 to group 3. Median gestational age at enrollment was 27 weeks. Among HBV-viremic women, mean antepartum HBV viral load change at week 8 was -0.26 log 10 international units/mL in group 1, -1.86 in group 2, and -1.89 in group 3. In those who were HBeAg-positive, HBeAg loss occurred in 44.4% at delivery. Two perinatal HBV transmissions occurred in group 2. During the antepartum period, one woman (2.4%) in group 1 had grade 3 or 4 ALT or AST elevations, two women (4.2%) in group 2, and three women (6.3%) in group 3. CONCLUSION: Over a short period of time, HBV DNA suppression was not different with one or two HBV-active agents. HbeAg loss occurred in a substantial proportion of participants. Perinatal transmission of HBV infection was low. Hepatitis B virus-active ART was well-tolerated in pregnancy, with few grade 3 or 4 ALT or AST elevations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov , NCT01061151.

rAAV-PHP.B escapes the mouse eye and causes lethality whereas rAAV9 can transduce aniridic corneal limbal stem cells without lethality.

Recently safety concerns have been raised in connection with high doses of recombinant adeno-associated viruses (rAAV). Therefore, we undertook a series of experiments to test viral capsid (rAAV9 and rAAV-PHP.B), dose, and route of administration (intrastromal, intravitreal, and intravenous) focused on aniridia, a congenital blindness that currently has no cure. The success of gene therapy for aniridia may depend on the presence of functional limbal stem cells (LSCs) in the damaged aniridic corneas and whether rAAV can transduce them. Both these concerns were unknown, and thus were also addressed by our studies. For the first time, we report ataxia and lethality after intravitreal or intrastromal rAAV-PHP.B virus injections. We demonstrated virus escape from the eye and transduction of non-ocular tissues by rAAV9 and rAAV-PHP.B capsids. We have also shown that intrastromal and intravitreal delivery of rAAV9 can transduce functional LSCs, as well as all four PAX6-expressing retinal cell types in aniridic eye, respectively. Overall, lack of adverse events and successful transduction of LSCs and retinal cells makes it clear that rAAV9 is the capsid of choice for future aniridia gene therapy. Our finding of rAAV lethality after intraocular injections will be impactful for other researchers developing rAAV-based gene therapies.

Anti-hepatitis B virus activity of lithospermic acid, a polyphenol from Salvia miltiorrhiza, in vitro and in vivo by autophagy regulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (the roots of S. miltiorrhiza Bunge, Danshen in Chinese), a traditional Chinese medicine, has been clinically used to prevent and treat various diseases, such as cardiovascular and cerebrovascular diseases, diabetes, and hepatitis B, in China and some other Asian countries. Lithospermic acid (LA), a polyphenol derived from S. miltiorrhiza, has been reported to exhibit multiple pharmacological properties, such as anti-inflammatory, anti-HIV, and anti-carbon tetrachloride-induced liver injury activities. However, little is known about the anti-hepatitis B virus (HBV) activity of LA. AIM OF THE STUDY: The study was projected to investigate the anti-HBV activity of LA in vitro (HepG2.2.15 and pHBV1.3-transfected HepG2 cells) and in vivo (pAAV-HBV1.2 hydrodynamic injection [HBV-HDI] mice) and explore the potential mechanism as well. MATERIALS AND METHODS: Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) contents were detected by ELISA kits. HBV DNA and hepatitis B core antigen (HBcAg) levels were evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry assay, respectively. The proteins in autophagy process, lysosomal acidic function, and autophagy-related signaling pathways were examined by Western blot. Transmission electron microscopy was used to observe the number of autophagosomes and autolysosomes. Confocal microscopy was applied to analyze the autophagic flux and lysosomal acidification, using mCherry-enhanced green fluorescent protein (EGFP)-microtubule-associated protein light chain (LC)3 and lysosomal probes, respectively. RESULTS: LA exhibited anti-HBV activity by inhibiting HBV DNA replication in HepG2.2.15 and pHBV-transfected HepG2 cells in dose- and time-dependent manners and hampering HBsAg and HBeAg levels in HepG2.2.15 cells to a certain extent. LA reduced HBV DNA, HBsAg/HBeAg, and HBcAg levels in the serum/liver tissues of HBV-HDI C57BL/6 mice during the 3-week treatment and suppressed the withdrawal rebound of HBV DNA and HBsAg in the mice serum. LA increased LC3-II protein expression and the number of autolysosomes/autophagosomes and promoted the degradation of sequestosome 1(p62) protein in vitro and in vivo. LA enhanced the co-localization of LC3 protein with autolysosomes, further confirming the ability of LA to induce a complete autophagy. Knockdown of autophagy-related gene (Atg) 7 or 5 in vitro and administration of 3-methyladenine (an autophagic inhibitor) in vivo disabled the inhibitory efficacy of LA on HBV DNA replication, suggesting that the anti-HBV efficacy of LA depended on its ability of inducing autophagy. LA could enhance lysosomal acidification and improve the function of lysosomes by promoting the protein expression of lysosomal-associated membrane protein (LAMP)-1, LAMP-2, and mature cathepsin D, which may contribute to the autophagic induction of LA. LA inhibited the activation of AKT and mammalian target of rapamycin (mTOR) induced by HBV, which was reversed by IGF-1 (an agonist of the PI3K/AKT/mTOR signaling pathway), indicating that LA elicited autophagy through hampering the PI3K/AKT/mTOR signaling pathway. CONCLUSION: We revealed the anti-HBV activity and mechanism of LA in vitro and in vivo. This study facilitates a new understanding of the anti-HBV potent components of S. miltiorrhiza and sheds light on LA for further development as an active constituent or candidate used in the therapy against HBV infection.

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies.

UNLABELLED: B virus (Macacine herpesvirus 1) can cause deadly zoonotic disease in humans. Molecular mechanisms of B virus cell entry are poorly understood for both macaques and humans. Here we investigated the abilities of clinical B virus isolates to use entry receptors of herpes simplex viruses (HSV). We showed that resistant B78H1 cells became susceptible to B virus clinical strains upon expression of either human nectin-2 or nectin-1. Antibody against glycoprotein D (gD) protected these nectin-bearing cells from B virus infection, and a gD-negative recombinant B virus failed to enter these cells, indicating that the nectin-mediated B virus entry depends on gD. We observed that the infectivity of B virus isolates with a single amino acid substitution (D122N) in the IgV-core of the gD ectodomain was impaired on nectin-1-bearing cells. Computational homology-based modeling of the B virus gD-nectin-1 complex revealed conformational differences between the structures of the gD-122N and gD-122D variants that affected the gD-nectin-1 protein-protein interface and binding affinity. Unlike HSV, B virus clinical strains were unable to use herpesvirus entry mediator (HVEM) as a receptor, regardless of conservation of the gD amino acid residues essential for HSV-1 entry via HVEM. Based on the model of the B virus gD-HVEM interface, we predict that residues R7, R11, and G15 are largely responsible for the inability of B virus to utilize HVEM for entry. The ability of B virus to enter cells of a human host by using a combination of receptors distinct from those for HSV-1 or HSV-2 suggests a possible mechanism of enhanced neuropathogenicity associated with zoonotic infections. IMPORTANCE: B virus causes brainstem destruction in infected humans in the absence of timely diagnosis and intervention. Nectins are cell adhesion molecules that are widely expressed in human tissues, including neurons and neuronal synapses. Here we report that human nectin-2 is a target receptor for B virus entry, in addition to the reported receptor human nectin-1. Similar to a B virus lab strain, B virus clinical strains can effectively use both nectin-1 and nectin-2 as cellular receptors for entry into human cells, but unlike HSV-1 and HSV-2, none of the clinical strains uses an HVEM-mediated entry pathway. Ultimately, these differences between B virus and HSV-1 and -2 may provide insight into the neuropathogenicity of B virus during zoonotic infections.

B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells.

UNLABELLED: Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-ΔgDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV-ΔgDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-ΔgDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-ΔgDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. IMPORTANCE: B virus is the only known simplexvirus that causes zoonotic infection, resulting in approximately 80% mortality in untreated humans or in lifelong persistence with the constant threat of reactivation in survivors. Here, we report that B virus lacking the gD envelope glycoprotein infects both human and monkey cells as efficiently as wild-type B virus. These data provide evidence for a novel mechanism(s) utilized by B virus to gain access to target cells. This mechanism is different from those used by its close relatives, HSV-1 and -2, where gD is a pivotal protein in the virus entry process. The possibility remains that unidentified receptors, specific for B virus, permit virus entry into target cells through gD-independent pathways. Understanding the molecular mechanisms of B virus entry may help in developing rational therapeutic strategies for the prevention and treatment of B virus infection in both macaques and humans.

Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

Herpes B virus, macacine herpesvirus 1, breaks simplex virus tradition via major histocompatibility complex class I expression in cells from human and macaque hosts.

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8(+) T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions.

[Epidemiological survey of a captive Chinese rhesus macaque breeding colony in Yunnan for SRV, STLV and BV].

Nonhuman primates are critical resources for biomedical research. Rhesus macaque is a popularly used laboratory nonhuman primate that share many characteristics with humans. However, rhesus macaques are the natural host of two exogenous retroviruses, SRV (simian type D retrovirus) and STLV (simian T lymphotropic virus). SRV and STLV may introduce potentially significant confounding factors into the study of AIDS model. Moreover, B virus (ceropithecine herpesvirus 1) is likely to harm not only rhesus macaque but also humans in experiments involving rhesus macaque. Yunnan province has large-scale breeding colonies of Chinese rhesus macaque. Therefore there is an urgent need for SPF Chinese rhesus macaque colonies. Here we investigated SRV, STLV and BV infections in 411 Chinese rhesus macaque by PCR technique. The results showed that the prevalence of SRV, STLV and BV among Chinese rhesus macaque breeding colony was 19.71% (81/411), 13.38% (55/411) and 23.11% (95/411), respectively. Comparison of viruses infection in different age-groups and male/female of Chinese rhesus macaque was also analyzed. This study will contribute to establishment of SPF Chinese rhesus macaque breeding colony.

Herpes B virus utilizes human nectin-1 but not HVEM or PILRα for cell-cell fusion and virus entry.

To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections.

Ulcerative cheilitis in a rhesus macaque.

A 2-year-old, female, simian immunodeficiency virus E543-infected rhesus macaque (Macaca mulatta) was presented for necropsy following euthanasia due to a history of diarrhea, weight loss, and a small, round ulcer along the left labial commissure. Histopathologic examination of the ulcer revealed infiltration by large numbers of degenerate and nondegenerate neutrophils and macrophages admixed with syncytial epithelial cells. Rare epithelial cells contained herpetic inclusion bodies. These cells stained positive for Human herpesvirus 1 via immunohistochemistry, and DNA sequencing confirmed the presence of closely related Macacine herpesvirus 1 (B virus).

[Construction of recombinant eukaryotic expression plasmid of murine chemokine MIP-1alpha and coimmunization with HSV-II DNA vaccine].

AIM: To construct the recombinant eukaryotic expression plasmid of murine macrophage inflammatory protein-1alpha (MIP-1alpha) and investigate the effect of MIP-1alpha as an adjuvant on immune response induced by herpes simplex virus type II glycoprotein D (HSV-II gD) DNA vaccine. METHODS: Using total RNA from RAW264.7 cells stimulated with LPS, the whole code sequence of murine MIP-1alpha was amplified by RT-PCR and inserted into pcDNA3 at Hind III/Xba I restriction sites. The recombinant eukaryotic expression plasmid Pm was transiently expressed in COS-7 cells and its specificity was demonstrated by RT-PCR and Boyden chemotaxis chamber assay. BALB/c mice were immunized with gD DNA vaccine and/or MIP-1alpha, and the effect of MIP-1alpha on gD DNA vaccine was evaluated by detecting anti-HSV-II antibody, antigen-specific lymphoproliferative responses, and examining survival rates after mice were challenged intravaginally with HSV-II. RESULTS: The recombinant eukaryotic expression plasmid of murine MIP-1alpha was constructed, and it was revealed that immune responses of HSV-II gD DNA vaccine were enhanced by coimmunization with MIP-1alpha. CONCLUSION: The murine MIP-1alpha can be used as an adjuvant of HSV-II gD DNA vaccine.

Infection of simian B lymphoblastoid cells with simian immunodeficiency virus is associated with upregulation of CD23 and CD40 cell surface markers.

Simian immunodeficiency virus (SIV) as well as human immunodeficiency virus (HIV) induce polyclonal B-cell activation and are associated with the appearance of lymphomas in their respective hosts in either the presence or the absence of other co-infecting viruses such as Epstein-Barr virus (EBV). However, the pathogenic role of these retroviruses in the development of lymphoproliferative disorders remains poorly understood. To explore the virus-B-cell interactions, two immortalized lymphoblastoid B-cell lines (SL-P1 and SL-691) were established from cynomolgus monkeys that were naturally co-infected with a simian type D retrovirus-2 (SRV-2) and with the herpes virus Macaca fascicularis (HVMF-1). We addressed their susceptibility to SIV infection and the phenotypic modifications associated with SIV infection. In response, both cell lines (1) were co-infected with HVMF-1 (latent infection) and with SRV-2 (productive infection), (2) had a transformed phenotype because they did not require exogenous growth factors, and (3) when injected into mice with severe combined immunodeficiency (SCID), generated serially transplantable tumors. The B-cell origin of SL cells was demonstrated by the presence of rearrangements of the IgH gene and by the expression of typical B-cell lineage markers, such as CD20. SL-P1 and SL-691 could be discriminated on the basis of different expressions of CD23 and CD40 and of kappa- and lambda-chains. Most importantly, SL-691 cells, but not SL-P1 cells, were susceptible to chronic noncytolytic SIV infection. This infection occurred in a CD4/CCR5/CXCR4-independent manner and was associated with the upregulated expression of CD23 and CD40 cell surface markers. In addition, CD20 expression, which progressively disappeared in SL-691 noninfected cells, was maintained in the SIV-infected counterpart. These findings support the hypothesis that SIV induce phenotypic perturbations in B cells that might eventually contribute to the development of lymphoproliferative disease.

One-step PCR to distinguish B virus from related primate alphaherpesviruses.

By adding betaine to the PCR mixture, we previously established a PCR method to amplify a DNA segment of the glycoprotein G gene of B virus (BV) derived from a rhesus macaque. We have found that DNA of other BV strains derived from cynomolgus, pigtail, and lion-tailed macaques can also serve as the template in our PCR assay. Under the same conditions no product was obtained with DNA of simian agent 8 of green monkeys and Herpesvirus papio 2 of baboons, or the human herpes simplex viruses types 1 and 2. Thus, this PCR method is useful to discriminate BV from other closely related primate alphaherpesviruses.

Rapid discrimination of monkey B virus from human herpes simplex viruses by PCR in the presence of betaine.

A PCR method to amplify DNA segments of the glycoprotein G gene of monkey B virus (BV) was achieved by adding betaine to the PCR mixture, in spite of the high G+C content of this gene. No product was obtained when DNA of human herpes simplex viruses (HSVs) was used as the template under the same conditions. Thus, this PCR method is useful in discriminating BV from HSVs.

Infectious simian varicella virus expressing the green fluorescent protein.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates closely resemble varicella-zoster virus (VZV) infection in humans. Such similarities provide a rationale to analyze SVV infection in primates as a model of varicella pathogenesis and latency. Thus, we constructed an SVV-expressing green fluorescent protein (SVV-GFP) by inserting the GFP gene into the unique short segment of the virus genome by homologous recombination. Analysis of recombinant viral DNA and the expressed proteins of plaque-purified SVV-GFP confirmed the location of the GFP insert and that the recombinant SVV expressed the 27 kDa GFP. Infection of monkey kidney cells in tissue culture with SVV-GFP revealed bright green fluorescence associated with the characteristic focal cytopathic effect produced by SVV infection. Microscopic examination of lung from a 3-month-old African green monkey 10 days after infection with SVV-GFP revealed bright green fluorescence in areas of acute necrotizing pneumonitis. SVV-GFP allows ready identification of cells infected with SVV both in vitro and in vivo, and will be useful for further analysis of varicella pathogenesis and latency in experimentally infected animals--studies not possible in humans.

Analysis of the genome of an Epstein-Barr-virus (EBV)-related herpesvirus in a cynomolgus monkey cell line (Si-IIA).

A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV)-related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

Genetic and functional complementation of the HSV1 UL27 gene and gB glycoprotein by simian alpha-herpesvirus homologs.

Utilizing co-transfection of DNA from glycoprotein gB- strain of HSV1 and cloned fragments of several simian alpha-herpesviruses containing the UL26, UL27 (gB glycoprotein), and UL28 gene homologs, replication-competent recombinant viruses were produced. Genetic analysis of one HSV1/SA8 recombinant (HSV1/SgB) demonstrated the presence of SA8 DNA comprising the entire UL27 (gB) gene and parts of the UL28 and UL26 ORFs in an otherwise HSV1 genome. The recombinant was shown to express the SA8 gB and p40 proteins (UL27 & UL26.5 gene products, respectively); all other proteins were indistinguishable from those of HSV1. The recombinant behaved like SA8 in gB-specific virus neutralization and cell surface antibody binding assays, while plaque morphology and replication kinetics were very similar to HSV1. Despite its overwhelming HSV1 genetic constitution, the recombinant displayed a pathogenic phenotype in mice very different from the parental HSV1. While HSV1 produced corneal disease in ocularly infected mice and readily spread to the nervous system. HSV1/SgB was markedly impaired in both respects. These results demonstrate the functional equivalency of the cercopithecine monkey virus gB glycoproteins and genes (including transcriptional regulatory elements) in HSV1, the functional nature of HSV1/SA8 chimeric UL28 and UL26 genes/proteins, and that UL28, gB and/or p40 proteins may effect the pathogenicity of HSV1.

Identification of simian varicella virus homologues of varicella zoster virus genes.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates resemble human varicella-zoster virus (VZV) infection. Further, the SVV and VZV genomes are similar in size and structure, show striking homology in their configuration and DNA sequences, and encode antigenically related polypeptides. Although the entire VZV genome is present during latency in human ganglia, transcription is limited. VZV genes 21, 29, 62 and 63 are transcribed during latency, while genes 4, 10, 40, 51 and 61 are not transcribed. The entire VZV genome has been sequenced, but the SVV genome has not. Thus, to analyze SVV genes transcribed during latency, we have begun to identify SVV homologues of the above VZV genes. We used nick-translated [32p]-labeled-VZV open reading frame (ORF)-specific probes to screen Southern blots containing EcoRI-digested SVV genomic DNA and recombinant clones of SVV EcoRI and BamHI DNA fragments spanning approximately 97% of the virus genome. We showed that SVV homologues of VZV ORFs 4, 10, 29, 40, 51 and 61 mapped to SVV DNA fragments EcoRI I, A, N, BamHI E, EcoRI D and E, respectively. We also confirmed earlier reports that SVV homologues of VZV genes 21 and 63 mapped to SVV EcoRI DNA fragments H and C, respectively. Viral genes on the SVV and VZV genomes seem to be collinear.

Identification and analysis of the simian varicella virus thymidine kinase gene.

The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.