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Amongst newly developed approaches to analyse molecular data, phylodynamic models are receiving much attention because of their potential to reveal changes to viral populations over short periods. This knowledge can be very important for understanding disease impacts. However, their accuracy needs to be fully understood, especially in relation to wildlife disease epidemiology, where sampling and prior knowledge may be limited. The release of the rabbit haemorrhagic disease virus (RHDV) as biological control in naive rabbit populations in Australia in 1996 provides a unique dataset with which to validate phylodynamic models. By comparing the results obtained for RHDV1 with our current understanding of the RHDV epidemiology in Australia, we evaluated the performances of these recently developed models. In line with our expectations, coalescent analyses detected a sharp increase in the virus trajectory in the first few months after the virus release, followed by a more gradual increase. The phylodynamic analyses with a birth-death tree prior generated effective reproductive number estimates (the average number of secondary infections per each infectious case, Re) larger than one for most of the epochs considered. However, the possible range of the initial Re included estimates lower than one despite the known rapid spread of RHDV1 in Australia. Furthermore, the analyses that took into account the geographical structuring failed to converge. We argue that the difficulties that we encountered most likely stem from the fact that the samples available from 1996 to 2014 were too sparse with respect to geographic and within outbreak coverage to adequately infer some of the model parameters. In general, while these Bayesian analyses proved to be greatly informative in some regards, we caution that their interpretation may not be straight forward and recommend further research in evaluating the robustness of these models to assumption violations and sensitivity to sampling regimes.

Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic to European rabbits. Until recently, only one serotype of RHDV was known, GI.1/RHDV. RHDV2/GI.2 is a novel virus that has rapidly spread and become the dominant pathogenic calicivirus in wild rabbits worldwide. It is speculated that RHDV2 has three competitive advantages over RHDV: (a) the ability to partially overcome immunity to other variants; (b) the ability to clinically infect young rabbits; and (c) a wider host range. These differences would be expected to influence virus transmission dynamics. We used markers of recent infection (IgM/IgA antibodies) to investigate virus transmission dynamics pre and post the arrival of RHDV2. Our data set contained over 3,900 rabbits sampled across a 7-year period at 12 Australian sites. Following the arrival of RHDV2, seasonal peaks in IgM and IgA seropositivity shifted forward one season, from winter to autumn and spring to winter, respectively. Contrary to predictions, we found only weak effects of rabbit age, seropositivity to non-pathogenic calicivirus RCV-A1 and population abundance on IgM/IgA seropositivity. Our results demonstrate that RHDV2 enters rabbit populations shortly after the commencement of annual breeding cycles. Upon entering, the population RHDV2 undergoes extensive replication in young rabbits, causing clinical disease, high virus shedding, mortality and the creation of virus-laden carcasses. This results in high virus contamination in the environment, furthering the transmission of RHDV2 and initiating outbreaks, whilst simultaneously removing the susceptible cohort required for the effective transmission of RHDV. Although RHDV may enter the population at the same time point, it is sub-clinical in young rabbits, causing minimal virus shedding and low environmental contamination. Our results demonstrate a major shift in epidemiological patterns in virus transmission, providing the first evidence that RHDV2's ability to clinically infect young rabbits is a key competitive advantage in the field.

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.

Rabbit haemorrhagic disease (RHD) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV), is an RNA virus of the family Caliciviridae, genus Lagovirus. Pathogenic (G1-G6 or variants GI.1a-GI.1d) and non-pathogenic strains (GI.4) have been characterized. In 2010, a new variant of RHDV, RHDV2/RHDVb/GI.2, was detected in France. GI.2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI.2 recombinant strains were detected, including triple recombinants. These recombinants possess a non-structural protein p16 related to a non-pathogenic strain. Evolutionary analyses were conducted on GI.2 VP60 sequences. Estimated time to the most recent common ancestor (tMRCA) suggests an emergence of GI.2 in July 2008, not distant from its first detection in 2010. This is the first study on GI.2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses' evolution.

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so-called ‘novel’ rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two-step in-depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future.

Summary Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Rabbit haemorrhagic disease virus (RHDV, or GI.1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT-qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies.

Mortality caused by rabbit haemorrhagic disease virus (RHDV) in wild rabbits is reduced in parts of Australia where the related, non-pathogenic calicivirus RCV-A1 is endemic. Laboratory experiments previously showed that prior infection with RCV-A1 enabled rabbits to better withstand subsequent infection with highly virulent RHDV, and this was assumed to explain higher survival. Here, we analyse serological data from the field suggesting that reduced mortality rates among wild rabbits may also result from rabbits previously infected with RCV-A1 having a reduced likelihood of RHDV infection. We discuss the possible mechanisms underlying this finding and its implications. The methods we describe for analysing field data gave far greater insights into epidemiological processes and virus interactions than gained from reporting basic seroprevalence rates alone.

Incursion of rabbit haemorrhagic disease virus (RHDV) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus) populations. Rabbit haemorrhagic disease virus 2 (RHDV2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD) do not distinguish between types of virus causing disease. Using RHDV2-specific RT-qPCR, sequencing of the VP60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV2, suggesting that RHDV2 may be replacing RHDV as the predominant cause of RHD in Sweden.