Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: DeltaNS1NS2, DeltaM2-2SH, DeltaM2-2NS2, DeltaSHNS1, DeltaSHNS2, and DeltaSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, DeltaM2-2NS2 was most attenuated. DeltaM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log(10) compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication.

Human metapneumovirus (hMPV), a recently identified paramyxovirus, is the causative agent of respiratory tract disease in young children. Epidemiological studies have established the presence of hMPV in retrospective as well as current clinical samples in Europe, USA, Canada, Hong Kong and Australia. The hMPV disease incidence rate varied from 7 to 12 %. This rate of disease attack places hMPV in severity between respiratory syncytial virus and human parainfluenza virus type 3, two common respiratory pathogens of young children, the elderly and immunosuppressed individuals. To evaluate the effectiveness and safety of future hMPV antiviral drugs, therapeutic and prophylactic monoclonal antibodies (mAbs), and vaccine candidates, it was necessary to identify small-animal and primate models that efficiently supported hMPV replication in the respiratory tract and produced neutralizing serum antibodies, commonly a clinical correlate of protection in humans. In this study, various rodents (mice, cotton rats, hamsters and ferrets) and two primate species, rhesus macaques and African green monkeys (AGMs), were evaluated for hMPV replication in the respiratory tract. The results showed that hamsters, ferrets and AGMs supported hMPV replication efficiently and produced high levels of hMPV-neutralizing antibody titres. Hamsters vaccinated with subgroup A hMPV were protected from challenge with subgroup A or subgroup B hMPV, which has implications for hMPV vaccine design. Although these animal models do not mimic human hMPV disease signs, they will nevertheless be invaluable for the future evaluation of hMPV antivirals, mAbs and vaccines.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10 7.2 and 10 5.7 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

A live attenuated bovine parainfluenza virus type 3 (PIV3), harboring the fusion (F) and hemagglutininneuraminidase (HN) genes of human PIV3, was used as a virus vector to express surface glycoproteins derived from two human pathogens, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). RSV and hMPV are both paramyxoviruses that cause respiratory disease in young children, the elderly, and immunocompromised individuals. RSV has been known for decades to cause acute lower respiratory tract infections in young children, which often result in hospitalization, while hMPV has only been recently identified as a novel human respiratory pathogen. In this study, the ability of bovine/human PIV3 to express three different foreign transmembrane surface glycoproteins and to induce a protective immune response was evaluated. The RNAdependent RNA polymerase of paramyxoviruses binds to a single site at the 3 end of the viral RNA genome to initiate transcription of viral genes. The genome position of the viral gene determines its level of gene expression. The promoter-proximal gene is transcribed with the highest frequency, and each downstream gene is transcribed less often due to attenuation of transcription at each gene junction. This feature of paramyxoviruses was exploited using the PIV3 vector by inserting the foreign viral genes at the 3 terminus, at position 1 or 2, of the viral RNA genome. These locations were expected to yield high levels of foreign viral protein expression stimulating a protective immune response. The immunogenicity and protection results obtained with a hamster model showed that bovine/human PIV3 can be employed to generate bivalent PIV3/RSV or PIV3/hMPV vaccine candidates that will be further evaluated for safety and efficacy in primates.Despite control of many infectious diseases in the industrialized world, acute viral respiratory tract infections remain a leading cause of illness and reason for hospitalization. Two paramyxoviruses, respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (hPIV3), are the causative agents of acute respiratory diseases of infancy and early childhood, resulting in 20 to 25% of pneumonia and 45 to 50% of bronchiolitis in hospitalized children (8). In addition, a recently identified human metapneumovirus (hMPV) appears to be associated with lower respiratory tract infections in children (25). Preliminary epidemiological reports have estimated an hMPV disease incidence rate of 7 to 10% in young children (5,11,16,18,25). The symptoms of hMPV infections are similar to those caused by RSV and hPIV3, and hospitalizations of young children with acute lower respiratory tract infections are necessary in some cases (17). Recently, Greensill et al. reported the detection of hMPV in bronchoalveolar lavage fluids from 21 of 30 infants (70%) ventilated for RSV bronchiolitis (7).RSV remains one of the most common respiratory pathogens afflicting infants, the elderly, and immunocompromised individuals. Hospitalization and immunoglobulin treatment are of...

cRespiratory syncytial virus (RSV) infects elderly (>65 years) adults, causing medically attended illness and hospitalizations. While RSV neutralizing antibody levels correlate inversely with RSV-associated hospitalization in the elderly, the role of RSVspecific T cells in preventing disease in the elderly remains unclear. We examined RSV-specific humoral, mucosal, and cellular immune profiles in healthy elderly (65 to 85 years) and young (20 to 30 years) adults. RSV neutralization antibody titers in the elderly (10.5 ؎ 2.2 log 2 ) and young (10.5 ؎ 2.1 log 2 ) were similar. In contrast, levels of RSV F protein-specific gamma interferon (IFN-␥)-producing T cells were lower in elderly (180 ؎ 80 spot-forming cells [SFC]/10 6 peripheral blood mononuclear cells [PBMC]) than in young adults (1,250 ؎ 420 SFC/10 6 PBMC). Higher levels of interleukin-13 (IL-13; 3,000 ؎ 1,000 pg/ml) in cultured PBMC supernatants and lower frequency of RSV F-specific CD107a ؉ CD8 ؉ T cells (3.0% ؎ 1.6% versus 5.0% ؎ 1.6%) were measured in PBMC from elderly than young adults. These results suggest that deficient RSV F-specific T cell responses contribute to susceptibility to severe RSV disease in elderly adults. Respiratory syncytial virus (RSV) causes annual outbreaks of respiratory disease. In North America and western Europe, these outbreaks are seasonal, occurring in winter and lasting for about 4 months. While the high global disease burden of RSV in young children and infants is well documented (1-5), the epidemiology of RSV illness in elderly adults is less well defined. Data from a variety of studies (6-14) suggest that in U.S. adults over 65 years of age, the overall annual incidence of RSV illness is ϳ3 to 4%, with an estimated annual RSV-associated hospitalization rate of ϳ0.1 to 0.4% and an estimated 10,000 RSV-associated deaths per year (Table 1).The immune correlates associated with increased susceptibility to severe RSV illness in the elderly are not well understood. Serum anti-RSV neutralizing antibody titers have been reported to inversely correlate with an increased risk of RSV-associated hospitalizations in the elderly (15). Other studies have found that the RSV-specific memory CD8 ϩ T cells are reduced in the peripheral blood of healthy elderly adults (16,17), and that a switch from a CD4 ϩ Th1 to a Th2 functional phenotype occurs with age (17). One report suggested that aging is associated with a defect in T cell responses to RSV, and this defect in cellular immunity is related to RSV disease susceptibility in older adults (18). These studies suggest that either waning RSV-specific neutralizing antibodies or declining cell-mediated immunity, or a combination of both, contribute to the greater severity of RSV disease in elderly compared to young adults.Our immune profiling studies revealed that plasma from healthy young and elderly adults had comparably high RSV neutralizing antibody titers. However, RSV F protein-specific memory CD4 ϩ and CD8 ϩ T cell responses were significantly lower in the elderly than young donors,...