Discriminating Tonate Virus from Dengue Virus Infection: A Matched Case-Control Study in French Guiana, 2003-2016.

Tonate virus (TONV) is an arbovirus discovered in 1973 in French Guiana (FG) belonging to the Venezuelan equine encephalitis virus complex, Alphavirus genus. Only few publications and cases have been reported in FG. The objectives of the present study were to describe the clinical picture of TONV and to compare its presentation with that of dengue virus (DENV). A retrospective study was performed in Cayenne hospital from 2003 to 2016 including all patients exclusively positive for TONV IgM and not for other alphaviruses. They were classified as high probability: typical clinical picture of arbovirus infection (i.e., fever, chills, headaches, muscle, and joint pains) and IgM seroconversion; medium probability: typical clinical picture + single positive IgM on a unique serum sample without control; and low probability: atypical clinical picture of infection and single positive IgM. Only patients with high and medium probability were included in the analysis and compared with a gender- and age-matched control group of DENV diagnosed by NS1 antigen (two controls per case). During the study period, 45 cases of TONV were included and compared with 90 cases of DENV. Twenty-eight (62.2%) were men; the median age was 34 years (IQ [22-49]). In the bivariate analysis, variables significantly associated with TONV versus DENV were the presence of cough (33.3% versus 10.3%) and anemia (32.5% versus 11.1%) and the absence of nausea (4.4% versus 32.2%), rash (2.2% versus 27.4%), fatigue (17.8% versus 41.0%), anorexia (6.7% versus 30.1%), muscle pain (42.2% versus 61.4%), headache (53.3% versus 70.8%), leukopenia (9.8% versus 44.4), and lymphopenia (42.5% versus 89.9%). There were no cases with severe neurological involvement, and there were no deaths. Tonate virus may be evoked as a cause of fever in patients living or returning from the Amazonian area. Positive TONV IgM does not prove the diagnosis and should not preclude from searching for alternative infectious diagnoses.

Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons.

In neonatal mice the A7(74) and L10 strains of Semliki Forest virus (SFV) are virulent. In 3- to 4-week-old mice the L10 strain is virulent, the A7(74) strain is avirulent. Following intraperitoneal inoculation of 3- to 4-week-old mice both strains produce a transient plasma viremia. This is cleared by IgM antibodies. IgG antibodies of all subclasses are produced. The distribution of viral RNA in the brain as determined by autoradiographic analysis of in situ hybridizations shows that in all cases virus is first apparent as small foci of infected cells around cerebral capillaries. In both neonatal and 3- to 4-week-old mice infected with L10 or neonatal mice infected with A7(74), infection spreads rapidly from the original foci to infect large areas throughout the brain. Both neurons and glial cells are infected resulting in pycnosis and death of the animals. In the brains of 3- to 4-week-old mice infected with A7(74) virus there is little spread from the original perivascular foci. Again neurons and oligodendrocytes are infected but cellular destruction is minimal. The same pattern of A7(74) infection is observed in 3- to 4-week-old athymic nu/nu mice and mice with severe combined immunodeficiency, indicating that failure to spread is not related to specific immune responses. Furthermore, in nu/nu and SCID mice the small restricted foci of A7(74) infection persist. Comparison of the replication of these two viruses by electronmicroscopy shows that although A7(74) virus replicates completely in the neurons of neonatal mice, the virus is unable to bud from the neurons of 3- to 4-week-old mice and aggregates of viral RNA and capsid accumulate. We conclude that there is an age-related restriction of A7(74) replication in mouse neurons and that this restriction is not associated with the maturity of virus-specific immune responses but probably reflects age-related changes in neurons.

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

Position-dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis.

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization.

Experimental infection of young turkeys with eastern equine encephalitis virus and highlands J virus.

Depression, somnolence, and increased mortality were observed in 2-week-old turkeys inoculated intramuscularly with either eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus. Mortality rates in EEE virus- and HJ virus-inoculated turkeys were 7/30 (23%) and 9/30 (27%), respectively; no sham-inoculated controls died. Both EEE virus- and HJ virus-inoculated turkeys developed viremia that lasted 2 days; peak mean titers were 5.5 and 3.2 log10 plaque-forming units per ml of blood, respectively. Pathologic changes in both EEE virus- and HJ virus-inoculated turkeys consisted primarily of multifocal necrosis in the heart, kidney, and pancreas, and lymphoid necrosis and depletion in the thymus, spleen, and bursa of Fabricius. The findings indicate that EEE virus and HJ virus are pathogenic for young turkeys.

High mortality of domestic turkeys associated with Highlands J virus and eastern equine encephalitis virus infections.

High mortality occurred in two flocks of commercial turkey hens placed in southern North Carolina in fall 1991. Daily mortality peaked at 3.19% in Flock 1 and 3.79% in Flock 2. Clinical signs included restlessness, somnolence, vocalization, and acute death. Gross lesions included atrophy of the bursa of Fabricius, thymus, and spleen, and watery intestinal contents. Microscopic changes included moderate to marked lymphocyte necrosis and depletion in the bursa, thymus, and spleen, widely scattered necrosis of pancreatic acinar cells, and mild villous atrophy and fusion in the jejunum and ileum with cuboidal to low columnar epithelial cells covering the villous tips. In Flock 1, at 27 days of age, reovirus and picornavirus particles were detected in the feces. One week later, togavirus-like particles were observed in fecal contents, and two of seven serum samples showed seroconversion to Highlands J virus. Eleven days later, five of six serum samples were positive for antibodies against Highlands J virus, with a fourfold increase in the geometric mean titer. In Flock 2, seroconversion to eastern equine encephalitis virus was observed in four of 10 serum samples 11 days after the onset of clinical signs. Based on the above observations, it is suspected that these alphaviruses were the cause of the clinical syndrome.

Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey.

An isolate of Getah virus was obtained from Culex mosquitos collected in Mao'an Village, Baoting County, Hainan Province, China, in 1964. The virus (strain M-1) replicated in laboratory-bred Aedes aegypti and Cx. fatigans (= quinquefasciatus), and was transmitted by laboratory-bred Ae. albopictus to healthy newborn albino mice. Skeletal muscles of newborn albino mice experimentally infected with the virus showed degeneration, atrophy, necrosis, and inflammatory changes of muscle fibers. Antibody prevalence in humans and animals ranged from 10.3% by neutralization tests of samples from healthy people in 1979 to 26.4% by CF tests of samples from people with febrile illnesses in 1982. The high prevalence of antibody in pigs, horses, and goats (17.6% to 37.5%) indicated that infection with Getah or a closely related virus is relatively common in domestic animals.

Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys.

Simian hemorrhagic fever (SHF) virus and a new strain of Ebola virus were isolated concurrently in recently imported cynomolgus monkeys (Macaca fascicularis) being maintained in a quarantine facility. Ebola virus had never been isolated in the U.S. previously and was presumed to be highly pathogenic for humans. A chronology of events including measures taken to address the public health concerns is presented. The clinicopathologic features of the disease were abrupt anorexia, splenomegaly, marked elevations of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, with less prominent elevations of blood urea nitrogen, creatinine, and other serum chemistry parameters. Histologically, fibrin deposition, hemorrhage, and necrosis of lymphoid cells and reticular mononuclear phagocytes were present in the spleens of SHF and of Ebola virus-infected animals. Intravascular fibrin thrombi and hemorrhage were also present in the renal medulla and multifocally in the gastrointestinal tract. Necrosis of lymphoid and epithelial cells was occasionally noted in the gastrointestinal tract. The histopathologic findings considered specific for Ebola virus infection include hepatocellular necrosis, necrosis of the zona glomerulosa of the adrenal cortex, and interstitial pneumonia, all of which were generally associated with the presence of 1 to 4 mu intracytoplasmic amphophilic inclusion bodies. The disease spread within rooms despite discontinuation of all direct contact with animals, and droplet or aerosol transmission was suspected. Antibody to Ebola virus developed in animal handlers but no clinical disease was noted, suggesting a less virulent strain of virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathomorphological and immunohistological findings in progeny of goats experimentally infected with pestiviruses.

A total of 25 pregnant goats without neutralizing antibodies against BVD virus were inoculated with two different pestivirus isolates at eight different stages of gestation. In both infection groups, various malformations were observed in fetuses and neonates. In three twins with neutralizing antibodies against BVD virus leukoencephalomalacia occurred, characterized by gelatinous transformation in the cerebral hemispheres. These lesions were comparable to alterations described in alternative pathology of Border disease in sheep. Although the immunohistological findings are characteristic for immunological tolerance and viral persistence, viable offspring persistently infected with pestivirus was not observed.

Macrophage function in the acute phase of lactic dehydrogenase virus-infection of mice: suppression of superoxide anion production in normal mouse peritoneal macrophages by interferon-alpha in vitro.

The effect of interferon (IFN)-alpha on the release of superoxide anions (O2-) by normal mouse macrophages (PEM) was examined. Sera from LDV-infected mice at 1 day, but not at 7 days post-infection, suppressed the O2- release by PEM. When PEM were exposed in vitro for 24 h to IFN-alpha, their capacity to release O2- was significantly suppressed. Progressive suppression of O2- release with increasing IFN-alpha concentration was observed. These results suggest that IFN-alpha in the circulation may be one of several suppressive factors on macrophage function in the early phase of infection and IFN-alpha may play a modulatory role in inflammation and immunity.

Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice.

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.

Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding.

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and E1 appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

Decrease in neutrophil migration induced by endotoxin and suppression of interleukin-1 production by macrophages in lactic dehydrogenase virus-infected mice.

Neutrophil (PMN) migration into the peritoneal cavity after intraperitoneal injection of lipopolysaccharide (LPS), chemotactic activity of PMN, interleukin-1 (IL-1) production by macrophages (M phi) and its ability to attract PMN in mice chronically infected with lactic dehydrogenase virus (LDV) were compared with those in uninfected control mice. PMN migration into the peritoneal cavity decreased in infected mice when LPS was injected intraperitoneally. PMN chemotactic activity did not show any difference following infection. To assess the mechanism of this decreased PMN migration, IL-1 production, which is responsible for PMN attraction, was studied in LDV-infected mice. IL-1 production by M phi derived from infected mice decreased and its ability to attract PMN was weak. IL-1 production by M phi from control and infected mice increased after treatment by indomethacin and LPS. PMN migration into the peritoneal cavity increased after treatment with indomethacin and LPS in both control and infected mice. However, the rate of increase of IL-1 production and PMN migration was greater in infected mice. These results suggest that the excess activation of cyclo-oxygenase-derived products (prostaglandins) in infected mice might be responsible for the suppression of IL-1 production by M phi, resulting in decreased PMN migration induced by endotoxin.

Stress-induced neuroinvasiveness of a neurovirulent noninvasive Sindbis virus in cold or isolation subjected mice.

Effects of cold or isolation stress on brain penetration by the neurovirulent noninvasive Sindbis virus strain (SVN) were studied in mice. SVN injected intracerebrally (i.c.) causes acute encephalitis and kills adult mice but is unable to invade the brain and kill when injected intraperitoneally (i.p.). Mice inoculated i.p. with SVN were exposed to cold stress or were singly housed. Both stress patterns induced SVN encephalitis and death in 42% (cold) and 37% (isolation) of the tested mice. No death was observed in the control injected mice. Brain virus levels were found to be more than 10(6) PFU in all dying mice. No virus was detected in the control group brains. The virus that was isolated from the brains of moribund mice demonstrated no changes in neuroinvasive and neurovirulent properties. We suggest a stress induced blood-brain-barrier opening with subsequent virus entrance as the mechanism of stress induced SVN encephalitis.

T-cells of virus-infected mice produce a lymphokine which activates the autoreactivity of intact mouse lymphocytes.

The role of lymphokines was estimated in induction of autoreactive T-cells during Langat virus infection in mice. It was shown that in vitro cultured splenocytes from virus-infected animal containing autoreactive lymphocytes (ARL) spontaneously produce a lymphokine which is capable to activate the autoreactivity of lymphocytes derived from the spleen of intact syngeneic mice. The capacity of this lymphokine to activate the autoreactivity of acceptor cells within 2 hr was demonstrated by local graft-versus host reaction (GVHR) in the donor-recipient system. According to their surface markers (theta-antigen expression, absence of immunoglobulins) the lymphokine activating autoreactivity (LAA) producers may belong to T-lymphocyte population. Autoreactivity could be induced by the lymphokine only if the LAA producers and acceptors were compatible by the major histocompatibility complex antigens.

Multiplication of virulent and demyelinating Semliki Forest virus in the mouse central nervous system: consequences in BALB/c and SJL mice.

The sites of multiplication in the mouse central nervous system (CNS) of the virulent L10 strain of Semliki Forest virus (SFV) and the L10-SFV-derived demyelinating M9 mutant were determined using both BALB/c and SJL mouse strains. In situ hybridization (ISH), using a cRNA probe to an SFV non-structural sequence, and immunogold-silver staining (IGSS), using polyclonal anti-SFV rabbit IgG, were the techniques utilized. For L10-SFV, viral RNA and antigen were detected in neurons and glial cells of both mouse strains. For BALB/c mice infected with M9-SFV, both neuronal and glial cell infection was less extensive than that obtained with L10. ISH or IGSS were generally not sensitive enough to detect viral RNA and antigen, respectively, in M9-SFV-infected SJL mice. M9-SFV multiplied to a similar titre in primary cultures of glial cells derived from either BALB/c or SJL mice. Following infection with M9-SFV, small plaques of demyelination in the CNS and occasional small aggregates of mononuclear leukocytes in the leptomeninges persisted for up to 12 months in SJL mice but not BALB/c mice. This was not associated with detectable persistence of infectious virus, viral antigen or viral RNA in the CNS.

Experimental pestivirus infections in newborn goat kids.

Two experiments were carried out in which 37 healthy newborn goat kids were inoculated with a non-cytopathic ovine (BDV) or a cytopathic bovine pestivirus (BVDV) by intramuscular or intracerebral injection. No kids showed signs of disease or gross lesions which could be attributed to these viruses, but inoculated kids had lower mean growth rates than the controls. Significant histological changes in the CNS of 14 kids were restricted largely to the white matter and consisted mainly of hypercellular foci comprising microglial/histiocytic cells and mild perivascular infiltration by mononuclear cells. Varying degrees of infiltration of the myocardium by lymphocytes and plasma cells were observed. All kids remained negative for neutralizing antibodies against pestivirus until 2 to 3 weeks after infection. Titres increased during the following weeks. Pestiviruses were recovered from kids necropsied 10 days after inoculation, but not from any kids killed 20 days after inoculation or later. Non-cytopathic virus was isolated from various tissues of four kids that had received BDV and three kids that had been given BVDV. Cytopathic viruses were not recovered from any kids. Mean white blood cell counts in all kids were within the normal range at 4 and 8 weeks after inoculation. The lymphocyte response to stimulation by phytohaemagglutinin was significantly increased on both sampling occasions in the BDV-inoculated kids, while in the BVDV-inoculated animals, a similar increase was seen only at 8 weeks.

Polymyositis in mice experimentally inoculated with Getah virus.

Mice inoculated intracerebrally with parent, large-plaque (LP) and small-plaque (SP) strains of Kanagawa strain of Getah virus showed clinically recumbency and paralysis. The LP strain caused recumbency more rapidly and killed mice more early after inoculation than the parent and SP strains. Microscopically, skeletal muscles of the whole body were involved showing degenerative or inflammatory changes. In mice inoculated with the parent or SP strains, there were degeneration and necrosis of the muscle fibers with inflammatory cell infiltration and regenerative reaction. The lesions were particularly conspicuous in muscles of the hind legs. In mice inoculated with the LP strain, most of the muscle fibers revealed degeneration and necrosis, but reactive changes were poor. In addition, the periosteum and muscular connective tissue were thickened with karyorrhexis. Electron microscopically, virus particles were recognized mainly in cisternae of sarcoplasmic reticulum in skeletal muscle fibers of mice inoculated with the LP strain, while they were rare in those of animals injected with the parent and SP strains. From these finding, it was suggested that Kanagawa strain of Getah virus has the virulence to skeletal muscles of mice.

Induction of membrane proliferation in mouse CNS by gold sodium thiomalate with reference to increased virulence of the avirulent Semliki Forest virus.

Separation of smooth membrane vesicles from whole mouse brain by isopycnic centrifugation in discontinuous sucrose density gradients show an increased membrane proliferation in gold sodium thiomalate (GSTM) treated mice. Induction of membrane proliferation by GSTM seems to be an important factor in converting the avirulent Semliki Forest virus infection into a lethal one.

Pathogenicity of Semliki Forest virus for the rat central nervous system and primary rat neural cell cultures: possible implications for the pathogenesis of multiple sclerosis.

The neurovirulent L10 strain of Semliki Forest virus (SFV) causes extensive neuronal damage in the central nervous system (CNS) of infected rats, and is probably the cause of death. The avirulent A7 and M9 strains do not cause extensive neuronal damage, but do induce immune-mediated CNS demyelination. In primary CNS cell cultures derived from rats, L10 multiplies more rapidly in neurons than avirulent strains, but infection with both virulent and avirulent strains causes depletion of oligodendrocytes from mixed glial cell cultures. It is proposed that the immune-mediated demyelination, which follows infection with avirulent strains, is induced by phagocytosis of myelin debris from infected oligodendrocytes, and the presentation of antigens derived from such debris to T-helper lymphocytes. Based on these and previous results, a scheme for the pathogenicity of defined strains of SFV is proposed. The applicability of this scheme to the understanding of human demyelinating disease such as multiple sclerosis is discussed.