Small Amounts Of Infectious Virus Research Articles

Spontaneous Reactivation of Herpes Simplex Virus Type 1 in Latently Infected Murine Sensory Ganglia

Careful studies of mouse trigeminal ganglia (TG) latently infected with herpes simplex virus type 1 (HSV-1) indicate the presence of productive cycle viral gene products and persistent immune response, suggesting ongoing spontaneous viral reactivation in these tissues. In the present study we set out to determine whether infectious virus is present in murine TG latently infected with HSV-1 (KOS). At 37 days after ocular inoculation we found a small amount of infectious virus in ca. 6% of latently infected murine TG. Furthermore, the amount of infectious virus that we detected (PFU per viral antigen-positive neuron) was similar to that detected in acutely infected ganglia. We conclude that spontaneous reactivation of infectious HSV-1 occurs in the mouse TG and is likely the principle cause of viral protein expression in these tissues. We next examined the role of latency-associated transcript (LAT) in spontaneous ganglionic reactivation by examining ganglia latently infected with KOS dlLAT1.8, a LAT deletion virus. Through the use of immunocytochemistry we found that KOS dlLAT1.8 had a rate of spontaneous ganglionic reactivation very similar to that of HSV-1 (KOS). Studying spontaneous ganglionic reactivation of HSV in the mouse TG allows a direct study of viral reactivation from latently infected neurons without the potential confounders and complicating downstream events that accompany the study of viral reactivation by explantation or peripheral viral shedding. Since most cases of human viral shedding and reactivation are not associated with a known trigger, spontaneous ganglionic reactivation of HSV-1 may be a better model of human disease than existing models.

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

Production of high-titer stocks of the English strain of rat cytomegalovirus

A simple technique to increase dramatically the yield of rat cytomegalovirus (RCMV) from infected monolayers of a rat embryo fibroblast-derived cell line is described. The method, which involves daily changes of the cell culture medium, can result in a 50 000-fold amplification of virus from cell monolayers inoculated with only a few RCMV particles. This modification of the standard in vitro culture technique to amplify viral yield can be used to increase the sensitivity of the plaque assay for detecting very small amounts of infectious virus in organ homogenates of RCMV infected animals.

Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake.

CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell.

Alteration of pulmonary macrophage function by respiratory syncytial virus infection in vitro.

Alveolar macrophages (AL) are the first line of defense against inhaled pathogens and are exposed to virus during the course of a respiratory syncytial virus (RSV) infection. Interference of virus with alveolar macrophage functions may contribute to the risk of acquiring secondary bacterial infections during or after respiratory tract infections with RSV or other viral agents. We studied whether murine AL get infected with RSV and whether they support viral replication in vitro. In addition, the effects of RSV on microbicidal and on immunoregulatory functions were examined. Only a subpopulation of AL expressed viral F proteins after exposure of these cells to RSV. Infected AL released only small amounts of infectious virus into the supernatant. The extent of virus replication in AL seemed to be dependent in part on the amount of IFN induced by the virus, as has been demonstrated by infection of lung tissue macrophages and AL in vitro. In general, RSV infection of pulmonary macrophages appeared to be abortive. Nevertheless, release of reactive oxygen intermediates, phagocytosis, and killing of protozoa were reduced in RSV-infected AL in comparison to noninfected AL. In contrast, RSV stimulated secretion of TNF-alpha, IL-1, and IL-6 in an infectious-dose dependent manner. Along with the increased cytokine release, accessory functions of AL were increased after RSV exposure. Thus, exposure of AL to RSV appeared to stimulate their immunoregulatory functions, whereas the microbicidal activity of these cells seemed to be severely diminished.

The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant

Herpes simplex virus type 1 (HSV-1) encodes a novel enzyme activity, the alkaline nuclease, whose precise role in the viral replication cycle remains obscure. The alkaline nuclease gene corresponds to the UL12 open reading frame, which is predicted to encode a protein of 626 amino acid residues. We describe the isolation and characterization of a null mutant of the gene for the viral alkaline nuclease in which 917 bp from the N-terminal half of the gene (corresponding to residues 70 to 375) were deleted and replaced by the insertional mutagen ICP6::lacZ. The resulting mutant virus, AN-1, was propagated in helper cells (S22) which express the wild-type version of the alkaline nuclease gene. Mutant AN-1 growth in Vero cells is severely restricted, although small amounts of infectious virus are produced. On the other hand, wild-type levels of viral DNA and late viral proteins are expressed in virus AN-1-infected Vero cells. These results indicate that the HSV-1 alkaline nuclease gene product is not essential for viral DNA synthesis but may play a role in the processing or packaging of viral DNA into infectious virions. Possible roles in the viral infectious cycle will be discussed.

Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization

Persistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i. and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i. a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects.

Pathological lesions in the lungs of ducks infected with influenza A viruses.

To determine histopathological damage in the respiratory tract, ducks were inoculated with five different influenza A viruses, including viruses virulent for other avian hosts. Lungs were collected for detection of virus and histopathological examination. Small amounts of infectious virus were recovered from lungs, and viral antigens were demonstrated by immunoperoxidase staining with monoclonal antibodies to the viral nucleoprotein. Although clinical signs were not detected, lungs of ducks infected with both virulent and avirulent viruses had mild pneumonia characterized by infiltrates of lymphocytes and macrophages. These findings show that although clinical signs are not evident, ducks may have damage to the respiratory tract during influenza.

Interaction of human adenovirus serotype 2 with human lymphoid cells

Human adenoviruses (e.g., Ad2, Ad5) establish chronic infections in human lymphoid-derived cell lines, including Raji and Jijoye ( R. E. Wallace, 1969, Proc. Soc. Exp. Bich Med. 130, 702–710; N. Faucon, G. Ogier, and Y. Chardonnet, 1982, J. Nad. Cancer Inst. 69, 1215–1220); however, the mechanisms by which chronic infections are established and maintained are not understood. When Raji or MOLT-3 cell cultures were infected with Ad2 at high multiplicity, these cell lines continued to grow exponentially and produced only small amounts of infectious virus. Virus-specific antigens, including the DNA-binding protein and hexon, were expressed in only 5% of the Ad2-inoculated cultures. All Raji and MOLT-3 cells were found to have adenovirus receptors, but the Ad2 virions that adsorbed to most Raji cells were sequestered in caps, suggesting that most cells fail to internalize adsorbed Ad2. Cell synchronization experiments showed a correlation between the proportion of cells that became productively infected and the proportion of cells in mitosis at the time of infection. In contrast, primary blood lymphocytes had few, if any, Ad2 receptors and were not productively infected by Ad2.

Measles Virus Strain-dependent Variation in Outcome of Infection of Human Blood Mononuclear Cells

Eight measles virus strains including four subacute sclerosing panencephalitis (SSPE) isolates were compared on the basis of their growth characteristics in human peripheral blood mononuclear cells and effect on mitogen-stimulated lymphocyte proliferation. The Edmonston strain virus and some other strains of measles virus replicated in phytohaemagglutinin (PHA)-stimulated lymphocytes and released high titres of infectious virus into the culture medium. Inhibition of DNA synthesis was relatively low at the beginning of lymphocyte proliferation and could be detected only at high multiplicity of infection. Three to 4 days after initiation of proliferation a strong inhibition was seen also in cultures infected with low doses of virus, and was apparently related to the production of high titres of new infectious virus. Some virus strains originally isolated from SSPE patients produced only a small amount of infectious virus in lymphocytes at 37 degrees C but had a very strong inhibitory effect on lymphocyte proliferation leading to early cell death. The inhibitory effect was found over a wide range of virus concentrations, but was strictly dose-dependent and no increase in inhibition with low multiplicities of infection could be seen with longer culture times. The amount of interferon induced by different strains varied from 400 to 6400 international units/ml but the amount of interferon produced in the cultures did not correlate with the inhibitory effect on proliferation or with the amount of released new infectious virus. The Hallé measles strain, originally isolated from an SSPE patient, differed most from the Edmonston strain in its characteristics, and was studied in greater detail. Although only a small amount of infectious virus was produced, the Hallé strain had a very strong inhibiting effect on the proliferation of PHA-stimulated lymphocytes, and extensive syncytium formation was seen leading to cell death within 3 days.

Latent infection of human ovarian teratocarcinoma cells with human cytomegalovirus. Brief report.

Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA1) cells, and maintained for more than 200 days. Infected cultures maintained at 34 degrees C (PA1CMV34) and 37 degrees C (PA1CMV37) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32 degrees C (PA1CMV32) and 39 degrees C (PA1CMV39) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p.i.) respectively. PA1CMV32 cultures shifted to 37 degrees C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA1 cells at 32 degrees C. In contrast, PA1CMV39 cells did not produce infectious virus even when cultured at 37 degrees C for more than 100 days after the temperature shift.

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA is involved in establishing and maintaining a persistent infection, the intracellular viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3′ end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.

Characterization of a hamster brain cell line persistently infected with measles virus.

A persistent infection of measles virus in hamster brain cell cultures was established. Hamster brain cells were transformed with a human papovavirus strain BK and infected with a wild-type measles virus in order to get the cell line persistently infected with measles virus. About 75 per cent of these M-HB/MVB-cells were measles virus-infected. The cells released only small amount of infectious virus and produced low levels of interferon-like activity into the growth medium. During the first 50 passages no large syncytia typical of a lytic measles virus infection were seen. The products of measles virus infection in the cells and in cell culture fluids were followed at two temperatures. Another cell line persistently infected with measles virus (Lu-carrier-cells, 28) was investigated in parallel. In both cell lines measles antigens were cytoplasmic, but during the observation period large amounts of measles nucleocapsid accumulated in the nuclei of the M-HB/MVB-cells. The virus-specific protein synthesis in M-HB/MVB-cells was weak and the intracellular amount of immunoreactive measles nucleocapsid was only 50 per cent (600 ng/10(5) infected cells) of the (1200 ng/10(5) cells) found in Lu-carrier-cells. Also the release of nucleocapsid was minimal in hamster brain cells. The decreased temperature had no clear-cut effect on virus-specific protein synthesis or on the release of the virus-specific products.

Atypical patterns of neural infection produced in mice by drug-resistant strains of herpes simplex virus.

Mice inoculated intracerebrally (i.c.) with a mutant strain of HSV were found to develop cataracts 1 to 2 months after inoculation. Cataract formation was subsequently shown to follow an acute retinitis which commenced within 1 week of inoculation. The mutant had been selected for high resistance to the nucleoside analogue acyclovir and has been shown previously to be defective in the induction of thymidine kinase and also to express an altered DNA polymerase. The LD50 for mice inoculated i.c. was greater than 10(5) p.f.u. compared with approx 7 p.f.u. for the parental strain. Studies of virus replication following i.c. inoculation with a sublethal dose of the mutant revealed that only small amounts of infectious virus were produced in the brain, but during a period from 6 to 12 days after inoculation vigorous replication occurred in retinal tissue, producing very high titres of virus.

Hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus, MHV-A59.

After intracerebral inoculation of mouse hepatitis virus, MHV‐A59 strain, into 3‐ to 5‐day‐old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of a considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus‐specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection.

Non-productive paramyxovirus infection: Nariva virus infection in hamsters.

The pathogenesis of infection with Nariva virus (NV)--recently classified as a paramyxovirus--was studied in the hamster, an animal closely related to the natural host. Intracranial inoculation of suckling hamsters produces an acute necrotizing encephalitis with large amounts of infectious virus and virus antigen in the brain. In contrast, weanling hamsters have only small amounts of infectious virus and only early in the disease, when they are well; later, when clinically ill, they have a non-productive infection with continuing evidence of viral antigen, but no detectable infectious virus. Weanlings die later than sucklings with less cerebral parenchymal necrosis. The integrity of the immune system affects the expression of NV since brain tissue from anti-lymphocyte serum (ALS) treated infected weanling hamsters have more infectious virus, and for longer periods, than brain tissue from untreated infected weanling hamsters. Changing susceptibility of the host's neural cells may also be involved in determining the course of the illness and expression of the virus since: 1) ALS treatment does not influence the clinical course of the disease or pathology, 2) ALS treated weanlings still have much lower levels of infectious virus than sucklings, 3) infected weanling and suckling hamsters have a similar time course of development of NV neutralizing antibody.

Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation

Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.

Response of simian virus 40-transformed cell lines and cell hybrids to superinfection with simian virus 40 and its deoxyribonucleic acid.

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.

Characterization of Defectiveness of Human Adenoviruses in Green Monkey Kidney Cells.

SummaryIn the absence of foreign helper viruses, human adenoviruses are defective in simian cells, but the degree of effectiveness varies with the adenovirus. Adenovirus 2 initiates an infection in the simian cells but there is no increase in virus titer. When simian cells are infected with adenovirus 7, a small amount of infectious virus is produced but the amount never exceeds the input level. Each cell in the population produces a small amount of virus. The increase in infectious virus is dependent upon DNA synthesis. The simian cells therefore appear to be “leaky” for some human adenovirus serotypes.

The Anamnestic Antibody Response to Japanese Encephalitis Virus in Monkeys and its Implications Concerning Naturally Acquired Immunity in Man

Summary Primary and anamnestic antibody responses to infectious Japanese encephalitis (JE) virus were studied in monkeys. The intradermal anamnestic dose of virus in suspensions of Japanese mosquitoes collected in nature was small, essentially equal to the intradermal infectious dose for susceptible monkeys, and well within the range of virus which vector mosquitoes might inoculate. Anamnestic antibody responses were uniformly measurable by hemagglutination-inhibition (HI) tests, usually detectable by complement fixation (CF) tests, but only infrequently demonstrable by virus-dilution neutralization tests. That anamnestic antibody responses resulted merely from virus antigen inoculated intradermally and did not depend on multiplication of virus in blood, skin or superficial tissues of previously infected monkeys was evidenced by a) lack of measurable viremia and virus proliferation in skin or superficial tissues and b) the ability of noninfectious, ultraviolet-irradiated virus to stimulate anamnestic antibody responses as readily as infectious virus. The fact that small amounts of infectious virus stimulated anamnestic antibody responses in previously infected, nonviremic monkeys, whereas large amounts of virus were available in viremic, susceptible monkeys to evoke primary antibody responses is compatible with the increased sensitivity of hosts to small “booster” injections of antigens; it does not necessarily infer that anamnestic antibody responses occur as a result of virus multiplication in previously infected hosts. Upon extrapolation to humans these findings suggest that a single endemic arthropod-borne virus can, upon transmission by vector mosquitoes, repeatedly stimulate anamnestic antibody responses in man and thus maintain detectable antibody levels and immunity throughout life. Moreover, in areas such as the Kanto Plain near Tokyo, Japan, where JE virus is the only known group B arthropod-borne virus, seasonal rises in HI and CF antibody titers to JE virus in persons with preseasonal homologous neutralizing (N) antibody need not imply that another group B virus exists in the same geographic region. HI, CF and N antibodies remained detectable in monkeys for at least 11 months after primary inapparent infection. HI and CF anamnestic antibody responses to large doses of JE virus were not significantly inhibited by high, preexisting HI, CF or N antibody levels in serum of hyperimmunized monkeys, but the effects of high antibody levels on anamnestic antibody responses to subsequent small virus doses were not conclusively ascertained.