Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe, and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT, and firstly determined and analyzed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0% to 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0% to 89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT-PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.
The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrheoa virus (PEDV). To that, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0 days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five month later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralisation test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14 days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection although the duration of the viral shedding and the total load of virus shed was significantly lower for previously challenged pigs (p<0.05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilising immunity is shorter than the productive life of pigs.
Leishmania donovani is the causative agent of historically anthroponotic visceral leishmaniasis (VL) on the Indian subcontinent (ISC). L. donovani is transmitted by the sand fly species Phlebotomus argentipes. Our collaborative group and others have shown that sand flies trapped outside in endemic villages have fed on cattle and dogs in addition to people. Domestic animals are reservoirs for L. donovani complex spp., particularly L. infantum, in other endemic areas. Multiple studies using quantitative PCR or serological detection methods have demonstrated that goats, cattle, rats and dogs were diagnostically positive for L. donovani infection or exposure in eastern Africa, Bangladesh, Nepal and India. There is a limited understanding of the extent to which L. donovani infection of domestic animals drives transmission to other animals or humans on the ISC. Evidence from other vector-borne disease elimination strategies indicated that emerging infections in domestic species hindered eradication. The predominant lesson learned from these other situations is that non-human reservoirs must be identified, controlled and/or prevented. Massive efforts are underway for VL elimination on the Indian subcontinent. Despite these herculean efforts, residual VL incidence persists. The specter of an animal reservoir complicating elimination efforts haunts the final push toward full VL control. Better understanding of L. donovani transmission on the Indian subcontinent and rigorous consideration of how non-human reservoirs alter VL ecology are critical to sustain elimination goals.
Current results do not provide conclusive evidence on the effect of BCG vaccination on COVID-19 alone or in combination with other factors. To address this limitation, in this study we used a citizen science initiative on the COVID-19 pandemic to collect data worldwide during October 2-30, 2020 (1,233 individuals) in a structured way for analyzing factors and characteristics of affected individuals in relation to BCG vaccination. For the first time, the results of our study suggested that vaccination with BCG may increase the risk for COVID-19 at certain age, particularly in individuals vaccinated at childhood. A reasonable explanation for this effect is the activation of certain innate immunity mechanisms associated with inflammatory reactions, which should be considered when analyzing the risks associated with this global pandemic. These factors should be considered when analyzing the risks associated with this global pandemic.
Level and duration of protective immunity against SARS-CoV-2 after primary infection is of crucial importance for preventive approaches. In order to provide evidence for the longevity of specific antibodies, we investigated the generation and maintenance of neutralizing antibodies of convalescent SARS-CoV-2-afflicted patients over a five month period post primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in-house enzyme-linked plaque-reduction neutralization assay. We present the successful application of an improved version of the plaque-reduction neutralization assay, which can be analyzed optometrically, significantly simplifying the interpretation of the results. Based on the results of the plaque-reduction neutralization assay, neutralizing antibodies were maintained in 85.3% of convalescent individuals without significant decay over five months. Furthermore, a positive correlation between severity of infection and neutralizing titer was shown. In conclusion, SARS-CoV-2-afflicted individuals have been proven to be able to establish and maintain neutralizing antibodies over a five months’ period after primary infection which allows to hope for long-lasting presumably protective humoral immunity after wild-type infection or even after vaccination.
African Swine Fever Virus (ASFV) is a highly contagious pathogen causing disease in pigs, commonly characterised by acute haemorrhagic fever. Prior to August 2018, African Swine Fever (ASF) had not been reported in Asia, but has since spread throughout China, Mongolia, Korea, Vietnam, Laos, Cambodia, Myanmar, the Philippines, Hong Kong, Indonesia, Timor-Leste and Papua New Guinea. Using data collated from reports of confirmed cases, we applied spatio-temporal analysis to describe ASFV spread throughout Asia, from 1 August 2018 (reported start date) to 31 December 2019. Analysis revealed a propagating epidemic of ASFV throughout Asia, with peaks corresponding to increased reports from China, Vietnam and Laos. Two clusters of reported outbreaks were found. During the epidemic, ASFV primarily spread from the North-East to the South-East: a larger, secondary cluster in the North-East represented earlier reports, whilst the smaller, primary cluster in the South-East was characterised by later reports. Significant differences in country-specific epidemics, morbidity, mortality and unit types were discovered, likely attributable to differences in prevention, surveillance and control measures. The initial number of outbreaks and enterprise size are likely predictors of the speed of spread and the effectiveness of ASFV stamping out procedures. Biosecurity methods, wild boar populations and the transportation of pigs and movement of infected fomites are discussed as likely risk factors for facilitating ASFV spread across Asia.
Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is endemic in poultry in the Middle East causing continuing outbreaks in poultry populations despite efforts to vaccinate. In the past, genotype 2.XXI (former 2.VI) was present in poultry in Egypt but has been replaced by genotype 2.VII. We investigated whether virus evolution contributed to superseding, and focused on the antigenic sites within the Heamagglutinin-Neuramindase (HN) spike protein. Full length sequences of a NDV genotype 2.VII isolate currently circulating in Egypt was compared to a genotype 2.XXI isolate that was present as co-infection with vaccine type viruses (2.II) in an historical isolate of the year 2011. Amino acid differences in the HN glycoprotein for both 2.XXI and 2.VII viruses amounted to 11,7% and 11,9 % compared to LaSota vaccine type. However, mutations within the globular head (aa 126-570), bearing relevant antigenic sites, were underrepresented (aa divergence of 8,8% and 8,1 % compared to 22,4% and 25,6% within the fragment encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1-125) for genotypes 2.XXI and 2.VII, respectively. Nevertheless, reaction patterns of HN-specific monoclonal antibodies revealed differences between vaccine type viruses and genotype 2.XXI and 2.VII viruses for specific epitopes. Accordingly, compared to Egyptian vaccine type isolates and the LaSota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes were found within the attachment protein. However, the same alterations in neutralization sensitive epitopes were present in old genotype 2.XXI as well as in newly emerged genotype 2.VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest, that factors other than antigenic differences within the HN-protein account for facilitating spread of genotype 2.VII while displacing genotype 2.XXI viruses in Egypt.
Avian influenza (AI) is a contagious disease of birds with zoonotic potential. AI virus (AIV) can infect most bird species, but clinical signs and mortality vary. Assessing the distribution and factors affecting AI incidence can direct targeted surveillance to areas at risk of disease outbreaks, or help identify disease hotspots or areas with inadequate surveillance. Using virus surveillance data from passive and active AIV wild bird surveillance, 20062020, we investigated the association between a range of landscape factors and game bird release and the presence of AIV. Furthermore, we assessed potential bias in the passive AIV surveillance data submitted by the public, via factors related to public accessibility. Lastly, we tested the AIV data for possible hot and cold spots within Denmark. The passive surveillance data was biased regarding accessibility to areas (distance to roads, cities and coast) compared to random locations within Denmark. We found significant effects of variables related to coast, wetlands and cities for the passive and active AIV surveillance data (P< 0.01), but found no significant effect of game bird release. We used these variables to predict the risk of AIV presence throughout Denmark, and found high-risk areas concentrated along the coast and fjords. For both passive and active surveillance data, low-risk clusters were mainly seen in Jutland and northern Zealand, whereas high-risk clusters were found in Jutland, Zealand, Funen and the southern Isles such as Lolland and Falster. Our results suggest that landscape affects AIV presence, as coastal areas and wetlands attract waterfowl and migrating birds and therefore might increase the potential for AIV transmission. These findings have enabled us to create risk maps of AIV incidence in wild birds and pinpoint high-risk clusters within Denmark. This will aid targeted surveillance efforts within Denmark and potentially aid in planning the location of future poultry farms.
Since 2014, highly pathogenic avian influenza H5N6 viruses have been responsible for outbreaks in poultry. In this study, four H5N6 virus strains were isolated from fecal samples of sick white ducks and dead chickens in Shandong in 2019. These H5N6 viruses were triple-reassortant viruses that have not been previously characterized. Their HA genes were derived from the H5 viruses and were closely related to the vaccine strain Re-11. Their NA genes all fell into the N6-like lineage and the internal gene were derived from H5N1 and H9N2 viruses. They all showed high pathogenicity in mice and caused lethal infection with high rates of transmission in chickens. Moreover, the SPF chickens inoculated with the current used vaccine in China were completely protected from these four H5N6 viruses. Our study indicated the necessity of continued surveillance for H5 IAV and the importance of timely update of vaccine strains in poultry industry.
An 11 years-old male mixed-breed cat, with exclusively indoor life, presented 3 cough episodes after the owners tested positive by RT-PCR for SARS-CoV-2. The house is inhabited by 5 people (3 adults and 2 children), and 2 of the adults have shown mild symptoms associated with throat discomfort. The cat was vaccinated, had no history of any previous disease, and tested negative for Feline Coronavirus (FeCoV), Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV). Rectal sample collected from the cat was positive for SARS-CoV-2 by RT-PCR. Viral genome sequences recovered from human and cat samples showed an average 99.4% sequence identity. This is the first report of genome sequences of SARS-CoV-2 recovered from a cat and its owner in Latin America.
PRV1 was first detected in deceased pigs from Hong Kong in 2013. It has since been detected in the USA, Chile and most recently in Hungary. Information on the pathogenicity and global spread is sparse, however it has been speculated to play a role in the porcine respiratory disease complex. In an effort to investigate the porcine virome, we screened 53 pig samples from 29 farms using SMg within the Dutch/German border region. In five farms we detected PRV1. qPCR confirmed the presence of the virus in 2 of these farms and found an additional 6 positive farms. Phylogenetic analysis found the closest match to the first detected PRV1 strain in Hong Kong. The Dutch/German region represents a major area of pig farming within Europe and could provide important information on the characterization and circulation of porcine viruses, such as PRV1. Together with the recent detection of PRV1 in Hungary, these findings suggest widespread of PRV1 in Central Europe, highlighting the need for further research on persistence, pathogenicity and transmission in Europe.
Advanced and accurate forecasting of COVID-19 cases plays a crucial role in planning and supplying resources effectively. Artificial Intelligence (AI) techniques have proved its capability in time series forecasting of the non-linear problems. In the present study, the relationship between weather factor and COVID-19 cases was assessed and also developed a forecasting model using long short term memory (LSTM), a deep learning model. The study found that the specific humidity has a strong positive correlation, whereas there is a negative correlation with maximum temperature and positive correlation with minimum temperature was observed in various geographic locations of India. The weather data and COVID-19 confirmed cases data (1st April-30th June 2020) was used to optimize univariate and multivariate LSTM time series forecast models. The optimized models were utilized to forecast the COVID-19 cases for the period 1st July 2020 to 31st July 2020 with 1 to 14 days of lead time. The results showed that the univariate LSTM model was reasonably good for the short term (1day lead) forecast of COVID-19 cases (relative error < 20%). Moreover, the multivariate LSTM model improved the medium-range forecast skill (1-7days) after including the weather factors. The study observed that the specific humidity played a crucial role in improving the forecast skill majorly in the West and northwest region of India. Similarly, the temperature played a significant role in model enhancement in the Southern and Eastern regions of India.
In the present study, the highly pathogenic bovine Deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real time PCR (qPCR). Overall, ddPCR detected BPV E5 DNA in ~61.3% of the blood samples examined, while real time qPCR revealed the virus in ~10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR revealed a single BPV genotype in ~59.2% and multiple genotypes in ~40.8% of goats harboring BPV DNA, while real time qPCR detected single genotypes in ~17% and multiple genotypes in ~1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes, and six (~14%) four genotypes. In contrast, real time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that the ddPCR technique has much higher sensitivity and specificity in the detection of these viruses, and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly identiﬁed swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets, leading to significant economic losses to the swine industry. Currently there are no suitable serological method to assess the infection of SADV-CoV and effectiveness of vaccines, making an urgent need to exploit effect enzyme-linked immunosorbent assay (ELISA) to compensate for this deficiency. In this study, an indirect ELISA (S-iELISA) based on recombinant spike (S) protein expressed in Baculovirus was developed and evaluated. The reaction conditions of S-iELISA were optimized and cut-off value determined as 0.3711 by analyzing OD450nm values of 40 SADS-CoV-negative sera confirmed by immunoinfluscent assay (IFA) and Western Blot. The coefficients of variation of 6 positive sera within and between runs of S-iELISA were both less than 10% and cross-reactivity assays demonstrated that S-iELISA was non-cross-reactive with other swine viruses’ sera. Furthermore, the overall coincidence rate between IFA and S-iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with 7 different OD450nm value sera showed that the OD450nm values tested by S-iELISA are positive correlated with virus neutralization. Finally, a total of 300 pig field serum samples were tested by S-iELISA and commercial kits of other swine enteroviruses showed that the IgG-positive for SADS-CoV, TGEV, PDCoV and PEDV were 81.7%, 54%, 65.3%, 6%, respectively. The results suggest this S-iELISA is specific, sensitive, repeatable and can be applied for vaccines evaluation and detection the SADS-CoV infection in swine industry.
Rabbit hemorrhagic disease virus 2 (RHDV2) is a newly emerging Lagovirus belonging to the family Caliciviridae. After its first discovery in 2010 in France, this highly pathogenic virus rapidly spread to neighboring countries and has become the dominant strain, replacing the classical RHDV1 strains. RHDV2 was first reported in North America in 2016 in Mont-Joli, Quebec, Canada and it was reported again in 2018 and 2019 on Vancouver, Island and the southeast mainland of British Columbia (BC). The whole genome sequence of the RHDV2 Quebec isolate resembled the RHDV-N11 isolate from Navarra, Spain identified in 2011 with 97% identity. The epidemiological investigation involved three hobby farms and one personal residence. In December and February 2018, high mortality was reported in first a private feral rabbit refuge and then, a large colony of feral rabbits on the Vancouver Island University Campus, Nanaimo, BC. The virus responsible showed only 93% identity to the Quebec RHDV2 isolate at the nucleotide level. Additional cases of RHDV2 on Vancouver Island and on the BC mainland affecting feral, captive domestic and commercial rabbits were reported subsequently. Vaccination was recommended to control the outbreak and an inactivated bivalent vaccine was made available to the private veterinary practices. In June 2019 an isolated RHDV2 outbreak was reported in an apartment building in Vancouver, BC. This virus showed only 97% identity to the RHDV2 isolate responsible for the BC outbreak in 2018 at the nucleotide level suggesting that it was an independent incursion. In October 2020, there are reports of partial recovery of the feral population in Nanaimo and to date there are no confirmed deaths of native rabbit species in BC.
In Southern Africa, the African Swine Fever (ASF) sylvatic cycle presents a permanent threat for the development of the pig farming industry. Warthogs (Phacochoerus africanus) and bushpigs (Potamochoerus larvatus), wild reservoirs of ASF are present in Ndumo Game Reserve (NGR), located in Northern KwaZulu Natal Province in South Africa and within 30 km of the locations of endemic ASF outbreaks in Mozambique where sylvatic disease transmission has been implicated. To assess if wild suids represent a risk of ASF virus spillover to domestic pigs in the neighbouring community, transect counts and fence patrols were conducted and camera traps were deployed in NGR to estimate wild suid abundance and incursions outside NGR boundaries. We searched for Ornithodoros ticks in 35 warthog burrows distributed across different NGR areas. Pig farmers (n=254) surrounding Mathenjwa Community were interviewed to gather information on interactions between domestic and wild suids and the occurrence of ASF. We conclude that NGR has established populations of bushpigs and warthogs, estimated at 5 and 3-5 individuals/km2 respectively. Both species move out of the reserve regularly (15.4 warthogs/day and 6.35 bushpigs/day), with a significant increase of movements during the dry season. Some farmers observed warthogs and bushpigs as far as 8 and 19 km from the reserve respectively, but no direct wild-domestic suids interactions nor any ASF outbreaks were reported. In addition, no soft ticks were found among the 35 warthog burrows. The absence of ticks in warthog burrows from NGR and the absence of reported outbreaks and familiarity with ASF in the surrounding farming area, suggest that a sylvatic cycle of ASF is at present unlikely in NGR. However, further research should be undertaken to confirm this by surveying a larger number of warthog burrows and monitoring potential antibodies in warthogs from NGR and domestic pigs in the neighbouring community.
Cryptosporidium parvum is a major zoonotic pathogen responsible for outbreaks of severe diarrhea in humans and calves. Almost all investigations of cryptosporidiosis outbreaks caused by C. parvum have focused on its IIa subtype family in industrialized nations. From December 2018 to April 2019, approximately 200 neonatal calves on a large cattle farm in Hebei Province, China presented watery diarrhea and over 40 died. To investigate the cause of the outbreak, 179 and 223 fecal specimens were collected during and after the diarrhea outbreak from the farm, including 40 and 56 from neonatal calves, respectively. Among them, 18 fecal specimens from ill calves during the peak of the outbreak were analyzed for four common enteric pathogens using enzymatic immunoassay (EIA), 75 additional specimens from neonatal calves were tested for rotavirus by EIA, and all specimens were analyzed for Cryptosporidium spp. using PCR and sequencing techniques. Of the initial 18 specimens from sick calves, ten were positive for C. parvum, five for rotavirus, and one for coronavirus. The overall prevalence of rotavirus in neonatal calves was 20.0% (15/75), with no significant differences during (21.6% or 8/37) and after (18.4% or 7/38) the outbreak. In contrast, the prevalence of C. parvum was significantly higher during the outbreak (60.0%, 24/40) than after the outbreak (30.4%, 17/56). C. parvum infection was associated with the occurrence of watery diarrhea in neonatal calves (odds ratio = 11.19), while no association was observed between C. bovis infection and diarrhea. All C. parvum isolates were identified as subtype IIdA20G1. Older animals were infected with C. bovis, C. ryanae, C. occultus, and C. andersoni. This is one of the few reports of outbreaks of severe diarrhea caused by C. parvum IId subtypes in calves. More attention should be directed toward preventing the dissemination of C. parvum in China.
African swine fever (ASF) has spread across many countries in Europe since the introduction into Georgia in 2007. We report here on the first cases of ASF in wild boar detected in Germany close to the border with Poland. In addition to the constant risk of ASF virus (ASFV) spread through human activities, movements of infected wild boar also represent a route of introduction. Since ASF emerged in Western Poland in November 2019, surveillance efforts, in particular examination of wild boar found dead, were intensified in the regions of Germany bordering with Poland. The first case of ASF in wild boar in Germany was therefore detected by passive surveillance and confirmed on 10th September 2020. By 24th September 2020, 32 cases were recorded. Testing of samples from tissues of carcasses in different stages of decomposition yielded cycle threshold values from 18 to 36 in the OIE-recommended PCR which were comparable between the regional and national reference laboratory. Blood swabs yielded reliable results, indicating that the method is suitable also under outbreak conditions. Phylogenetic analysis of the ASFV whole-genome sequence generated from material of the first carcass detected in Germany, revealed that it groups with ASFV genotype II including all sequences from Eastern Europe, Asia and Belgium. However, some genetic markers including a 14 bp tandem repeat duplication in the O174L gene were confirmed that have so far been detected only in sequences from Poland (including Western Poland). Epidemiological investigations that include estimated postmortem intervals of wild boar carcasses of infected animals suggest that ASFV had been introduced into Germany in the first half of July 2020 or even earlier.