Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.

We demonstrated the presence of two adeno-associated viruses (AAVs), designated AAV10 and AAV11, in cynomolgus monkeys by isolating and sequencing the entire viral coding regions from the monkey DNA. AAV10 and AAV11 capsid proteins shared 84% and 65%, respectively, of amino acids with AAV2. A phylogenetic analysis of AAV capsid proteins showed that AAV10 and AAV11 resembled most AAV8 and AAV4, respectively. To characterize the capsid protein, we pseudotyped an AAV2 vector with the monkey AAV capsid proteins and examined the resulting pseudotypes AAV2/10 and AAV2/11, in comparison with the AAV2 vector, for their host ranges in cell lines and tissue tropism in mice. AAV2/10 and AAV2/11 transduced primate cells less efficiently than AAV2. Whereas AAV2 transduced undifferentiated C2C12 mouse myoblasts more efficiently than differentiated ones, AAV2/10 and AAV2/11 transduced the undifferentiated myoblasts less efficiently than differentiated ones. Three weeks after injection to the muscle of the hind legs, AAV2/10 and AAV2 induced transgene expression similarly, but AAV2/11 did not transduce the skeletal muscle. Six weeks after systemic administration, transduced vector DNA was detected by PCR in the liver and spleen of mice inoculated with AAV2, in the liver, heart, muscle, lung, kidney, and uterus of mice with AAV2/10, and the muscle, kidney, spleen, lung, heart, and stomach of mice with AAV2/11. Mouse antisera against capsid protein VP2 of the three AAVs neutralized the respective vector particles in a type-specific manner. The results indicate that AAV10 and AAV11 capsid proteins, which are antigenically distinct from each other and AAV2, are likely to determine their host ranges and tissue tropism that are different from AAV2s, suggesting that cynomolgus AAVs could provide a broader choice of pseudotype AAV vectors for gene therapy.

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The sequence of 3,687 nucleotides from the 3' end of the Sendai virus genome (Z strain) was determined by a molecular cloning technique followed by rapid sequence analysis. Two large open reading frames, one consisting of 1,572 nucleotides and the other of 1,704 nucleotides, were observed in the region, that is OP-1 and OP-2 from the 3' end of the genome. The amino acid sequences of the gene products were predicted from the observed sequence. Determination of amino acid compositions of viral proteins, P, HN, Fo, NP and M, led us to conclude that NP and P are the gene products of OP-1 and OP-2, respectively. An additional open reading frame consisting of 612 nucleotides (OP-3) was discovered in the 3' most proximal region of OP-2. The predicted product of OP-3 was considered to be viral non-structural protein C. The leader sequence of 51 nucleotides at the 3' terminal of the genome and consensus sequences at 3' and 5' ends of each gene for proteins NP and P were identified.

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4°C and to enter the cytoplasm after subsequent incubation at 37°C. Papillomaviruses, whose nonenveloped, icosahedral, 55-nmdiameter virions are made of 72 pentameric capsomeres composed of the structural proteins L1 and L2 at an estimated molar ratio of 30 to 1, have been found in various animal species, including humans (26). Human papillomaviruses (HPVs) have to date been classified into more than 80 genotypes, which constitute two groups: cutaneous and mucosal HPVs (26). HPVs infecting the cutaneous epithelium such as types 1, 2, 4, and 8 mainly cause skin warts (26). HPVs infecting the mucosal epithelium such as types 6 and 11 cause benign condyloma, but types 16, 18, and 33 cause cervical cancer (14, 26). Among the nine major types of HPV associated with cervial cancer, HPV16 is the most prevalent type, constituting approximately 50% of cases (14, 27).Because it is almost impossible to efficiently grow HPVs in cell cultures, except HPV18 in a raft culture (16), surrogate systems have been developed for production of capsids (particles without viral DNA) and for assay of viral infectivity. The capsids produced in surrogate systems resemble morphologically and immunlogically the natural virions (7,11,12,22). When L1 alone is expressed in eukaryotic cells by recombinant baculovirus or vaccinia virus (7,11,12,22), L1 can self-assemble to form icosahedral particles (L1 capsids). When L2, which is not required for assembly, is coexpressed with L1, both L1 and L2 are incorporated into the particles (L1-L2 capsids) (7,12). Furthermore, infectious HPV pseudovirions are produced in cultured cells (19,24). Also, pseudovirions are constructed in vitro from disassembled capsids and a plasmid capable of expressing a reporter gene, and their infectivity can be assayed in COS-1 cells (9). The capsids and pseudovirions and natural bovine papillomavirus type 1 (BPV1) virions, isolated from cutaneous legions, have been used for studies of adsorption of papillomaviruses and of neutralization of their infectivity.Viral infection in vitro is supposed to start from attachment of virions to the cell surface (20,25). The adsorption appears to occur from binding of L1 to cell recep...

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanningplot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P < 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.

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