Transmission of viral pathogens in a social network of university students: the eX-FLU study.

Previous research on respiratory infection transmission among university students has primarily focused on influenza. In this study, we explore potential transmission events for multiple respiratory pathogens in a social contact network of university students. University students residing in on-campus housing (n = 590) were followed for the development of influenza-like illness for 10-weeks during the 2012-13 influenza season. A contact network was built using weekly self-reported contacts, class schedules, and housing information. We considered a transmission event to have occurred if students were positive for the same pathogen and had a network connection within a 14-day period. Transmitters were individuals who had onset date prior to their infected social contact. Throat and nasal samples were analysed for multiple viruses by RT-PCR. Five viruses were involved in 18 transmission events (influenza A, parainfluenza virus 3, rhinovirus, coronavirus NL63, respiratory syncytial virus). Transmitters had higher numbers of co-infections (67%). Identified transmission events had contacts reported in small classes (33%), dormitory common areas (22%) and dormitory rooms (17%). These results suggest that targeting person-to-person interactions, through measures such as isolation and quarantine, could reduce transmission of respiratory infections on campus.

Specificity and kinetics of norovirus binding to magnetic bead-conjugated histo-blood group antigens.

AIMS: To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo-blood group antigens (HBGA)-conjugated magnetic beads. METHODS AND RESULTS: HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM-MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15min of incubation with PGM-MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real-time RT-PCR analyses suggest that NoV could be concentrated as much as 1000-fold at neutral pH, up to 3·4-fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0-4·2. Variation between GI and GII viral binding to the PGM-MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM-MB, ranging from 28-fold (GI.4) to 1502-fold (GI.1). Eleven of 13 GII strains were concentrated from 30-fold (GII.5) to 1014-fold (GII.4, lab strain) by PGM-MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery. CONCLUSIONS: All GI and 85% of GII NoVs tested could be captured and concentrated by PGM-MB method. The binding occurred rapidly and was enhanced at low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.

Sindbis virus mutants selected for rapid growth in cell culture display attenuated virulence in animals.

Mutants of Sindbis virus were selected for rapid growth in baby hamster kidney (BHK) cell cultures and screened for attenuation of virulence in suckling mice. Comparisons among independently isolated virulent and attenuated strains, as well as a classical reversion analysis, showed that accelerated penetration of BHK cells was correlated with attenuation in vivo. Both phenotypic changes resulted from a reorganization of virion structure as detected by monoclonal antibodies. These results suggest that mutants selected for rapid growth in cell culture may be useful as attenuated vaccines and for studies of the molecular basis of virus pathogenesis.

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus.

The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Coronavirus genome structure and replication.

In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. Their lengths range from 27,317 nt for HCoV-229E to 31,357 nt for the murine hepatitis virus-A59, establishing the coronavirus genome as the largest known among RNA viruses. The basic organization of the coronavirus genome is shared with other members of the Nidovirus order (the torovirus genus, also in the family Coronaviridae, and members of the family Arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mRNA synthesis (transcription) (an estimated 14-16 end products for coronaviruses) are encoded within the 5'-proximal two-thirds of the genome on gene 1 and the (mostly) structural proteins are encoded within the 3'-proximal one-third of the genome (8-9 genes for coronaviruses). Genes for the major structural proteins in all coronaviruses occur in the 5' to 3' order as S, E, M, and N. The precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. This chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (DI) RNA molecules], the minimum sequence requirements for autonomous replication of an RNA replicon, and the importance of gene order in genome replication.

Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs.

The genomes of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) have been generated with a novel construction strategy that allows for the assembly of very large RNA and DNA genomes from a panel of contiguous cDNA subclones. Recombinant viruses generated from these methods contained the appropriate marker mutations and replicated as efficiently as wild-type virus. The MHV cloning strategy can also be used to generate recombinant viruses that contain foreign genes or mutations at virtually any given nucleotide. MHV molecular viruses were engineered to express green fluorescent protein (GFP), demonstrating the feasibility of the systematic assembly approach to create recombinant viruses expressing foreign genes. The systematic assembly approach was used to develop an infectious clone of the newly identified human coronavirus, the serve acute respiratory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an infectious clone within 2 months of identification of the causative agent of SARS, providing a critical tool to study coronavirus pathogenesis and replication. The availability of coronavirus infectious cDNAs heralds a new era in coronavirus genetics and genomic applications, especially within the replicase proteins whose functions in replication and pathogenesis are virtually unknown.

Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus.

UNLABELLED: Zika virus (ZIKV) is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV) infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73%) failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], <1:100 serum dilution; 18%) or moderate to high (EC50, >1:100 serum dilution; 9%) levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV. IMPORTANCE: ZIKV is an emerging arbovirus that has been associated with severe neurological birth defects and fetal loss in pregnant women and Guillain-Barré syndrome in adults. Currently, there is no vaccine or therapeutic for ZIKV. The identification of a class of antibodies (envelope dimer epitope 1 [EDE1]) that potently neutralizes ZIKV in addition to all four DENV serotypes points to a potential immunotherapeutic to combat ZIKV. This is especially salient given the precedent of antibody therapy to treat pregnant women infected with other viruses associated with microcephaly, such as cytomegalovirus and rubella virus. Furthermore, the identification of a functionally conserved epitope between ZIKV and DENV raises the possibility that a vaccine may be able to elicit neutralizing antibodies against both viruses.

A new quaternary structure epitope on dengue virus serotype 2 is the target of durable type-specific neutralizing antibodies.

UNLABELLED: Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE: Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines.

Isolation and identification of six Pneumocystis carinii genes utilizing codon bias.

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.

Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription.

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70-82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro.

Serial passage of a multidrug-resistant clone of Plasmodium falciparum in concentrations of mefloquine hydrochloride ranging from 30 to 2,400 ng/ml resulted in the derivation of increasingly resistant parasite lines in vitro. Parasite lines isolated in mefloquine concentrations greater than 300 ng/ml demonstrated increased vacuolization, enhanced pigment production, and increased growth rates as compared with the progenitor clone, W2-mef. Although microdilution incorporation assays demonstrated that the 50% inhibitory concentration (IC50) of mefloquine were similar for all lines, the IC90, IC95, and IC99 levels were significantly increased. Growth rate assays performed in 5% hematocrit suspensions demonstrated different levels of mefloquine resistance among these lines. Under these conditions the most resistant line, Mef 2.4, grew efficiently in approximately 10-fold higher concentrations of mefloquine than the progenitor clone W2-mef. Analysis of drug susceptibility profiles to mefloquine hydrochloride, chloroquine diphosphate, quinine sulfate, and halofantrine hydrochloride indicated that selection for high levels of mefloquine resistance had resulted in significant increases in resistance to halofantrine and increased sensitivity to chloroquine. The phenotypic changes demonstrated in the most resistant line, Mef 2.4, reflect a multidrug resistant-like phenotype, and appear to mimic changes recently reported in drug susceptibility profiles of recrudescent isolates following mefloquine treatment failures in Thailand.

A strong association between mefloquine and halofantrine resistance and amplification, overexpression, and mutation in the P-glycoprotein gene homolog (pfmdr) of Plasmodium falciparum in vitro.

Stepwise selection for increased mefloquine resistance in a line of Plasmodium falciparum in vitro resulted in increased resistance to halofantrine and quinine, increased sensitivity to chloroquine, and amplification and overexpression of the P-glycoprotein gene homolog (pfmdr1). A point mutation (tyrosine to phenylalanine) noted at amino acid 86 in pfmdr1 in the mefloquine-resistant line W2mef was amplified in more resistant lines derived from it by in vitro selection pressure with mefloquine. Conversely, lines selected for increased chloroquine resistance exhibited a revertant phenotype that was sensitive to mefloquine and halofantrine. These lines also demonstrated increased sensitivity to quinine, loss of amplification of pfmdr1, loss of the mefloquine/halofantrine phenylalanine-86 mutation, and selection for a tyrosine-86 mutation previously associated with chloroquine resistance. These findings provide strong evidence for pfmdr1 mediating cross-resistance to halofantrine and mefloquine in P. falciparum in vitro.

Detection of hepatitis A virus and other enteroviruses in water by ssRNA probes.

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA encoding the 5'-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65 degrees C), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500-1000 infectious units of the 7 strains of HAV tested. Conversely, the CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected enteroviruses in samples of surface water and drinking water that were negative for cytopathic effects in inoculated cell cultures.

Evolution and persistence mechanisms of mouse hepatitis virus.

We established and characterized persistently-infected DBT cells with mouse hepatitis virus to study the molecular mechanisms of MHV persistence and evolution in vitro. Following infection, viral mRNA and RF RNA were coordinately reduced by about 70% as compared to acute infection suggesting that the reduction in mRNA synthesis was due to reduced levels of transcriptionally active full length and subgenomic length negative-stranded RNAs. Although the rates of mRNA synthesis were also reduced, the relative percent molar ratio of the mRNAs and RF RNAs were similar to those detected during acute infection. In contrast to the finding during BCV persistence, analysis of the MHV leader RNA indicated that the leader RNA and leader/body junction sequences were extremely stable. These data suggested that polymorphism and mutations resulting in intraleader ORFs was not required for MHV persistence. Conversely MHV persistence was significantly associated with a A to G mutation at nt 77 in the 5' end untranslated region (UTR) of the genomic RNA.

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.

Altered proteolytic processing of the polymerase polyprotein in RNA(-) temperature sensitive mutants of murine coronavirus.

We examined the synthesis and processing of the polymerase polyprotein in RNA(-) temperature sensitive mutant of murine coronavirus strain A59. These temperature sensitive mutants of MHV-A59 synthesize viral RNA at the permissive temperature (33.0 degrees C), but are unable to synthesize viral RNA at the nonpermissive temperature (39.5 degrees C). The ts mutants have been mapped to five different complementation groups in the polymerase gene. The 5'-most complementation groups, Groups A and B, map to a region encoding an autoproteinase responsible for the cleavage of p28, the amino-terminal product of the polymerase polyprotein. We screened six temperature sensitive mutants to determine if there was an alteration in the proteolytic processing of the polymerase polyprotein, particularly in the cleavage of the p28 protein. Two mutants, tsNC9 and tsLA16, had altered proteolytic products at both the permissive and nonpermissive temperatures. One Group B temperature sensitive mutant, designated tsNC11, was defective in the production of p28 protein at the nonpermissive temperature. To further localize the site of the mutation in tsNC11, RNA representing the 5'-most 5.3 kb region of the polymerase gene was transfected into tsNC11-infected cells and virus production monitored. The transfected RNA was able to complement the defect in tsNC11, resulting in viral RNA synthesis and production of viral particles at the nonpermissive temperature. These results indicate that a gene product from the 5.3 kb region of gene 1 is required for coronavirus RNA synthesis.

Studies into the mechanism for MHV transcription.

Previous studies have demonstrated that the MHV genome is divided into seven transcriptional units which are transcribed from highly conserved intergenic start sites (UCU/CAAAC) into mRNA containing a common leader RNA at the 5' end and a coterminal 3' end. In this manuscript, we provide evidence that an additional transcriptional unit is encoded at the 3' end of the MHV genome and is transcribed from a perfect intergenic region into a leader-containing approximately 800 nt mRNA. This mRNA could potentially encode a small 17-18 kDa protein which is identical to the C-terminal third of the nucleocapsid gene.

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.

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.

Electrocardiographic changes following rabbit coronavirus-induced myocarditis and dilated cardiomyopathy.

Rabbit Coronavirus (RbCV) infection was divided into two phases based upon day of death and pathologic findings. During the acute phase (days 2-5) heart weights (HW) and heart weight-to-body weight (HW/BW) ratios were increased with striking dilation of the right ventricle. These changes as well as increased dilation of the left ventricle were especially pronounced during the subacute phase (days 6-12). Myocytolysis, pulmonary edema, and degeneration and necrosis of myocytes, were seen during both phases. Myocarditis, pleural effusion, calcification of myocytes, and congestion in the liver and lungs were seen in the subacute phase. Electrocardiograms (ECGs) exhibited low voltage, nonspecific ST-T wave changes, sinus tachycardia, occasional ventricular and supraventricular premature complexes and 2(0) AV block consistent with myocarditis and heart failure. Forty-one percent of the survivors exhibited increased HW and HW/BW ratios, biventricular dilation, interstitial and replacement fibrosis, myocyte hypertrophy and myocarditis. ECGs exhibited nonspecific ST-T wave changes, sinus arrhythmia, occasional ventricular and supraventricular premature complexes and 2(0) AV block. These data suggest that RbCV infection may result in viral myocarditis and heart failure with a proportion of survivors progressing into DCM.