P.H. Russell

Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies

Newcastle disease (ND) virus (APMV-1) isolates submitted to the International Reference Laboratory for ND were characterised antigenically by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Since the availability of the mAbs 1526 viruses have been examined using a panel of nine mAbs and 818 with an extended panel of 26 mAbs. Using the nine mAb panel a total of 14 different patterns was seen and viruses grouped by the same pattern showed relationships with each other which were either biological, temporal or geographical or more than one of these. There was a marked tendency of viruses placed in the same group to show similar virulence for chickens. Extension of the panel to 26 mAbs produced 39 distinct patterns, although some of these were seen with only a single virus. Again, viruses inducing similar binding patterns shared similar properties and some binding patterns were specific for viruses causing discrete epizootics. Cluster analysis of the mAb binding patterns did not produce concise, discrete groupings, but did emphasise some relationships between virus properties and antigenicity. Examples of the usefulness of this approach were the ability to link two important outbreaks to the contamination of stored food by infected feral pigeons, and the demonstration of two separate viruses responsible for outbreaks in countries of the European Union during 1991 to 1994 thus preventing erroneous epizootiological tracing.

Mammalian granulocyte–macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a ‘danger’ signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with β‐galactosidase (β‐gal). A second pUC‐based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to β‐gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co‐administered β‐gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as ‘danger’ signals. In addition, plasmid DNA expressing mouse granulocyte–macrophage colony‐stimulating factor (GM‐CSF) had a marked effect on cytotoxic T‐cell‐like activity in fish by reducing the average number of myofibres that expressed β‐gal, 28 days after co‐injection with plasmid DNA expressing β‐gal. Although the mechanism by which the mouse GM‐CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

The safety and longevity of DNA vaccines for fish

A plasmid that contained the cytomegalovirus (CMV)‐promoter‐driven lacZ reporter gene (pCMV‐lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22°. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coliβ‐galactosidase (β‐gal), which was in the injected myofibres, was detected in all the fish at 4–21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to β‐gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to β‐gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune‐like antibodies to itself or to single‐ or double‐stranded salmon testis DNA. Plasmids can therefore induce long‐term foreign protein expression whilst inducing humoral and cell‐mediated immunity without autoimmunity or integration in goldfish.

Poxviral disease in red squirrels Sciurus vulgaris in the UK: spatial and temporal trends of an emerging threat.

The squirrel poxvirus (SQPV) is the probable mediator of apparent competition between the introduced invading gray squirrel (Sciurus carolinensis) and the red squirrel (Sciurus vulgaris) in the UK, and modeling studies have shown that this viral disease has had a significant impact on the decline of the red squirrel in the UK. However, given our limited understanding of the epidemiology of the disease, and more generally the effects of invasive species on parasite ecology, there is a need to investigate the transmission dynamics and the relative pathogenicity of the virus between species. We aimed to increase our knowledge of these processes through an empirical study in which we: (i) used pathological signs and transmission electron microscopy (TEM) to diagnose SQPV disease in red squirrels found dead during scanning surveillance between 1993 and 2005; (ii) detected antibody to SQPV using an enzyme-linked immunosorbent assay (ELISA) in the same animals; and (iii) mapped cases of the disease, and the gray squirrel distribution, using a geographical information system. We analyzed the distribution of cases of SQPV disease according to woodland type, a measure of squirrel density. SQPV disease occurred only in areas of England also inhabited by seropositive gray squirrels, and as the geographical range of gray squirrels expanded, SQPV disease occurred in these new gray squirrel habitats, supporting a role for the gray squirrel as a reservoir host of the virus. There was a delay between the establishment of invading gray squirrels and cases of the disease in red squirrels which implies gray squirrels must reach a threshold number or density before the virus is transmitted to red squirrels. The spatial and temporal trend in SQPV disease outbreaks suggested that SQPV disease will have a significant effect on Scottish populations of red squirrels within 25 years. The even spread of cases of disease across months suggested a direct rather than vector-borne transmission route is more likely. Eight juvenile and sub-adult free-living red squirrels apparently survived exposure to SQPV by mounting an immune response, the first evidence of immunity to SQPV in free-living red squirrels, which possibly suggests a changing host-parasite relationship and that the use of a vaccine may be an effective management tool to protect remnant red squirrel populations.

Use of monoclonal antibodies in the characterisation of avian paramyxovirus type 1 (Newcastle disease virus) isolates submitted to an international reference laboratory.

A total of 106 Newcastle disease viruses submitted to the International Reference Laboratory at the Central Veterinary Laboratory, Weybridge from field investigations in 15 different countries was characterised using pathogenicity index tests in chickens and mouse monoclonal antibodies raised against NDV-Ulster 2C (Russell and Alexander, Archives of Virology, 75: 243, 1983) and pigeon isolate 617/83. These isolates could be placed into six distinct groups on the basis of their reaction with the monoclonal antibodies although four isolates gave ambiguous results and remained untyped. Forty isolates, obtained from chickens (21), pigeons (16), a duck (1), a sparrow (1) and a kestrel (1), were indistinguishable from isolates which were responsible for the recent panzootic in pigeons. Twenty-one isolates from domestic poultry, one isolate from a pheasant and one from a chicken in quarantine were identified as vaccinal virus of Bi or La Sota type. Thirty-five isolates placed in the same monoclonal antibody group were velogenic viruses. These had been obtained from domestic poultry in Italy, Austria, Mauritius and Saudi Arabia during 1983-1985, commercial pigeons in Hong Kong in 1986 and exotic birds in Italy, Great Britain and the Federal Republic of Germany during 1981-1985. This group was distinguishable from velogenic viruses responsible for disease outbreaks in poultry during the 1970s. Two lentogenic isolates from commercial ducks in England showed different monoclonal antibody binding patterns both of which have been associated with feral ducks. An isolate from chickens in Italy was also placed in one of these groups. A single isolate from a loon (Gavia sp) in the USA showed a monoclonal antibody binding pattern which had not been seen previously. In addition, 11 vaccinal or laboratory viruses were received for confirmatory characterisation which was greatly aided by the use of the monoclonal antibodies.

A microwell immunoperoxidase test for screening hybridomas and for diagnosing Newcastle disease virus and Sendai virus.

A cell counting assay was developed to measure the infectivity of Sendai virus and Newcastle disease virus by indirect immunoperoxidase. It was used to monitor monoclonal antibodies against Newcastle disease virus, antibody to Sendai virus in mice and tracheal infectivity in chickens.

The characterization of monoclonal antibodies to Newcastle disease virus

Monoclonal antibodies to the haemagglutinin-neuraminidase (HN), fusion (F), polymerase and nucleocapsid polypeptides of Newcastle disease virus were prepared. Two epitopes were recognized on the HN polypeptide: one was associated with inhibition of haemagglutination and poor neutralization and the other with good neutralization and no inhibition of haemagglutination. The most effective neutralizing antibody was that produced against the F polypeptide. The poorer neutralization associated with the antibody against the HN epitope was augmented by antiglobulin or complement. The monoclonal antibodies that inhibited haemagglutination also inhibited neuraminidase activity when fetuin but not neuraminyl lactose was the substrate.

DNA is as effective as protein at inducing antibody in fish

Antiviral vaccines are needed for fish. 50 microg plasmid DNA in saline by the intramuscular route and 10 microg beta-gal protein in a commercial oil adjuvant by the peritoneal route induced serum antibody of the same titre and avidity in goldfish. The DNA expressed beta-gal under control of the immediate early promoter/enhancer gene of human cytomegalovirus. Commercial bacterin vaccines are administered to fish by the intraperitoneal route with oil and this route for DNA induced only 2-fold less antibody than DNA by the intramuscular route. Bacterin vaccines and antiviral plasmid DNA could therefore be co-injected into the peritoneum of fish in an oil adjuvant as a single dose.

Local antibody forming cell responses to the Hitchner B1 and Ulster strains of Newcastle disease virus.

The Hitchner B1 and Ulster strains of Newcastle disease virus (NDV) replicated to high titre in the Harderian gland (HG) after eye-drop infection. The Harderian gland then became the major site of antiviral IgA-antibody-forming cells (AFC) in the body and their number correlated to the level of antiviral IgA antibody in the tears. The spleen, HG and femoral bone marrow all contained comparable levels of antiviral IgG-AFC and IgM-AFC after two intra-ocular inoculations of virus, whereas the caecal tonsil and bursa contained few AFC despite the local replication of the Ulster strain of NDV leading to high titres of virus in the faeces. Vaccines of the Hitchner B1 strain of NDV were much less effective at inducing antibody by the intranasal compared with intra-ocular route and no virus was re-isolated after intranasal vaccination. The intravenous inoculation of inactivated Iscoms of NDV could stimulate the spleen, but not the Harderian gland to the same extent as a live virus.

Emerging epidemic diseases of frogs in Britain are dependent on the source of ranavirus agent and the route of exposure

A series of transmission studies was conducted to investigate the aetiology, or aetiologies, of emerging fatal epidemic disease syndromes affecting the common frog (Rana temporaria) in Britain. The syndromes, characterized by skin ulceration or systemic haemorrhages, were induced upon exposure to lesion homogenates or cultured ranavirus. The re-isolation of ranavirus from experimentally affected frogs fulfilled Kochs postulates. Aeromonas hydrophila, previously associated with similar lesions, was not significant to disease development. Unexpectedly, disease outcomes were influenced by both the source of agent and the route of exposure, indicating that different ranaviruses with different tissue tropisms and pathogeneses (possibly similar to quasi-species in RNA virus populations) are circulating in the British common frog population. Our findings confirm that ranavirus disease has emerged as an important cause of amphibian mortality in Britain.