The importance of accounting for testing and positivity in surveillance by time and place: an illustration from HIV surveillance in Japan.

The number of tests performed is an important surveillance indicator. We illustrate this point using HIV surveillance data, focusing on Tokyo and Okinawa, two prefectures with high HIV notification rates in Japan. Restricting to data reported from local public health centres and affiliate centres where testing data are accessible, we assessed HIV surveillance data during 2007-2014, based on the annual HIV notification rate (per 100 000 population), HIV testing rate (per 100 000 population) and proportion testing HIV-positive (positivity). Nationally, testing activity and positivity showed an inverse relationship; in 2008, the testing rate peaked, but positivity was lowest. While notification rates were higher for Tokyo (median = 0.98, range = 0.89-1.33) than Okinawa (median = 0.61, range = 0.42-1.09), Okinawa had slightly higher testing rates (median = 187, range = 158-274) relative to Tokyo (median = 172, range = 163-210). Positivity was substantially lower in Okinawa (median = 0.34%, range = 0.24-0.45%) compared with Tokyo (median = 0.57%, range = 0.46-0.67%). Relative to the national testing rate (median = 85, range = 80-115) and positivity (median = 0.34%, range = 0.28-0.36%), Tokyo had higher positivity, despite more testing. In 2014 in Okinawa, all three indicators increased, providing a strong reason to be concerned as positivity increased despite more testing. Together with other information, accounting for testing and positivity improve interpretation of surveillance data to guide public health assessments.

Rapid appearance of secondary immune responses and protection from acute CD4 depletion after a highly pathogenic immunodeficiency virus challenge in macaques vaccinated with a DNA prime/Sendai virus vector boost regimen.

Heterologous prime/boost regimens are AIDS vaccine candidates because of their potential for inducing cellular immune responses. Here, we have developed a prime/boost regimen leading to rapid control of highly pathogenic immunodeficiency virus infection in macaques. The strategy, priming by an env and nef deletion-containing simian-human immunodeficiency virus (SHIV) proviral DNA followed by a single booster with a Gag-expressing Sendai virus (SeV-Gag), efficiently induced virus-specific T cells, which were maintained for more than 3 months until challenge. While all naive control macaques showed acute CD4(+) T-cell depletion at week 2 after an intravenous SHIV89.6PD challenge, all the macaques vaccinated with the prime/boost regimen were protected from depletion and showed greatly reduced peak viral loads compared with controls. Vaccination with the DNA alone or SeV-Gag alone was not enough to confer the consistent protection from the depletion, although it led to efficient secondary CD8(+) T-cell responses at week 2 after challenge. At week 1, a difference in the secondary responses between the protected and the unprotected macaques was clear; rapid augmentation of virus-specific CD8(+) T cells was detected in the former but not in the latter. Thus, our results indicate the importance of rapid secondary responses for reduction in the peak viral loads and protection from acute CD4(+) T-cell depletion.

Postbinding fusion function contributed by a chimeric murine leukemia virus envelope protein.

We previously obtained a chimeric Friend murine leukemia virus (FMLV) envelope protein (Env) in which the whole receptor-binding domain (RBD) was replaced with a surface domain of human CD4. Here, we examined if the postbinding fusion function of the CD4-Env chimera still remains to be intact. While a pseudotype MLV bearing CD4-Env showed no infectivity, NIH 3T3 cells could be infected with a pseudotype MLV bearing both CD4-Env and a mutant FMLV Env defective in postbinding fusion function. The pseudotype MLV showed no infectivity on HeLa cells but on the FMLV receptor (mCAT1)-expressing HeLa cells. In NIH 3T3 cells, the R-peptide-deleted CD4-Env could not induce syncytia by itself but did so in co-operation with the fusion-deficient Env. Syncytia induced by the coexpression were not observed in HeLa cells but in the mCAT1-expressing HeLa cells. These results indicate that the CD4-Env could contribute postbinding fusion function in the membrane fusion process triggered by FMLV RBD-mCAT1 interaction.

Induction of protective immunity against pathogenic simian immunodeficiency virus by a foreign receptor-dependent replication of an engineered avirulent virus.

In AIDS vaccine strategies, live attenuated vaccines can confer good resistance against pathogenic virus infections but have the potential risk of inducing disease, whereas safer replication-negative strategies such as DNA vaccinations have so far failed to prevent the disease onset. Here, we developed a novel DNA vaccine strategy to induce restricted replication of an avirulent virus and evaluated it in a simian immunodeficiency virus (SIV) infection model. We generated a chimeric SIV, FMSIV, by replacing SIV env with ecotropic Friend murine leukemia virus (FMLV) env to confine its replication to FMLV receptor (mCAT1)-expressing cells. In primate cells lacking mCAT1, FMSIV did not replicate unless mCAT1 was introduced exogenously. Vaccination to macaques with both the FMSIV DNA and the mCAT1-expression plasmid DNA induced SIV Gag-specific cellular immune responses and resistance against pathogenic SIV(mac239) challenge more efficiently than the replication-negative control vaccination with the FMSIV DNA alone. This strategy may be useful for development of safe and effective vaccines against various kinds of pathogenic viruses.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus.

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIV(DH12)-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIV(DH12) infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4(+) T lymphocytes. Although the CD4(+) T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIV(DH12R), induced marked and rapid CD4(+) cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIV(DH12R) indicated: (i) the input cloned SHIV remained the predominant virus during the first 5-7 months of infection; (ii) variants bearing only a few of the SHIV(DH12R) consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIV(DH12R) ever developed.

Retroviruses and autoimmune diseases.

The possible involvement of retroviruses in the development of autoimmune diseases has long been discussed. Exogenous retroviral infections and aberrant endogenous retroviral expressions can possibly induce immune dysfunction directly or indirectly. In addition to observations of autoimmune disease-like features in animal models, indirect evidences implicating retroviruses in human autoimmune diseases have been shown in a number of reports. However, direct evidence for the etiologic role of retroviruses in human autoimmune diseases has not yet been obtained.

Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques.

Parenteral administration of a mouse anti-human CD8 monoclonal antibody (MAb) to rhesus macaques resulted in a transient depletion of CD8+ cells in both the peripheral blood and lymphoid tissues. When administered during primary chimeric simian/human immunodeficiency virus infections, the CD8 MAb caused marked elevations of plasma and cell-associated virus levels in both the peripheral blood and lymphoid tissues and led to prolonged depletion of CD4 cells. Taken together, these results directly demonstrate that CD8+ T lymphocytes are actively involved in controlling the acute phase of primate lentivirus infections.

Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing.

Chimeric simian-human immunodeficiency viruses (SHIVs) carrying envelope glycoproteins derived from a T cell-macrophage dual-tropic primary isolate (human immunodeficiency virus type 1 [HIV-1] strain DH12) were constructed. When inoculated into macaque monkeys, SHIV(MD14) carrying simian immunodeficiency virus-derived nef established significantly higher virus loads than did SHIV(MD1), which contains the HIV-1 nef gene. Three patterns of CD4 cell depletion were observed in infected monkeys: exponential and irreversible loss to undetectable levels within 10 weeks of infection; marked reduction during acute infection followed by partial recovery and stabilization (lasting from 10 weeks to > 1 year), with a later decline to undetectable levels in some animals; and a transient loss during acute infection. The induced immunodeficiency was accompanied by CD4 cell counts of < 50 cells/microL and was associated with Pneumocystis carinii pneumonia, cytomegalovirus meningoencephalitis, lymphoid depletion, and thymic atrophy.

Reactivation of HIV type 1 in chronically infected chimpanzees following xenostimulation with human cells or with pulses of corticosteroid.

Following resolution of a primary HIV-1 infection initially induced by inoculating a mixture of three different virus strains, a chimpanzee was exposed to both immunostimulatory and immunosuppressive agents in an attempt to assess the contributions of different components of the immune system in suppressing circulating virus. The infusion of human leukocytes as an xenogeneic stimulus induced the replication of one of the input virus strains that had not previously been isolated or detected by PCR. The administration of high-dose, 17-day courses of corticosteroids resulted in coordinate and transient increases of each of the three viruses present in the original inoculum and elevation of HIV-1-specific ELISA antibody levels. Steroids administered to a second chimpanzee, chronically infected with a single HIV-1 isolate, also induced elevations of cell-associated virus. These results highlight the intimate relationship between immune system activation/immunosuppression and HIV replication in an animal model.

Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus.

Moloney murine leukemia virus (MLV) having the gag coding region alone, G3.6, produced a low level of mRNA (1/10 of the wild-type level). Ligation of 441 nucleotides (nt) containing a splice acceptor (SA) site to the downstream portion of the remaining gag region restored the level of the unspliced message, simultaneously activating a cryptic splice donor (SD) site in the middle of the p30 coding region (between nt 1596 and 1597). Ligation of the 441 nt in the same site in the inverted orientation also increased the level of the unspliced message, activating the same SD site (between nt 1596 and 1597) and a new SA site just in front of the inserted 441 nt (between nt 4770 and 4771). Deletion or inversion of the 441-nt SA sequence from the wild-type MLV or from int in-frame deletion or int frameshift mutant MLVs of nearly full size resulted in the loss of spliced mRNA and concomitantly in a severe reduction of the unspliced mRNA, particularly at 37 degrees C. Deletion of the 5' SD site did not result in the reduction of the unspliced-mRNA level. When the gag region in G3.6 was replaced with a Neo(r) coding region, the level of expression was high. The data taken together suggest that the presence of an SA signal is necessary for high-level expression of unspliced mRNA encoding Gag or Gag-Pol.

Targeted infection of a retrovirus bearing a CD4-Env chimera into human cells expressing human immunodeficiency virus type 1.

We constructed a hybrid Moloney murine leukaemia virus (MoMLV) bearing both a chimeric CD4 and the wild-type MoMLV envelope protein (Env) on its surface. The chimeric molecule, consisting of a surface domain of CD4 and the C-terminal two-thirds of MLV Env, was expressed on the cell surface. When expressed in MoMLV-infected cells, the CD4-Env chimera was incorporated into the virion as efficiently as the wild-type MoMLV Env. The hybrid MoMLV could infect human HeLa cells (although not with high efficiency) only if the cells were expressing human immunodeficiency virus type 1 genome. This method of ligand incorporation into a virion may lead to a development of a cell-specific retroviral vector for targeting gene therapy.

Interaction between the dominant negative mutant and the wild-type envelope proteins of Friend murine leukemia virus.

Interaction between the previously obtained dominant negative mutant, referred to as fcr (T. Matano, T. Odawara, M. Ohshima, H. Yoshikura, and A. Iwamoto, J. Virol. 67:2026-2033, 1993), and the wild-type envelope proteins (Env) of Friend murine leukemia virus was examined. The wild-type Env was bound to the fcr mutant Env and trapped in the endoplasmic reticulum. The virus receptor was not involved in this interaction.

Adaptation of skeletal muscle in limb lengthening: a light diffraction study on the sarcomere length in situ.

Adaptation of skeletal muscle during limb lengthening was assessed by measurement of the length of sarcomeres in situ. An external fixator was applied to the rabbit radius and ulna to elongate the forelimb by 3.5 mm, while allowing all the joints to be free. At regular intervals after the operation, the extensor digitorum lateralis muscle of the fifth digit was exposed in situ and the length of the sarcomeres was measured by a laser diffraction technique. The sarcomeres, which had stretched to 3.51 microns immediately after elongation of the bone, became shorter with the passage of time. On postoperative day 9, the length was 3.10 microns, which was similar to the length of the unstretched muscle. These results indicated structural adaptation of the muscle to a new length and could explain why the efficiency of muscle function is maintained after limb lengthening. When these findings are combined with our previous results, it appears that stretch-induced changes in sarcomere length are common in immobilized and nonimmobilized muscles.

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.