Vladimir Savić

Genome Analysis Linking Recent European and African Influenza (H5N1) Viruses

Although linked, these viruses are distinct from earlier outbreak strains.

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.

Short Communication: Chicken Anemia Virus and Infectious Bursal Disease Virus Interfere with Transcription of Chicken IFN-α and IFN-γ mRNA

Chicken anemia virus (CAV) and infectious bursal disease virus (IBDV) are the two most important viruses that cause immunosuppression in commercial chickens. Because inapparent, subclinical infections by these viruses cause immunosuppression, there is need for assessment of the immune status of chickens. Interference with induction of transcription for chicken interferon-α (ChIFN-α) and ChIFN-γ was noted after subclinical infections with either CAV or IBDV. Because the immunosuppressive viruses of chickens may interfere with transcription for ChIFN-α and ChIFN-γ, we propose using this interference to assess the immune status of chickens.

Characterization of velogenic Newcastle disease viruses isolated from dead wild birds in Serbia during 2007.

Avian paramyxoviruses type 1 or Newcastle disease viruses (NDV) are frequently recovered from wild birds and such isolates are most frequently of low virulence. Velogenic NDV are usually recovered from poultry and only occasionally from wild birds. Five NDV isolates were obtained from carcasses of four wild bird species during 2007 in Serbia: Mallard (Anas platyrhynchos), Eurasian Sparrowhawk (Accipiter nisus), feral Rock Pigeon (Columba livia), and Eurasian Collared Dove (Streptopelia decaocto). All the isolates have a typical fusion protein cleavage site motif of velogenic viruses (112R-R-Q-K-R-F117). The highest homology (99%) for the nucleotide sequences spanning the M and F gene of the studied isolates was with the genotype VII NDV isolate Muscovy duck/China(Fujian)/FP1/02. Phylogenetic analysis based on a partial F gene sequence showed that the isolates from wild birds cluster together with concurrent isolates from poultry in Serbia within the subgenotype VIId, which is the predominant pathogen involved currently in Newcastle disease outbreaks in poultry worldwide. It is unlikely that the wild birds played an important role in primary introduction or consequent spread of the velogenic NDV to domestic poultry in Serbia, and they probably contracted the virus from locally infected poultry.

Immune status assessment by abundance of IFN-alpha and IFN-gamma mRNA in chicken blood.

Avian diseases, including such viral infection as infectious bursal disease, infectious anemia, and Mareks disease, often cause immunosuppression, leading to more severe infection, problems with secondary infection, and inadequate responses to vaccination. Immunosuppression thus causes serious economic losses in commercial poultry production. To date, methods for assessing immune status have been too slow to be of practical help. Reasoning that immunosuppression should be reflected by reduced production of interferons (IFN) in response to a viral antigen, we have developed competitive nucleic acid hybridization microtiter plate assays for chicken IFN-alpha (ChIFN-alpha) and ChIFN-gamma mRNA. To evaluate the assay, chickens were challenged with inactivated Newcastle disease virus (iNDV). Whole blood samples were collected at various times subsequently and preserved with a cationic detergent. Later, total RNA was extracted, and mRNA for both ChIFN-alpha and ChIFN-gamma was measured. Both rose from undetectable levels to reach a peak by 4 h, remained high for about 3 days, and fell to undetectable levels by day 5. Results were similar in chickens aged between 1 and 28 days. In later experiments, blood was collected 4 h after viral challenge. When chickens were immunosuppressed by administering 4-5 mg cyclophosphamide (CY) daily for 3 days and challenged with iNDV, they transcribed less ChIFN-alpha and ChIFN-gamma mRNA, and their antibody response was impaired. Our results suggest that suspected immunosuppression in a commercial flock could be assessed within 2-3 days by challenging birds with iNDV and measuring the amounts of ChIFN-alpha and ChIFN-gamma mRNA in blood obtained 2-4 h later.

Genetic analysis of Newcastle disease virus strains isolated in Bosnia-Herzegovina, Croatia, Slovenia and Yugoslavia, reveals the presence of only a single genotype, V, between 1979 and 2002.

Newcastle disease (ND) epizootics in some European countries after the World War II were caused by ND virus (NDV) of multiple genotypes (IV-VIIa) occurring sequentially and/or simultaneously. This study was carried out to characterise the genetic composition of NDV strains during the outbreaks in the territory of the former Yugoslavia in order to enhance our understanding of the relationships of past epizootics in Europe. Sixty-eight NDV strains isolated between 1979 and 2002 were analysed by restriction enzyme digestion and partial sequencing of the fusion protein gene. All isolates were placed in genotype V, an exotic type, that was introduced to western Europe in 1970. Residue substitution analysis has allowed the recognition of four genetic variants, Vb1-Vb4, and the tracing of their movements. Vb1, a dominant variant in Bulgaria from the late 1970s, was also wide spread in the former Yugoslavia throughout the period under investigation. Vb2, a variant occurring in the neighbouring countries in the early 1970s could be the founder of the epidemic in Yugoslavia and it was present up to the late 1980s. Variants Vb3 and Vb4 could be found only after 1987. In conclusion, the ND outbreaks in Yugoslavia were part of the epizootic wave due to genotype V viruses that started in western Europe in 1970 and became endemic in the region. Inter-country transmission occurred for all variants, and Vb3 and Vb4 might have evolved during the endemic period.

Multiple Introduction of Asian H5N1 Avian Influenza Virus in Croatia by Wild Birds During 2005–2006 and Isolation of the Virus from Apparently Healthy Black-Headed Gulls (Larus ridibundus)

This study describes the introduction and spread of avian influenza A (H5N1) subtype in Croatia. Seventeen isolates were identified during the period from October 2005 to March 2006, all originating from wild birds. The full-length nucleotide sequence analysis of the hemagglutinin (HA) gene of seven representative isolates revealed that three distinct genetic strains involved in the outbreaks, implicating at least three independent introductions of the virus into Croatia during a relatively short period of time. All three genetic strains belonged to clade 2.2 (Qinghai-like viruses) and each strain displayed significant similarity to concurrent H5N1 viruses from other European countries. The dominant strain of the virus was present in all four affected areas and in all three bird species (mute swan, mallard, and black-headed gull), indicating cross-species transmission of the virus. Two other genetic strains were found, together with the dominant strain, only in a marsh at the Adriatic coast during late February and early March 2006, which could be associated with frozen water surfaces in the continental part of Croatia as well as in Eastern Europe in early 2006 and the movement of birds toward warmer areas. This is also the first isolation of highly pathogenic avian influenza virus of H5N1 subtype from apparently healthy black-headed gulls.

Influence of environmental and nutritional stressors on yolk sac utilization, development of chicken gastrointestinal system and its immune status

The impact of practically all the everyday stress factors occurring in the first days of the chick life on the rate of the utilisation of the yolk sac content, on the chicken immune status and on the growth of the animals body and intestinal masses was investigated. The impact of the stress was investigated during the first five days of chicks life. The chicks were divided into five groups, each comprising 44 birds. The first chick group was exposed to moderate cold (2–3°C below the optimal temperature for the chick age). The second group was exposed to moderate heat (2–3 °C above the optimal temperature for the age). The third and fourth group were deprived of feed and drinking water for 12 hours and 24 hour, respectively. The fifth chick group was the control group: the birds in this group had optimal environmental conditions and received feed and drinking water immediately upon entering the trial. Ten chicks were taken every day and weighed. Their blood samples were obtained for the assessment of infectious bursal disease virus antibody titres using ELISA method. After that, the birds were sacrificed, their intestinal masses weighed and the quantity of resorbed yolk measured. The greatest body and intestinal masses were found in the control animals. The body and intestinal masses were the lowest in the group which was denied water and food for 24 hours. The resorption of the yolk sac content was approximately equal in all the chick groups, but the resorption rate varied by days. The highest resorption was observed in the chick groups on the day when feed was denied to them, so that this high resorption could be associated with the birds body energy requirements. The resorption of the remaining yolk is associated with feed intake: in chicks to which feed is withheld the yolk is resorbed through the yolk sac wall directly into the blood, which increased its usability. When energy requirements are not satisfied, the chicks body probably utilises resorbed antibodies from the yolk remnants, as their glycoprotein composition is suitable for this purpose.

Immune suppression of commercial broilers in Croatia, Slovenia, and Bosnia and Herzegovina from 1981 to 1991.

A continuous decline in immune responses to Newcastle disease (ND) vaccine was observed in commercial broiler flocks in Croatia, Slovenia, and Bosnia and Herzegovina beginning in 1982. Floating mean haemagglutination inhibition (HI) titres declined from log(2) 4 in 1983 to a low of log(2) 2.4 in 1986, then were log(2) 2.9 in 1990. Several causes of the decline were discounted, leaving mycotoxins in feed and infection with chicken anaemia virus (CAV) as the two most likely causes. Mycotoxins in feed could not be evaluated retrospectively, but archival tissues were available from Croatia and Slovenia. Tissue sections were examined by in situ hybridization for CAV. Whereas only one chicken from early in the decade was infected, all but one of the chickens from late in the decade were. The increase in CAV detection correlated inversely with ND HI titres. Whereas this correlation does not establish cause and effect, CAV cannot be eliminated as a contributory cause of immune suppression.

Abundance of IFN-α and IFN-γ gene transcripts and absence of IL-2 transcripts in the blood of chickens vaccinated with live or inactivated NDV.

As immune responses to live and inactivated vaccines might differ, temporal responses of broiler chickens to vaccination were examined on the basis of the abundance in the circulating blood of gene transcripts of IFN-α, IFN-γ and IL-2, critical cytokines for immune responses. Blood samples were collected 6, 12 and 24 hours, and 7 and 14 days following vaccination with either live or inactivated Newcastle disease virus, La Sota strain, at 14 days of age, and the abundance of transcripts for each cytokine was assayed by real-time RT-PCR. Physiological saline and vaccine emulsion without viral antigen were administered to control groups for live and inactivated vaccine groups, respectively. The abundance of IFN-γ transcripts was elevated during the early times following vaccination and had reached baseline by the seventh day but the abundance of IFN-α transcripts remained elevated. Transcripts for neither IFN gene were detected in the control birds. The abundance of transcripts for each IFN was not different between the two vaccinated groups at any time. Transcripts for IL-2 were detected only in spleens from chicken embryos that had been inoculated with the live virus. The results show that cells stimulated to produce IFN-α and IFN-γ enter the circulating blood but those stimulated to produce IL-2 do not, or in very low number, and the IFN responses to both vaccines are the same.