Paola K. Vaz

NetB, a New Toxin That Is Associated with Avian Necrotic Enteritis Caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

Gammaherpesvirus infection in a free-ranging eastern grey kangaroo (Macropus giganteus).

A gammaherpesvirus was detected by polymerase chain reaction (PCR) in ocular, nasal and oropharyngeal swab samples collected from an adult free-ranging male eastern grey kangaroo (Macropus giganteus) with clinical signs of severe respiratory disease. This is the first time a gammaherpesvirus has been detected in a free-ranging macropod in Australia. The nucleotide sequence of a conserved region of the DNA polymerase gene of the detected virus showed a high degree of identity to a gammaherpesvirus recently detected in a zoological collection of eastern grey kangaroos in North America. The detection of this gammaherpesvirus in a free-ranging, native eastern grey kangaroo provides evidence that this species is a natural host.

Horizontal transmission dynamics of a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus and the effect of vaccination on transmission of virulent virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R₀, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R₀ was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs.

Full genome analysis of Australian infectious bronchitis viruses suggests frequent recombination events between vaccine strains and multiple phylogenetically distant avian coronaviruses of unknown origin

Abstract Australian strains of infectious bronchitis virus (IBV) have been evolving independently for many years, with control achieved by vaccination with local attenuated strains. Previous studies have documented the emergence of recombinants over the last 20 years, with the most recent one, Ck/Aus/N1/08, detected in 2008. These recombinants did not appear to be controlled by the vaccines currently in use. In this study we sequenced the complete genomes of three emergent Australian strains of IBV (IBV/Ck/Aus/N1/88, IBV/Ck/Aus/N1/03 and IBV/Ck/Aus/N1/08) and a previously incompletely characterised vaccine strain, IBV/Ck/Aus/Armidale, and compared them to the genome of the vaccine strain VicS. We detected multiple recombination events throughout the genome between wild type viruses and the vaccine strains in all three emergent isolates. Moreover, we found that strain N1/88 was not entirely exogenous, as was previously hypothesised. Rather, it originated from a recombination event involving the VicS vaccine strain. The S glycoprotein genes of N1/88 and N1/03 were known to be genetically distinct from previously characterised circulating strains and from each other, and the original donors of these genes remains unknown. The S1 glycoprotein gene of N1/88, a subgroup 2 strain, shares a high nucleotide identity with the sequence of the S1 gene of the recent isolate N1/08. As the subgroup 2 strains have not been isolated for at least 20 years, it appears likely that an unknown avian coronavirus that was the donor of the S1 glycoprotein sequence of N1/88 in the 1980s is still recombining with IBV strains in the field.

Detection of a Novel Gammaherpesvirus in Koalas (Phascolarctos cinereus)

A novel gammaherpesvirus was detected in wild koalas (Phascolarctos cinereus) captured at different locations during 2010. Sequence analysis of the DNA polymerase gene revealed that the virus was genetically distinct from all known gammaherpesviruses. This is the first herpesvirus to be definitively identified in the Vombatiforme suborder (koalas and wombats).

Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR—restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 102 or 103 plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 104 PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

Natural recombination in alphaherpesviruses: Insights into viral evolution through full genome sequencing and sequence analysis.

Recombination in alphaherpesviruses was first described more than sixty years ago. Since then, different techniques have been used to detect recombination in natural (field) and experimental settings. Over the last ten years, next-generation sequencing (NGS) technologies and bioinformatic analyses have greatly increased the accuracy of recombination detection, particularly in field settings, thus contributing greatly to the study of natural alphaherpesvirus recombination in both human and veterinary medicine. Such studies have highlighted the important role that natural recombination plays in the evolution of many alphaherpesviruses. These studies have also shown that recombination can be a safety concern for attenuated alphaherpesvirus vaccines, particularly in veterinary medicine where such vaccines are used extensively, but also potentially in human medicine where attenuated varicella zoster virus vaccines are in use. This review focuses on the contributions that NGS and sequence analysis have made over the last ten years to our understanding of recombination in mammalian and avian alphaherpesviruses, with particular focus on attenuated live vaccine use.

Prevalence and Clinical Significance of Herpesvirus Infection in Populations of Australian Marsupials

Herpesviruses have been reported in several marsupial species, but molecular classification has been limited to four herpesviruses in macropodids, a gammaherpesvirus in two antechinus species (Antechinus flavipes and Antechinus agilis), a gammaherpesvirus in a potoroid, the eastern bettong (Bettongia gaimardi) and two gammaherpesviruses in koalas (Phascolarctos cinereus). In this study we examined a range of Australian marsupials for the presence of herpesviruses using molecular and serological techniques, and also assessed risk factors associated with herpesvirus infection. Our study population included 99 koalas (Phascolarctos cinereus), 96 eastern grey kangaroos (Macropus giganteus), 50 Tasmanian devils (Sarcophilus harrisii) and 33 common wombats (Vombatus ursinius). In total, six novel herpesviruses (one alphaherpesvirus and five gammaherpesviruses) were identified in various host species. The overall prevalence of detection of herpesvirus DNA in our study population was 27.2% (95% confidence interval (CI) of 22.6–32.2%), but this varied between species and reached as high as 45.4% (95% CI 28.1–63.7%) in common wombats. Serum antibodies to two closely related macropodid herpesviruses (macropodid herpesvirus 1 and 2) were detected in 44.3% (95% CI 33.1–55.9%) of animals tested. This also varied between species and was as high as 92% (95% CI 74.0–99.0%) in eastern grey kangaroos. A number of epidemiological variables were identified as positive predictors for the presence of herpesvirus DNA in the marsupial samples evaluated. The most striking association was observed in koalas, where the presence of Chlamydia pecorum DNA was strongly associated with the presence of herpesvirus DNA (Odds Ratio = 60, 95% CI 12.1–297.8). Our results demonstrate the common presence of herpesviruses in Australian marsupials and provide directions for future research.

Detection and identification of a Gammaherpesvirus in Antechinus spp. in Australia

Abstract We detected herpesvirus infection in a male yellow-footed antechinus (Antechinus flavipes) and male agile antechinus (Antechinus agilis) during the period of postmating male antechinus immunosuppression and mortality. Histopathologic examination of tissues revealed lesions consistent with herpesvirus infection in the prostate of both animals. Herpesvirus virions were observed by transmission electron microscopy in the prostate tissue collected from the male yellow-footed antechinus. Herpesvirus DNA was detected in prostate, liver, lung, kidney, spleen, and ocular/nasal tissues using a pan-herpesvirus PCR targeting the viral DNA polymerase. Nucleotide sequencing identified a novel herpesvirus from the Gammaherpesvirinae subfamily that we have tentatively designated dasyurid herpesvirus 1 (DaHV-1).