Alan D Radford

Application of next-generation sequencing technologies in virology

The progress of science is punctuated by the advent of revolutionary technologies that provide new ways and scales to formulate scientific questions and advance knowledge. Following on from electron microscopy, cell culture and PCR, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. Possibilities for these methodologies are only limited by our scientific imagination and, to some extent, by their cost, which has restricted their use to relatively small numbers of samples. Challenges remain, including the storage and analysis of the large amounts of data generated. As the chemistries employed mature, costs will decrease. In addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. An exciting era of viral exploration has begun, and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health.

Feline Herpesvirus Infection: ABCD Guidelines on Prevention and Management:

Overview Feline viral rhinotracheitis, caused by feline herpesvirus (FHV), is an upper respiratory tract disease that is often associated with feline calicivirus and bacteria. In most cats, FHV remains latent after recovery, and they become lifelong virus carriers. Stress or corticosteroid treatment may lead to virus reactivation and shedding in oronasal and conjunctival secretions. Infection Sick cats shed FHV in oral, nasal and conjunctival secretions; shedding may last for 3 weeks. Infection requires direct contact with a shedding cat. Disease signs Feline herpesvirus infections cause acute rhinitis and conjunctivitis, usually accompanied by fever, depression and anorexia. Affected cats may also develop typical ulcerative, dendritic keratitis. Diagnosis Samples consist of conjunctival, corneal or oropharyngeal swabs, corneal scrapings or biopsies. It is not recommended that cats recently vaccinated with a modified-live virus vaccine are sampled. Positive PCR results should be interpreted with caution, as they may be produced by low-level shedding or viral latency. Disease management ‘Tender loving care’ from the owner, supportive therapy and good nursing are essential. Anorexic cats should be fed blended, highly palatable food - warmed up if required. Mucolytic drugs (eg, bromhexine) or nebulisation with saline may offer relief. Broad-spectrum antibiotics should be given to prevent secondary bacterial infections. Topical antiviral drugs may be used for the treatment of acute FHV ocular disease. The virus is labile and susceptible to most disinfectants, antiseptics and detergents.

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

Feline immunodeficiency. ABCD guidelines on prevention and management.

Overview Feline immunodeficiency virus (FIV) is a retrovirus closely related to human immunodeficiency virus. Most felids are susceptible to FIV, but humans are not. Feline immunodeficiency virus is endemic in domestic cat populations worldwide. The virus loses infectivity quickly outside the host and is susceptible to all disinfectants. Infection Feline immunodeficiency virus is transmitted via bites. The risk of transmission is low in households with socially well-adapted cats. Transmission from mother to kittens may occur, especially if the queen is undergoing an acute infection. Cats with FIV are persistently infected in spite of their ability to mount antibody and cell-mediated immune responses. Disease signs Infected cats generally remain free of clinical signs for several years, and some cats never develop disease, depending on the infecting isolate. Most clinical signs are the consequence of immunodeficiency and secondary infection. Typical manifestations are chronic gingivostomatitis, chronic rhinitis, lymphadenopathy, weight loss and immune-mediated glomerulonephritis. Diagnosis Positive in-practice ELISA results obtained in a low-prevalence or low-risk population should always be confirmed by a laboratory. Western blot is the ‘gold standard’ laboratory test for FIV serology. PCR-based assays vary in performance. Disease management Cats should never be euthanased solely on the basis of an FIV-positive test result. Cats infected with FIV may live as long as uninfected cats, with appropriate management. Asymptomatic FIV-infected cats should be neutered to avoid fighting and virus transmission. Infected cats should receive regular veterinary health checks. They can be housed in the same ward as other patients, but should be kept in individual cages.

Feline Infectious Peritonitis ABCD Guidelines on Prevention and Management

Overview Feline Coronavirus infection is ubiquitous in domestic cats, and is particularly common where conditions are crowded. While most FCoV-infected cats are healthy or display only a mild enteritis, some go on to develop feline infectious peritonitis, a disease that is especially common in young cats and multi-cat environments. Up to 12% of FCoV-infected cats may succumb to FIP, with stress predisposing to the development of disease. Disease signs The ‘wet’ or effusive form, characterised by polyserositis (abdominal and/or thoracic effusion) and vasculitis, and the ‘dry’ or non-effusive form (pyogranulomatous lesions in organs) reflect clinical extremes of a continuum. The clinical picture of FIP is highly variable, depending on the distribution of the vasculitis and pyogranulomatous lesions. Fever refractory to antibiotics, lethargy, anorexia and weight loss are common non-specific signs. Ascites is the most obvious manifestation of the effusive form. Diagnosis The aetiological diagnosis of FIP ante-mortem may be difficult, if not impossible. The background of the cat, its history, the clinical signs, laboratory changes, antibody titres and effusion analysis should all be used to help in decisionmaking about further diagnostic procedures. At the time of writing, there is no non-invasive confirmatory test available for cats without effusion. Disease management In most cases FIP is fatal. Supportive treatment is aimed at suppressing the inflammatory and detrimental immune response. However, there are no controlled studies to prove any beneficial effect of corticosteroids.

Feline Leukaemia: ABCD Guidelines on Prevention and Management

Overview Feline leukaemia virus (FeLV) is a retrovirus that may induce depression of the immune system, anaemia and/or lymphoma. Over the past 25 years, the prevalence of FeLV infection has decreased considerably, thanks both to reliable tests for the identification of viraemic carriers and to effective vaccines. Infection Transmission between cats occurs mainly through friendly contacts, but also through biting. In large groups of non-vaccinated cats, around 30–40% will develop persistent viraemia, 30–40% show transient viraemia and 20–30% seroconvert. Young kittens are especially susceptible to FeLV infection. Disease signs The most common signs of persistent FeLV viraemia are immune suppression, anaemia and lymphoma. Less common signs are immune-mediated disease, chronic enteritis, reproductive disorders and peripheral neuropathies. Most persistently viraemic cats die within 2–3 years. Diagnosis In low-prevalence areas there may be a risk of false-positive results; a doubtful positive test result in a healthy cat should therefore be confirmed, preferably by PCR for provirus. Asymptomatic FeLV-positive cats should be retested. Disease management Supportive therapy and good nursing care are required. Secondary infections should be treated promptly. Cats infected with FeLV should remain indoors. Vaccination against common pathogens should be maintained. Inactivated vaccines are recommended. The virus does not survive for long outside the host.

Lethal outbreak of disease associated with feline calicivirus infection in cats

Recently, in the USA, virulent mutants of feline calicivirus (FCV) have been identified as the cause of a severe and acute virulent systemic disease, characterised by jaundice, oedema and high mortality in groups of cats. This severe manifestation of FCV disease has so far only been reported in the USA. However, in 2003, an outbreak of disease affected a household of four adult cats and an adult cat from a neighbouring household in the UK. Three of the adult cats in the household and the neighbouring cat developed clinical signs including pyrexia (39·5 to 40·5°C), lameness, voice loss, inappetence and jaundice. One cat was euthanased in extremis, two died and one recovered. A postmortem examination of one of the cats revealed focal cellulitis around the right hock and right elbow joints. The principal finding of histopathological examinations of selected organs from two of the cats was disseminated hepatocellular necrosis with mild inflammatory infiltration. Immunohistology identified FCV antigen in parenchymal and Kupffer cells in the liver of both animals and in alveolar macrophages of one of them. In addition, calicivirus-like particles were observed by electron microscopy within the hepatocytes of one cat. FCV was isolated from two of the dead cats and from the two surviving cats. Sequence analysis showed that they were all infected with the same strain of virus, but that it was different from strains of FCV associated with the virulent systemic disease in cats in the USA. The outbreak was successfully controlled by quarantine in the owner’s house.

Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats.

Feline calicivirus (FCV) is a respiratory pathogen of cats that is capable of causing persistent infections. This study examined the evolution of a hypervariable region of the FCV capsid gene both during 90 passages in cell culture and during replication in persistently infected cats. This region of the capsid protein is known to contain neutralization epitopes and may be a target for immune evasion during virus persistence in the host. Sequence analysis showed that FCV exists as a quasispecies which evolved both in cell culture and in persistently infected cats. Changes involved both loss of sequence present in the infecting isolate and a gain of both synonymous and non-synonymous nucleotide substitutions to generate sequences not detected within earlier isolates. Overall, these changes led to a reduction in population heterogeneity over time. Where virus populations were highly homogeneous allowing a consensus sequence to be determined, evolution rates for the consensus sequence ranged from 0.10-1.07 substitutions per nucleotide per year. Marked changes in virus neutralization profiles were seen in isolates obtained sequentially from a persistently infected cat. This was not the case with cell culture passaged virus, suggesting that the individual amino acid changes found only in virus from persistently infected cats may significantly alter the antigenic profile of FCV, and may be the result of immune selection.

Feline panleukopenia. ABCD guidelines on prevention and management

Overview Feline panleukopenia virus (FPV) infects all felids as well as raccoons, mink and foxes. This pathogen may survive in the environment for several months and is highly resistant to some disinfectants. Infection Transmission occurs via the faecal-oral route. Indirect contact is the most common route of infection, and FPV may be carried by fomites (shoes, clothing), which means indoor cats are also at risk. Intrauterine virus transmission and infection of neonates can occur. Disease signs Cats of all ages may be affected by FPV, but kittens are most susceptible. Mortality rates are high - over 90% in kittens. Signs of disease include diarrhoea, lymphopenia and neutropenia, followed by thrombocytopenia and anaemia, immunosuppression (transient in adult cats), cerebellar ataxia (in kittens only) and abortion. Diagnosis Feline panleukopenia virus antigen is detected in faeces using commercially available test kits. Specialised laboratories carry out PCR testing on whole blood or faeces. Serological tests are not recommended, as they do not distinguish between infection and vaccination. Disease management Supportive therapy and good nursing significantly decrease mortality rates. In cases of enteritis, parenteral administration of a broad-spectrum antibiotic is recommended. Disinfectants containing sodium hypochlorite (bleach), peracetic acid, formaldehyde or sodium hydroxide are effective.

Molecular phylogenetic and evolutionary analyses of Muar strain of Japanese encephalitis virus reveal it is the missing fifth genotype

Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.