Hellenic Society of Virology

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Hellenic Virology

Volume 4, Number 2, 1999

Abstracts

Biology of enteroviruses: structure, function, pathogenesis and evolution. Part II.
N. Siafakas, A. Georgopoulou, P. Markoulatos, N. Spyrou
Dept. of Virology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece
Hellenic Virology, 1999, 4(2): 65-80.
The entrance of enteroviruses to the host cell is carried out through receptor-mediated endocytosis. A series of biochemical events will lead to the release of the genetic material to the cytoplasm, where the translation of the positive-sense genome to a large polyprotein will be facilitated. Molecular studies have shown that the synthesis of the polyprotein is effected by the binding of ribosomes to specific sequences within the 5'-UTR of the enteroviral genome. These sequences are termed IRES/Internal Ribosome Entry Site, or RLP/ Ribosome Landing Pad, and it has been suggested that key-elements of the IRES are widespread throughout much of the 5'-UTR. The oral-faecal route is the primary path of enterovirus infection, with the exeption of Coxsackie A21 wich is transmitted mostly through respiratory system secretions and the enterovirus 70, which is secreted in tears and is transmitted through tears and faeces. Following initial multiplication of enteroviruses, possibly in the lymphatic tissue of the pharynx and the intestine, viremia may be established which in its turn will lead to further multiplication of the virus in cells of the reticuloendothelial system and finally, in the target organ (spinal cord and brain, meninges, myocardium, skin). Isolation and serotypic identification of enteroviruses are extremely important for the discovery of new enterovirus types or strains, the study of the relationships between certain enterovirus serotypes and several diseases and their manifestations, the epidemiological surveillance of populations, the correct identification of wild-type polioviruses and the diagnosis of enterovirus infections in neonates and immunocompromised patients. New molecular techniques for the typing of enteroviruses are appearing in the foreground, gradually replacing the conventional methods of serological identification which rely on the use of mixtures of anti-sera that contain different combinations of serotype-specific meutralising sera. The efforts for the typing of the isolated enteroviruses have revealed the existence of genetic and biological diversity, which is due to the high error-frequency of viral RNA-polymerase that leads correspondingly to a high rate of point mutations, and to the recombination of control elements of the genetic material between homologous viruses. A third possible way of enteroviral evolutionary variability is the introduction of genetic material of cellular origin to the virus genome.
Molecular differentiation of polioviruses and coxsackie B viruses: applications in clinical diagnosis
A. Georgopoulou (1,2), N. Siafakas (1), P. Markoulatos (1), N. Spyrou (1), N. Vamvakopoulos (2)
(1) Dept. of Virology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece (2) Dept. of Virology & Genetics, University of Thessaly Medical School, Papakyriazi 22, 41222 Larissa, Greece
Hellenic Virology, 1999, 4(2): 81-93.
Reference Polio and Coxsackie B enteroviruses have been genotyped by reverse transcriptase polymerase chain reaction amplification using a common enteroviral consensus primer pair designated UC53/UG52, followed by single restriction endonuclease digestion of the generated amplicons with HaeIII, DdeI and NcoI and electrophoretic analysis of the resulting products (RT-PCR/RFLP). Coxsackie B2, Coxsackie B6 and wild type poliovirus 2 (strain MEF) reference strains were genotyped according to their RT-PCR/DdeI pattern alone. Sabin 3 reference strain was genotyped according to its RT-PCR/HaeIII, or NcoI pattern alone and reference strain Coxsackie B1 according to its RT-PCR/NcoI pattern alone. Further comparison of the RT-PCR/RFLP patterns between all three enzymes differentiated ten of the twelve reference strains examined, with the exception of vaccine poliovirus 1 (Sabin 1) and wild type poliovirus 1 (Mahoney). A Table-based display method was introduced to facilitate and simplify assignment of unknown clinical samples. This sequential approach, managed the accurate genotyping of all ten clinical specimens examined, as their Table-based assignments matched the assignments made by electrophoretic analysis of their RT-PCR/RFLP patterns relative to a selected set of similarly processed candidate reference strains, also identified from the Tables. Our findings support the use of the presently introduced, non-isotopic molecular genotyping method for rapid, sensitive and accurate diagnosis of single Polio and Coxsackie B enteroviral infections.
The use of PCR for the earliest sheep poxvirus detection in cell cultures inoculated with clinical specimen material
O. Mangana-Vougiouka (1), P. Markoulatos (2), G. Koptopoulos (3), K. Nomikou (1), N. Bakandritsos (4), O. Papadopoulos (3)
(1) Dept. of Virology, Institute of Infectious & Parasitic Diseases, Center of Athens Veterinary Institutions, 25 Neapoleos St., 15310 Ag. Paraskevi, Greece (2) Dept. of Virology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece (3) Dept. of Microbiology & Infectious Diseases, Veterinary Faculty, Aristotelian University, 54006 Thessaloniki, Greece and (4) National Agriculture Research Foundation, Institute of Veterinary Research of Athens, 25 Neapoleos St., 15310 Ag. Paraskevi, Greece
Hellenic Virology, 1999, 4(2): 94-99.
The polymerase chain reaction (PCR) was applied for the earliest possible sheeppox virus detection and identification in cell cultures inoculated with clinical specimens. Seventeen skin samples with lesions suspected of sheeppox virus were examined. They were ground, passaged and titrated in primary lamb testis (LT) cell cultures. As negative controls, five skin samples with orf virus lesions and three skin biopsies from normal sheep were examined. The Greek sheeppox virus strain 281 and orf virus strain 176 were also included. PCR for sheeppox virus was positive on the first day of the first passage for all clinical specimens, whereas cytopathogenic effect appeared on the 2nd to 5th day after inoculation of the first, second or third passages. All isolates were also identified by the VNT using homologous antisera. Thus virus isolation and identification by VNT can take from 15 to 30 days. By PCR no reaction was observed with the negative control samples. These results indicate that PCR can be used for the early and specific diagnosis of sheeppox virus from suspected clinical specimens once inoculated in LT cells.
Protection of neonatal pigs from Aujeszky's disease virus (Suid herpesvirus 1) by administration of virus-specific non-neutralizing monoclonal antibodies
S.K. Kritas (1), H.J. Nauwynck (2), M.B. Pensaert (2), S.C. Kyriakis (3)
(1) Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, 43100 Karditsa, Greece (2) Laboratory of Virology, Faculty of Veterinary Medicine, University of Gent, 9820 Merelbeke, Belgium and (3) Clinic of Productive Animal Medicine, Faculty of Veterinary Medicine, University of Thessaloniki, 54006 Macedonia, Greece
Hellenic Virology, 1999, 4(2): 100-107.
The purpose of this study was to investigate the mechanisms by which, passively administered-antibodies protect neonatal pigs against neurological disease caused by Aujeszky's disease virus (Suid herpesvirus 1, SHV-1). Neonatal pigs without specific maternal immunity (1st group), pigs injected with ascites fluid containing non-neutralizing antibodies against gE glycoprotein of SHV-1 (2nd group), and pigs with low levels of specific maternal immunity (3rd group) were inoculated with 107,0 TCID50 of the Ka strain of SHV-1 and were examined with respect to the development of neurological signs. Also, virus invasion and spread in 3 levels of the olfactory and trigeminal nervous pathways was studied at 5 days post inoculation by virus isolation in cell culture. The pigs of the 1st group have developed neurological symptoms and died by 4 to 5 days post inoculation, whilst no pig of the other two groups have shown any neurological sign. Invasion and spread of the virus in the neuronal levels of the olfactory pathway was similar for all the pigs of the three groups. However, the spread of the virus in the neuronal levels of the trigeminal pathway was diminished in the pigs of the 2nd and 3rd groups when compared to the pigs of the 1st group. Findings by immunohistochemical examination of the trigeminal ganglion was suggestive for an antibody-dependent defence mechanism at that site. Conclusively, the antibodies are involved in protective mechanisms of the nervous tissue of the host species agaist spread of HSV-1, and particularly within the trigeminal pathway.
Invasion and spread of a gG-deleted mutant of Aujeszky's disease virus (Suid herpesvirus 1) in the trigeminal nervous pathway of neonatal pigs
S.K. Kritas (1), H.J. Nauwynck (2), M.B. Pensaert (2), S.C. Kyriakis (3)
(1) Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, 43100 Karditsa, Greece (2) Laboratory of Virology, Faculty of Veterinary Medicine, University of Gent, 9820 Merelbeke, Belgium and (3) Clinic of Productive Animal Medicine, Faculty of Veterinary Medicine, University of Thessaloniki, 54006 Macedonia, Greece
Hellenic Virology, 1999, 4(2): 108-116.
The purpose of this study was to evaluate the role that viral glycoprotein gG plays in neuropathogenicity and neuroinvasion of suid herpesvirus 1 (SHV-1 or pseudorabies or Aujeszky's disease virus) in the host species. One-week-old conventional pigs and without antibodies against SHV-1 were intranasally inoculated with 107.0 TCID50 of the Ka strain of SHV-1 or its single deleted gG- mutant. The neural invasion and replication of the strains were examined in the trigeminal nervous pathway (nasal mucosa, trigeminal ganglion= 1st level, pons/medulla = 2nd level and cerebellum/thalamus= 3rd level) by virus titration in cell culture and immunohistochemistry. The pigs inoculated with the Ka parental strain have developed neurological disease and died within 5 days post inoculation, whilst none of the Ka gG- inoculated pigs have developed nervous disorders. The Ka strain replicated at high titers in the nasal mucosa and invaded in all neuronal levels of the pathway showing high virus titers. The Ka gG-deleted strain have shown reduced replication at the site of nasal mucosa and delayed invasion in the trigeminal pathway with titers being significantly lower in neural tissues compared to the parental strain Conclusively, the results of this study suggest that gG glycoprotein plays a role in the neuropathogenicity of SHV-1 in pigs by affecting its neural invasion and spread in the trigeminal nervous pathway.
Invasion and spread of a gG-deleted mutant of Aujeszky's disease virus (Suid herpesvirus 1) in the olfactory nervous pathway of neonatal pigs
S.K. Kritas (1), H.J. Nauwynck (2), M.B. Pensaert (2), S.C. Kyriakis (3)
(1) Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, 43100 Karditsa, Greece (2) Laboratory of Virology, Faculty of Veterinary Medicine, University of Gent, 9820 Merelbeke, Belgium and (3) Clinic of Productive Animal Medicine, Faculty of Veterinary Medicine, University of Thessaloniki, 54006 Macedonia, Greece
Hellenic Virology, 1999, 4(2): 117-125.
The purpose of this study was to evaluate in pigs, the role that viral glycoprotein gG plays in neuropathogenicity and invasion of suid herpesvirus 1 (SHV-1 or pseudorabies or Aujeszky's disease virus) in the olfactory nervous pathway. One-week-old conventional pigs and without antibodies against SHV-1 were intranasally inoculated with 107.0 TCID50 of the Ka strain of SHV-1 or its single deleted gG- mutant. The neural invasion and replication of the strains were examined in 3 neuronal levels of the olfactory pathway (olfactory mucosa= 1st level, olfactory bulb= 2nd level and lateral olfactory gyrus, rostral perforated substance and piriform lobe = 3rd levels) by virus titration in cell culture and immunohistochemistry. The pigs inoculated with the Ka parental strain have developed neurological disease and died within 5 days post inoculation, whilst none of the Ka gG- inoculated pigs have developed nervous disorders. The Ka strain replicated at high titers in the olfactory mucosa and invaded in all neuronal levels the pathway showing high virus titers. The Ka gG-deleted strain have shown reduced replication at the site of olfactory mucosa and delayed invasion in the olfactory pathway with titers being significantly lower in neural tissues compared to the parental strain. Conclusively, the results of this study suggest that gG glycoprotein plays a role in the neuropathogenicity of SHV-1 in pigs by affecting its neural invasion and spread in the olfactory nervous pathway. The pattern of invasion and spread of these strains in the olfactory pathway of pigs was similar to that previously observed in the trigeminal pathway. Therefore, the type of nervous pathway appears not to influence the neuropathogenesis of SHV-1 and its gG-deleted mutants in the pig.
Laboratory surveillance of influenza viruses in Southern Greece during 1996-1998
E. Plakokefalos (1), P. Markoulatos (1), S. Bizta (1), E. Xanthou (2), M. Theodoridou (2), N. Spyrou (1)
(1) Dept. of Virology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521 Athens, Greece (2) Children's Hospital of Athens - Ag. Sofia, Thivon-Levadias St., 11527 Athens, Greece
Hellenic Virology, 1999, 4(2): 126-130.
Influenza virus is the causative agent for the epidemic respiratory illness in humans. Due to the evolutionary process of antigenic drift and antigenic shift in their external molecules of hemmaglutinin (HA) and neuraminidase (NA), influenza viruses, especially type A, escape the immune response of the organism, causing epidemics. The timely identification of circulating variant strains of influenza, with epidemic or pandemic potential is crucial in the surveillance of Influenza and contributes to the protection of public health. At the National Influenza Center of Southern Greece, during the period 1996-1998, 197 clinical specimens (nasal and pharyngeal swabs) were examined for isolation of influenza viruses. The specimens were collected from adults (18-65 years) and children up to 12 years old with manifested influenza like symptoms. In children the symptoms further included bronchitis, pharyngitis and nausea. All clinical specimens were cultured in 9 day-old embryonated hens' eggs. Viral isolates were identified by the Hemmaglutination-Inhibition (HAI), Immunocapture-ELISA and RT-PCR methods. During 1996-1997, 90 clinical samples were examined and 11 viral strains were isolated and identified as influenza type A, subtype H3N2, (12,2%). In 1997-1998, 107 samples were examined and 14 viral strains were isolated and identified, (13,1%). Of these, 7 were identified as type A, subtype H3N2, 4 as type B and 3 were mixed infections with influenza type A(H3N2) and type B. All isolates from the surveillance period 1996-1997 were antigenically similar to the variant strain A/South Africa/1147/96(H3N2). In Southern Greece, A/South Africa//1147/96 circulated from December 1996 until January 1998, causing moderate to acute influenza symptoms. Influenza type A isolates from 1997-1998 were anigenically similar to A/Sydney/5/97(H3N2), the prototype vaccine strain for 1998-1999, and to A/South Africa/1147/96. A/Sydney/5/97-like viruses, which dominated worldwide in early 1998, were first isolated form the population of Southern Greece in February 1998. All Influenza B strains isolated during 1997-1998 were B/Beijing/184/93-like, the protoype vaccine strain for 1998-1999. All B viruses were isolated from children up to 12 years old. The viral strains from the mixed infections were A/Sydney/5/97-like and B/Beijing/184/93-like. A phylogenetic analysis of the HA and NA genes is being carried out in order to determine the antigenic drift of our influenza isolates. At the same period, 470 serological examinations were performed for the detection of antibodies (IgG, IgM) against Influenza types A and B. Positive detections were found in 74 cases, (15,7%). More specifically, 42 sera were positive for influenza type A, (8,9%) and 32 sera were found positive for influenza type B, (6,8%).