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Known human coronaviruses (hCoV) usually cause mild to moderate upper-respiratory tract illnesses, except SARS-CoV and MERS-CoV, which, in addition to mild illness can also be associated with severe respiratory diseases and high mortality rates. Well-characterized multiplexed serologic assays are needed to aid in rapid detection and surveillance of hCoVs. The present study describes development and evaluation of a multiplexed magnetic microsphere immunoassay (MMIA) to simultaneously detect immunoglobulin G (IgG) antibodies specific for recombinant nucleocapsid proteins (recN) from hCoVs 229E, NL63, OC43, HKU1, SARS-CoV, and MERS-CoV. We used paired human sera to screen for IgG with reactivity against six hCoVs to determine assay sensitivity, specificity and reproducibility. We found no signal interference between monoplex and multiplex assay formats (R2 range = 0.87–0.97). Screening of paired human sera using MMIA, resulted in 92 of 106 (sensitivity: 86%) as positive and 68 of 80 (specificity: 84%) as negative. This study serves as a proof of concept that it is feasible to develop and use a multiplexed microsphere immunoassay as a next generation screening tool for use in large scale seroprevalence studies of hCoVs.

Background Although there is evidence of person-to-person transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. Methods A seroepidemiological investigation was conducted among MERS-CoV case patients (cases) and their household contacts to investigate transmission risk in Abu Dhabi, United Arab Emirates. Cases diagnosed between 1 January 2013 and 9 May 2014 and their household contacts were approached for enrollment. Demographic, clinical, and exposure history data were collected. Sera were screened by MERS-CoV nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. Results Thirty-one of 34 (91%) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. MERS-CoV antibodies were detected in 13 of 24 (54%) case patients with available sera, including 1 severely symptomatic, 9 mildly symptomatic, and 3 asymptomatic case patients. No serologic evidence of MERS-CoV transmission was found among 105 household contacts with available sera. Conclusions Transmission of MERS-CoV was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. These results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission., Transmission of Middle East respiratory syndrome coronavirus was not documented in this seroepidemiologic follow-up investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. This may have implications for isolation policies to reduce risk of transmission to others.

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies.

In 2014, the Centers for Disease Control and Prevention conducted conveyance contact investigations for 2 Middle East respiratory syndrome cases imported into the United States, comprising all passengers and crew on 4 international and domestic flights and 1 bus. Of 655 contacts, 78% were interviewed; 33% had serologic testing. No secondary cases were identified.

Infections occurred exclusively among personnel who had close contact with MERS-CoV patients., Healthcare settings can amplify transmission of Middle East respiratory syndrome coronavirus (MERS-CoV), but knowledge gaps about the epidemiology of transmission remain. We conducted a retrospective cohort study among healthcare personnel in hospital units that treated MERS-CoV patients. Participants were interviewed about exposures to MERS-CoV patients, use of personal protective equipment, and signs and symptoms of illness after exposure. Infection status was determined by the presence of antibodies against MERS-CoV. To assess risk factors, we compared infected and uninfected participants. Healthcare personnel caring for MERS-CoV patients were at high risk for infection, but infection most often resulted in a relatively mild illness that might be unrecognized. In the healthcare personnel cohort reported here, infections occurred exclusively among those who had close contact with MERS-CoV patients.

Casual contact was not associated with transmission, and serologic methods were more sensitive than real-time reverse transcription-PCR., Risk factors for human-to-human transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) are largely unknown. After MERS-CoV infections occurred in an extended family in Saudi Arabia in 2014, relatives were tested by using real-time reverse transcription PCR (rRT-PCR) and serologic methods. Among 79 relatives, 19 (24%) were MERS-CoV positive; 11 were hospitalized, and 2 died. Eleven (58%) tested positive by rRT-PCR; 8 (42%) tested negative by rRT-PCR but positive by serology. Compared with MERS-CoV–negative adult relatives, MERS-CoV–positive adult relatives were older and more likely to be male and to have chronic medical conditions. Risk factors for household transmission included sleeping in an index patient’s room and touching respiratory secretions from an index patient. Casual contact and simple proximity were not associated with transmission. Serology was more sensitive than standard rRT-PCR for identifying infected relatives, highlighting the value of including serology in future investigations.

Early detection and adherence to infection prevention recommendations are necessary to avoid transmission., Middle East respiratory syndrome coronavirus (MERS-CoV) infections sharply increased in the Arabian Peninsula during spring 2014. In Abu Dhabi, United Arab Emirates, these infections occurred primarily among healthcare workers and patients. To identify and describe epidemiologic and clinical characteristics of persons with healthcare-associated infection, we reviewed laboratory-confirmed MERS-CoV cases reported to the Health Authority of Abu Dhabi during January 1, 2013–May 9, 2014. Of 65 case-patients identified with MERS-CoV infection, 27 (42%) had healthcare-associated cases. Epidemiologic and genetic sequencing findings suggest that 3 healthcare clusters of MERS-CoV infection occurred, including 1 that resulted in 20 infected persons in 1 hospital. MERS-CoV in healthcare settings spread predominantly before MERS-CoV infection was diagnosed, underscoring the importance of increasing awareness and infection control measures at first points of entry to healthcare facilities.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal MERS-CoV infection is unknown. We describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of MERS-CoV in the world, which was related to a hospital outbreak in the United Arab Emirates in April 2014. The main histopathologic finding in the lungs was diffuse alveolar damage. Evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. Double staining immunoassays that used anti-MERS-CoV antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of MERS-CoV antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. No evidence of extrapulmonary MERS-CoV antigens were detected, including the kidney. These results provide critical insights into the pathogenesis of MERS-CoV in humans.

We found that this virus may be detected in mildly ill and asymptomatic case-patients., In January 2013, several months after Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia, Abu Dhabi, United Arab Emirates, began surveillance for MERS-CoV. We analyzed medical chart and laboratory data collected by the Health Authority–Abu Dhabi during January 2013–May 2014. Using real-time reverse transcription PCR, we tested respiratory tract samples for MERS-CoV and identified 65 case-patients. Of these patients, 23 (35%) were asymptomatic at the time of testing, and 4 (6%) showed positive test results for >3 weeks (1 had severe symptoms and 3 had mild symptoms). We also identified 6 clusters of MERS-CoV cases. This report highlights the potential for virus shedding by mildly ill and asymptomatic case-patients. These findings will be useful for MERS-CoV management and infection prevention strategies.

Middle East respiratory syndrome coronavirus, MERS-CoV, was identified in Saudi Arabia in 2012, and as of January 29, 2018, there were 2,123 laboratory-confirmed MERS-CoV cases reported to WHO (WHO, 2018, https://www.who.int/emergencies/mers-cov/en/). Multiple studies suggest that dromedary camels are a source for human MERS-CoV infection. MERS-CoV-specific antibodies have been detected in the serum of dromedary camels across Northern Africa and across the Arabian Peninsula. Israel's geographic location places Israel at risk for MERS-CoV infection. To date, MERS-CoV-related illness has not been reported and the burden of MERS-CoV infection in the Israeli population is unknown. The seroprevalence of MERS-CoV-specific antibodies in Israeli dromedary camels is unknown. The objective of this study was to determine the prevalence of MERS-CoV seropositivity in dromedary camels in Israel. The prevalence of MERS-CoV antibodies in Israeli camels was examined in 71 camel sera collected from four farms across Israel by MERS-CoV-specific microneutralization (Mnt) assay and confirmed by MERS-CoV-specific immunofluorescence assay (IFA). Although this study cannot rule out potential antibody cross-reactivity by IFA, the presence of bovine coronavirus-specific antibodies do not appear to impact detection of MERS-CoV antibodies by Mnt. MERS-CoV neutralizing antibodies were detectable in 51 (71.8%) camel sera, and no association was observed between the presence of neutralizing antibodies and camel age or gender. These findings extend the known range of MERS-CoV circulation in Middle Eastern camels. The high rate of MERS-CoV-specific antibody seropositivity in dromedary camels in the absence of any reported human MERS cases suggests that there is still much to be learned about the dynamics of camel-to-human transmission of MERS-CoV.

The first ever case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was reported in September 2012. This report describes the approaches taken by CDC, in collaboration with the World Health Organization (WHO) and other partners, to respond to this novel virus, and outlines the agency responses prior to the first case appearing in the United States in May 2014. During this time, CDC's response integrated multiple disciplines and was divided into three distinct phases: before, during, and after the initial activation of its Emergency Operations Center. CDC's response to MERS-CoV required a large effort, deploying at least 353 staff members who worked in the areas of surveillance, laboratory capacity, infection control guidance, and travelers' health. This response built on CDC's experience with previous outbreaks of other pathogens and provided useful lessons for future emerging threats.

Despite 61 contacts with unprotected exposure, no secondary cases occurred., In May 2014, a traveler from the Kingdom of Saudi Arabia was the first person identified with Middle East respiratory syndrome coronavirus (MERS-CoV) infection in the United States. To evaluate transmission risk, we determined the type, duration, and frequency of patient contact among health care personnel (HCP), household, and community contacts by using standard questionnaires and, for HCP, global positioning system (GPS) tracer tag logs. Respiratory and serum samples from all contacts were tested for MERS-CoV. Of 61 identified contacts, 56 were interviewed. HCP exposures occurred most frequently in the emergency department (69%) and among nurses (47%); some HCP had contact with respiratory secretions. Household and community contacts had brief contact (e.g., hugging). All laboratory test results were negative for MERS-CoV. This contact investigation found no secondary cases, despite case-patient contact by 61 persons, and provides useful information about MERS-CoV transmission risk. Compared with GPS tracer tag recordings, self-reported contact may not be as accurate.

To determine how long antibodies against Middle East respiratory syndrome coronavirus persist, we measured long-term antibody responses among persons serologically positive or indeterminate after a 2012 outbreak in Jordan. Antibodies, including neutralizing antibodies, were detectable in 6 (86%) of 7 persons for at least 34 months after the outbreak.

The Second Year of Life project of the Global Health Security Agenda aims to improve immunization systems and strengthen measles and rubella surveillance, including building laboratory capacity. A new laboratory assessment tool was developed by the Centers for Disease Control and Prevention to assess the national laboratory in Ghana to improve molecular surveillance for measles and rubella. Results for the tool showed that the laboratory is well organized, has a good capacity for handling specimens, has a good biosafety system, and is proficient for diagnosis of measles and rubella by serologic analysis. However, there was little knowledge about molecular biology and virology activities (i.e., virus isolation on tissue culture was not available). Recommendations included training of technical personnel for molecular techniques and advocacy for funding for laboratory equipment, reagents, and supplies.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the “out” position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182–186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log 10 -fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine. IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab′) 2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab′) 2 forms of an anti-G protein monoclonal antibody (MAb), 131-2G, on the humoral and cellular adaptive immune responses to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab′) 2 form of 131-2G does not decrease virus replication, but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75, and 95 days postinfection (p.i.) with or without prior treatment with 131-2G. The ratios of Th2 to Th1 antibody isotypes at each time p.i indicated that both forms of MAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1 to IgG2A antibody titer was 3-fold to 10-fold higher for untreated than MAb-treated mice. There was also some increase in IgG (22% ± 13% increase) and neutralization (32% increase) in antibodies with MAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly ( P ≤ 0.001) decreased the percentage of interleukin-4 (IL-4)-positive CD4 and CD8 cells in RSV-stimulated spleen cells at all times p.i., while the percentage of interferon gamma (IFN-γ) T cells significantly ( P ≤ 0.001) increased ≥75 days p.i. The shift from a Th2- to a Th1-biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (T-bet) (≥45% versus IMPORTANCE The data in this report suggest that the RSV G protein not only contributes to disease but also dampens the host immune response to infection. Both effects of G likely contribute to difficulties in achieving an effective vaccine. The ability of MAb 131-2G to block these effects of G suggests that inducing antibodies similar to 131-2G should prevent disease and enhance the adaptive immune response with later RSV infection. The fact that 131-2G binds to the 13-amino-acid region conserved among all strains and that flanking sequences are conserved within group A or group B strains simplifies the task of developing a vaccine to induce 131-2G-like antibodies. If our findings in mice apply to humans, then including the 131-2G binding region of G in a vaccine should improve its safety and efficacy.

Background. The Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered September 2012 in the Kingdom of Saudi Arabia (KSA). The first US case of MERS-CoV was confirmed on 2 May 2014. Methods. We summarize the clinical symptoms and signs, laboratory and radiologic findings, and MERS-CoV–specific tests. Results. The patient is a 65-year-old physician who worked in a hospital in KSA where MERS-CoV patients were treated. His illness onset included malaise, myalgias, and low-grade fever. He flew to the United States on day of illness (DOI) 7. His first respiratory symptom, a dry cough, developed on DOI 10. On DOI 11, he presented to an Indiana hospital as dyspneic, hypoxic, and with a right lower lobe infiltrate on chest radiography. On DOI 12, his serum tested positive by real-time reverse transcription polymerase chain reaction (rRT-PCR) for MERS-CoV and showed high MERS-CoV antibody titers, whereas his nasopharyngeal swab was rRT-PCR negative. Expectorated sputum was rRT-PCR positive the following day, with a high viral load (5.31 × 106 copies/mL). He was treated with antibiotics, intravenous immunoglobulin, and oxygen by nasal cannula. He was discharged on DOI 22. The genome sequence was similar (>99%) to other known MERS-CoV sequences, clustering with those from KSA from June to July 2013. Conclusions. This patient had a prolonged nonspecific prodromal illness before developing respiratory symptoms. Both sera and sputum were rRT-PCR positive when nasopharyngeal specimens were negative. US clinicians must be vigilant for MERS-CoV in patients with febrile and/or respiratory illness with recent travel to the Arabian Peninsula, especially among healthcare workers.

This report highlights the first two cases of MERS coronavirus in the United States. Although these patients were not transplant recipients, it is important for transplant professionals to be aware of this infection and to consider it when evaluating patients with respiratory illnesses and travel to the Arabian peninsula.

In April 2012, the Jordan Ministry of Health investigated an outbreak of lower respiratory illnesses at a hospital in Jordan; 2 fatal cases were retrospectively confirmed by real-time reverse transcription polymerase chain reaction (rRT-PCR) to be the first detected cases of Middle East respiratory syndrome (MERS-CoV).Epidemiologic and clinical characteristics of selected potential cases were assessed through serum blood specimens, medical record reviews, and interviews with surviving outbreak members, household contacts, and healthcare personnel. Cases of MERS-CoV infection were identified using 3 US Centers for Disease Control and Prevention serologic tests for detection of anti-MERS-CoV antibodies.Specimens and interviews were obtained from 124 subjects. Seven previously unconfirmed individuals tested positive for anti-MERS-CoV antibodies by at least 2 of 3 serologic tests, in addition to 2 fatal cases identified by rRT-PCR. The case-fatality rate among the 9 total cases was 22%. Six subjects were healthcare workers at the outbreak hospital, yielding an attack rate of 10% among potentially exposed outbreak hospital personnel. There was no evidence of MERS-CoV transmission at 2 transfer hospitals having acceptable infection control practices.Novel serologic tests allowed for the detection of otherwise unrecognized cases of MERS-CoV infection among contacts in a Jordanian hospital-associated respiratory illness outbreak in April 2012, resulting in a total of 9 test-positive cases. Serologic results suggest that further spread of this outbreak to transfer hospitals did not occur. Most subjects had no major, underlying medical conditions; none were on hemodialysis. Our observed case-fatality rate was lower than has been reported from outbreaks elsewhere.

To investigate potential transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) to health care workers in a hospital, we serologically tested hospital contacts of the index case-patient in Saudi Arabia, 4 months after his death. None of the 48 contacts showed evidence of MERS-CoV infection.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Increased airway resistance and increased airway mucin production are two manifestations of RSV infection in children. RSV rA2-line19F infection induces pulmonary mucous production and increased breathing effort in BALB/c mice and provides a way to assess these manifestations of RSV disease in an animal model. In the present study, we investigated the effect of prophylactic treatment with the F(ab′) 2 form of the anti-G protein monoclonal antibody (MAb) 131-2G on disease in RSV rA2-line19F-challenged mice. F(ab′) 2 131-2G does not affect virus replication. It and the intact form that does decrease virus replication prevented increased breathing effort and airway mucin production, as well as weight loss, pulmonary inflammatory-cell infiltration, and the pulmonary substance P and pulmonary Th2 cytokine levels that occur in mice challenged with this virus. These data suggest that the RSV G protein contributes to prominent manifestations of RSV disease and that MAb 131-2G can prevent these manifestations of RSV disease without inhibiting virus infection.

Defective interfering viral particles have been reported as important determinants of the course of viral infection, and they can markedly temper the virulence of the infection. Here, we describe a simple method, based on limiting dilution, for the removal of defective interfering particles from RSV. This method results in a high-titer viral preparation from both HEp-2 and Vero cell lines. We evaluated two concentrations of sucrose to stabilize the virus preparation, and demonstrate that RSV is stable when prepared and stored in 25 % sucrose at -152 °C. In addition, this chapter describes some commonly used methods of RSV titration, detection using microtitration and quantitative real-time RT-PCR, and the use of immunostaining for antigenic characterization.

Defective interfering viral particles have been reported as important determinants of the course of viral infection, and they can markedly temper the virulence of the infection. Here, we describe a simple method, based on limiting dilution, for the removal of defective interfering particles from RSV. This method results in a high-titer viral preparation from both HEp-2 and Vero cell lines. We evaluated two concentrations of sucrose to stabilize the virus preparation, and demonstrate that RSV is stable when prepared and stored in 25 % sucrose at -152 °C. In addition, this chapter describes some commonly used methods of RSV titration, detection using microtitration and quantitative real-time RT-PCR, and the use of immunostaining for antigenic characterization.

Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus-host interactions. However, NHBE cells are expensive, difficult to culture, and vary with the source patient. An alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as Calu-3, an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. The results show that Calu-3 fully polarize when grown on permeable supports as liquid-covered cultures. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The data demonstrate that polarized Calu-3 may serve as a useful in vitro model to study host responses to RSV infection.

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues (507)PAT(509) and V(349), mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P(507) and A(508)). The epitope corresponding to mAb 540C is a conformational one, involving residues L(504) and N(505). In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Since the introduction of the first applications of immunoglobulin M (IgM) determinations in diagnostic virology, a variety of methods have been developed and applied. These methods can generally be separated into three groups: (i) those based on comparing IgM titers before and after chemical inactivation of serum IgM, (ii) those based on the physicochemical separation of IgM from other serum Ig classes, and (iii) those based on solid-phase immunologic detection of IgM antibodies. This chapter discusses the relative merits of each of these approaches. Physicochemical separation methods were originally developed to separate IgM antibodies from other serum Igs to facilitate assay by conventional serological tests, e.g., complement fixation (CF) and hemagglutination inhibition (HI) assays. The major distinguishing features of solid-phase immunoassays are the choice of indicator label and solid phase. Solid-phase immunoassays can be further differentiated into indirect and reverse, or “capture,” forms, based on the orientation of the immunoreactants on the solid phase. The indirect and capture formats have advantages and disadvantages that are described in detail. Compared to the whole virus antigen-based IgM immunoassay, the recombinant protein-based assay offers several distinct advantages. First, the use of infectious virus and special safety precautions used for antigen production are not required. Second, recombinant proteins can be easily standardized and quality controlled. Third, recombinant antigen production is efficient and relatively economical, thus eliminating the generally high production costs associated with virus cultivation.

Therapeutic treatment with a non-neutralizing monoclonal antibody (mAb) (131-2G) specific to respiratory syncytial virus (RSV) G glycoprotein mediates virus clearance and decreases leukocyte trafficking and interferon gamma (IFN-γ) production in the lungs of RSV-infected mice. Its F(ab′)2 component only mediates decreased leukocyte trafficking and IFN-γ production without reducing virus replication. Thus, this mAb has two independent actions that could facilitate treatment and/or prevention of RSV infection by reducing both virus replication and virus-induced pulmonary inflammation.

Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children

In 2002, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known CoVs and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine respiratory CoV [PRCV]) was mediated through the N but not the spike protein, whereas weaker cross-reactivity occurred with feline (feline infectious peritonitis virus) and canine CoVs. Using Escherichia coli -expressed recombinant SARS-CoV N protein and fragments, the cross-reactive region was localized between amino acids (aa) 120 to 208. The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted specifically with SARS convalescent-phase sera but not with negative human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted with antisera to animal CoVs, whereas the fragment comprising aa 360 to 412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of HCoV-NL63 with group 1 CoVs was localized to aa 1 to 39 and at least one other antigenic site in the N-protein C terminus, differing from the cross-reactive region identified in SARS-CoV N protein. The observed cross-reactivity is not a consequence of a higher level of amino acid identity between SARS-CoV and porcine CoV nucleoproteins, because sequence comparisons indicated that SARS-CoV N protein has amino acid identity similar to that of infectious bronchitis virus N protein and shares a higher level of identity with bovine CoV N protein within the cross-reactive region. The TGEV and SARS-CoV N proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV-specific antibody assays for screening animal and human sera.

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Four sporadic cases of SARS-associated coronavirus infection were identified through collaboration of four laboratories., Four cases of severe acute respiratory syndrome (SARS) that occurred from December 16, 2003, to January 8, 2004, in the city of Guangzhou, Guangdong Province, China, were investigated. Clinical specimens collected from these patients were tested by provincial and national laboratories in China as well as members of the World Health Organization SARS Reference and Verification Laboratory Network in a collaborative effort to identify and confirm SARS-associated coronavirus (SARS-CoV) infection. Although SARS-CoV was not isolated from any patient, specimens from three patients were positive for viral RNA by reverse transcription–polymerase chain reaction assay, and all patients had detectable rises in SARS-CoV–specific antibodies. This study shows the effectiveness of a collaborative, multilaboratory response to diagnose SARS.

Transient CD154 blockade at the onset of Theiler's murine encephalomyelitis virus-induced demyelinating disease ameliorated disease progression for 80 days, reduced immune cell infiltration, and transiently increased viral loads in the central nervous system. Peripheral antiviral and autoimmune T-cell responses were normal, and disease severity returned to control levels by day 120.