Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the ⌬SH virus was only marginally less efficient than that of wild-type HMPV, whereas the ⌬G and ⌬M2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The ⌬G and ⌬M2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [r⌬M2-1, r⌬M2-2, and r⌬M2(1؉2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The r⌬M2-2 mutants exhibited a two-to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with r⌬M2-1 or r⌬M2(1؉2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with r⌬M2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV r⌬M2-2 virus is a promising and highly attenuated HMPV vaccine candidate.Human metapneumovirus (HMPV) was first identified in 2001 in The Netherlands from infants and children with acute respiratory tract disease (38) and is now recognized to be worldwide in prevalence (22,41). HMPV resembles human respiratory syncytial virus (HRSV) with regard to disease signs and the ability to infect and cause disease in infants as well as in individuals of all ages (7,18,20,29,32,39,41). The contribution of HMPV to respiratory tract disease remains to be fully defined but appears to be sufficient to warrant the development of a vaccine, especially for the pediatric population. Reverse genetic systems were recently developed for HMPV, allowing the generation of infectious virus from cDNA and providing an important tool for characterizing HMPV biology and for designing live-attenuated HMPV vaccines (5, 25).HMPV has a negative-strand RNA genome of approximately 13 kb (4, 37). It has been classified, together with avian metapneumovirus, in the Metapneumovirus genus, Pneumovirus subfamily, Paramyxovirus family, of the order Mononegavirales. The Pneumovirus subfamily also contains the genus Pneumovirus, represented by HRSV. The Metapneumovirus gene order is N-P-M-F-M2-SH-G-L. By analogy to HRSV, the predicted HMPV proteins are the following: the nucleocapsid protein N, which encapsidates the RNA genome and, together with the phosphoprotein P and the RNA polymerase protein L, forms the ribonucleoprotein co...

Chimeric versions of recombinant human metapneumovirus (HMPV) were generated by replacing the nucleoprotein (N) or phosphoprotein (P) open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. In Vero cells, AMPV replicated to an approximately 100-foldhigher titer than HMPV. Surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11-and 25-fold higher, respectively, than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. AMPV and the N and P chimeras were evaluated for replication, immunogenicity, and protective efficacy in hamsters. AMPV was attenuated compared to HMPV in this mammalian host on day 5 postinfection, but not on day 3, and only in the nasal turbinates. In contrast, the N and P chimeras were reduced approximately 100-fold in both the upper and lower respiratory tract on day 3 postinfection, although there was little difference by day 5. The N and P chimeras induced a high level of neutralizing serum antibodies and protective efficacy against HMPV; AMPV was only weakly immunogenic and protective against HMPV challenge, reflecting antigenic differences. In African green monkeys immunized intranasally and intratracheally, the mean peak titer of the P chimera was reduced 100-and 1,000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold in the lower respiratory tract. Both chimeras were comparable to wild-type HMPV in immunogenicity and protective efficacy. Thus, the P chimera is a promising live HMPV vaccine candidate that paradoxically combines improved growth in vitro with attenuation in vivo.Human metapneumovirus (HMPV) is an enveloped RNA virus of the genus Metapneumovirus, subfamily Pneumovirinae, family Paramyxoviridae, order Mononegavirales. HMPV was first described in 2001 (34) and is now regarded as an important viral respiratory pathogen of worldwide distribution (9,20,35). Its clinical impact warrants the development of a vaccine, particularly for the pediatric population. Avian metapneumovirus (AMPV) is a closely related virus that causes respiratory disease in turkeys, chickens, and other birds (24).HMPV has a nonsegmented negative-strand RNA genome of approximately 13.3 kb that contains eight genes in the order 3Ј-N-P-M-F-M2-SH-G-L-5Ј (5, 33). As is the case for other members of Mononegavirales, the nucleoprotein N, the phosphoprotein P, the RNA-dependent RNA polymerase L, and the viral genomic RNA form the ribonucleoprotein complex, which is the minimal replication/transcription unit. The other genes encode matrix protein M, fusion protein F, M2 mRNA, small hydrophobic glycoprotein SH of unknown function, and putative attachment glycoprotein G. The genomic RNA and its positive-sense replicative intermediate, the antigenomic RNA, are tightly encapsidated by the N protein. The N and L proteins are among the more highly conserved proteins between different Paramyxoviridae species, whereas P is som...

Abstract. Microtubule associated protein tau (MAPT) encodes the microtubule associated protein tau, the primary component of neurofibrillary tangles found in Alzheimer's disease and other neurodegenerative disorders. Mutations in the coding and intronic sequences of MAPT cause autosomal dominant frontotemporal dementia . MAPT is also a candidate gene for progressive supranuclear palsy and hereditary dysphagic dementia. A human PAC (201 kb) and a mouse BAC (161 kb) containing the entire MAPT and Mtapt genes, respectively, were identified and sequenced. Comparative DNA sequence analysis revealed over 100 conserved non-repeat potential cis-acting regulatory sequences in or close to MAPT. Those islands with greater than 67% nucleotide identity range in size from 20 to greater than 1700 nucleotides. Over 90 single nucleotide polymorphisms were identified in MAPT that are candidate susceptibility alleles for neurodegenerative disease. The 5Ј and 3Ј flanking genes for MAPT are the corticotrophin-releasing factor receptor (CRFR) gene and KIAA1267, a gene of unknown function expressed in brain.

The contribution of cleavage activation of the fusion F protein of human metapneumovirus (HMPV) to replication and pathogenicity in rodents and nonhuman primates was investigated. Recombinant HMPVs were generated in which the naturally occurring trypsin-dependent cleavage sequence (R-Q-S-R2) was replaced by each of three sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multibasic sequences derived from avian metapneumovirus type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R2). The third one (R-Q-P-R) was derived from a recently described trypsin independent HMPV isolate (J. H. Schickli, J. Kaur, N. Ulbrandt, R. R. Spaete, and R. S. Tang, J. Virol. 79:10678-10689, 2005). To preclude the possibility of conferring even greater virulence to this significant human pathogen, the modifications were done in an HMPV variant that was attenuated by the deletion of two of the three envelope glycoproteins, SH and G. Each of the introduced cleavage sequences conferred trypsin independent F cleavage and growth to HMPV in vitro. However, they differed in the efficiency of trypsin independent growth and plaque formation in vitro: R-R-R-R > R-K-A-R > R-Q-P-R > R-Q-S-R. The R-R-R-R mutant was the only one whose growth in vitro was not augmented by added trypsin, indicative of highly efficient trypsin independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract between the mutants, and virus was not detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence.