Genome organization of the Kresse strain of porcine parvovirus: identification of the allotropic determinant and comparison with those of NADL-2 and field isolates.

The Kresse strain of porcine parvovirus (PPV) was cloned into pUC19, and independent infectious clones were sequenced. The PPV Kresse and NADL-2 strains, which have different pathogenicities, shared an identical genomic organization and a high degree of sequence identity. Partial genomes (1.5 or 1.6 kb) of 15 field isolates were also amplified by PCR in regions with significant sequence differences between the laboratory strains. Five amino acid differences were consistently present within the VP1/VP2 coding region of the Kresse strain and virulent field isolates. A number of inconsistent point mutations were also found throughout the genomes of field isolates. In addition, among those with the vaccine amino acid profile, all but one isolate (IAF-3) contained a 127-bp noncoding direct repeat downstream of the capsid protein gene. The one exception was also the only vaccine-type PPV obtained from a mummified fetus. In order to identify genetic elements responsible for the distinct tropism (and possibly the pathology) of the Kresse strain, in vitro cell systems which differentiated the virulent from the vaccinal strains were established. Subsequently, chimeric infectious clones of the Kresse and NADL-2 strains were used to identify the allotropic determinant located in the VP1/VP2 region. The transfer of the BglII fragment of the Kresse genome, containing three amino acid differences, into the NADL-2 background, or the opposite construct, caused the phenotype of the target genome to revert to that of the parent strain of the BglII fragment. Prediction of the localization of amino acid differences on the basis of canine parvovirus capsid structure indicates that each is located on or near the outer surface of the virion. In particular, the position of one mutation (S-436-->P) maps by analogy to the threefold spike, the most accessible region of the capsid.     

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.     

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5'' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.     

Coexistence of multiple strains of porcine parvovirus 2 in pig farms

The porcine parvovirus 2 (PPV2) genome was first identified in 2001 in Myanmar. Recently, the PPV2 genome has been found in several other countries. In this study, the prevalence of PPV2 in Japanese domestic pigs was investigated and found to be 58% (69/120) in healthy domestic pigs and 100% (69/69) in sick domestic pigs. Sequencing and phylogenetic analysis of the PCR products of the VP1 gene and an almost full length PPV2 clone indicated that diverged PPV2 strains exist in Japan. Clearly distinct strains of PPV2 were detected in 7 of the 10 pig farms.     

Duplication of noncoding sequences in polyomavirus specifically augments the development of thymic tumors in mice.

A 40-base-pair duplication of noncoding sequences in polyomavirus specifically augmented the development of thymic epitheliomas following inoculation of virus into newborn mice. Virus strains carrying only one copy of this sequence induced a full spectrum of tumors except for overt thymic tumors. This 40-base-pair repeat, on the early side of the replication origin, constituted a tissue-specific regulatory determinant for tumor induction.     

The structure of porcine parvovirus: comparison with related viruses

The structure of baculovirus-expressed porcine parvovirus (PPV) capsids was solved using X-ray crystallography and was found to be similar to the related canine parvovirus (CPV) and minute virus of mice (MVM). The PPV capsid protein has 57 % and 49 % amino acid sequence identity with CPV and MVM, respectively, but the degree of conservation of surface-exposed residues is lower than average. Consequently, most of the structural differences are on the surface and are the probable cause of the known variability in antigenicity and host range. The NADL-2 and Kresse strains of PPV have distinct tissue tropisms and pathogenicity, which are mediated by one or more of the amino acid residues 381, 386, and 436. These residues are on or near the surface of the virus capsid, where they are likely to be associated with virus-cell interactions.     

Fine mapping and sequencing of a variable segment in the inverted repeat region of varicella-zoster virus DNA.

A strain variation in the internal and terminal repeats which bind the short unique sequence of varicella-zoster virus (VZV) DNA was found to be due to an insertion or deletion of DNA sequences at a single site. DNA sequence analysis showed that the nucleotide sequence CCGCCGATGGGGAGGGGGCGCGGTACC is tandemly duplicated a variable number of times in different VZV strains and is responsible for the observed variation in mobilities of restriction fragments from this region of VZV DNA. The variable region sequence shares some homology with tandemly repeated regions in the a and c sequences of herpes simplex virus type 1 and probably exists in a noncoding region of the VZV genome.     

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.     

Prevalence and genomic characterization of porcine parvoviruses detected in Chiangmai area of Thailand in 2011

Porcine parvovirus (PPV) causes reproductive failure in sows and has spread worldwide. Several new types of porcine parvoviruses have recently been identified in pig herds. The prevalence of five porcine parvoviruses in the Chiangmai area of Thailand was studied. The prevalence in 80 pigs was 53% for PPV (PPV‐Kr or ‐NADL2 being the new abbreviations), 83% for PPV2 (CnP‐PARV4), 73% for PPV3 (P‐PARV4), 44% for PPV4 (PPV4), and 18% for PBo‐likeV (PBoV7). Over 60% of the pigs carried more than three of the five porcine parvoviruses and occurrence together of the two pairs of viral genes, PPV1/PPV3 and PPV2/PBo‐likeV were observed. Phylogenetic analyses for PPV2 and PPV3 indicated the existence of only two major clades of PPV2 and one major clade of PPV3.     

In vitro exposure of preimplantation porcine embryos to porcine parvovirus

Early porcine embryos at the four- to eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos with virus. Treatment of embryos by microinjection of virus or incubation in media with virus did not significantly inhibit in vitro development of the embryos when compared with untreated controls. RNA-DNA hybridization was used to identify the presence of virus associated with embryos. It was found that PPV-DNA was present in viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos.     

猪圆环病毒2型与猪细小病毒共感染对猪肺泡巨噬细胞吞噬功能及其干扰素表达水平的影响

【目的】分析猪圆环病毒2型(PCV2)与猪细小病毒(PPV)共感染对猪肺泡巨噬细胞(PAM)吞噬功能及其干扰素表达水平的影响,为进一步阐明猪断奶后多系统衰减综合征的发病机制提供实验依据。【方法】将48头5周龄健康仔猪随机分为PCV2组、PPV组、PCV2/PPV组和对照组,每组12头。PCV2和PPV组经口鼻途径分别接种PCV2或PPV,PCV2/PPV组同时接种PCV2和PPV,对照组接种细胞营养液。感染后3、7、14和35d(dpi)从每组随机选择3头剖杀,检测PAM的存活率、吞噬活性及其α1型干扰素(IFN-α1)、γ干扰素(IFN-γ)mRNA的表达水平。【结果】PCV2组和PCV2/PPV组PAM的存活率于3、7、14dpi均低于对照组,35dpi均与对照组PAM的存活率接近,PCV2与PCV2/PPV两组之间差异不显著(P>0.05);3个病毒感染组PAM的吞噬功能均显著下降(P<0.01),其中PCV2/PPV组的吞噬功能低于PCV2组(P<0.05);PCV2/PPV组PAM中IFN-α1和IFN-γ mRNA的水平持续低于PCV2、PPV组和对照组的水平(P<0.01)。【结论】PCV2与PPV共感染没有引起PAM的活力进一步下降,但导致其吞噬功能及IFN-α1和IFN-γmRNA的表达水平持续降低。     

Genome sequence of Chinese porcine parvovirus strain PPV2010

Porcine parvovirus (PPV) isolate PPV2010 has recently emerged in China. Herein, we analyze the complete genome sequence of PPV2010. Our results indicate that the genome of PPV2010 bears mixed characteristics of virulent PPV and vaccine strains. Importantly, PPV2010 has the potential to be a naturally attenuated candidate vaccine strain.     

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.     

The PagN protein mediates invasion via interaction with proteoglycan

Streptomyces linear plasmids start replication at centrally located loci, usually consisting of iterons and adjacent rep genes. Here, we identified four new replication loci from Streptomyces linear plasmids. A discontinuous locus, consisting of two genes and iterons separated by two nonessential genes, was required for replication of pRL2 in both linear and circular modes. A temperature-sensitive plasmid, pRL4, contained a replication locus, a noncoding sequence and a SAP1.35 -like gene. A telomere-adjacent locus, another noncoding sequence and SAP1.1 -like gene, was identified for replication of the large plasmid pFRL2. The replication locus of pSHK1 consisted of SCP1- rep -like genes and iterons. These results indicate an unexpected variety of components, positions and combinations of replication loci among Streptomyces linear plasmids.     

Yersinia enterocolitica 03 findings on porcine tongues in comparison with yersiniosis incidence in man in Czechoslovakia

Positive isolations of Yersinia obtained in repeated bacteriological examinations of porcine tongues at three slaughter-houses in Prague and a single examination at the slaughter-house at Kladno were compared with notified yersiniosis morbidity. The incidence of illnesses caused by Y. enterocolitica 03 does not exceed values of 4.5/100,000 and 3.5/100,000 population in the Czech and Slovak Socialist Republics, respectively, and is equal to a sixtieth part of the notified shigellosis and salmonellosis morbidity. Cultivation of 334 pooled samples consisting of 1142 porcine tongues yielded 12 strains (1.05%) of Y. enterocolitica 03, five strains (0.44%) of Y. pseudotuberculosis and 55 strains (4.82%) of other Yersinia organisms (indole-positive serotypes). Because of the low isolation rates obtained for the individual Yersinia species, Y. enterocolitica 03 in particular, the isolation efficiency of different cultivation techniques and culture media was statistically evaluated for all Yersinia organisms jointly. Primary cultivation on deoxycholate-citrate medium yielded five of the 12 Y. enterocolitica 03 strains isolated. The other Yersinia strains grew only after preliminary propagation. Yersinia pseudotuberculosis grew almost exclusively (4 out of 5 strains) on McConkey's agar.