Electronic Library of Scientific Literature - © Academic Electronic Press

Acta virologica

Volume 45 / 2001 / number 5-6

• EARLY SHUTOFF OF HOST PROTEIN SYNTHESIS IN CELLS INFECTED WITH HERPES SIMPLEX VIRUSES
• HUMAN IMMUNODEFICIENCY VIRUS 1 STRAINS RESISTANT TO NUCLEOSIDE INHIBITORS OF REVERSE TRANSCRIPTASE IN ISOLATES FROM THE CZECH REPUBLIC AS MONITORED BY LINE PROBE ASSAY AND NUCLEOTIDE SEQUENCING
• SIGNIFICANCE OF ANTI-INTERFERON-ALPHA2 AND SICAM-1 ACTIVITIES IN THE SERA OF VIRAL HEPATITIS B AND C PATIENTS TREATED WITH HUMAN RECOMBINANT INTERFERON- ALPHA2
• EFFICACY OF ALPHA-INTERFERON THERAPY OF CHRONIC HEPATITIS PATIENTS INFECTED WITH WILD TYPE HEPATITIS B VIRUS AND HBEAG-MINUS MUTANT
• EVALUATION OF A DIPSTICK ELISA AND A RAPID IMMUNOCHROMATOGRAPHIC TEST FOR DIAGNOSIS OF DENGUE VIRUS INFECTION
• ISOLATION OF CHIKUNGUNYA VIRUS FROM AEDES AEGYPTI MOSQUITOES COLLECTED IN THE TOWN OF YAWAT, PUNE DISTRICT, MAHARASHTRA STATE, INDIA
• THE UL9 ORI-BINDING PROTEIN OF HERPES SIMPLEX VIRUS 1: ITS EXPRESSION AND LOCALIZATION IN VERO CELLS
• CLONING AND SEQUENCING OF THE COAT PROTEIN GENE OF TOBACCO RINGSPOT VIRUS ISOLATES FROM UK AND IRAN
• CHARACTERIZATION OF SRI LANKA RABIES VIRUS ISOLATES USING NUCLEOTIDE SEQUENCE ANALYSIS OF NUCLEOPROTEIN GENE
• IDENTIFICATION OF RICE BLACK STREAKED DWARF VIRUS IN DIFFERENT CEREAL CROPS WITH DWARFING SYMPTOMS IN CHINA
• Contents and indices of volume 45

EARLY SHUTOFF OF HOST PROTEIN SYNTHESIS IN CELLS INFECTED WITH HERPES SIMPLEX VIRUSES

Summary. – Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.

Key words: HSV-1; HSV-2; host protein synthesis; early shutoff; vhs gene; vhs protein
Acta virologica 45: 269 – 277, 2001

HUMAN IMMUNODEFICIENCY VIRUS 1 STRAINS RESISTANT TO NUCLEOSIDE INHIBITORS OF REVERSE TRANSCRIPTASE IN ISOLATES FROM THE CZECH REPUBLIC AS MONITORED BY LINE PROBE ASSAY AND NUCLEOTIDE SEQUENCING

Summary. – The genetic resistance to nucleoside inhibitors of the reverse transcriptase (RT) of human immunodeficiency virus 1 (HIV-1) isolates in the Czech Republic was examined by a line probe assay (LiPA) and nucleotide sequencing. The results of LiPA analysis of 294 blood specimens obtained from 156 patients revealed a high incidence of mutations in the RT gene related to resistance to various drugs (67.3%) in various combinations. Mutations in RT gene (M41L, K70R and T215Y/F) conferring the resistance to zidovudine (ZDV) were most frequent (62.6%), that (M184V) responsible for the resistance to lamivudine (3TC) was less frequent (33.7%), while those linked to the resistance to dideoxyinosine (ddI) and dideoxyinosine together with dideoxycytidine (ddI/ddC) were rather rare (6.5% and 5.1%, respectively). LiPA gave a high rate of uninterpretable results due to codon hybridization failure, especially in HIV-1 isolates of non-B subtype. Thirty-two specimens were analyzed also by direct sequencing of a part of RT gene. The results obtained by LiPA and the sequencing were highly concordant for codons successfully analyzed by both methods, but the sequencing provided information also about the codons that could not be analyzed by LiPA. A high prevalence of resistant strains in the Czech Republic and their heterogeneity justifies a regular HIV-1 resistance testing. LiPA turned out as a fast, powerful and most reliable tool for such a purpose. However, due to an increasing diversity of HIV-1 strains circulating in the Czech Republic, LiPA cannot replace the nucleotide sequence analysis.

Key words: human immunodeficiency virus 1; reverse transcriptase; antiviral drugs; resistant mutants; line probe assay; nucleotide sequencing
Acta virologica 45: 279 – 286, 2001

SIGNIFICANCE OF ANTI-INTERFERON-ALPHA2 AND SICAM-1 ACTIVITIES IN THE SERA OF VIRAL HEPATITIS B AND C PATIENTS TREATED WITH HUMAN RECOMBINANT INTERFERON- ALPHA2

Summary. – In this study the presence of an IFN-binding activity in the sera of patients with chronic viral hepatitis B or C treated with rIFN-alpha2 was screened by a radioimmune assay (RIA) using radiolabeled rIFN- alpha2. Incidence of an anti-IFN activity was compared with hepatitis B virus (HBV) or hepatitis C virus (HCV) serum markers as hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies to HBsAg (anti-HBsAg), antibodies to HBeAg (anti-HBeAg), seroconversion, HBV DNA, HCV RNA, and serum soluble intracellular adhesion molecule 1 (sICAM). Injections (intramuscular ) of rIFN- alpha2 caused an anti-rIFN activity formation in 8 (27.6%) of 29 patients with chronic active hepatitis B (CAH-B) and in 8 (30.8%) of 26 patients with chronic active hepatitis C (CAH-C). The presence of the anti-rIFN activity in CAH-B patients correlated frequently with the persistence of HBsAg, HBeAg and HBV-DNA, while its absence was often accompanied by the anti-HBeAg and anti-HBsAg seroconversion, respectively, and HBV-DNA negativity. In two CAH-C patients who became HCV RNA-negative no anti-IFN activity was found. Levels of serum sICAM-1 in CAH-B patients responding to the IFN treatment were higher than those in non-responders or in which the anti-IFN activity was present. The anti-IFN activity may negatively influence the effect of the IFN therapy of CAH-B or CAH-C patients at early stages of the therapy. The appearance of the anti-IFN activity at the end of a long-term IFN therapy does not seem to influence the outcome of the therapy. sICAM-1 may be involved in the process of CAH-B reactivation and IFN-triggered cytotoxicity during the IFN therapy.

Key words: interferon- alpha2; interferon therapy; anti-interferon activity; viral hepatitis B, viral hepatitis C; sICAM-1
Acta virologica 45: 287 – 292, 2001

EFFICACY OF ALPHA-INTERFERON THERAPY OF CHRONIC HEPATITIS PATIENTS INFECTED WITH WILD TYPE HEPATITIS B VIRUS AND HBEAG-MINUS MUTANT

Summary. – The aim of this study was to evaluate the efficacy of alpha-interferon (alpha-IFN) treatment of 56 chronic hepatitis B (HB) patients positive for HB e antigen (HBeAg), which were previously not treated with alpha-IFN (group A). Seven of them, which did not respond to initial alpha-IFN treatment, were subjected to additional treatment with alpha-IFN (group B). Another 7 patients with chronic HB caused apparently by an HBeAg-minus HB virus (HBV) mutant represented group C. In the alpha-IFN treatment, 5 megaunits (MU) of alpha-IFN were administered subcutaneously three times a week for six months. A trend of improvement of important markers of the disease in the treated patients could be seen with increasing time after completion of the treatment even though it was not statistically significant. In group A, the absence of serum HBV DNA was found in 43% of the patients at the end of the treatment, in 41% 6 months later, and in 46% 12 months later. At the same time intervals group A showed negative HBeAg in 36%, 39% and 46%, positive anti-HBeAg in 36%, 38%, and 46%, negative HBsAg in 9%, 11%, and 14%, and normal level of alanine transaminase (ALT) in 23%, 39%, and 44%, respectively. A trend toward better results of alpha-interferon therapy for the group A patients displaying lower baseline viremia and higher ALT activity could be seen; however, this relationship was not statistically significant. Groups B and C were too small for statistical analysis. Nevertheless, 4 of 7 patients of group B were negative for HBV DNA 12 months after the treatment and HBV DNA was eliminated during the treatment in all patients of group C; however, 3 patients relapsed after the treatment.

Key words: chronic hepatitis B; hepatitis B virus; wild type virus; HBeAg-minus mutant virus; alpha-interferon
Acta virologica 45: 293 – 297, 2001

EVALUATION OF A DIPSTICK ELISA AND A RAPID IMMUNOCHROMATOGRAPHIC TEST FOR DIAGNOSIS OF DENGUE VIRUS INFECTION

Summary. – Here we report standardization of a dipstick enzyme-linked immunosorbent assay (ELISA, Dipstick ELISA) and its comparative evaluation with a commercial Rapid PanBio Immunochromatographic test (IC test) for detection of Dengue (DEN) virus-specific IgM and IgG antibodies in patient sera. Among crude and purified viral antigens prepared from mouse brains or cell cultures, a DEN virus type 2 antigen purified from cell cultures by sucrose density gradient centrifugation was found superior in terms of the signal/noise (S/N) ratio in the assay system. The sensitivity of detection of the virus by specific IgM antibody was improved by removal of IgG from patient sera prior to testing. The evaluation of the Dipstick ELISA by use of 156 serum samples revealed an overall accordance of 96% and 93% with the IC test in detection of IgM antibodies to DEN viruses (IgM antibodies) and IgG antibodies to DEN viruses (IgG antibodies), respectively. The sensitivity of the Dipstick ELISA and the IC test with reference to the µ-capture ELISA was 83% and 87%, respectively, with a specificity of 98% in both cases. The sensitivity of the Dipstick ELISA with reference to the IC test in detecting IgM and IgG antibodies was 84% and 94%, respectively, and the specificity of the Dipstick ELISA was 98% and 92%, respectively.

Key words: Dengue; infection; virus; antigen; antibody; detection; dipstick ELISA; µ-capture ELISA; immunochromatographic test
Acta virologica 45: 299 – 304, 2001

ISOLATION OF CHIKUNGUNYA VIRUS FROM AEDES AEGYPTI MOSQUITOES COLLECTED IN THE TOWN OF YAWAT, PUNE DISTRICT, MAHARASHTRA STATE, INDIA

Summary. – Chikungunya (CHIK) virus is prevalent throughout Southeast Asia and Africa. It has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic occurring in 1971. During a recent outbreak of Dengue (DEN)-like illness in eastern India, Aedes aegypti mosquitoes collected from the affected area were positive for CHIK virus. Evidence of dual infection with CHIK and DEN type1 virus was also obtained. A widely circulating low-virulent CHIK virus is a possible explanation for the epidemiological pattern of the CHIK virus disease in this region.

Key words: Aedes aegypti; Chikungunya virus; Dengue virus
Acta virologica 45: 305 – 309, 2001

THE UL9 ORI-BINDING PROTEIN OF HERPES SIMPLEX VIRUS 1: ITS EXPRESSION AND LOCALIZATION IN VERO CELLS

Summary. – The ori-binding protein (OBP), an early protein which is encoded by the herpes simples virus 1 (HSV-1) UL9 gene and initiates the replication of viral DNA, was expressed in Escherichia coli, purified on an avidin resin and used for preparation of a mouse antiserum to OBP (OBP antiserum). Expression and localization of OBP in HSV-1-infected Vero cells was assessed by reverse transcription–polymerase chain reaction (RT-PCR) and indirect immunofluorescence test. RT-PCR revealed the presence of abundant UL9 transcripts from 3 to 12 hrs post infection (p.i). Traces of UL9 mRNA were detected already at 1.5 hr p.i. The OBP antiserum detected clumps of irregularly shaped structures in the nuclei of infected Vero cells first at 4 hrs p.i. These nuclear structures peaked at 5–6 hrs p.i. and later on (at 8–12 hrs p.i.) they changed into fine granules filling the whole nucleus.

Key words: herpes simplex virus 1; UL9 gene; ori-binding protein; OBP; expression; E. coli; fusion protein; RT-PCR, OBP antiserum; immunofluorescence
Acta virologica 45: 311 – 317, 2001

CLONING AND SEQUENCING OF THE COAT PROTEIN GENE OF TOBACCO RINGSPOT VIRUS ISOLATES FROM UK AND IRAN

Summary. – The coat protein (CP) gene and the 3' untranslated region (UTR) of genomic RNA 2 of Tobacco ringspot virus (TRSV, the genus Nepovirus, subgroup a) isolates from the UK and Iran were cloned from total viral RNA and sequenced. Comparison of these isolates with an isolate from the USA revealed a high degree of nucleotide and amino acid identity which extends the knowledge of molecular relationships between these three TRSV isolates. The UK isolate shared the highest nucleotide identity (95%) with the US isolate as compared to a lower identity with the Iranian isolate (92%). The highest identity (98%) was found between the UK and US isolates at amino acid level. Comparative analysis of the Iranian, UK and US isolates revealed some differences concerning some members of other subgroups of nepoviruses. For example, the N-terminal FDAYXR and the C-terminal FYGRXS motifs conserved among some nepoviruses, which occur adjacent to each other in folded CP molecules, were not detected in the Iranian, UK or US TSRV isolates. These isolates shared similarity only with Tomato ringspot virus (TomRSV) belonging to the subgroup c of nepoviruses. Another similarity of these isolates with TomRSV and Raspberry ringspot virus (RRSV) was the presence of a 34 nucleotide (nt) long sequence within the 3'-UTR.

Key words: Tobacco ringspot virus; coat protein; gene; nucleotide sequence; amino acid sequence
Acta virologica 45: 319 – 326, 2001

CHARACTERIZATION OF SRI LANKA RABIES VIRUS ISOLATES USING NUCLEOTIDE SEQUENCE ANALYSIS OF NUCLEOPROTEIN GENE

Summary. – Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, 1 jackal and 1 water buffalo were collected in 1995–1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription–polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

Key words: rabies virus; isolates; Sri Lanka; nucleoprotein gene; nucleotide sequence; phylogenetic analysis; RT-PCR
Acta virologica 45: 327 – 333, 2001

IDENTIFICATION OF RICE BLACK STREAKED DWARF VIRUS IN DIFFERENT CEREAL CROPS WITH DWARFING SYMPTOMS IN CHINA

Summary. – This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription–polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1–89.6% homology at nucleotide level and 92.3–92.9% and 95.8–98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0–95.4% homology at nucleotide level and 96.2–97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments of the Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.

Key words: maize rough dwarf virus; rice black streaked dwarf virus; Chinese isolates; homology; phylogenetic analysis
Acta virologica 45: 335 – 339, 2001

Electronic Library of Scientific Literature - © Academic Electronic Press