DEMONSTRATION OF DIFFERENT MODES OF CELL DEATH UPON HERPES SIMPLEX VIRUS 1 INFECTION IN DIFFERENT TYPES OF ORAL CELLS
C.-R. HUANG, S.-S. LIN, M.-Y. CHOU, C.-C. HO, L.WANG, Y.-L. LEE, C.-S. CHEN, C.-C. YANG
1 Institute of Immunology,
2 Department of Dentistry, Chung Shan Medical University Hospital,
3 School of Dentistry,
4 School of Medical Technology,
5 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China;
6 Department of Pharmacy, Kuang Tien General Hospital, Taichung, Taiwan, Republic of China
Received June 21, 2004; accepted January 3, 2005
Summary. – The effects of Herpes simplex virus 1 (HSV-1) infection on five different types of oral cancerous cells (neck metastasis of gingival carcinoma (GNM) cells and tongue squamous cells of carcinoma (TSCCa) and non-cancerous cells (buccal mucosal fibroblasts (BF), gingival fibroblasts (GF), oral submucosal fibrosis cells (OSF)) and one type of non-oral cancerous cells (KB cells) were investigated. In HSV-1-infected cells the cell viability, CPE, viral antigens accumulation, caspase-3 activity, annexin V binding and DNA fragmentation were estimated. Three different forms or pathways of cell death were considered: apoptosis (the presence or rise of caspase-3 activity, DNA fragmentation and annexin V binding), slow cell death (the presence or rise of DNA fragmentation, the absence or decline of caspase-3 activity and annexin V binding), and necrosis (the absence of decline of caspase-3 activity, DNA fragmentation and annexin V binding). The viability of all cell types, except for KB cells, was reduced by the infection. CPE and viral antigens data demonstrated that all six types of cells could be infected with HSV-1. Upon HSV-1 infection there occurred (i) a classical apoptosis in GF cells, (ii) apoptosis in the early phase of infection and necrosis in the late phase of infection in GNM and TSCCa cells, (iii) slow cell death followed by necrosis in BF and OSF cells (however, these cells showed a different type of CPE), (iv) a classical slow cell death in KB cells. It is hypothesized that HSV-1 infection has a potential to induce several distinct pathways leading to cell death or several forms of cell death. Moreover, more than one pathway may be involved in the death of particular cell type. As HSV-1 was demonstrated to infect different oral and non-oral cells and cause different pathways or forms of cell death, the safety of using HSV-1 as a vector for gene therapy should be re-considered.
Key words: apoptosis; phosphatidylserine; DNA fragmentation; necrosis; slow cell death
Acta virologica 49: 7 – 15, 2005
EFFICIENT INTRANASAL IMMUNIZATION OF NEWBORN MICE
WITH RECOMBINANT ADENOVIRUS EXPRESSING ROTAVIRUS
PROTEIN VP4 AGAINST ORAL ROTAVIRUS INFECTION
X. LIU1, T. YANG2, Q.-M. SUN1, M.-S. SUN1*
1 Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming 650118, Yunnan Province, P.R. China;
2 Department of Phytochemistry, Institute of Botany, Chinese Science Academy, Kunming, Yunnan Province, P.R. China
Received February 2, 2004; accepted February 16, 2005
Summary. – Efficacy of passive protection of newborn mice against rotavirus infection by the rotavirus VP4 protein expressed by an adenoviral vector in mice was studied. The VP4 gene was inserted into the E1 region of adenoviral vector pJM17. Recombinant adenovirus Ad5/VP4 was grown in 293 cells. Intramuscular (i.m.), oral or intranasal (i.n.) immunization of newborn mice with Ad5/VP4 resulted in appearance of VP4- specific antibodies. Specific IgG antibodies were detected in the serum and intestine specimens of i.m. vaccinated mice. Oral immunization elicited serum IgG antibodies and intestinal IgG and IgA antibodies. Compared with i.m. and oral applications, i.n. immunization led to higher levels of serum IgG and intestinal IgG and IgA antibodies. Pups were challenged twice with simian rotavirus SA11 strain orally at the days 7 and 8 after birth. Pups born to i.n. immunized dams achieved 100% protection from rotavirus-induced diarrhea after both challenges. The protection of pups born to orally immunized dams was 80%, while only 30% of pups born to i.m. immunized dams were protected after both challenges. I.n. immunization was most efficient in inducing rotavirus VP4-specific serum, intestinal and milk IgG or IgA in mice that protected newborn mice completely.
Key words: rotavirus; antibodies; adenoviral vector; diarrhea; immunization; mice; protection; VP4 protein
Acta virologica 49: 17 – 22, 2005
GROWTH OF CYANOPHAGE N-1 UNDER THE INFLUENCE OF HEAVY
METAL IONS
T.A. SARMA*, SUKHRISH P. KAUR
Department of Botany, Punjabi University, Patiala - 147 002, India
Received January 7, 2004; accepted February 15, 2005
Summary. – The growth of cyanophage N-1 in the cyanobacterium Nostoc muscorum under the influence
of heavy metal ions, namely Co2+, Cr6+, Cu2+, Mn2+ and Ni2+ has been studied. One-step growth experiments
revealed that heavy metal ions extended the latent period by 1–2 hrs with a concomitant decrease in the phage
burst size. The latter was reduced in the order Cu2+/Mn2+, Ni2+, Co2+ and Cr6+. The treatment of the phageinfected
bacteria with heavy metal ions did not induce mutations affecting either the phage plaque morphology
or burst size. The final phage titer after such a treatments was lowest with Co2+, Cu2+ and Cr6+. The inhibition
of the phage growth under the influence of heavy metal ions is discussed in context with the interaction of
cyanophage N-1 with the photosynthetic reactions in the host bacteria.
Key words: cyanophage N-1; heavy metal ions; growth; Nostoc muscorum; photosynthesis
Acta virologica 49: 23 – 28, 2005
LONG-TERM ANALYSIS OF THE RESISTANCE DEVELOPMENT IN
HIV-1 POSITIVE PATIENTS TREATED WITH PROTEASE AND REVERSE TRANSCRIPTASE INHIBITORS: CORRELATION OF THE GENOTYPE AND DISEASE PROGRESSION
J. VÁCLAVÍKOVÁ1,4, J. WEBER1, L. MACHALA2, M. REINIŠ3, M. LINKA3, M. BRÙÈKOVÁ3, J. VANDASOVÁ3, M. STAÒKOVÁ2, J. KONVALINKA1,4,*
1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague, Czech Republic;
2 AIDS Centre at the Clinic of Infectious Diseases, Prague, Czech Republic;
3 National Institutes of Public Health, National Reference Laboratory for AIDS, Prague, Czech Republic;
4 Department of Biochemistry, School of Science, Charles University, Hlavova 2030, 128 43 Prague, Czech Republic
Received May 18, 2004; accepted February 16, 2005
Summary. – In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART)
in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence
common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse
transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease
in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of
specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged
therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and,
consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations
in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet
temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.
Key words: drug resistance; HAART; HIV-1; inhibitors; protease; reverse transcriptase; sequence variability
Acta virologica 49: 29 – 36, 2005
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COMPARISON OF THREE TECHNIQUES FOR DETECTION OF
GRAPEVINE LEAFROLL-ASSOCIATED VIRUS 1
P. KOMÍNEK*, M. BRYXIOVÁ
Research Institute of Crop Production, Drnovská 507, 161 06 Prague 6, Czech Republic
Received July 15, 2004; accepted February 14, 2005
Summary. – Thirty seven plants of grapevine from the Research Station of Viticulture, Karlštejn was
examined for the presence of leafroll viruses. Grapevine leafroll-associated virus 1 (GLRaV-1) was detected in
the grapevines plants tested using double-antibody sandwich ELISA (DAS-ELISA), RT-PCR and molecular
hybridization with non-radioactive RNA probes. Both molecular methods were based on a detection of the
GLRaV-1 heat-shock protein 70 (HSP70) gene and showed a higher sensitivity in the detection of GLRaV-1
compared to DAS-ELISA. RNA probes are considered more suitable for the GLRaV-1 detection, as their
application can overcome potential minor sequence variability, which may cause the detection by RT-PCR less
reliable, especially when the variability occurs in the genome region targeted by RT-PCR primers. Based on
additional DAS-ELISA, a mixed infection of GLRaV-1 and Grapevine leafroll-associated virus 3 (GLRaV-3)
occurred frequently, while a mixed infection of GLRaV-1 and Grapevine virus A (GVA) or Grapevine fleck
virus (GFkV) or a multiple infection of GLRaV-1, GLRaV-3 and GFkV occurred rarely in the tested plants.
A mixed infection of all the four viruses mentioned above was not observed.
Key words: Grapevine leafroll-associated virus 1; ampelovirus; HSP 70 gene; RT-PCR; non-radioactive
probe; DAS-ELISA
Acta virologica 49: 37 – 43, 2005
HERPES SIMPLEX VIRUS 1 INHIBITS APOPTOSIS THROUGH A CASPASE-3-DEPENDENT PATHWAY IN PRIMARY CULTURES OF CORTICAL NEURONAL CELLS OF FETAL MICE
J.W. WANG, D.X. WANG*, R.J. WANG, W.R. LI, H.Z. TUO, Z.J. FENG
Department of Neurology, Beijing Friendship Hospital-Affiliate of Capital University of Medical Sciences, Beijing 100050,
P.R. China
Received March 1, 2004; accepted February 17, 2005
Summary. – We could induce apoptosis in primary cultures of cortical neurons of fetal mice with ceramide
or sorbitol. The induction was accompanied by an increase in caspase-3 (CAS-3) activity and depolarization of
the inner mitochondrial membrane of neuronal cells which both could be reversed by Herpes simplex virus 1
(HSV-1) infection. We conclude tha HSV-1 infection inhibited the apoptosis, induced in neuronal cells by
sorbitol or ceramide, via a CAS-dependent pathway.
Key words: apoptosis; caspase-3; Herpes simplex virus 1; cortical neurons
Acta virologica 49: 45 – 49, 2005
VARIABILITY OF G1 GENE OF HANTAVIRUSES OCCURRING IN THE HUBEI PROVINCE, P.R. CHINA FROM 1985 TO 2000
Q. LI, Z.Q. YANG*, H. QU, H. XIAO
National Key Laboratory of Virology, Institute of Virology, Wuhan University School of Medicine, 115, Dong-Hu Road, Wuhan, Hubei 430071, P.R. China
Received July 13, 2004; accepted February15, 2005
Summary. – AWe studied variability of G1 gene of hantaviruses occurring in the Hubei province, P.R.
China. Serum samples were collected from 229 patients with hemorrhagic fever with renal syndromes (HFRS)
during 1985–1989 and 1996–2000 and were tested by RT-PCR for the presence of Hantaan and Seoul viruses
(HTNVs, SEOVs) and by restriction fragment length polymorphism (RFLP) analysis for the respective pattern.
Out of 229 sera 166 (72.5%) were hantavirus-positive by RT-PCR, including 124 from 1985–1989 and 42 from
1996–2000, with HTNVs in majority (80.1%) and SEOVs in minority (19.9%). By RFLP analysis, four types
of RFLP pattern were recognized. In the 133 HTNV isolates the A pattern was most predominant (62.5%),
while the remaining patterns B, C, and D were present in minority. This kind of the RFLP pattern distribution
was observed regardless the year of virus isolation. In contrast, only one type of RFLP pattern was obtained
from 33 SEOVs, but this was different from that of R22 virus. Our results indicate that temporal factor, represented
by years 1985–2000 seems to be too short to affect markedly the genetic makeup of the hantaviruses investigated.
Key words: epidemiology; evolution; genetic variability; hantaviruses; Hantaan virus; Seoul virus;
RT-PCR; RFLP
Acta virologica 49: 51 – 57, 2005
MOLECULAR CHARACTERIZATION OF INFECTIOUS BURSAL
DISEASE VIRUS ISOLATES FROM NEPAL BASED ON
HYPERVARIABLE REGION OF VP2 GENE
K. SHARMA, M. HAIR-BEJO*, A.R. OMAR, I. AINI
Faculty of Veterinary Medicine, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
Received June 7, 2004; accepted February 16, 2005
Summary. – Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from
a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene
(1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were
compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison
revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast,
classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed
a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other
and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino
acids substitutions at positions 300 (E?A), 308 (I?F) and 334 (A?P) within the HV region were common for
both the isolates. The amino acids substitutions at positions 27 (S?T), 28 (I?T), 31 (D?A), 36 (H?Y), 135
(E?G), 223 (G?S), 225 (V?I), 351 (L?I), 352 (V?E) and 399 (I?S) for NP1SSH and at position 438 (I?S)
for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity
(97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude
that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.
Key words: Infectious bursal disease virus; VP2 gene; nucleotides sequence; deduced amino acids sequence;
phylogenetic analysis; Nepal
Acta virologica 49: 59 – 64, 2005
NUCLEOTIDE SEQUENCE ANALYSIS OF THE L1 LOOP VARIABLE
REGION OF HEXON GENE OF FOWL ADENOVIRUS 4 ISOLATES
FROM INDIA
M. PARTHIBAN, S. MANOHARAN, P. ROY, N.D.J. CHANDRAN, A.W. ARUNI, A. KOTEESWARAN
Vaccine Research Centre-Viral Vaccines, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 051, India
Received May 4, 2004; accepted February 16, 2005
Summary. – Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, India were analyzed. The L1 loop variable region of hexon gene of these isolates was amplified by PCR and sequenced. The nucleotide sequences (442 bp) and deduced amino acid sequences of the four isolates were compared with those of other isolates of FAV4. The nucleotide sequences of the four isolates had a 98% homology with other Indian isolates and a 96% homology with Belgian and Russian isolates. The amino acid sequences of the four Indian isolates had a more than 98% homology with other Indian isolates and a more than 92% homology with Belgian and Russian isolates. Hence, the variable of L1 loop region of hexon gene was found to be highly homologous in all the FAV4 isolates tested both at nucleotide and amino acid level.
Key words: Fowl adenovirus 4; hexon gene; hydropericardium syndrome; Indian isolates; L1 loop; PCR;
nucleotide sequencing; phylogenetic analysis
Acta virologica 49: 65 – 68, 2005