Performance characteristics of two real-time TaqMan polymerase chain reaction assays for the detection of WSSV in clinically diseased and apparently healthy prawns.

This study aimed to generate data on performance characteristics for 2 real-time TaqMan PCR assays (CSIRO and WOAH WSSV qPCRs) for the purposes of (1) detection of white spot syndrome virus (WSSV) in clinically diseased prawns and (2) detection of WSSV in apparently healthy prawns. Analytical sensitivity of both assays was 2 to 20 genome copies per reaction, and analytical specificity was 100% after testing nucleic acid from 9 heterologous prawn pathogens and 4 prawn species. Results obtained after testing more than 20 000 samples in up to 559 runs with the CSIRO WSSV qPCR and up to 293 runs with the WOAH WSSV qPCR demonstrated satisfactory repeatability for both assays. Both assays demonstrated median diagnostic sensitivity (DSe) 100% (95% CI: 94.9-100%) when testing clinically diseased prawns. When 1591 test results from apparently healthy prawns were analysed by Bayesian latent class analysis, median DSe and diagnostic specificity (DSp) were 82.9% (95% probability interval [PI]: 75.0-90.2%) and 99.7% (95% PI: 98.6-99.99%) for the CSIRO WSSV qPCR and 76.8% (95% PI: 68.9-84.9%) and 99.7% (95% PI: 98.7-99.99%) for the WOAH WSSV qPCR. When both assays were interpreted in parallel, median DSe increased to 98.3 (95% PI: 91.6-99.99%), and median DSp decreased slightly to 99.4% (95% PI: 97.9-99.99%). Routine testing of quantified positive controls by laboratories in the Australian laboratory network demonstrated satisfactory reproducibility of the CSIRO WSSV qPCR assay. Both assays demonstrated comparable performance characteristics, and the results contribute to the validation data required in the WOAH validation pathway for the purposes of detection of WSSV in clinically diseased and apparently healthy prawns.

At Least Three Doses of Leading Vaccines Essential for Neutralisation of SARS-CoV-2 Omicron Variant.

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Type I Hypersensitivity in Ferrets Following Exposure to SARS-CoV-2 Inoculum: Lessons Learned.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

Serological Evidence of Foot-and-Mouth Disease Infection in Goats in Lao PDR.

Foot and Mouth Disease (FMD) causes significant economic loss in Lao PDR (Laos) and perpetuates the cycle of smallholder poverty mainly through large ruminant productivity losses, increased costs of production and potential limitations to market access for trade in livestock and their products. Goats are emerging as an important livestock species in Laos, and there is an increasing trend in the number of households with goats, often farmed alongside cattle and buffalo. Although an FMD susceptible species, very little is known about the role of goats in the epidemiology of the disease in Laos. A cross-sectional seroprevalence study was conducted by detecting antibodies to the non-structural proteins (NSP), an indication of a previous infection, and serotype-specific structural proteins (SP) that could be due to vaccination or infection. The study commenced in late 2017 and sera were collected from 591 goats in 26 villages of northern, central and southern Laos. For a subset of sera samples, paired oral swab samples were also collected by a simple random sampling method to detect the prevalence of FMD virus infection at the time of collection. The NSP seroprevalence in the provinces of Borkeo and Xayabouli in the north was 42 and 8%, respectively and in Khammoune in the center, it was 20%. In the other five provinces, Luang Namtha and Luang Prabang (northern Laos), Xieng Khouang and Savannaket (central Laos), and Champasak (southern Laos), the seroprevalence was close to zero. The multivariable analysis indicated that age (p < 0.001) was positively associated with animal-level seropositivity and males were less likely to be seropositive than females (OR: 0.29; 95%CI: 0.10-0.83; p = 0.017). Continued sero-surveillance for FMD in goats is recommended to improve our understanding of their role in the epidemiology of FMD in the region and to extend support to FMD control decisions, particularly regarding vaccination.

Probe capture enrichment next-generation sequencing of complete foot-and-mouth disease virus genomes in clinical samples.

Next-generation sequencing (NGS) techniques offer an unprecedented "step-change" increase in the quantity and quality of sequence data rapidly generated from a sample and can be applied to obtain ultra-deep coverage of viral genomes. This is not possible with the routinely used Sanger sequencing method that gives the consensus reads, or by cloning approaches. In this study, a targeted-enrichment methodology for the simultaneous acquisition of complete foot-and-mouth disease virus (FMDV) genomes directly from clinical samples is presented. Biotinylated oligonucleotide probes (120 nt) were used to capture and enrich viral RNA following library preparation. To create a virus capture panel targeting serotype O and A simultaneously, 18 baits targeting the highly conserved regions of the 8.3 kb FMDV genome were synthesised, with 14 common to both serotypes, 2 specific to serotype O and 2 specific to serotype A. These baits were used to capture and enrich FMDV RNA (as cDNA) from samples collected during one pathogenesis and two vaccine efficacy trials, where pigs were infected with serotype O or A viruses. After enrichment, FMDV-specific sequencing reads increased by almost 3000-fold. The sequence data were used in variant call analysis to identify single nucleotide polymorphisms (SNPs). This methodology was robust in its ability to capture diverse sequences, was shown to be highly sensitive, and can be easily scaled for large-scale epidemiological studies.

Virulence beneath the fleece; a tale of foot-and-mouth disease virus pathogenesis in sheep.

Foot-and-mouth disease virus (FMDV) is capable of infecting all cloven-hoofed domestic livestock species, including cattle, pigs, goats, and sheep. However, in contrast to cattle and pigs, the pathogenesis of FMDV in small ruminants has been incompletely elucidated. The objective of the current investigation was to characterize tissue- and cellular tropism of early and late stages of FMDV infection in sheep following three different routes of simulated natural virus exposure. Extensive post-mortem harvest of tissue samples at pre-determined time points during early infection (24 and 48 hours post infection) demonstrated that tissues specifically susceptible to primary FMDV infection included the paraepiglottic- and palatine tonsils, as well as the nasopharyngeal mucosa. Additionally, experimental aerosol inoculation of sheep led to substantial virus replication in the lungs at 24-48 hours post-inoculation. During persistent infection (35 days post infection), the paraepiglottic- and palatine tonsils were the only tissues from which infectious FMDV was recovered. This is strikingly different from cattle, in which persistent FMDV infection has consistently been located to the nasopharyngeal mucosa. Analysis of tissue sections by immunomicroscopy revealed a strict epithelial tropism during both early and late phases of infection as FMDV was consistently localized to cytokeratin-expressing epithelial cells. This study expands upon previous knowledge of FMDV pathogenesis in sheep by providing detailed information on the temporo-anatomic distribution of FMDV in ovine tissues. Findings are discussed in relation to similar investigations previously performed in cattle and pigs, highlighting similarities and differences in FMDV pathogenesis across natural host species.

Risk factors for emergence of exotic foot-and-mouth disease O/ME-SA/Ind-2001d on smallholder farms in the Greater Mekong Subregion.

Foot-and-mouth disease (FMD) is a significant endemic transboundary animal disease in Lao People's Democratic Republic (Lao PDR) and throughout the Greater Mekong Subregion (GMS). The disease has been shown to perpetuate the cycle of smallholder poverty through reduced animal production, plus limitations on market access for trading in livestock and their products. Despite significant national and multilateral efforts to control FMD over the past two decades, endemic FMD viruses (FMDVs) continue to circulate in Lao PDR. Further, the threat from new and emerging FMDVs is increasing as transboundary movements in the region intensify in response to increasing regional demand for meat. Although the economic impacts of FMD on smallholder farmers in Lao PDR are significant, studies investigating household-level risk factors for FMD are lacking. Following an outbreak of a novel FMDV (O/ME-SA/Ind2001d) in Lao PDR in 2015, a questionnaire and serological study were conducted in Naxaythong District to identify household-level risk factors associated with this outbreak, as well as endemic circulating viruses in the outbreak area. Data were analysed using a multivariable generalised estimating equation (GEE) model with a logit link function and associations were calculated as odds ratios (OR) with 95% confidence intervals (CI95%). After adjusting for other variables, the practice of quarantining new livestock for a minimum of two weeks prior to introduction to a herd was found to be a significant protective factor during the 2015 outbreak (OR 0.225, CI95% [0.06, 0.88], p-value 0.003). In addition, households owning one or more animals with titres to the non-structural proteins of FMDV, indicating prior infection, had 5.5 times the odds (CI95% [6.16, 49.11], p-value <0.001) of sharing communal grazing land with neighbouring villages. These findings indicate that implementing basic on-farm biosecurity and improved husbandry measures to minimise FMDV circulation at the household level are important and reinforce the need to enhance the education of smallholder farmers in infectious disease control.

Protective effects of high-potency FMDV O1 Manisa monovalent vaccine in cattle challenged with FMDV O/SKR/2010 at 7 or 4 days post vaccination.

Serotype O foot-and-mouth disease (FMD) virus belonging to the SEA topotype continues to be a significant problem in the Eastern Asia region, with outbreaks in Japan and South Korea resulting in the culling of over 3.5 million cattle and pigs in recent years. High-potency O1 Manisa vaccine was previously shown to provide protection in cattle 21days post vaccination (dpv) following challenge with a representative virus, O/SKR/2010. This study tested the ability of the O1 Manisa vaccine to protect cattle from infection and disease with the O/SKR/2010 virus within just 4 or 7days post vaccination. The vaccine protected 50% of cattle from clinical disease when administered 7days prior to challenge, but was not protective with just 4days between vaccination and challenge. Viraemia was significantly reduced in animals challenged 7 dpv but not 4 dpv, compared to unvaccinated controls, however, there were no effects on the level of virus detected in nasal and oral secretions regardless of vaccination time. The level of neutralising antibodies detected in cattle challenged 7 dpv correlated with protection from clinical disease. All animals seroconverted to FMDV non-structural proteins, suggesting no sterile protection. An equal number of animals became persistently infected in both vaccine groups. The results indicated that high-potency O1 Manisa vaccine administered just 7days prior to challenge should provide partial protection of cattle if an outbreak of O/SKR/2010, or related viruses, occurs, and would be useful to limit spread of FMDV when used in conjunction with other control measures.

The B-cell response to foot-and-mouth-disease virus in cattle following vaccination and live-virus challenge.

Antibodies play a pivotal role against viral infection, and maintenance of protection is dependent on plasma and memory B-cells. Understanding antigen-specific B-cell responses in cattle is essential to inform future vaccine design. We have previously defined T-cell-dependent and -independent B-cell responses in cattle, as a prelude to investigating foot-and-mouth-disease-virus (FMDV)-specific B-cell responses. In this study, we have used an FMDV O-serotype vaccination (O1-Manisa or O SKR) and live-virus challenge (FMDV O SKR) to investigate the homologous and heterologous B-cell response in cattle following both vaccination and live-virus challenge. The FMDV O-serotype vaccines were able to induce a cross-reactive plasma-cell response, specific for both O1-Manisa and O SKR, post-vaccination. Post-FMDV O SKR live-virus challenge, the heterologous O1-Manisa vaccination provided cross-protection against O SKR challenge and cross-reactive O SKR-specific plasma cells were induced. However, vaccination and live-virus challenge were not able to induce a detectable FMDV O-serotype-specific memory B-cell response in any of the cattle. The aim of new FMDV vaccines should be to induce memory responses and increased duration of immunity in cattle.

Clinical and virological dynamics of a serotype O 2010 South East Asia lineage foot-and-mouth disease virus in sheep using natural and simulated natural inoculation and exposure systems.

Within-host infection dynamics of a recent field isolate of foot-and-mouth disease virus (FMDV), serotype O, topotype South East Asia, lineage Myamar'98 were evaluated in sheep using four different systems for virus exposure. Two novel, simulated natural, inoculation systems consisting of intra-nasopharyngeal (INP) deposition and aerosol inoculation were evaluated in comparison with two conventional systems: coronary band inoculation and direct contact exposure. All four exposure systems were efficient in generating consistently severe, generalized FMD with synchronous clinical characteristics within exposure groups, indicating that this Myanmar98 strain is highly virulent in sheep. Clinical and virological dynamics were similarly rapid following INP- and coronary band inoculation, with both systems leading to significantly earlier detection of virus shedding when compared to aerosol inoculation and contact exposure. The data presented herein support application of the two optimized simulated natural inoculation systems as valid alternatives to conventionally used exposure systems for studies of FMDV pathogenesis and vaccinology in sheep. Furthermore, the data suggest that targeted exposure of the ovine pharynx is highly efficient for generating consistent FMDV infection, which supports critical involvement of this anatomic region as a site of primary virus replication in sheep.

Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B.

An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection.

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency, emergency vaccine.

In 2009-2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD50) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n=8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24h prior to contact with the vaccinated animals, or unvaccinated controls (n=4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.

Emergence of antigenic variants with in serotype A foot and mouth disease virus in India and evaluation of a new vaccine candidate panel.

Emergence of genetically and antigenically divergent lineages/genotypes and poor intergenotypic antigenic coverage is a major concern in serotype A foot-and-mouth-disease virus (FMDV) in India. In 2009, to cover antigenic diversity emerged in serotype A virus field isolates, IND40/2000 was selected as the new vaccine strain for incorporation in the trivalent FMD vaccine formulation used in India. Although current vaccine strain (IND40/2000) covers most isolates antigenically, a few VP3(59)-deletion group isolates showed low r-value in routine vaccine matching exercise. The VP3(59)-deletion group within genotype 18 emerged first in late part of 2002 and in 2007 causing outbreaks along with non-deletion isolates of the same genotype. In case of emergence or re-emergence of more antigenically divergent isolates in future, a need for a new vaccine candidate to cover maximum isolates of both deletion and non-deletion group may arise. Four alternate candidate vaccine strains (IND281/2003, IND195/2007, IND360/2007 and IND123/2008) were selected based on set criteria and antigenic relationships with field isolates sampled between 2002 and 2009 were analyzed using a micro-neutralization test. Phylogenetic analysis based on capsid region of serotype A isolates revealed existence of two broad distinct clusters (VP3(59)-deletion and non-deletion group) within genotype 18. The VP3(59)-deletion group has diversified genetically with time giving rise to three different sub-lineages (clade18a, 18b and 18c). The present study indicates that the virus candidates IND281/2003 (VP3(59)-deletion group) and IND195/2007 (non-deletion group) can be used as an adjunct or alternative strain to currently used vaccine strain IND40/2000 in case of emergence of more antigenically divergent isolates in future.

Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR.

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.

Serotyping of foot-and-mouth disease virus by antigen capture-ELISA using monoclonal antibodies and chicken IgY.

Laboratory detection of specific foot-and-mouth disease virus (FMDV) is routinely carried out by ELISA and RT-PCR. Identification and serotyping of FMDV by ELISA requires polyclonal antibodies raised in rabbits and guinea pigs. The polyclonal antibodies have certain disadvantages such as batch to batch variation, inconsistent yields of antibodies and limited quantity of serum obtained from individual animals. This paper describes a method wherein monoclonal antibodies and chicken IgY were used in an antigen capture-ELISA for serotyping of thirty tongue epithelial samples and sixty tissue culture fluids. The results were compared with the routine antigen detection ELISA. The present study indicated that monoclonal antibodies and chicken IgY can substitute conventional polyclonal antibodies for routine serotyping of FMDV.