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[Changes in cellular microfilaments in viral infection].

The effect of disorders in actin microfilament polymerization by cytochalasin B (CB) on the development of cytopathic changes was studied in two models. CB in concentrations of 0.1-1.0 microgram/ml enhanced 3-5-fold the formation of symplasts in a line of kidney cells from green monkey 4647 infected with carnivore distemper virus and inhibited this process in higher concentrations (2.5-5.0 micrograms/ml). The addition of CB in concentrations 5-10 micrograms/ml into the medium of a line of Chinese striped hamster cells infected with Sindbis virus led to disorders in the permeability of plasmolemma and destruction of cells although virus reproduction and production of virus antigens remained uninvolved. The above results not only demonstrate the role of actin microfilaments in the development of cytopathic lesions caused by paramyxo- and alphaviruses but also allow the assessment of separate stages of these processes.

Actin Cytoskeleton↗

Potential role of paramyxoviruses in multiple sclerosis.

At the present time, the cause of multiple sclerosis (MS) remains unknown, although it seem likely that one or more infectious agents triggers the disease, perhaps through an autoimmune mechanism. In this article, evidence linking the paramyxoviruses, canine distemper, and measles to MS is reviewed.

Animals↗

Absence of paramyxovirus RNA in cultures of pagetic bone cells and in pagetic bone.

It has been proposed that Paget's disease of bone is caused by the infection of bone cells with one or several paramyxoviruses. In this study we used the polymerase chain reaction (PCR), which allows the detection of very low levels of a target nucleic acid sequence, to study cultures of pagetic bone cells and samples of pagetic bone. Oligonucleotide primers were designed to flank a sequence of the nucleocapsid genome of measles virus and canine distemper virus (CDV). Within this fragment there were contrasting restriction endonuclease sites specific to measles or CDV that allowed identification of the original template. We were unable to detect paramyxovirus RNA in four strains of human bone cells outgrown from pagetic bone and one strain derived from an uninvolved site of a patient with Paget's disease. Furthermore, paramyxovirus sequences were not detected in cDNA prepared from six samples of pagetic bone biopsies. The work presented here further questions the role of measles and CDV in the abnormal remodeling observed in Paget's disease.

Aged↗

Morbilliviruses and human disease.

Morbilliviruses are a group of viruses that belong to the family Paramyxoviridae. The most instantly recognizable member is measles virus (MV) and individuals acutely infected with the virus exhibit a wide range of clinical symptoms ranging from a characteristic mild self-limiting infection to death. Canine distemper virus (CDV) and rinderpest virus (RPV) cause a similar but distinctive pathology in dogs and cattle, respectively, and these, alongside experimental MV infection of primates, have been useful models for MV pathogenesis. Traditionally, viruses were identified because a distinctive disease was observed in man or animals; an infectious agent was subsequently isolated, cultured, and this could be used to recapitulate the disease in an experimentally infected host. Thus, satisfying Koch's postulates has been the norm. More recently, particularly due to the advent of exceedingly sensitive molecular biological assays, many researchers have looked for infectious agents in disease conditions for which a viral aetiology has not been previously established. For these cases, the modified Koch's postulates of Bradford Hill have been developed as criteria to link a virus to a specific disease. Only in a few cases have these conditions been fulfilled. Therefore, many viruses have over the years been definitely and tentatively linked to human diseases and in this respect the morbilliviruses are no different. In this review, human diseases associated with morbillivirus infection have been grouped into three broad categories: (1) those which are definitely caused by the infection; (2) those which may be exacerbated or facilitated by an infection; and (3) those which currently have limited, weak, unsubstantiated or no credible scientific evidence to support any link to a morbillivirus. Thus, an attempt has been made to clarify the published data and separate human diseases actually linked to morbilliviruses from those that are merely anecdotally associated.

Animals↗

The removal of viruses during the purification of equine antisera using filtration aids Hyflo Super-Cel and Fulmon Super A.

The manufacturing process in Australia for equine antisera against various venoms/toxins is based primarily on ammonium sulphate precipitation of pepsin-digested IgG, whereby Fc and F(ab')(2)fragments are separated. The capacity of the process to remove non-enveloped and enveloped model viruses was assessed using a scaled-down process. Each virus was added to mid-process samples from equine plasma before the material was applied to Hyflo Super-Celtrade mark filtration followed by Fulmonttrade mark Super A filtration. Samples were analysed pre- and post-filtration and the log clearance of the viruses calculated. The mean clearance factors for viral load of canine adenovirus type II (CAV(2)), poliovirus type 1 (PV1), infectious bovine rhinotracheitis virus (IBR) and canine distemper virus (CDV) were 5.3 logs, 4.2 logs, 5.7 logs and 4. 0 logs respectively. Clearance results as virus is adsorbed to the filtration aids which are removed from the process, thereby demonstrating improved viral safety of equine antisera produced by CSL.

Adenoviruses, Canine↗

Extinction times and phase transitions for spatially structured closed epidemics.

This paper considers the time to extinction for a stochastic epidemic model of SEIR form without replacement of susceptibles. It first shows how previous rigorous results can be heuristically explained in terms of the more transparent dynamics of an approximating deterministic system. The model is then extended to include a host population structured into patches, with weak nearest-neighbour mixing of infection. It is shown, by considering the approximating deterministic system, that the expected time to extinction in a population of n + 1 patches each of size N is of the form a log N + bn, provided that N > Nc where Nc is a critical patch size below which transits are unlikely to occur. This corresponds to the simple decomposition of the time of an epidemic into the time it takes to spread through one patch plus the time it takes to transit to each of n successive patches. Expressions for this threshold and the coefficients of the time to extinction are given in terms of the transmission parameters of infection and the coupling strength between patches. These expressions are compared with numerical results using parameters relevant to a study of phocine distemper virus in North Sea seals, and the agreement is found to be good for large and small N. In the region when N approximately Nc, where transits may or may not occur, interesting transitional behaviour is seen, leading to a non-monotonicity of the extinction time as a function of N.

Animals↗

Nucleotide sequence of cDNA to the rinderpest virus mRNA encoding the nucleocapsid protein.

The full-length cDNA corresponding to the mRNA encoding the nucleocapsid protein (NP) of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The gene of RV-NP was composed of 1683 nucleotides and contained a single large open reading frame, which is capable of encoding a protein of 525 amino acids with a molecular weight of 58,241 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of measles virus (MV) and canine distemper virus (CDV). The nucleotide sequence of the coding region of RV-NP (53-1630) revealed a homology of 68.1% and 63.0% with MV and CDV-NP, respectively. Relatively moderate homologies of 68.7% (MV) and 64.3% (CDV) were found at nucleotides 53-592. The highest homology of 75.3-74.3% was equally present between RV and both MV and CDV in the middle region at nucleotides 593-1312. The homologies of the predicted amino acids in this region were 88.3% (MV) and 86.3% (CDV). Relatively low (MV) or little (CDV) homology was detected in the last 318 nucleotides toward the 3' terminus (1313-1630). The predicted secondary structures of amino acids at the C terminus differed between the three viruses.

Amino Acid Sequence↗

Antarctic seals carry antibodies against seal herpesvirus.

Weddell seals in the Antarctica had high neutralizing antibody titres to seal- and feline herpesvirus and none against phocine distemper virus. Crab-eater seals were free of antibodies. This suggests an evolutionary wide spread of seal herpesvirus.

Animals↗

Comparison of morbillivirus proteins by limited proteolysis.

Proteins of a number of measles and SSPE virus strains have been compared by limited proteolysis and they appear to be largely conserved amongst the various strains. Viruses derived from SSPE cannot be distinguished from other measles viruses by this technique. Small differences in the digest patterns of the M proteins have been observed between the Edmonston and other measles virus strains. Furthermore, in some strains where the M proteins migrate slower in SDS-PAGE the limited proteolysis patterns are slightly different from those in other MV and SSPE virus strains. The limited proteolysis pattern of some canine distemper virus (CDV) proteins have been determined and nucleocapsid breakdown products have been identified in infected cells. Comparisons of proteins of four strains of CDV have shown that these, too, are largely conserved, although the digest of proteins of CDV appear to show more pronounced differences than those present in the MV and SSPE virus group. Limited proteolysis can be used readily to distinguish MV from CDV isolates.

Chemical Phenomena↗

Lenticular lesions in harbour seals (Phoca vitulina).

Lenticular lesions were found in 21 of 44 mainly younger harbour seals investigated during the epidemic caused by phocine distemper virus (PDV) in 1988/89. Malformations and cataracts varied in quality and degree and were histologically characterized by proliferative and/or degenerative lenticular alterations. Except for two animals, both eyes were affected symmetrically. For that reason, local exogenous causes are improbable. There was very little evidence for infectious agents isolated from the seals investigated (viruses, in particular PDV, bacteria, mycoplasma, parasites). Environmental, dietary and hereditary aspects were also considered, but no final conclusions could be made as to the involvement of these factors in the genesis of lens lesions in harbour seals.

Animals↗

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA.

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.

Amino Acid Sequence↗

Immune responses in common and grey seals during the seal epizootic.

Serological surveys of common and grey seals were performed to monitor the spread of the phocid distemper outbreak in 1988 and its effects on the host immune response. The disease affected mainly common seals; grey seals were largely unaffected but did have anti-CDV antibodies which developed during 1988. Canadian seals, both common and grey, showed evidence of a morbillivirus infection but without the apparent mortality observed in British common seals. Common seal circulating immunoglobulin levels were not greatly affected during the outbreak, although there was some reduction in the most severely affected pups. Vaccination of common seals with CDV antigens elicited high titres of antibodies and produced clinical protection. Vertical transmission of host immunity was shown in grey seals although the longevity of protection has not been determined. The large population of common seals which are not immune emphasises that such an epizootic could have serious consequences again in the future.

Animals↗

Diseases and environmental contaminants in seals from the Baltic and the Swedish west coast.

Investigations have shown that Baltic grey seal (Halichoerus grypus) and ringed seal (Phoca hispida) suffer from a disease complex described as a primary lesion in the adrenals causing secondary reactions in various other organs. Studies on historical Baltic grey seal skull bone material show that the prevalence of affected animals started to increase after World War II. The disease complex explains the dramatic decrease in the Baltic grey and ringed seal population during the 1960s and 1970s and is believed to be caused by environmental pollutants. In 1988, about 60% of the harbor seal population (Phoca vitulina) along the Swedish west coast and in the southwestern part of the Baltic died in the PDV epizootic (Phocine Distemper Virus). Whether the course of the epizootic was altered by environmental pollutants is still an open question. Studies on historical harbor seal skull bone material from both the Baltic and the Swedish west coast show that the incidence of skull bone lesions has also increased in these populations since World War II, indicating the presence of unnatural stress factors. After the epizootic, the harbor seal populations both in the Baltic and along the Swedish west coast have increased in number. Chemical analysis of tissues has been performed on the three seal species collected in various areas of the Baltic and the Swedish west coast. The concentrations of 17 metals and non-metal elements, sDDT and PCBs, DDE and PCB methylsulfones, toxaphene, chlordanes, polybrominated diphenyl ethers, PCDDs and PCDFs have been determined in selected groups of seals in order to determine spatial, species and age variations in concentrations. Furthermore, healthy animals have been compared to diseased animals. Spatial variation was found mostly within the group of organohalogenated compounds, a group of contaminants where a strong covariation between the various compounds was also found. On the basis of the analytical results as well as the pathological findings on Baltic seals, the group of DDE and PCB methyl sulfones is tentatively suggested to be more important in explaining the disease complex than coplanar structures including dioxins.

Adrenal Gland Diseases↗

Relative immunocompetence of the newborn harbour seal, Phoca vitulina.

The immune system of many mammalian species is not fully developed at birth, with newborns obtaining temporary immunological protection from maternal antibodies. Little is known of the immune system of the harbour seal, and developmental aspects of its immune system have not been systematically studied. We collected blood and milk samples from nine free-ranging mother-pup pairs throughout the lactation period on Sable Island, Canada, in an effort to characterise developmental aspects of the immune system of this newborn pinniped. Pup lymphocytes responded stronger to the mitogens concanavalin A, phytohaemagglutinin, and pokeweed mitogen than the lymphocytes of their mothers. In contrast to newborn cats and dogs, newborn seal pups developed high specific antibody responses after immunisation with an inactivated rabies vaccine. Circulating levels of total IgG in newborn pups were low (3% of maternal levels), but increased rapidly after colostrum intake (to 65% of maternal levels after 15 days). A similar pattern of increase in pup serum was observed for phocine distemper virus specific antibodies which had been detected in the serum and milk of mothers, suggesting that the transfer of colostral antibodies is an important feature of temporary protection for the pup. We speculate that the relative immunocompetence of the harbour seal at birth reflects an adaptation to its relatively short nursing period and limited maternal care.

Animals↗

Rapid isolation of morbillivirus nucleocapsid for genomic RNA cDNA cloning and the production of specific core protein antisera.

A procedure is described for the rapid isolation of canine distemper virus nucleocapsid, free from contaminating viral non-core and host cellular proteins. Nucleocapsid isolated in this manner is amenable to ultrastructural evaluation, protein isolation for the production of monospecific hyperimmune serum, and genomic RNA isolation for cDNA cloning. Nucleocapsid (NC) and a defective NC variant (Df-NC) isolated from 5.5 x 10(7) Vero cells infected with Ond-CDV is readily visualized on cesium gradients. The calculated density for NC is 1.2976 +/- 0.0033 g/ml and 1.2458 +/- 0.0056 g/ml for Df-NC. Ultrastructurally, NC appears as long uninterrupted strands, 1.6 +/- 0.1 microns in length, 21.2 +/- 1.7 nm in diameter, with well defined capsid subunits. Df-NC are truncated with a uniform length of 85.8 +/- 7.1 nm and a 24.5 +/- 1.3 nm diameter. A total of 2.1 +/- 0.2 mu of NC protein is obtained for every 1 x 10(6) cells infected; 89.7% of this mass is represented by a 61 kDa protein (N), 8.4% by a 75 kDa protein (P), and 1.9% by a 160-200 kDa protein (L), which is in agreement with the NC constituency of other paramyxoviruses. Viral N and P proteins, purified by 7.5% SDS-PAGE, were used in the production of hyperimmune serum. Specificity was demonstrated by Western blot analysis. Both antisera were capable of detecting viral antigen in persistently and lytically CDV infected cells by indirect immunofluorescence. A single high molecular weight species of nucleic acid was isolated from purified nucleocapsids compatible with a 14.6 kb morbillivirus genome. Although the efficiency of RNA extraction from purified NC was low (14.2%), sufficient RNA was obtained for gel analysis and the establishment of genomic RNA cDNA clones.

Antibodies, Viral↗

Sensitive detection of morbillivirus cell-free transcription in a direct RNase protection assay.

Cell-free measurement of viral transcription is necessary to determine if alterations of in situ levels of viral mRNA represent altered mRNA production or stability. Conditions for cell-free genomic transcription have been developed for the morbilliviruses canine distemper virus (CDV) and measles virus (MV), although the means for detecting nascent transcripts in these assays are insensitive in some cell systems. This work describes a technique in which CDV cell-free transcription reactions are modified so that non-radiolabeled transcripts are produced, precluding the need for limiting nucleotide concentrations in the reaction mixtures and allowing nucleotide concentrations which support optimal polymerase activities. Cell-free transcripts are then detected using anti-sense gene-specific riboprobes in an RNase protection assay (RPA). This approach is more sensitive than conventional slot blot analyses in detecting radiolabeled nascent transcripts and is efficacious in cell systems supporting inherently low levels of virus gene expression. Preformed viral RNA is distinguished from RNA synthesized during the course of the cell-free reactions by using a direct RPA (i.e., hybridization of target RNA prior to phenol/chloroform extraction). The use of this approach will expand the range of virus-host systems in which determinants of morbillivirus transcription can be characterized.

Animals↗

Electroencephalogram and evoked potentials in the primate model of viral encephalitis.

Squirrel monkeys with induced canine distemper virus (CDV) encephalitis showed characteristic clinical signs such as seizures or myoclonus, with EEGs showing periodic synchronized discharge (PSD). Histopathologically, there was gliosis and neuronal degeneration diffusely distributed in both the gray and white matters in the subacute phase, the lesions resembling those found in childhood acute viral encephalitis and subacute sclerosing panencephalitis (SSPE). The auditory brain-stem response (ABR) changes were remarkable in the subacute phase, and the recovery of latency was correlated with survival. The visual evoked potential (VEP) abnormalities disappeared in the acute phase and appeared again with delayed latency in the subacute stage. One monkey which did not show clinical signs but had the pathological changes showed the VEP abnormality. These data suggest that the evoked potentials are helpful in judging the prognosis, and that this model is useful for the analysis of the pathogenesis of viral encephalitis.

Animals↗

Induction of systemic immune responses to measles virus synthetic peptides administered intranasally.

A systemic antibody response was induced when a chimeric peptide containing two copies of a promiscuous T-cell epitope and one copy of a B-cell epitope (TTB) from the fusion protein of measles virus (MV) was administered to mice intranasally without adjuvant. A higher antibody titre was produced when the peptide was administered intranasally with cholera toxin B subunit (CTB) as an adjuvant and these antibodies crossreacted with the MV. Furthermore, splenocytes from intranasally immunized mice proliferated in vitro in the presence of the TTB peptide. The immune response following intranasal immunization with the peptide was influenced by the MHC haplotype of the strain of mice used. Thus CBA and BALB/c mice were high responders whereas C57BL/6 mice were low responders. Although peptide administered intranasally with CTB to CBA mice induced an immune response, no significant protection was observed against intra-cranial challenge with canine distemper virus which is antigenically related to MV.

Administration, Intranasal↗