MHV subgenomic negative strand function.
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Biomedical subjects
R S Baric
Publications and source records attributed to R S Baric.
A simple strategy to assemble infectious RNA and DNA clones.
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Detection of norwalk-like virus in shellfish implicated in illness.
In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.
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Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model.
A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of approximately 28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.
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Subgenomic negative-strand RNA function during mouse hepatitis virus infection.
Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.
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ECG changes after rabbit coronavirus infection.
This study examines the electrocardiographic (ECG) changes following rabbit coronavirus (RbCV) infection. We have shown that infection with RbCV results in the development of myocarditis and congestive heart failure and that some survivors of RbCV infection go on to develop dilated cardiomyopathy in the chronic phase. Serial ECGs were recorded on 31 RbCV-infected rabbits. Measurements of heart rate; P-R interval; QRS duration; QTc interval; and P-, QRS-, and T-wave voltages were taken. The recordings were also examined for disturbances of conduction, rhythm, and repolarization. The acute and subacute phases were characterized by sinus tachycardia with depressed R- and T-wave voltages as well as disturbances of conduction, rhythm, and repolarization. In most animals in the chronic phase, the sinus rate returned to near-baseline values with resolution of the QRS voltage changes. The ECG changes observed during RbCV infection are similar to the spectrum of interval/segment abnormalities, rhythm disturbances, conduction defects, and myocardial pathology seen in human myocarditis, heart failure, and dilated cardiomyopathy. Because animals often died suddenly in the absence of severe clinical signs of congestive heart failure during the acute phase, RbCV infection may increase ventricular vulnerability, resulting in sudden cardiac death. RbCV infection may provide a rare opportunity to study sudden cardiac death in an animal model in which the ventricle is capable of supporting ventricular fibrillation, and invasive techniques monitoring cardiac function can be performed.
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A method to detect low levels of enteric viruses in contaminated oysters.
Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-microl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.
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Persistent infection promotes cross-species transmissibility of mouse hepatitis virus.
Persistent infection with mouse hepatitis virus (MHV) strain A59 in murine DBT (delayed brain tumor) cells resulted in the emergence of host range variants, designated V51A and V51B, at 210 days postinfection. These host range mutants replicated efficiently in normally nonpermissive Chinese hamster ovary (CHO), in human hepatocarcinoma (HepG2), and to a lesser extent in human breast carcinoma (MCF7) cell lines. Little if any replication was noted in baby hamster kidney (BHK), green African monkey kidney (COS-7), feline kidney (CRFK), and swine testicular (ST) cell lines. By fluorescent antibody (FA) staining, persistent viruses V10B and V30B, isolated at days 38 and 119 days postinfection, also demonstrated very low levels of replication in human HepG2 cells. These data suggest that persistence may rapidly select for host range expansion of animal viruses. Pretreatment of HepG2 cells with a polyclonal antibody directed against human carcinoembryonic antigens (CEA) or with some monoclonal antibodies (Col-1, Col-4, Col-12, and Col-14) that bind human CEA significantly inhibited V51B infection. Under identical conditions, little or no blockade was evident with other monoclonal antibodies (kat4c or Col-6) which also bind the human CEA glycoproteins. In addition, an antibody (EDDA) directed against irrelevant antigens did not block V51B replication. Pretreatment with the Col-4 and Col-14 antibodies did not block Sindbis virus replication in HepG2 cells or MHV infection in DBT cells, suggesting that one or more CEA glycoproteins likely functioned as receptors for V51B entry into human cell lines. To test this hypothesis, the human biliary glycoprotein (Bgp) and CEA genes were cloned and expressed in normally nonpermissive BHK cell lines by using noncytopathic Sindbis virus replicons (pSinRep19). By growth curves and FA staining, human CEA and to a much lesser extent human Bgp functioned as receptors for V51B entry. Furthermore, V51B replication was blocked with polyclonal antiserum directed against human CEA and Bgp. Under identical conditions, the parental MHV strain A59 failed to replicate in BHK cells expressing human Bgp or CEA. These data suggest that MHV persistence may promote virus cross-species transmissibility by selecting for virus variants that recognize phylogenetic homologues of the normal receptor.
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Genetic complementation among three panels of mouse hepatitis virus gene 1 mutants.
Temperature-sensitive (ts) mutant viruses have been useful for the study of replication processes in many viral systems. To determine how our panel of MHV-JHM-derived RNA- ts mutants (Robb et al., 1979) is genetically related to other panels of MHV RNA- ts mutants, we tested our mutants for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). These three ts mutant panels together comprise eight genetically distinct complementation groups. Considerable genetic similarity was observed among the three mutant panels. Only three complementation classes are unique to their particular mutant panel, and genetically equivalent mutants were not observed within the other two mutant panels. There are two overlapping complementation groups between the mutant sets derived from MHV-A59 and four overlapping complementation classes between the MHV-JHM panel and the MHV-A59 panels. Two complementation groups had representative mutants in all three mutant panels. One of these latter complementation classes demonstrated nonreciprocal complementation patterns consistent with intragenic complementation.
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Human papillomavirus and head and neck cancer: epidemiology and molecular biology.
BACKGROUND: Human papillomaviruses (HPV) are known to cause cancers of the cervix and other anogenital tract sites. Molecular biology has provided some evidence as to the specific mechanisms involved in the HPV-related carcinogenesis. Epidemiologic and molecular biology studies have also suggested that HPV infection may be associated with cancers of the head and neck. METHODS: This review summarizes the biology of HPV and its potential etiologic role in head and neck cancer. Published reports were used to determine the prevalence of HPV in benign, precancerous, and neoplastic lesions of the oral cavity, pharynx, and larynx. The prevalence was also examined by head and neck site, HPV type, and method of HPV detection. In addition, the occurrence of HPV in normal head and neck tissue, epidemiologic factors related to HPV infection, and clinical implications are discussed. RESULTS: Overall, the frequency of HPV in benign and precancerous lesions ranged from 18.5% to 35.9%, depending upon the detection methodology. Based upon the most sensitive method of detection, polymerase chain reaction (PCR), the overall prevalence of HPV in head and neck tumors was 34.5% (416 of 1205 tumors). The majority of HPV-positive tumors contained the "high risk" HPV types 16 (40.0%) and 18 (11.9%). Among head and neck sites, HPV was most often detected in tumors of the oral cavity (59%), followed by the pharynx (43%), and larynx (33%). The frequency of HPV positivity in oral samples from healthy individuals ranged from 1% to 60%. A limited number of descriptive and analytic epidemiologic studies have indicated that age (<60 years) and sex (male) were associated with the presence of HPV in the tumor, whereas tobacco and alcohol use were not. The relationship between HPV and survival is unclear, with few comprehensive studies currently available. CONCLUSIONS: The prevalence of HPV, particularly the high-risk types, suggests a potential etiologic role for the virus in head and neck cancer. Molecular biology has provided important data on the interaction of the HPV oncoproteins with genes important in cell cycle control. Nonetheless, more basic research is needed to describe the physical state of the virus in a variety of cell types and the interaction with other genes. In addition, epidemiologic research is required to further understand the association between HPV and demographic and other risk factors as well as possible routes of transmission. Finally, much work is warranted to provide a definitive assessment of the prognostic significance of HPV in head and neck cancer.
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Virus-receptor interactions and interspecies transfer of a mouse hepatitis virus.
Molecular mechanisms regulating virus xenotropism and cross-species transmission are poorly understood. Host range mutants (MHV-H2) of mouse hepatitis virus (MHV) strains were isolated from mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine astrocytoma (DBT) cells. MHV-H2 was polytrophic, replicating efficiently in normally nonpermissive BHK cells, Syrian and Chinese hamster (DDT-1 and CHO) cells, human adenocarcinoma (HRT), primate kidney (VERO) and in murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK), and porcine testicular (ST) cell lines. To study the effects of xenotrophic spread on virus receptor-interactions in the original host, murine DBT cells were pretreated with a monoclonal antibody (MAb) CC1, directed against the MHV receptor, MHVR, a biliary glycoprotein (Bgp1a). Under treatment conditions that completely ablated the replication of the parental MHV strains, CC1 antireceptor antibodies did not block MHV-H2 replication. Following expression of MHVR in normally nonpermissive ST and CRFK cells, infection with the parental MHV strains, but not MHV-H2 was observed. To characterize the molecular basis preventing the interaction between MHV-H2 and MHVR, revertants of MHV-H2 (MHV-H2R6, MHV-H2R11) were isolated following a persistent MHV-H2 infection in DBT cells. These revertant viruses efficiently recognized MHVR, however infection of murine cells was resistant to MAb CC1 blockade. In addition, MHV-H2 and the revertant viruses efficiently recognized other Bgp receptors for docking and entry. These data suggest that interspecies transfer may remodel normal virus-receptor interactions that may result in altered virulence, tropism or pathogenesis in the original host.
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Human biliary glycoproteins function as receptors for interspecies transfer of mouse hepatitis virus.
A variant Mouse Hepatitis virus (MHV), designated MHV-H2, was isolated by serial passage in mixed cultures of permissive DBT cells and nonpermissive Syrian Hamster Kidney (BHK) cells. MHV-H2 replicated efficiently in hamster, mouse, primate kidney (Vero, Cos 1, Cos 7), and human adenocarcinoma (HRT) cell lines but failed to replicate in porcine testicular (ST), feline kidney (CRFK), and canine kidney (MDCK) cells. To understand the molecular basis for coronavirus cross-species transfer into human cell lines, the replication of MHV-H2 was studied in hepatocellular carcinoma (HepG2) cells which expressed high levels of the human homologue of the normal murine receptor, biliary glycoprotein (Bgp). MHV-H2 replicated efficiently in human HepG2 cells, at low levels in breast carcinoma (MCF7) cells, and poorly, if at all, in human colon adenocarcinoma (LS 174T) cell lines which expressed high levels of carcinoembryonic antigen (CEA). These data suggested that MHV-H2 may utilize the human Bgp homologue as a receptor for entry into HepG2 cells. To further study MHV-H2 receptor utilization in human cell lines, blockade experiments were performed with a panel of different monoclonal or polyclonal antiserum directed against the human CEA genes. Pretreatment of HepG2 cells with a polyclonal antiserum directed against all CEA family members, or with a monoclonal antibody, Kat4c (cd66abde), directed against Bgp1, CGM6, CGM1a, NCA and CEA, significantly reduced virus replication and the capacity of MHV-H2 to infect HepG2 cells. Using another panel of monoclonals with more restricted cross reactivities among the human CEA's, Col-4 and Col-14, but not B6.2 B1.13, Col-1, Col-6 and Col-12 blocked MHV-H2 infection in HepG2 cells. These antibodies did not block sindbis virus (SB) replication in HepG2 cells, or block SB, MHV-A59 or MHV-H2 replication in DBT cells. Monoclonal antibodies Col-4, Col-14, and Kat4c (cd66abde) all reacted strongly with human Bgp and CEA, but displayed variable binding patterns with other CEA genes. Following expression of human Bgp in normally nonpermissive porcine testicular (ST) and feline kidney (CRFK) cells, the cells became susceptible to MHV-H2 infection. These data suggested that phylogenetic homologues of virus receptors represent natural conduits for virus xenotropism and cross-species transfer.
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Receptor homologue scanning functions in the maintenance of MHV-A59 persistence in vitro.
Cell lines and viruses were isolated from mouse hepatitis virus (MHV-A59) persistently-infected DBT cells at different times postinfection. Cloned cell lines had cured virus infection, displayed low levels of MHVR receptor expression and were progressively more resistance to MHV infection. MHV persistence was likely maintained by epigenetic expression of the MHVR receptor in subsets of these resistant cells and by the emergence of persistent viruses characterized by high affinity MHVR receptor usage. Persistent viruses also displayed higher affinity for alternative biliary glycoprotein receptors suggesting that receptor homologue scanning functioned in the maintenance of persistence. Importantly, persistent viruses isolated after 210 days postinfection efficiently replicated in human HepG2 cells, a hepatocarcinoma cell line, suggesting that persistence promotes the interspecies transfer of MHV.
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Host-derived intracellular immunization against mouse hepatitis virus infection.
Mouse hepatitis virus (MHV) utilizes the murine biliary glycoprotein receptor (BgpA) for entry into susceptible cells. The Bgp1 gene was transfected into a murine DBT cell clone that expressed little if any BgpA receptor and resisted wild-type MHV infection. Clones which expressed low levels of receptor were efficient hosts for MHV-A59 replication. Clones which expressed 20- to 69-fold more BgpA receptor than controls were also susceptible to MHV-A59 infection, yet few infectious progeny virions were released. Pretreatment of clones with monoclonal antibody CC1, which binds to the N-terminus of BgpA, blocked MHV-A59 replication in DBT clones that expressed wild-type levels of receptor protein. In the overexpressing cell clones, however, CC1 pretreatment reversed the BgpA overexpression blockade and increased virus titers by 3-4 logs. BgpA overexpression inhibited the formation of infectious extracellular and intracellular virions, but levels of virus transcription were equivalent to those of controls. Ultrastructural studies revealed normal cell cytopathology in both the MHV-A59-infected controls and the overexpressing cell clones. Although equivalent numbers of virions were released compared with the control, peplomer spike glycoproteins were rarely evident in virions derived from the BgpA-overexpressing cell clones. Consonant with these findings, purified virions from BgpA-overexpressing cell clones demonstrated a 70% reduction in the amount of S glycoprotein, but not of N or M proteins. These data suggest that BgpA receptor overexpression establishes an intracellular trap which blocks MHV replication during the maturation and release of infectious progeny virions.
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Detection of low levels of enteric viruses in metropolitan and airplane sewage.
To detect less prevalent viruses, such as wild-type polioviruses in sewage from a highly immunized community, a method was developed to efficiently recover viruses and remove PCR inhibitors. The method consisted of initial separation of solids from liquid, followed by solvent extractions, polyethylene glycol precipitations, Sephadex G-200 chromatography, and guanidinium isothiocyanate (GIT) extraction. To elute viruses from the separated solids, 0.5 M threonine (pH 7.5) was as efficient as 3% beef extract but conferred no PCR inhibition. In samples that were concentrated approximately 1,000-fold, 21% of the initially seeded viruses were recovered. When poliovirus type 3 (PV3) Sabin strain at low levels and PV1 LSc strain at high levels were seeded in raw sewage, PV3 was specifically detected in the final sample concentrates at sensitivities of 14 PFU by direct PCR and 0.7 PFU by GIT extraction-PCR. While applying the method to international airplane sewage, which contains high levels of solids as well as commercial sanitizers, 44% (7 of 16) of the samples were found to harbor enteroviruses by both cell culture infectivity and pan-enterovirus PCR analyses. Nucleotide sequencing of the PCR products revealed that multiple enterovirus genotypes were amplified from each final sewage concentrate, whereas the fewer virus genotypes detected by cell culture infectivity were probably the better growing strains. By this method, we demonstrated that air travel may contribute to the intercontinental dissemination of enteric pathogens.
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Episodic evolution mediates interspecies transfer of a murine coronavirus.
Molecular mechanisms permitting the establishment and dissemination of a virus within a newly adopted host species are poorly understood. Mouse hepatitis virus (MHV) strains (MHV-A59, MHV-JHM, and MHV-A59/MHV-JHM) were passaged in mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine DBT cells. From MHV-A59/MHV-JHM mixed infection, variant viruses (MHV-H1 and MHV-H2) which replicated efficiently in BHK cells were isolated. Under identical treatment conditions, the parental MHV-A59 or MHV-JHM strains failed to produce infectious virus or transcribe detectable levels of viral RNA or protein. The MHV-H isolates were polytrophic, replicating efficiently in normally nonpermissive Syrian hamster smooth muscle (DDT-1), Chinese hamster ovary (CHO), human adenocarcinoma (HRT), primate kidney (Vero), and murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK) and porcine testicular (ST) cell lines. The variant virus, MHV-H2, transcribed seven mRNAs equivalent in relative abundance and size to those synthesized by the parental virus strains. MHV-H2 was an RNA recombinant virus containing a crossover site in the S glycoprotein gene. At the molecular level, episodic evolution and positive Darwinian natural selection were apparent within the MHV-H2 S and HE glycoprotein genes. These findings differ from the hypothesis that neutral changes are the predominant feature of molecular evolution and argue that changing ecologies actuate episodic evolution in the MHV spike glycoprotein genes that govern interspecies transfer and spread into alternative hosts.
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Molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence.
Persistent infection of murine astrocytoma (DBT) cells with mouse hepatitis virus (MHV) has been established. From this in vitro virus-host system, persistence is mediated at the level of cellular MHV receptor (MHVR) expression and increased virus virulence. MHV persistence selects for resistant host cell populations which abate virus replication. Reductions in MHVR expression were significantly associated with increased host resistance, and transfection of MHVR into resistant host cells completely restored the capacity of cells to support efficient replication of MHV strain A59. The emergence of resistant host cells coselected for variant viruses that had increased avidity for MHVR and also recognized different receptors for entry into resistant cells. These data illustrate that MHV persistence in vitro provides a model to identify critical sites of virus-host interaction at the cellular level which are altered during the evolution of host cell resistance to viral infection and the coevolution of virus virulence.
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Echocardiographic changes following rabbit coronavirus infection.
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