Cloning of the genome of cricket paralysis virus: sequence of the 3′ end

DNA complementary to the single-stranded RNA genome of the insect picornavirus, cricket paralysis virus (CrPV), was cloned into the plasmid pBR322 by a hybrid RNA-cDNA cloning strategy. Positive CrPV-specific clones were selected by colony hybridization and characterised by restriction enzyme mapping. Overlapping clones spanning 7.5 kb of the estimated 8.5 kb genome were obtained, the largest being 7.0 kb. Comparison of the restriction enzyme map with those of mammalian picornaviruses revealed no conserved pattern of cleavage sites. The CrPV-cDNA was sequenced using the M13-dideoxy chain-terminating method although the chemical method of Maxam and Gilbert was employed to complete gaps in the sequence. The sequence of the 3′-terminal 1600 nucleotides is presented and is compared with those of mammalian picornaviruses. Computer comparisons of the CrPV sequence and those of mammalian picornaviruses revealed no significant homology between either the nucleotide or the predicted amino acid sequence of this region.     

On the variability of the 3′ terminal sequence of the turnip mosaic virus genome

Summary The sequence of the 3-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212 nts upstream of the 3 poly(A)-tract. Determination of the N-terminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the 3-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212 nts) than the 3-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3-NTR of the TuMV-Chi isolate which also measures 212 nts. The 3-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3-NTR of TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp 12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3-NTR of approximately 212 nucleotides in length.     

Mapping of the functional domains of the α 4 protein of herpes simplex virus 1

Summary Truncated 4 genes were introduced into BHK tk^– cells along with the neomycin phosphotransferase gene, that confers resistance to the eukaryotic antibiotic G 418, driven by the HSV-1 tk promoter ( tk^–neo^r). Stably transformed cell lines were obtained and studied for the ability of the resident truncated 4 genes to regulate the expression of the tk^–neo^r, and for the ability of the truncated 4 polypeptides to localize to the nuclei of transformed cells. The results indicated that the domain(s) for gene induction and for nuclear localization of the 4 protein are located within the N-terminal 288 amino acids of the protein.     

Effects of the polysaccharides from Spirulina platensis on the activities of hepatitis B virus

It has been well known that hepatitis B virus(HBV) infection can result in acute or chronichepatitis as well as hepatocirrhosis and/or hepato-carcinomainsome cases ,especiallyforthe chron-ic patients [1] . Few drugs can efficiently curechronic hepatitis B (CHB) . Up to now,only in-terferon-αandlamivudine (3TC) were certified byFDAfortreatment CHB.Someinvestigates showedthat the percentage of response to IFN-αwas nomore than 30 %after one year of treatment . Al-though copies of HBV …     

Analysis of the genetic and the corresponding antigenic variability of the VP1 3′ end of ECHO virus type 11 and ECHO virus type 30

The enteroviruses (EV), RNA viruses belonging to the Picornaviridae family, have a high genetic variability due to the absence of the efficient proofreading and post replicative repair activities associated with the RNA polymerase. In the present work, we studied the genetic and the antigenic variability of ECHO virus types 11 (E11) and 30 (E30), which are the most isolated echoviruses serotypes in clinical and environmental samples. We established on the 3′ end of the VP1 gene, consensus sequences of E11 and E30 by alignment of 67 E11 and 247 E30 published sequences in GenBank. Our results of sequences comparison showed that the majority of the mutational sites are situated on the third nucleotide of the codon. These mutations were without consequence on the antigenic sequences of the VP1 protein. Thus, E11 and E30 have a high genetic variability (1/3 of the nucleotides are variable), but a relative antigenic conservation. The analysis of the intertypic antigenic variability between E11 and E30 was obtained by the alignment of the corresponding amino acids sequences relative to the N-terminal part of the VP1 protein. Two discriminating parts were highlighted, probably representing antigenic sites for neutralisation antibodies.     

Fabrication of oligonucleotide microarray for the detection of Japanese encephalitis virus

A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application.     

A broader definition of ‘the virus species’

Summary. We propose that the formal definition of a virus species by the International Committee on Taxonomy of Viruses (ICTV) should be broadened by removing the restrictive word “polythetic” from the current definition, so that any characters can be used to define species. This change will bring the definition of virus species into line with the species definitions of cellular organisms and broaden the range of characters available for describing virus species.     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

On the antigenic variation of the influenza A virus“*”

Summary A sensitive micro antibody absorption test has been developed for use in studies on the antigenic structure of the influenza virus. The antigenic structure of the A-prime strains isolated in Hungary in the period 1949–54, as determined in tests against strain-specific sera, suggested that these strains originated or developed from the strains which had been isolated in Hungary at earlier dates.The experimental evidence obtained permits one to draw the conclusion that the variation of the antigen of the influenza A virus takes place not by a rearrangement of already present antigenic parts, but by an appearance of new and disappearance of old antigens. Changes in the viral antigen were demonstrable also within a single epidemic.The strains of influenza A virus can be classified by the use of strainspecific sera prepared with due regard to the origin of antigens within single strains.     

Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.     

Utilization of the embryonated egg for in vivo evaluation of the anti-influenza virus activity of neuraminidase inhibitors

Previous studies have shown that embryonated egg provides a convenient and easy to use system for in vivo screening of anti-influenza virus inhibitors. However, it is not known whether this model is suitable for testing neuraminidase (NA) inhibitors, too. Therefore, the present study describes the evaluation of the ion-channel blockers amantadine and rimantadine in comparison with the NA inhibitors oseltamivir and zanamivir by using the influenza A virus hen’s egg model. The treatment was started immediately before or after the challenge dose was placed on the chorioallantoic membrane (CAM). Differences between the survival rate of treated and untreated chick embryos infected with influenza A virus were analyzed statistically. As result, the survival rate of chick embryos could be significantly increased when the treatment with amantadine, rimantadine, oseltamivir, or zanamivir was started before the CAM was inoculated with one egg infective dose 50% (EID₅₀) influenza A virus. When the drugs were administered shortly after viral inoculation, significant antiviral efficacy was shown for rimantadine, oseltamivir, and zanamivir. Antiviral efficacy could be demonstrated exclusively for both oseltamivir and zanamivir after the embryos were infected with higher challenge doses of 10² EID₅₀influenza A virus. In conclusion, the NA inhibitors oseltamivir and zanamivir have a significantly better antiviral activity against influenza A virus than amantadine and rimantadine tested in embryonated hen’s eggs. Therefore, this model can be a valuable alternative approach for in vivo pre-testing anti-influenza virus activity of NA inhibitors.     

Identification and characterization of the short variable region of the Japanese encephalitis virus 3′ NTR

Since the 1980s, the Japanese encephalitis virus (JEV) variants with slightly short variable regions (VR) of the 3′ non-translated region (NTR) have been found; however, the implications of these short VR remain unclear. We recently identified two novel types of short VR (5 and 9 nt shorter than that of major group of genotype I JEV strains) of genotype I JEV isolates. To elucidate the impact of these short VR on the replication and virulence of JEV, we generated five recombinant JEV viruses: M41-d5 and M41-d9 have deletions in the VR that correspond to those observed in some recent JEV isolates, M41-d5d9 has both the 5- and 9-nt deletions in the VR, M41-d27 has a large deletion that encompasses both the 5- and 9-nt deletion regions, and M41-a13 has a 13-nt sequence insertion of the genotype III JEV strain Beijing-1 into the parent genotype I JEV strain Mie/41/2002 genome. The recombinant viruses and the parent virus, except for the M41-d27 mutant, showed similar growth properties in mammalian and mosquito cell lines. Mouse challenge experiments indicated that no significant differences among the recombinant viruses M41-d5d9, M41-d27, M41-a13, and the parent virus. Our results suggest that the short VR in JEV 3′ NTR do not affect its growth in vitro or its pathogenicity in mice.     

Classic paper: Are the chickenpox virus and the zoster virus identical?

As early as 1943, the German physician Helmut Ruska visualized the virus of varicella and zoster (at that time, he was not completely certain whether the virus was the same) by the newly developed electron microscope; he is regarded as the discoverer of this virus. Here, we present a translation of his classical paper into the English language. In our introduction and commentary to his paper, we discuss the significance of Helmut Ruska's work for the development of virology, his distinction between the varicella, zoster, and herpes virus group on one hand and poxviruses on the other, as well as the development of imaging techniques which have refined or substituted for electron microscopy of viruses and virus‐infected cells.     

A conserved sequence motif at the 5′ terminus of the Southampton virus genome is characteristic of the Caliciviridae

We have determined the 5 terminal cDNA sequence for the genome of Southampton virus, a recently characterized, human, small round-structured virus (SRSV). Genomic RNA was extracted directly from a stool sample and amplified by RT-PCR by homopolymer tailing of the 3 terminus of the cDNA. The additional sequence increases the overall length of the Southampton virus genome by 12 nucleotides, resulting in a significant change to the genome organization by extending the first large open reading frame (ORF) by 51 amino acids. The 5 terminal bases pGpT and the presence of conserved genome and putative subgenomic RNA terminal motifs are now prominent features shared between the human SRSV Southampton virus and the animal caliciviruses rabbit hemorrhagic disease virus and feline calicivirus.     

What is the role of serology for the study of chronic hepatitis B virus infection in the age of molecular biology?

To assess quantitative serology in chronic hepatitis B virus (HBV) infection, testing by novel immunoassays has been carried out on 202 specimens from untreated patients and in 83 samples from 10 patients with chronic hepatitis B treated with lamivudine. Serum samples were assayed for quantitative HBsAg, in comparison with quantitative HBV-DNA, and for anti-HBc IgM and the avidity index (AI) of total anti-HBc antibodies. The AI was high (mean: 0.93 +/- 0.19) in all groups, confirming the consistency of this procedure in chronic HBV infections. A low-level positivity (2-28 Paul-Ehrlich units/ml) for IgM anti-HBc was detectable both in HBeAg-positive and in HBeAg-negative untreated chronic hepatitis cases (mean S/CO values by the Abbott Architect assay: 0.51 +/- 0.12 and 0.48 +/- 0.10, respectively; correlation between assays: r = 0.685), while treated patients (mean: 0.20 +/- 0.15) and inactive carriers (mean: 0.17 +/- 0.21), were generally negative for IgM. The levels of HBsAg (IU/ml) showed a weak correlation with HBV-DNA (IU/ml). A difference in HBsAg levels was found between inactive carriers (1,935 +/- 2,887 IU/ml) and chronic hepatitis B cases, either treated (5,199 +/- 9,259 IU/ml) or untreated (14,596 +/- 15,227 IU/ml). Pre-treatment levels of HBsAg in patients undergoing lamivudine treatment were correlated with a sustained response to therapy over 13-33 months (mean: 27.3) of follow-up: mean HBsAg values were 1,576 + 1,487 IU/ml in five responders and 6,063 + 5,142 in five nonresponders or breakthrough responders (P < 0.05). The availability of standardized quantitative immunoassays for HBsAg and anti-HBc IgM may be considered in addition to quantitative HBV-DNA in the staging and monitoring of chronic HBV infection.     

Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).