Virus Isolation Research Articles

Isolation and Identification of Lumpy Skin Disease Virus.

Lumpy skin disease virus (LSDV), a member of the capripoxvirus genus, induces lumpy skin disease (LSD) in cattle and buffalo populations. The initial documentation of LSD dates back to 1929 in Zambia, with subsequent rapid dissemination within the cattle community in Africa and Asia. Described as the "smallpox" equivalent in cattle, LSD manifests through clinical features such as fever, extensive cutaneous nodules, body wasting, lymph node enlargement, and skin edema. Isolation of LSDV constitutes an important step in evaluating virulence and implementing disease control measures. Skin samples obtained from afflicted cattle serve as the primary source for LSDV isolation due to its tropism for epidermal cells. Other tissues, including blood, lung, spleen, and saliva samples, are also employed for viral isolation. This chapter presents protocols for the convenient isolation of LSDV, relying on the plaque formation phenotype and utilizing MDBK cells. Emphasis is placed on stringent quality control methodologies to identify isolated viruses through multiple techniques, facilitating further investigations such as the in vitro growth of isolated LSDV.

Construction and Isolation of Recombinant Vaccinia Virus by Homologous Recombination Using Fluorescent Protein Markers.

Genetic modification of vaccinia virus (VACV) is a fundamental and valuable research technique in elucidating the function of VACV genes, as well as the development as vaccine vectors for other infectious diseases, oncolytic therapeutics for cancers, and protein expression systems in mammalian cells. Because of the large size of poxvirus genome and noninfectious feature of the naked viral DNA, construction of recombinant VACV relies on intracellular homologous recombination between transfected DNA and replicating viral DNA in infected cells occurred in VACV infected cells. The efficiency of homologous recombination event for vaccinia virus is relatively low, and recombinant viruses only account for 0.1% of progeny viruses. Therefore, fluorescent protein markers are often included in the transfected DNA to facilitate the selection and screening of recombined viruses. Here we provide a detailed procedure for the design, generation, isolation, and detection of recombinant VACV by homologous recombination using fluorescent protein markers.

A new isolate of mungbean yellow mosaic India virus in Vigna mungo L. reported from a Dayalbagh field, Agra.

Black gram (Vigna mungo L.) plants showing yellow mosaic symptoms during 2019-2022 crop seasons were collected randomly from a Dayalbagh field, Agra Region of Uttar Pradesh, India. Total genomic DNA was isolated from the infected leaf samples by the Cetyltrimethylammonium bromide (CTAB) method and subjected to PCR. After viral confirmation, the viral genome was amplified by rolling circle amplification following the standard protocol. The DNA A and DNA B subgenomes were cloned individually as a PstI and BamHI fragment in the pUC18 vector. Positive clones were subjected to DNA sequencing. The results revealed that DNA A and DNA B show the closest nucleotide identity with "mungbean yellow mosaic India virus-[Mungbean], DNA-A, the complete sequence" (GeneBank Accession No AF416742.1) with 98.14% identity, and "mungbean yellow mosaic India virus isolate Mu1-Dholi segment DNA-B, the complete sequence" (GeneBank Accession No MW814723.1) with 97.94% identity, respectively. The new isolate of mungbean yellow mosaic India virus (MYMIV) shows sequence similarity with the coat protein gene of various strains of MYMIV. In the new isolate of MYMIV, a point mutation wasobserved at the 2036th nucleotide of DNA B, which disrupts the reading frame to introduce a stop codon and thus leading to a decrease in the size of the movement protein gene. In the present study we are reporting the whole genome sequence of the MYMIV Dayalbagh isolate for the first time.

Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by inhibiting cathepsin L and furin

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19) pandemic, posing serious threats to global health. Effective broad-spectrum antiviral drugs for the treatment of COVID-19 are not sufficiently available. In the present study, we investigated the antiviral activity of the natural lignan diphyllin (PubChem CID 100492) against different SARS-CoV-2 variants and explored the underlying molecular mechanisms. We found that diphyllin dose-dependently inhibits the SARS-CoV-2 spike (S)-mediated entry into different types of cells. The potent inhibition was evident against spike proteins derived from the original SARS-CoV-2 and from variants of concern such as Alpha, Beta, Delta or Omicron. Accordingly, diphyllin also significantly inhibited the in vitro infection of a clinical SARS-CoV-2 virus isolate. Mechanistically, diphyllin simultaneously inhibited the endosomal entry of SARS-CoV-2 by neutralizing the endosomal acidification and reducing the activity of the cysteine protease cathepsin L (CTSL) as well as S-meditated cell surface entry by impairing furin activity. Collectively, our findings establish diphyllin as novel inhibitor of CTSL and furin proteases, resulting in a double-pronged attack on SARS-CoV-2 entry along endosomal as well as cell surface routes. Therefore, diphyllin has the potential to be advanced as an inhibitor of SARS-CoV-2 entry.

Detection of Hepatitis C Virus Infection from Patient Sera in Cell Culture Using Semi-Automated Image Analysis

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed to optimize cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81, and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wild-type isolates. RT-qPCR-based quantification of HCV infections using patient sera suffered from a high background in the daclatasvir-treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis, the method revealed no background in daclatasvir-treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation between serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR-based quantification of viral genomes.

A new record of avian reovirus genogroup clusters isolated and molecularly characterized in chickens in Egypt

BackgroundThe global poultry industry has been experiencing increasing condemnation at slaughterhouses since 2011, due to the emergence of avian reovirus (ARV) variant strains. Recently seven ARV genogroup clusters were identified based on the σC sequence of the S1 segment that was used as a genetic marker to characterize and classify ARV.ResultsAmong ninety-seven suspected broilers, breeders, and commercial grandparent’s homogenates, ARV was PCR confirmed in thirty-five (36%). Samples were collected from different governorates in Egypt, including Giza–Ismailia–Sharqia–Menoufia, and Behira, between 2020 and 2023. A viral isolation trial was performed on weak positive samples, successfully isolating sixteen samples on the LMH cell line forming the characteristic giant cells of ARV infection. Partial sequencing and phylogenetic analysis of the σC gene of ten ARV PCR positive samples revealed the circulation of ARV genogroup clusters 1, 2, 4, and 5, eight samples being distinguished from commercially available vaccine strains with a low percentage of nucleotides and amino acid identities.ConclusionsThis research demonstrated that the ARV isolates were distinct compared to commercially available vaccine strains based on partial σC sequence. Detection, isolation, and classification of ARV as genogroups 1, 2, 4, and 5 are recorded. A vigilant surveillance to evaluate the ARV situation in Egypt is urgent. We recommend re-evaluating the ARV commercial vaccines by challenging them with circulating Egyptian field isolates, as well as their cross-protection. In addition, the development of autologous vaccines from both classical and variant strains to provide optimal protection.

4D-DIA-Based Quantitative Proteomic Analysis Reveals the Involvement of TRPV2 Protein in Duck Tembusu Virus Replication

Duck Tembusu virus (DTMUV), a novel positive-sense RNA virus, has caused significant economic losses in the poultry industry of Eastern and Southeast Asia since its outbreak in 2010. Furthermore, the rapid transmission and potential zoonotic nature of DTMUV pose a threat to public health safety. In this study, a 4D-DIA quantitative proteomics approach was employed to identify differentially expressed cellular proteins in DTMUV-infected DF-1 cells, which are routinely used for virus isolation and identification for DTMUV, as well as the development of vaccines against other poultry viruses. One hundred fifty-seven differentially expressed cellular proteins were identified, including 84 upregulated and 73 downregulated proteins at 48 h post-infection, among which CXCL8, DDX3X, and TRPV2 may play crucial roles in viral propagation. Notably, for the upregulated protein TRPV2, the DTMUV replication was inhibited in TRPV2-low-expressing DF-1 cells. In summary, our research represents the application of 4D-DIA quantitative proteomics to analyze the proteomic landscape of DTMUV-infected poultry cells. These findings may provide valuable insights into understanding the interaction mechanism between DTMUV and poultry cells, as well as the identification of disease-resistant host factors in poultry breeding research.

Cache Valley virus isolation from a horse in Veracruz State, Mexico

Cache Valley virus (CVV) was isolated from a clinically presenting horse in the Mexican Gulf region. CVV is linked to neuroinvasive disease in humans and fetal demise and defects in sheep. This is the first association between CVV and disease in horses. Implications for public and veterinary health are discussed.

Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities at Antarctic stations

BackgroundThe Antarctic environment is susceptible to the introduction of non-native species due to its unique ecosystem, which has evolved under geographical isolation and extreme climatic conditions over an extended period. The recent introduction of the non-native winter crane fly, Trichocera maculipennis, to maritime Antarctica may pose a potential threat to the Antarctic ecosystem. In this study, we evaluated the possibility of the mechanical transmission of viruses by T. maculipennis.MethodsWe assessed the potential for the mechanical transmission of viruses using next-generation sequencing (NGS), quantitative PCR (qPCR), and virus isolation methods from T. maculipennis (Tm)-related samples (Tm body-wash fluid and Tm body-ground samples) collected from habitats and sewage treatment facilities located at three research stations in Antarctica.ResultsVirome analysis detected the genomic fragments of human adenovirus (AdV) and human endogenous retrovirus (HERV) in Tm-related samples. These viruses are commonly found in human feces. In addition, plant viruses, such as pepper mild mottle virus (PMMoV) and cucumber green mottle mosaic virus (CGMMV), both known indicators of enteric viruses, were identified in all Tm-related samples, likely originating from wastewater. However, the low quantities of AdV and HERV genomes detected in Tm-related samples through qPCR, coupled with the non-viability of AdV in virus isolation tests, indicate that T. maculipennis has limited potential for mechanical transmission under the conditions in the studies.ConclusionsOur study represents the first evaluation of the potential risk of non-native species serving as vectors for viral pathogens in Antarctica. Although the viruses detected were in relatively low quantities and non-viable, this study highlights the importance of further evaluating the risks associated with non-native species, particularly as the likelihood of their introduction increases to Antarctica due to climate change and increased human activity.Graphical abstract

The Growing Phenomenon of ‘Frozen’ Virus Genome Sequences and Their Likely Origin in Research Facility Escapes

‘Frozen’ virus genome sequences are sampled from outbreaks and have unusually low sequence divergence when compared to genome sequences from historical strains. A growing number of ‘frozen’ virus genome sequences are being reported as virus genome sequencing becomes more common. Examples of ‘frozen’ sequences include the 1977 H1N1 ‘Russian’ flu; Venezuelan Equine Encephalitis Virus from Venezuela and Colombia in 1995; E71 sequences from a Hand, Foot and Mouth outbreak in 2007–2009 in China; and a polio strain isolated in 2014 from Anhui, China. The origin of these ‘frozen’ sequences has been attributed to escapes from research facilities and often appears to be associated with vaccine work. Consequently, a new paradigm for pathogen emergence appears in operation, that involves laboratory research or vaccine production which utilizes ‘live’ virus isolates of historical strains. The accidental release and re-emergence of such strains are straightforward to detect from their genome sequences and should spur the routine sequencing and publication of all known pathogenic viral strains undergoing experimentation, or being used for vaccine manufacture, in order to facilitate tracing. However, it is noted that novel pathogenic viruses accidentally released into the population from research facilities are harder to detect if their sequence has first not been made public, which should prompt the routine sequencing and reporting of all novel pathogenic viruses before experimentation.

Detection and Molecular Characterization of Avian Encephalomyelitis Virus in India: A Preliminary Report

Tremovirus A, previously recognized as Avian encephalomyelitis virus (AEV) is, one of the viruses that have been reported to be associated with nervous and enteric disorders in young birds. In Kerala, there has been an increase in the cases of nervous and enteric diseases in chickens. The detection and characterization of other viruses associated with nervous and enteric viruses in chickens has been established in Kerala. While there have been reports of nervous and enteric diseases in chickens in the region, no previous research had focused on AEV as the causative agent. A total of 50 samples were tested to detect AEV by reverse-transcriptase polymerase chain reaction (RT-PCR) targeting the 5' untranslated region (5'UTR) of AEV. Of the samples tested, one (2%) was found to be positive for the virus. The most immunogenic protein of AEV is VP1 protein forming the major capsid protein. The VP1gene segment of the detected virus isolate was amplified and molecular characterization was done. The phylogenetic analysis of the VP1 gene sequence revealed that the virus was identical to AEV viruses detected in Hungary, China, and Hong Kong (64% similarity). The current isolate showed a nonsynonymous mutation of I184V which was noticed only in Hungary isolate. The results of the study indicate that AEV is rarely present in chickens of Kerala, India, and that its occurrence is very low. Since the virus is capable of inducing nervous signs, mortality, and economic losses, measures are to be taken to control the spread of the virus in chickens in Kerala, India.

Field-Based cDNA-Biosensor for Accurate Detection of Canine Distemper Virus in Tissue Samples.

Canine distemper, a viral disease with a global impact on various animals including dogs, foxes, wolves, lions, and leopards, requires early diagnosis for effective treatment and outbreak control. Common laboratory methods, such as enzyme-linked immunosorbent assay, polymerase chain reaction, and viral isolation, face challenges such as extended turnaround times, high costs, and the expertise required. This study has developed a field-based biosensor for detecting the canine distemper virus (CDV), utilising a screen-printed carbon electrode (SPCE) and a computer-assisted portable potentiostat. A 30-mer oligonucleotide capture probe, designed using Primer3 Plus software version 3.3.0, detected hybridisation with the CDV complementary strand through electrochemical analysis via differential pulsed voltammetry. The developed biosensor exhibited good linearity in quantifying the target analyte concentration (0.1 to 12.8µM), with a detection limit of 0.05µM, indicating high sensitivity. Specificity tests using complementary and non-complementary sequences confirmed the biosensor's accuracy. The electrode can be reused up to eight times with a residual capacity of 93.72 ± 5.45% after regeneration using a 50mM NaOH solution. The developed biosensor was also used to detect CDV in biological samples after RNA extraction and amplification. Results from the biosensor aligned with those from reverse transcriptase polymerase chain reaction (RT-PCR) findings, showing 100% agreement. These findings support the potential development of a field-deployable portable device for effectively diagnosing canine distemper in biological samples.

Isolation, Identification, and Molecular Evolutionary Analysis of Infectious Hematopoietic Necrosis Virus (IHNV) from Farmed Rainbow Trout in Southwest China

IHNV troubled the salmon breeding industry in China for years, and it often causes large-scale deaths. To study the disease characteristics of IHNV and its epidemiology in China， this study isolated and sequenced the Whole genome of an IHNV strain (IHNV-SCCD) (GenBank No： OQ801357) from farmed diseased rainbow trout in Chengdu and compared 99 IHNV strains from China by mega.11. The IHNV-SCCD strain was isolated by using EPC cells. The complete genome sequence was determined and deposited in the NCBI database. Phylogenetic analysis showed that IHNV-SCCD belongs to the typical J gene group, recently with Sichuan strains. Except for BjLL strain and LN12-17 strain, the remaining 98 strains followed a single evolutionary path. Note that IHNV in Liaoning province is the fastest evolutionary. In addition to the geographical differences, the Xinjiang strains were closely related to the Yunnan strains, and some strains from Beijing were closely related to some strains from Sichuan. There were two evolutionary branches in Gansu and Qinghai; QH17, isolated from Qinghai, was most closely related to SX1704 from Shaanxi. These results suggest that the prevalence of IHNV in China was extremely wide, and the evolution rate was fast. It is necessary to strengthen the monitoring and prevention methods.

Validation of a reduction in time for avian influenza virus isolation using specific pathogen-free embryonated chicken eggs.

The international gold standard for avian influenza virus (AIV) diagnosis is virus isolation (VI) in specific pathogen-free embryonated chicken eggs (ECEs). AIV isolation typically involves a 6-day turnaround, during which premises under suspicion for notifiable AIV infection are held under restriction regardless of molecular diagnoses, often with significant welfare implications. A reduction in time for negation by VI was investigated following experimental inoculation of AIV from known-positive original clinical material into ECEs. VI data derived from more than 600 case investigations from epizootics of high-pathogenicity AIV (HPAIV) in Great Britain since 2016 and from low-pathogenicity AIV (LPAIV) cases in Great Britain since 2014 were examined to support a reduction in test timing using alternative regimens. HPAIVs were isolated during the first passage, and for LPAIV VI, the second passage could be reduced to 2 days. Power analysis showed that the benefit of reducing the number of days outweighed the risk of missing a positive isolate. Limited data were available from experimental inoculations. This truncated methodology, which enables an earlier release of restrictions, may substantially ease the economic implications of restriction. It could also reduce bird welfare implications and improve international standards without loss of test performance.

Genome sequences of recombinant African swine fever virus isolated from domestic pigs in Vietnam

ABSTRACT African swine fever is a highly contagious and devastating disease of pigs. Here, we present the draft genome assemblies of three Vietnamese African s wine fever virus (ASFV) isolates: rASF1/2-avac02, rASF1/2-avac03, and rASF1/2-avac07. All isolates were closely related to the Chinese recombinant ASFV strains of genotypes I and II.

Characterization of bean common mosaic virus isolates infecting three leguminous bean crops from South and Southeast Asia.

Bean common mosaic virus (BCMV) is causing economically important diseases in leguminous crops worldwide. In this study, BCMV isolates from country bean (CB, Lablab purpureus), yard-long bean (YLB, Vigna unguiculata) and rajma bean (RB, Phaseolus vulgaris) collected from Bangladesh, Nepal and Cambodia were characterized. Samples tested positive for BCMV in serological assays were subjected to high-throughput sequencing to generate near full-length genome sequences. In pair-wise comparisons of the polyprotein open reading frame, thirteen BCMV isolates from Bangladesh, Cambodia, and Nepal showed sequence identity of 92.1 to 98.8% at the nucleotide and 94.2 to 99% at the amino acid level among themselves and with corresponding sequences of BCMV reported previously. In phylogenetic analyses using the global BCMV sequences, they segregated into five distinct lineages, with RB isolates from Nepal clustering with US1/NL1-clade of common bean isolates from different countries, YLB isolates aligning with blackeye cowpea strain sequences reported from China, and CB isolates from Nepal and Bangladesh clustering with soybean isolates from China. One YLB isolate from Nepal was identified as a putative recombinant. None of the BCMV sequences aligned with isolates representing the RU1 or PStV clades. In grow-out tests, seed samples from local markets showed 14.3 to 38.1% transmission efficiency rate of BCMV with CB seed lots and from 9.5% to 33.3% with YLB seed lots.

Isolation and phylogenetic analysis of camel contagious ecthyma virus in Morocco

Camel contagious ecthyma is a highly infectious viral skin disease that affects camels and causes economic losses. This study reports the isolation and the phylogenetic analysis of contagious ecthyma virus among camels in Morocco. The disease was detected in four among fifteen camels with severe papules on the lips and nares. Samples of skin crusts were collected and pooled for virus isolation and titration, PCR testing, and histopathological examination. PCR was used to amplify the B2L gene and the resulting product was sequenced and analyzed genetically. The study's findings indicate the presence of characteristic microscopic changes of poxvirus infection in the examined tissues and the virus was isolated on two cells (lambs testis and Vero) and showed distinct growth patterns. The virus grew rapidly on TA cells and delayed growth on Vero cells.The third passage showed cytopathic effects characterized by cell aggregation. Sequence analysis of the B2L gene revealed 100 % similarity to the camel contagious Ecthyma virus isolated from Ethiopia. The camel virus isolates can be classified into two genetic clades according to the B2L gene sequence: the Asian lineage, which includes isolates from Saudi Arabia, Bahrain, and India, and the African lineage, which includes isolates from the Sudan.In conclusion, this is the first instance of the camel contagious ecthyma virus being identified in North Africa in a herd of camels following exposure to stress. Moreover, the progression of the disease was closely monitored from onset to recovery in a setting without the bacterial complication often observed in the field. The virus exhibited opportunistic behavior, exploiting the stress response. Further studies are warranted to evaluate the pathogenicity of the virus in camels and to genetically characterize the circulating virus from different regions. This will be highly beneficial in the development of an appropriate vaccine.

Abstract C035: Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms

Abstract Despite the ability to induce strong clinical responses, therapies that target mutational drivers of oncogenic signaling are not curative in advanced cancers, as a subset of cancer cells is capable of surviving therapy and evolving resistance. While therapy resistance is commonly viewed as a cell-intrinsic phenomenon, multiple factors produced by tumors can also confer strong therapy resistance. In contrast to the detailed elucidation of molecular mediators of cell-intrinsic and stroma-mediated (SM) resistance, the relative importance of cell-intrinsic and SM resistance to in vivo therapeutic responses remains poorly defined. To address this gap of knowledge, we used a well-characterized experimental model of ALK+ lung cancer H3122 cell line that exemplifies a duality observed across common models of targetable cancers. Under standard stroma-free in vitro cultures, H3122 cells can survive under therapeutically relevant concentrations of ALK inhibitors (ALKi), eventually acquiring cell-intrinsic therapy resistance. At the same time, co-culture with stromal fibroblasts drastically reduces the sensitivity of H3122 cells to ALKi, indicating the relevance of SM resistance. To understand the impact of SM resistance on in vivo responses, we sought to identify the mechanism(s) responsible for the therapy-protective effects of stromal fibroblasts, subsequently interrogating the impact of the disruption of this mechanism on in vivo therapeutic responses. Our in vitro studies with a large panel of primary stromal fibroblast isolates identified the HGF-cMET axis as the major mechanism responsible for ALKi desensitization. Surprisingly, xenograft validation studies demonstrated a weak effect of HGF-cMET modulation. Histological analyses of tumor tissues revealed that this relative weakness is attributable to a strong, spatially limited HGF-independent component of SM resistance. Mechanistic follow-up demonstrated that the HGF-independent component of SM resistance integrates the effect of multiple well-known juxtacrine and paracrine-acting mechanisms. Whereas the multifactorial nature of stroma-mediated resistance prevented a clear assessment of its relative contribution to in vivo therapy responses, our spatial analyses indicate that SM resistance dominates the ability of tumors to avoid therapeutic elimination. Surprisingly, we found that SM resistance was also a substantial contributor to tumor relapse, indicating that in vivo therapy resistance can integrate both cell-intrinsic and cell-extrinsic effects. This duality of therapy resistance and the multifactorial underpinning of both cell-intrinsic and stroma-mediated resistance present a challenge to therapeutic strategies focused on identifying and targeting specific resistance mechanisms. Our studies suggest that this limitation can be overcome by shifting the therapeutic focus towards shared orthogonal therapeutic sensitivities of tumor cells within residual disease. Citation Format: Bina Desai, Tatiana Miti, Sandhya Prabhakaran, Daria Miroshnychenko, Menkara Henry, Viktoriya Marusyk, Chandler Gatenbee, Marilyn Bui, Jacob Scott, Philipp M. Altrock, Eric Haura, Alexander R.A. Anderson, David Basanta, Andriy Marusyk. Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C035.

In Silico Genomic Analysis of Avian Influenza Viruses Isolated From Marine Seal Colonies.

Genetically diverse avian influenza viruses (AIVs) are maintained in wild aquatic birds with increasingly frequent spillover into mammals, yet these represent a small proportion of the overall detections. The isolation of AIVs in marine mammals, including seals, has been reported sporadically over the last 45 years. Prior to 2016, all reports of AIVs detected in seals were of low-pathogenicity AIVs. In spite of this, the majority of reported AIV outbreaks caused fatal respiratory diseases, with harbor seals particularly susceptible to infection. The H5 clade 2.3.4.4b highly pathogenic AIV (HPAIV) was detected in seals for the first time in 2016. Recently, many cases of mass seal die-offs have occurred because of 2.3.4.4b HPAIV and are attributed to spillover from wild bird species. The potential for seal-to-seal transmission has been considered after the mass mortality of southern elephant seals off the coast of Argentina. Close contact between seals and wild birds, the rapid evolution of H5N1 AIVs, and the possibility of efficient mammal-to-mammal transmission are increasing concerns due to the potential for the establishment of a marine mammal reservoir and public health risks associated with the pandemic potential of the virus. This manuscript details the detection of AIVs in the seal population, comparing interesting features of various subtypes with an emphasis on avian-to-mammal-to-mammal transmission. Phylogenetic characterizations of the representative seal isolates were performed to demonstrate the relationships within the different virus isolates. Furthermore, we demonstrate that the reassortment events between different LPAIVs occurred before and after the viruses reached the seal population. The reassortment of viral segments plays an important role in the evolution of influenza viruses. Taken together, these data report on the 45 year history between seals and AIVs.

Seroprevalence and associated risk factors of peste des petits ruminants in sheep and goats in three districts of the Central Oromia Region, Ethiopia.

Peste des petits ruminants (PPR) is a viral disease that affects domestic and wild small ruminants and camels in Africa, the Middle East, and Asia. Following the successful eradication of rinderpest, the World Organization for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) have undertaken to eradicate PPR by 2030. Regular surveillance and monitoring of the disease in various regions of Ethiopia are crucial to achieving this goal. This study aimed to estimate the prevalence of PPR, assess community awareness of PPR infection, and examine the associated risk factors of the disease in selected districts of the Central Oromia Region, Ethiopia. The study collected 384 serum samples from 73 flocks containing 217 sheep and 167 goats using a multi-stage sampling technique. A competitive enzyme-linked immunosorbent assay (c-ELISA) was used to detect antibodies against the PPR virus. Additionally, a pre-tested questionnaire was used to gather information on community awareness and potential risk factors for PPRV infection in the study area. The study found that the overall prevalence of PPR in flocks was 71.2% [95% confidence interval (CI): 59.4%-81.2%]. The prevalence of PPR at the animal level was 50% (95% CI: 44.9%-55.1%), with sheep having a prevalence of 54.4% (95% CI: 47.0%-60.6%) and goats having a prevalence of 44.3% (95% CI: 36.6%-52.2%). The study also found that districts, flock size, and agroecology were independent predictors of PPRV seropositivity in sheep, whereas districts, origin, and mixed species were independent predictors of PPRV seropositivity in goats. The study revealed a high prevalence of PPR in sheep and goats in the study area. To prevent the spread of the disease, the study suggests quarantining animals before introducing them to districts, regular PPR vaccination, and isolation and molecular characterization of the PPR virus circulating in the study area.