Phosphorylation of the mouse hepatitis virus nucleocapsid protein

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.     

Comparative analysis of RNA genomes of mouse hepatitis viruses.

The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.     

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.     

Synthetic gene for the hepatitis C virus nucleocapsid protein.

A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E. coli. To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides. The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase. A special mechanism was designed to exchange the templates during the polymerase reaction. The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide. When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer. The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR. The data verify that this method is efficient. The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component. The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence. The synthetic gene expressed in E. coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.     

Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

Differentiation of lactococci by rRNA gene restriction analysis

Strains of the subspecies of Lactococcus lactis could be differentiated by rRNA gene restriction fragment length polymorphisms (RFLP). 16S rRNA-specific oligonucleotide as well as polynucleotide DNA probes were used for the detection of restriction fragments. In addition, a site-specific probe was designed for the intergenic spacer region of 23S and 5S rRNA genes. For all lactococcal strains the putative presence of six rRNA operons was confirmed. A non-radioactive hybridization assay was used based on hybrid detection by chemiluminescence. Specific patterns were found for any of the strains investigated. Subspecies-specific restriction fragments could be identified in addition to the strain-specific patterns.     

An evaluation of a water purification system for use in animal facilities

A commercially available water purification system was evaluated for its ability to minimize chemical and microbial contaminants. The reduction or removal of these impurities from the drinking water of experimental animals would reduce experimental variability. 3 strains of bacteria were collected from the processed water. An increase in the total number of bacteria was observed the longer the filters remained in use. Determinations of heavy metals in water samples before and after processing were made for lead, zinc, copper, nickel, manganese, iron, arsenic and mercury. Calcium and magnesium levels were also determined. The concentrations of these inorganic chemicals were reduced by the purification process except at 2 time points in which desorption of the chemical could have occurred. Bacterial colonization and desorption of these chemicals were controlled by installing new filter cartridges. Volatile halocarbon concentrations were determined for water samples before and after purification. All volatile halocarbons analyzed were less than 10 ppb before and after purification at all time points. Other organic chemicals were greatly reduced by the purification process. In a study of contaminants associated with installation of the unit, it was found that flushing the unit for 8 days reduced lead and methyl ethyl ketone concentrations to insignificant levels. The purification system was found to be effective in providing high quality drinking water as verified by a microbial and chemical testing program.     

Sequence analysis of the nucleocapsid gene of feline coronaviruses circulating in Italy

Molecular analysis of the N genes of feline coronaviruses (FCoV) strains detected in naturally infected cats were carried out to investigate the genetic diversity among these viruses. Phylogeny showed a general clustering trend on the basis of geographic origin rather than on virulence characteristics. The analysis of the pattern of nucleotide substitutions disclosed "hot spots" sites which may represent immunological domains. In conclusion, our results demonstrate that the N gene does not carry mutations associated with the pathotypical switch FECV --> FIPV. During persistent infection, the individual qualitative immune response might address the accumulations of mutations in the N gene and the development of FIP.     

Phylogenetic analysis of envelope glycoprotein (E1) gene of rubella viruses prevalent in Japan in 2004

We performed a molecular epidemiological study on the envelope glycoprotein gene (E1 gene) obtained by PCR amplification from specimens of 17 rubella patients in certain areas (Gunma, Saitama, and Kagoshima prefectures, and Tokyo metropolitan area) in Japan in 2004. In these sequences of partially amplified DNAs (283 bases) within the E1 gene, no nucleotide substitution was observed. They were classified into genotype 1D of clade 1 in the constructed phylogenetic tree. One amino acid substitution was found between the amino acid sequence predicted from these DNAs and those of Japanese strains [To-336 vaccine strain (To-336 vac) and its wild progenitor (To-336 wt)]. The results suggest that the rubella viruses (RV) prevalent in certain areas of Japan in 2004 were highly homologous and were closely related with Japanese vaccine strain.     

Identification by nucleotide sequence analysis of a goat pseudoglobin gene

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.     

Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles

SARS-CoV is a newly identified coronavirus that causes severe acute respiratory syndrome (SARS). Currently, there is no effective method available for prophylaxis and treatment of SARS-CoV infections. In the present study, the influence of small interfering RNA (siRNA) on SARS-CoV nucleocapsid (N) protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein (EGFP) fusion protein were observed. A candidate siRNA was proved to down-regulate N and EGFP expression actively in a sequence-specific manner. The expression vector of this siRNA was constructed and confirmed to reduce N and EGFP expression efficiently in both cultured cells and adult mouse muscles. Our findings suggest that the siRNA should provide the basis for prophylaxis and therapy of SARS-CoV infection in human.