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Novel coronavirus SARS-CoV-2, designated as COVID-19 by the World Health Organization (WHO) on the February 11, 2020, is one of the highly pathogenic β‐coronaviruses which infects human. Early diagnosis of COVID-19 is the most critical step to treat infection. The diagnostic tools are generally molecular methods, serology and viral culture. Recently CRISPR-based method has been investigated to diagnose and treat coronavirus infection. The emergence of 2019-nCoV during the influenza season, has led to the extensive use of antibiotics and neuraminidase enzyme inhibitors, taken orally and intravenously. Currently, antiviral inhibitors of SARS and MERS spike proteins, neuraminidase inhibitors, anti-inflammatory drugs and EK1 peptide are the available therapeutic options for SARS-CoV-2 infected individuals. In addition, Chloroquine, which was previously used for malarial and autoimmune disease, has shown efficacy in the 2019-nCoV infection treatment. In severe hypoxaemia, a combination of antibiotics, α-interferon, lopinavir and mechanical ventilation can effectively mitigate the symptoms. Comprehensive knowledge on the innate and adaptive immune responses, will make it possible to propose potent antiviral drugs with their effective therapeutic measures for the prevention of viral infection. This therapeutic strategy will help patients worldwide to protect themselves against severe and fatal viral infections, that potentially can evolve and develop drug resistance, and to reduce mortality rates., Graphical abstract Image 1, Highlights • Novel coronavirus SARS-CoV-2 which was named as COVID-19 by the WHO on the February 11, 2020. • 2019-nCoV can cause severe pulmonary infection, respiratory failure, along with organ damage and dysfunction. Early diagnosis of 2019 novel coronavirus disease (COVID-19) is crucial for disease control and treatment. • Due to appearance of the 2019-nCoV pneumonia during the influenza season, orally and intravenously antibiotics and neuraminidase inhibitors such as oseltamivir are has been widely used. • Nowadays, Griffithsin, a spike protein inhibitor, nucleoside analogues (Remdesivir, favipiravir and ribavirin), protease inhibitors (lopinavir/ritonavir), neuraminidase inhibitors (oseltamivir), anti-inflammatory drugs and EK1 peptide, Umifenovir, RNA synthesis inhibitors, Chinese traditional medicine (ShuFengJieDu and Lianhuaqingwen Capsule), could be the drug treatment options for new respiratory infectious diseases such as 2019-nCoV. • Chloroquine is a widely-used anti-malarial and autoimmune disease drug which clinically has potential applicable against the 2019-nCoV infection. Western-type antiviral therapies with antibiotics, α-interferon and lopinavir as well as support therapies including oxygen and mechanical ventilation have also been administered to the treatment of COVID-19 patients, according to the severity of hypoxaemia. • Using accumulated knowledge of the innate immune response system, will make it possible to propose the potent antiviral drugs with its effective therapeutic measures for detection and prevention of viral infection.

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (

Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.

HEp-2 cells that survived a lytic infection with Human Respiratory Syncytial Virus (HRSV) were grown to obtain a persistently infected culture that produced relatively high amounts of virus (106–107 pfu/ml) for more than twenty passages. The cells in this culture were heterogeneous with regard to the expression of viral antigens, ranging from high to undetectable levels. However, all cell clones derived from the persistent culture did not produce infectious virus or viral antigens and grew more slowly than the original uninfected HEp-2 cells. When these “cured” cell clones were infected with wild-type HRSV, delayed virus production and reduction in the number and size of syncytia were observed compared to lytically infected HEp-2 cells. Most significantly, differences in gene expression between persistently and lytically infected cultures were also observed, including genes that encode for cytokines, chemokines and other gene products that either promote cell survival or inhibit apoptosis. These results highlight the significantly different responses of the same cell type to HRSV infection depending on the outcome of such infection, i.e., lytic versus persistent.

A virus pseudotyped with glycoprotein of vesicular stomatitis virus (VSV-G) can enter various cell types at a relatively high titer. We observed that the amount of viral antigen from VSV-G pseudotyped human immunodeficiency virus type 1 (HIV-1) producing cells was much higher than that from their non-pseudotyped counterparts. This enhanced viral antigen production was not observed when we used HIV-1 pol mutant, viral enzyme inhibitors, HIV Env protein, or VSV-G fusion defective mutants. The transfection experiment using GFP-expressing virus showed time-dependent expansion of GFP-positive cells and viral DNA integration. These results suggested that the increase in viral antigen yield was caused by the release of a progeny virus following retro-transduction by the pseudotyped virus of the cells within the transfected cell culture. The infectivity as well as the amount of VSV-G on virus particles per unit of viral antigen was significantly different before and after the onset of the yield enhancement. This suggests that results of infection assays of the virus pseudotyped with VSV-G may be affected by the occurrence of such enhancement. This means that, while pseudotyping with VSV-G is a simple and effective method, this procedure should be carefully considered when the virus is produced for infectivity assays.

Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations. © 2006 Elsevier Inc. All rights reserved.

Feline immunodeficiency virus infection of cats provides a model to elucidate mechanisms of lentiviral pathogenesis. We isolated a non-domestic FIV from a Pallas' cat, FIV-Oma, which replicates in feline PBMCs and CRFK cells. To gain insights into FIV pathogenesis, we compared rates of viral replication and apoptosis of FIV-Oma with FIV-PPR in the MYA-1 T-cell line. To minimize heterogeneity of virus, infections were initiated with virus derived from molecular clones. Viral DNA and RNA levels, assessed by qPCR and qRT-PCR, apoptosis, and supernatant reverse transcriptase were slower in FIV-Oma infections. Immunostaining for cellular Gag showed that few cells were productively infected. The majority of cells infected with either virus instead became apoptotic. Apoptosis was detectable within 6 h PI, suggesting activation of a signaling pathway. We propose that apoptosis is due to interaction of virus with cells, and is the usual outcome of infection by cytopathic FIVs in these cells.

Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) at primary infection and may reactivate as herpes zoster. VZV is a T-lymphotropic virus in vivo. To investigate the T-cell tropism of VZV, we constructed a recombinant virus expressing green fluorescent protein (VZV-GFP) under the CMV IE promoter. Coculture of VZV-GFP-infected fibroblasts with II-23 cells, a CD4-positive human T-cell hybridoma, resulted in transfer of virus to II-23 cells. II-23 cells are susceptible to VZV-GFP infection as demonstrated by expression of immediate/early (IE62), early (ORF4), and late (gE) genes. Recovery of infectious virus was limited, with only 1 to 3 in 106 cells releasing infectious virus by plaque assay, indicating that transfer of virus results in a limited productive infection. In vitro infection of II-23 cells will be useful for further analysis of VZV tropism for T-lymphocytes.

In an earlier study we found that pigtailed macaques (Macaca nemestrina) that were experimentally infected with human immunodeficiency virus type 1 (HIV-1) initially became viremic and seroconverted, but HIV-1 replication diminished markedly over time. In an attempt to develop a longer term pathogenic model, blood from HIV-1-infected macaques was serially transfused into three groups of naive macaques. Transfer was successful through two transfusions as shown by repeated virus isolations and confirmed by the development of cell-free plasma viremia and by seroconversion. Three to five weeks after transfusion, plasma levels of HIV-1 RNA from several macaques in the first two groups exceeded those of the initially inoculated macaques. However, animals in the third group had diminished RNA levels, were virus culture negative, and did not seroconvert. Sequence analyses of env-region clones from infected animals revealed only minimal changes over the course of the passages. These results confirm HIV-1 replication inM. nemestrinaduring the acute phase of infection. However, adaptation of HIV-1 to a macaque-pathogenic variant did not occur during serial passage, possibly because the animals were able to restrict HIV-1 replication below a level required for a pathogenic variant to emerge. Whether such containment is a function of the host's immune response or a virus cell incompatibility remains to be determined.

A replication-defective mutant of herpes simplex virus 2 (HSV-2) was engineered by replacing the ICP8 gene of HSV-2 strain 186 with an ICP8–lacZ fusion gene from the herpes simplex virus 1 (HSV-1) HD-2 mutant strain. The resulting virus, HSV-2 5BlacZ, is defective for growth in Vero cells but is capable of growth in a cell line that expresses HSV-1 ICP8. In Vero cells, the mutant virus is defective for DNA synthesis but is able to express many viral proteins at levels similar to those of wild-type virus, including several of the late kinetic class. SDS–PAGE and Western blot analysis demonstrated the expression of glycoproteins B and D by 5BlacZ in Vero cells. Initial studies have shown that immunization with 5BlacZ protects guinea pigs from intravaginal HSV-2 challenge. Immunized animals had less severe genital skin disease and reduced replication of the challenge virus in the genital tract during primary infection and reduced episodes of recurrent disease. Thus, HSV-2 ICP8 shows gene regulatory properties similar to those of HSV-1 ICP8, and this HSV-2 ICP8 mutant virus shows a phenotype similar to those of HSV-1 ICP8 mutant strains. Replication-defective mutants of HSV-2 offer a potential vaccine approach for immune intervention against HSV-2 genital disease and latent infection.

We have reported that infection of fetal or neonatal rhesus macaques with attenuated SIVmac1A11 results in transient viremia, anti-SIV antibody responses, weak or absent cytotoxic T-lymphocyte responses, and no clinical disease. In light of these results, we hypothesized that congenital infection with SIVmac1A11 produced immune tolerance to SIV. To test this hypothesis, at approximately 1 year of age, five rhesus macaques infected with SIVmac1A11 as fetuses (n= 3) or newborns (n= 2) and five naive juvenile rhesus macaques were challenged orally with pathogenic SIVmac251. The five naive animals became persistently viremic after oral SIVmac251 inoculation. In contrast, one of three monkeys inoculated with SIVmac1A11in uteroand one of two animals inoculated with SIVmac1A11 at birth were virus culture negative. Virus was isolated from PBMC of the other animals infected with SIVmac1A11in uteroor at birth. However, one animal had a substantially lower viral load than the control animals. These results suggest that SIV-specific immunity rather than tolerance results from congenital infection with attenuated SIVmac and that this immunity is sufficient to provide some protection from pathogenic virus challenge. These results also demonstrate that SIV can be transmitted orally in 6- to 17-month-old rhesus monkeys.

Pig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing the crmA gene; recombinant cowpox virus A602, lacking the crmA gene; or cowpox virus A604, a revertant of virus A602, expressing the crmA gene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that the crmA gene is necessary to inhibit processes of apoptosis induced in LLC-PK1 cells by infection with cowpox virus. Thus in cells of certain types, the crmA gene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replication in vivo, potentially facilitating both virus trafficking and interference with antiviral immune defenses.

We have investigated the effect of a monoclonal antibody on influenza virus release and the cell surface expression of M2, comparing virus strains which were observed previously to be sensitive (A/Udorn) or resistant (A/WSN and A/Udorn variants) to growth inhibition by M2 antibody 14C2. Incubation of A/Udorn virus-infected cells in the presence of the inhibitory M2 antibody resulted in a significant reduction in the yield of virus, as measured by infectivity assays as well as by the release of purified virions. The release of A/Udorn virus was not inhibited by the presence of monovalent 14C2 Fab, in contrast to IgG, indicating that a bivalent structure is essential for 14C2 antibody-mediated viral growth restriction. The level of M2 surface expression in A/Udorn virus-infected MDCK cells was found to be reduced to approximately 60% of control levels in cells incubated with the 14C2 antibody. In contrast, M2 surface expression levels in A/WSN virus-infected cells were decreased by only approximately 5-15%, and A/WSN virus assembly appeared to be unaffected by the M2 antibody treatment. M2 antigen associated with cell membranes and virus particles was redistributed into clusters after M2 antibody treatment in infected cells. Incubation in the presence of the 14C2 antibody also reduced M2 surface expression by approximately 40-50% in cells infected with a recombinant vaccinia virus that expresses the M2 A/Udorn protein. These results demonstrate that M2 antibody reduces the level of influenza virus particle formation in a single cycle of infection and suggest that inhibition of A/Udorn virus replication by the 14C2 antibody is related to the reduced cell surface expression and redistribution of the M2 protein induced by the antibody treatment.

Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from cell culture-adapted rr/cpe- HAV. To determine which mutations in one rr/cpe+ virus, HM175/18f, determine enhanced replication in BS-C-1 cells, a series of chimeric viruses was rescued from infectious cDNAs in which HM175/18f genomic segments were placed within the background of a related rr/cpe- virus, HAV/7. Chimeric viruses containing the P2 region of HM175/18f produced replication foci in BS-C-1 cells that were larger than HAV/7, but not as large as HM175/18f virus. Enhanced viral replication required mutations in both 2B and 2C proteins, suggesting that these proteins remain closely associated during replication. Mutations in 5′ nontranslated RNA (5′NTR) or P3 proteins had no independent effect, but acted cooperatively with mutations in P2 proteins to enhance replication and render the virus capable of conventional plaque formation. Cytopathic effects correlated with viral replication capacity and were not the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5′NTR, P2, and P3 segments. These results suggest novel interactions between the 5′NTR and P2 proteins during HAV replication and provide useful new infectious cDNA clones.

Human herpesvirus-6 (HHV-6) can enhance the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) in cells doubly infected by HIV-1 and HHV-6. HHV-6 enhances transcription of the HIV-1 long terminal repeat, and several HHV-6 trans -activator genes have been identified. Since HIV-1 and HHV-6 have similar cellular tropism, and since HIV-1 trans -activates other herpesviruses, a reciprocal interaction between the two viruses is possible. Interactions between HIV-1 and HHV-6 were analyzed in human umbilical cord blood (CB) lymphocytes and in a T-cell line by transfaction and infection experiments. CB cells dually infected with HIV-1 and HHV-6 showed an increase in HHV-6 infectious titer, an increase in HHV-6-specific immediate early RNA, and an increase in HHV-6 protein synthesis. Similarly, T-lymphocyte cells transfected with the entire HIV-1 proviral genome displayed an increase in HHV-6. When T-cells were transfected with a plasmid containing the HIV-1 tat gene under control of the simian virus (SV40) promoter and infected with HHV-6, higher levels of HHV-6 proteins and infectious virus were detected. Therefore, the presence of HIV-1 gene products, such as tat, can lead to an activation of HHV-6 expression. Since HHV-6 is cytopathic, its activation by HIV-1 may accelerate the depletion of CD4 + T-cells in infected individuals.

Human immunodeficiency virus type 1 (HIV-1) typically evolves from a macrophage-tropic, noncytopathic virus at early asymptomatic stages of infection to a T-cell-tropic, cytopathic, and syncytia-inducing virus population as humans progress to AIDS. This suggests that changes in virus phenotype may influence disease. Because simian immunodeficiency virus (SIV) infection in macaques is a common model system for HIV-1 pathogenesis, we determined whether SIV infection in macaques that develop simian AIDS is associated with a similar shift in viral tropism, replication, and cytopathic properties. The virus that infected the monkeys (SIVMneCL8) and predominated at early times in infection is a macrophage-tropic virus that replicates with relatively low efficiency in human T cell lines. The variant populations that arise in macaques as they progress to AIDS are more infectious for human T cell lines, exhibiting enhanced replication in CEM×174 cells and an expanded host range that includes Molt-4 Clone 8 cells. Infections starting with equal doses of the viruses demonstrated that the late variants are cytopathic and syncytia-inducing compared to SIVMneCL8, but the variants replicate less efficiently in primay macaque macrophages. V3 sequences were generally conserved between the early and the late variants, suggesting that changes in SIVMne tropism, replication, and cytopathicity were apparently not due to alterations in V3. This study demonstrates important similarities in the phenotypic viral changes that accompany development of AIDS in SIV and HIV-1 infections and suggest that SIV may provide a model system for determining whether the rapidly replicating, T-cell-tropic cytopathic variants present late in infection and disease are indeed important in determining progression to AIDS.

Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse.

The murine coronavirus, mouse hepatitis virus, JHM strain, induces a retinal degenerative disease in adult BALB/c mice. Coronavirus infections are highly species specific with virus exhibiting a strong tissue and cell specificity. In this report we evaluated the cellular basis of JHM virus retinal tropism. Retinal cultures and retinal pigment epithelial (RPE)-retinal mixed cell cultures were prepared from eyes obtained from Balb/c mice. The ability of JHM virus to infect and replicate in these retinal cultures was evaluated by light microscopy, immunofluorescent staining, electron microscopy, and virus isolation. Cytopathology was not observed and virus could not be detected in supernatant fluid in retinal cultures. However, low levels of infectious virus could be detected within the cells for the first 4 days. This observation suggested that cell-to-cell interactions may be critical since virus particles and virus antigens can be seen in vivo within the neural retina and the RPE. In contrast to the retinal cultures, retinal-RPE mixed cultures were supportive to JHM virus replication. Syncytial cytopathology was observed for the first 4 days and virus was isolated from supernatant fluids. By electron microscopy, virus was found intracellularly within vacuoles and extracellularly at the plasma membrane. After Day 4, a persistent virus infection was established in which cells produced virus for 5 weeks without cytopathic effects or cell death. Double-labeling immunofluorescent studies of retinal-RPE mixed cultures showed that the virus antigen was co-expressed with a Muller cell marker, glutamine synthetase. This cell is the most prominent glial element in the retina. These studies demonstrate that JHM virus is capable of establishing a persistent virus infection in mixed retinal (Muller)-RPE cell cultures. Moreover, these data suggest that cell-to-cell interactions influence the establishment of coronavirus infections in the retina.

It has been reported that cell clones isolated at different passages from a culture of HEp-2 cells infected persistently with human respiratory syncytial virus (HRSV) were cured of the virus. Further studies on one of these clones (31C1) are reported here, showing that 31C1 cells can still be infected by HRSV but release low amounts of virus to the culture supernatant, develop smaller and less numerous syncytia than the original HEp-2 cells, and display only a weak innate immune response to the infection. Accordingly, uninfected 31C1 cells, but not clones derived from uninfected HEp-2 cells, express low levels of TLR3 and RIG-I. In addition, 31C1 cells are partly resistant to apoptosis. These results indicate that persistent infection of HEp-2 cells by HRSV has selected cell variants, with changes affecting cell survival, virus growth and the innate immune response that may be valuable for studies of virus–cell interaction.

The influenza A/chicken/Pennsylvania/1/83 (H5N2) virus is the first known example of an influenza virus isolated from a natural infection which contained primarily defective interfering particles (T. M. Chambers and R. G. Webster, J. Virol. 61, 1517-1523, 1987). In chickens, coinoculation of this virus together with the closely related but highly virulent influenza A/chicken/Pennsylvania/1370/83 virus results in reduced mortality compared to virulent virus infection alone (Bean et al., J. Virol. 54, 151-160, 1985). The biological basis of this protective effect has not been established. Protective activity required greater than or equal to 100-fold excess input of protecting virus over virulent virus, functioned effectively during the first generations of virulent virus multiplication, and also functioned against an antigenically heterologous (H7N7) virulent influenza virus. Protection was correlated with the complete inhibition of virulent virus spread to the brain of infected chickens. Plaque-purified chicken/Pennsylvania/1/83 virus depleted of defective interfering particles, and beta-propiolactone-inactivated virus, had no protective effect. These characteristics are consistent with the hypothesis that protection was the result of defective interfering particle-mediated interference with virulent virus multiplication within the respiratory tract of the chicken.

The cold-adapted reassortant of influenza A, which is a candidate live virus vaccine, interfered with the replication of parental wild-type virus in mixed infections of either MDCK cells or embryonated eggs. The interference occurred at either the permissive or nonpermissive temperature for the cold-adapted virus. In doubly infected cells, the yield of the wild-type virus was reduced by as much as 3000-fold and the protein synthesis phenotype expressed was that of the cold-adapted virus. The interference was detected even when infection with wild-type virus was carried out at a 9-fold excess or 2 hr before infection with the cold-adapted virus. As well as interfering with its wild-type parental virus, the cold-adapted virus also inhibited the replication of a heterologous influenza A subtype. In addition to its immunogenic potential, the ability to interfere with the replication of wild-type viruses is a desirable trait for any live, attenuated virus vaccine.

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.

Pseudorabies virus (PRV) early protein 0 (EP0) is the homolog of varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein and herpes simplex virus type 1 (HSV-1) ICP0. A PRV EP0 deletion mutant grows poorly in cell culture, suggesting that EP0 plays a critical role in the viral replicative cycle. In this study, we have shown that the growth defect of an EP0 deletion mutant was complemented in Vero cells expressing VZV ORF61 protein or HSV-1 ICP0. In transient expression assays PRV EP0, like VZV ORF61 protein and HSV-1 ICP0, transactivates a variety of promoters from PRV, VZV, HSV, and unrelated viruses. These data indicate that PRV EP0 is functionally homologous to VZV ORF61 protein and HSV1 ICP0.

Natural isolates of measles virus readily infect several lymphocyte cell lines. These viruses appear to use a receptor other than CD46, the molecule to which most laboratory strains of virus bind. Methods used to identify and characterize this lymphocyte receptor for measles virus are described in this study. A binding assay with a soluble form of measles virus H protein demonstrated that B-cell lines, activated with Epstein–Barr virus, or T cells, transformed with human T-cell leukemia virus, exhibit this receptor on their cell surfaces. On the other hand, resting lymphocytes, monocytes, or immature leukocytes either failed to express or possessed reduced levels of this receptor. A cDNA library derived from B95-8 marmoset B-cell lines was used to identify this receptor through expression cloning. This molecule was shown to be CDw150, which is also known as the signaling lymphocytic activation molecule (SLAM). When the lymphocyte receptor was expressed in Chinese hamster ovary (CHOP) or human embryonic kidney (293T) cells, these cells became susceptible to lymphotropic as well as laboratory strains of measles virus. Binding assays confirmed that either lymphotropic or laboratory strains of measles virus could adhere to human or marmoset CDw150, but interaction with the mouse homolog was weak. These infections were independent of the presence of CD46 on the host cell surface. Interaction of measles virus with CDw150(SLAM) could explain the immunosuppressive properties of this virus.

Several examples of human hepatitis B virus strains with enhanced replicationin vitrohave been described. To understand whether this characteristic could be a cause of liver disease, we have studied a variant of the closely related duck hepatitis B virus (DHBV) that had enhanced levels of cccDNA accumulation, previously shown to be cytopathicin vitro,as a model for the pathogenesis of analogous viruses in humans.In vivoliver damage caused by this variant (G133E) occurred only during the first 2 weeks p.i., after which time cccDNA levels and liver histology returned to near normal despite continued virus replication. To determine whether recovery was due to the emergence of noncytopathic revertant, we tested whether wild-type virus would have a selective advantage in competition with the cytopathic mutant in a fully infected liver. In a mixed infection of ducklings with G133E and a small amount of wild-type virus, the wild-type virus was detected as the predominant genotype after recovery of normal liver histology. Two candidate revertant viral genomes were cloned directly from the serum virus of G133E-infected birds after recovery and tested for (i) control of cccDNA levels in primary hepatocyte cultures and (ii) their ability to compete with wild-type virus in a mixed infection. At least one noncytopathic revertant was identified by these two criteria. The results support the conclusion that the recovery from liver damage in G133E-infected ducklings was due to the emergence of spontaneous noncytopathic revertants rather than to host suppression of virus cytotoxicity. The results indicate that acute liver injury may result from infection with a cytopathic hepadnavirus but that such viruses may be rapidly replaced by noncytopathic variants during persistent infection.

Early events in the infection of the human T-lymphotropic virus type-I (HTLV-I)-positive MT-4 cell line by the acquired immune deficiency syndrome (AIDS) retrovirus HTLV-III were investigated. The virus was adsorbed completely to the cells within 60 min incubation after inoculation of the virus. Then, infected MT-4 cells started to produce the HTLV-III-specific antigens between 12 and 24 hr postinfection. Synthesis of the viral antigens consisting of 120K, 46K, 24K, and 17K polypeptides was suppressed by the treatment of the virus-infected MT-4 cells with cytosine arabinoside (Ara-C) or by the treatment of the virus with anti-HTLV-III-positive sera. The progeny of the virus released from the infected MT-4 cells was titrated by a newly developed plaque-forming assay method and reverse transcriptase activity. The maximum activity of HTLV-III (3 × 10 5 PFU/ml) was observed on Days 4 and 5 p.i. Most of the viral activities in this preparation were ascribed to HTLV-III, and not to HTLV-I. No phenotypic mixing between HTLV-III and HTLV-I was discerned, although MT-4 cells were HTLV-I-producer cell line. Thus, HTLV-III-infected MT-4 cells are thought to be useful in further study of the interaction between host cells and the virus, and appear to be a good viral source for the analysis of the virus.

Supernatant fluids from mouse cultures productively infected with Friend leukemia virus contain, in addition to virus, large quantities of the major virion glycoprotein (gp71) in soluble form. The release of this antigen in cell culture is of practical value for its large-scale isolation in nondenatured form. It may also have bearing on how the immune system of the mouse deals with virus infection and leukemogenesis.

A group of in vitro -maintained mouse leukemia cell lines has been used to analyze a murine leukemia virus S + L − infectious center assay. The rate of virus production by leukemia cell lines was determined by two methods and was compared to the observed incidence of infectious centers for each cell line in an effort to define the exact nature of an infectious center. The results show that detection of virus-producing cells in the infectious center assay is dependent on the rate of virus production by individual cells. Although 100% of high-virus-producer cells give positive infectious center plaques, only 0.01–1% of low virus producers are detectable. The results roughly fit the prediction that a virus producer cell is scored as an infectious center if it secretes 1 PFU of virus within 40 hr after seeding onto the target cell culture.

N-Acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) has been shown to be a high-affinity receptor determinant for attachment of influenza C virus to erythrocytes (G. N. Rogers, G. Herrler, J. C. Paulson, and H-D. Klenk, 1986, J. Biol. Chem. 261, 5947-5951). In this report the nature of the cell surface receptor for influenza C virus on tissue culture cells was analyzed. Pretreatment with either neuraminidase or neuraminate 9-O-acetylesterase was found to render LLC-MK2 cells resistant to infection by influenza C virus as evidenced by the failure to detect virus release into the medium by hemagglutination titration. Susceptibility to infection was fully restored after incubation of neuraminidase-treated cells with bovine brain gangliosides known to contain Neu5,9Ac2. These results indicate that (i) Neu5,9Ac2 is the primary receptor determinant required for influenza C virus to attach to tissue culture cells and to initiate infection and (ii) gangliosides containing this type of sialic acid are potential receptors for influenza C virus. Several cell lines which are resistant to infection by this virus were able to release influenza C virus into the medium provided they were incubated with bovine brain gangliosides prior to virus infection. This result indicates that lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus.

The effect of purified mouse interferon on the replication of AKR mouse leukemia virus (MLV) in a clonal line of AKR cells was studied, with emphasis on comparing the effect of interferon on the replication of exogenous virus, activation of endogenous virus by IdUrd, and production of virus by chronically infected cells. It was found that interferon inhibited replication of virus in all three systems. Interferon did not abort exogenous infection or virus induction by IdUrd, but only delayed appearance of infectious virus. Virus production by chronically infected cells also was suppressed in the presence of interferon; however, after removal of interferon, rapid recovery of virus production occurred. Under conditions where interferon treatment inhibited virus yield as measured both by infectious virus and virion-associated reverse transcriptase activity, no significant inhibition of synthesis of virus specific (gs) antigen was observed (as measured by immunofluorescence and radioimmunoassay for p30 protein). These results indicate that unlike its effect on the majority of viruses, interferon does not inhibit MLV by a general inhibition of viral protein synthesis; rather, it appears to inhibit one or more of the later steps in MLV replication which occur after the expression of viral gs antigen. The interferon block of acute exogenous or IdUrd-induced endogenous infection appeared to occur prior to virus assembly, resulting in a marked decrease in the number of free and cell-associated particles. In the chronic infection, however, the interferon treatment only partially prevented assembly and release of virus particles, but these had markedly reduced infectivity.

A subacute sclerosing panencephalitis strain of measles virus was found to be composed of two viral variants distinquishable by their cytopathic effects in Vero cells. One of the variants was similar to defective cell-associated measles virus strains, whereas the other was highly productive of viral progeny but did not induce cell fusion. Cloning of the variants by an agarose plaque procedure revealed a consistent and rapid interconversion of the variants into one another. While the mechanism of this interconversion remains unknown, analysis of the expression of viral antigens by the variants using indirect immunofluorescence with monoclonal antibodies specific for measles structural antigens suggested that the interconversion involved modulation of the expression of the viral matrix or M antigen.

Half-lives of the interfering activity of a human defective interfering (DI) influenza virus and of viral RNA in MDCK cells inoculated with noninfectious DI virus were 25 and 17 days, respectively, and of viral RNA in cells inoculated with noninfectious uv-irradiated standard virus was 21 days. In neither case was there evidence of virus replication (de novo synthesis of viral proteins, haemagglutinin, or infectivity). The half-life in BHK cells was shorter, although still considerable, showing evidence of a host contribution to stability. Implications of the putative persistence of influenza virus genes in vivo to the natural history of the virus are discussed.

A type A influenza virus was isolated from a pig in 1979 in Thailand. Hemagglutinin and neuraminidase antigens of this virus were similar to those of human H1N1 virus isolated also in Thailand shortly before. Genetic relatedness between the human and swine viruses was demonstrated by the electrophoretic pattern of RNA on urea-polyacrylamide gel and by oligonucleotide mapping of RNA, suggesting that the swine virus had been derived from human H1N1 virus.

Replication of a temperature-sensitive mutant of rubella virus, ts-201, occurred normally at 33° but was inhibited at 3.85° in BSC-1 cells. Temperature sensitivity of the mutant did not appear to be due to the failure of adsorption of the virus to the cells nor due to defects in structural components of virion. The mutant, however, was shown to be defective in inducing viral RNA synthesis, viral antigen(s) synthesis and interference to Newcastle diseases virus. Temperature-shift experiments on the subsequent replication of the virus and on the induction of interference suggested that the induction of interference is an early viral gene function and closely related to subsequent replication of the virus.

The growth of mouse encephalomyelitis (ME) virus was studied on monolayers of Earle's L cells. Cell-associated virus (CAV) was 60 to 1000 times higher in titer than released virus and could not be neutralized with ME antiserum. This indicates that the infectious particle is completed internally. Fluorescent antibody to ME virus showed that the virus antigen is formed in the cytoplasm. Concentration of viral antigen in peripheral nodules could be correlated with release of virus from infected cells.

Strains of mumps, parainfluenza 2, and Newcastle disease virus (NDV) induced cytoplasmic inclusion body formation and other cytopathic changes in one or more of the following mammalian cell types: HEP-2, L, dog kidney, guinea pig kidney, and monkey kidney. Influenza strains produced no inclusions, except for suggestive changes seen with influenza B in monkey kidney. Strains of mumps which had not been passed more than seven times in eggs, the parainfluenza 2 virus, and Newcastle viruses of moderate to high virulence for chickens were particularly prone to produce serially transmissible cytopathic changes.

Diploid Chinese hamster cells are less susceptible to herpes simplex virus than are primary cells derived from young rabbit kidneys. Study of the most sensitive Chinese hamster cell-virus system revealed a 12-hour latent period; at that time, intranuclear inclusion bodies and both intranuclear and intracytoplasmic virus antigen could be detected. Newly synthesized infectious virus was detected 2 hours later and continued to increase until 24 hours after inoculation of the cultures. A study of the replicative site of virus nucleic acid failed to reveal specific sites of synthesis on the chromosomes. Nevertheless, virus replication is necessary for the induction of the chromosomal aberrations previously observed for noninfective virus was unable to induce such chromosomal changes. However, the aberrations are not caused by synthesis of the viral nucleic acid at or near the broken or uncoiled areas on the chromosomes.

De-embryonated eggs were prepared by a special technique of agar filling so that the ectodermal side of the chorioallantoic membrane (CAM) was utilized for infection. When herpes virus was inoculated by placing a virus-smeared cover slip onto the CAM, approximately 40% of virus was adsorbed to the CAM cells within 2 hours and the viral growth was similar to that in the intact egg CAM previously studied. In the initial stage of infection, it was impossible to separate residual active virus from the cells completely even after fifty repeated washings with saline; nor was the virus completely neutralized by the addition of a potent antiserum. However, the residual virus was as sensitive to ultraviolet (UV) irradiation as was free virus and could be reduced to an undetectable level by a mild UV irradiation followed by ten washings. Most of the eggs so treated kept their virus-replicating potentials unchanged and demonstrated the eclipse phase directly. Newly formed virus seemed to appear within and outside infected cells 10–11 hours after inoculation.

The inactivation of influenza virus by ultraviolet light is a one-hit process. Multiple infection of cells with irradiated preparations leads to the production of virus. These preparations do not contain sufficient residual active virus to account for this yield. The amount and the quality (as indicated by ID50:HA ratio) of the virus produced depends on the degree of irradiation and the number of irradiated particles per cell. Response curves of the cooperative type are obtained indicating that multiplicity reactivation is occurring. Multiplicity reactivation of influenza virus appears to be a very efficient process, and no strain differences were detected. The predictions of the recombinational theory of multiplicity reactivation, proposed by Luria and Dulbecco (1949) for a unit number of six agree well with the observed data, except that maximum yields for the experimental system are never obtained under conditions of reactivation. Incomplete virus formation occurs under conditions of multiplicity reactivation. There is no evidence of an incomplete virus gradient under these conditions. As the irradiation dose increases, the quality of progeny produced on reactivation progressively deteriorates, until an irradiation dose is reached at which only noninfectious hemagglutin is produced, and beyond which reactivation ceases.

The multiplication of three strains of influenza virus has been investigated by performing single-cycle yield experiments in pieces of surviving allantois of uniform size. This system provides a simple and reproducible means of determining the relation between input multiplicity and the nature of virus yield. The strains tested differ in their ability to produce incomplete virus, thus confirming the existence of an incomplete virus gradient. For strains MEL and BEL, a multiplicity of two or more infectious units per cell results in incomplete virus formation, whereas for strain LEE, multiple infection does not result in incomplete virus formation. It is suggested that incomplete virus formation is the result of a cooperative process between infecting virus particles. The existence of grades of incompleteness is shown. The response curve obtained for an incomplete virus preparation suggests that some incomplete particles are capable of further limited multiplication.

Clonal strains of human epidermoid carcinoma No. 2 cells have been isolated that differ from one another in morphology and nutritional requirements. The strains also differ in their response to infection with measles virus. One line of cells produced more than 10,000 times as much virus as some other lines. The number of virus particles essential for initiating infection of the various clones differed by as much as a thousandfold, and insusceptibility was correlated with a delayed appearance in cytopathic effect. The cytopathic response also differed qualitatively among several clonal types. It was found possible to synchronize cell division of the clone exhibiting the shortest generation time. Attempts to synchronize the slower-growing cultures were unsuccessful. The clone that has been synchronized is also the most sensitive to infection with measles virus. Finally, there appears to be a correlation between generation time, time required for cytopathic response, and amount of measles virus produced 7 days after infection.

Adsorption of guinea pig erythrocytes to monkey kidney cells in tissue culture infected with influenza virus was found by electron microscopy to occur by two processes. In one, the red cells were attached to virus filaments protruding from the host cell cytoplasmic membrane, and the red cell and host cell were separated by a distance of up to 1μ. This type of attachment is similar to that seen in hemagglutination. In the other, there was direct and close binding of the erythrocytes to the infected monkey kidney cell in the absence of detectable virus filaments or particles. The significance of the finding of an apparent change in the immunologically specific structure of the surface of the host cell following infection by influenza virus is discussed.

Zoster virus infects human embryonic lung cells, but subsequently no free virus appears in the culture. When seeded onto cellular monolayers infected cells produce plaques under agar. A linear relationship has been established between the number of infected cells seeded and the number of plaques that develop. The spread of zoster virus has been followed by calculating the production of plaque-forming cells and correlating this with immunofluorescent detection of zoster antigen. Eight to sixteen hours are required for the virus to infect neighboring cells. The virus then spread from cell to cell throughout the culture; maximum infection was obtained 40–48 hours after inoculation. This spread was accompanied by cytopathic changes typical of zoster infection. Iododeoxyuridine prevents spread of zoster virus from cell to cell, but does not eradicate the infection since virus antigen and plaque-forming cells can be detected as long as 5 days after inoculation of the cultures in the presence of 100 μg of IUDR per milliliter.

The growth of measles virus in Hep-2 cultures has been investigated. Maturation of the first new virus occurred 15–18 hours after inoculation, but no virus was released into the fluid phase for 27–30 hours. Even after this time the major part of the active virus remained with the cells. At 37° the virus had a half-life of about 2 hours. Thus there was little accumulation of live virus in tissue culture fluid but during the period of active virus production there was an equilibrium between release and decay. The virus was relatively stable when suspended in media at 0° of pH 6 to 9, but titer was rapidly lost at 4.5 and lower.

We have previously reported that foot-and-mouth disease virus (FMDV) can enter an Fc receptor (FcR)-expressing cell line by antibody-dependent enhancement. Since FMDV can establish a persistent infection in animals in the presence of high levels of neutralizing antibodies (carrier state), we examined macrophages for their ability to be infected by the virus in the presence of antibody. The murine macrophage cell line P388D1 or porcine macrophage-monocytes isolated from peripheral blood were incubated with antibody-complexed virus. Under these conditions, host protein synthesis was rapidly inhibited in both cell types, but not in cells incubated either with virus alone or with imine-inactivated antibody-complexed virus. Virus-specific structural and nonstructural proteins were synthesized in antibody-complexed virus-infected P388D1 cells, while only nonstructural proteins were detected in porcine macrophage cultures. Negative-strand RNAs were detected in both cell types, indicating that RNA replication had taken place. Cultures of P388D1 cells transfected with viral RNA produced very low levels of infectious virus, and infection with virus-antibody complexes, followed by a brief wash with pH 6.0 buffer to remove residual input virus, allowed the detection of low levels of productive replication. Thus, macrophages can be infected with FMDV via FcR-mediated adsorption, and infection of these cells could contribute to pathology or provide a reservoir of infectious virus in carrier animals.