Hem1 inborn errors of immunity: waving goodbye to coordinated immunity in mice and humans.

Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-of-function mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC.

Long-term monitoring of immune response to recombinant lumpy skin disease virus in dairy cattle from small-household farms in western Thailand.

Lumpy skin disease (LSD) was firstly reported in Thailand in 2021 which affected the cattle industry. However, there is limited information on the immune response of LSDV infection in Thailand where recombinant vaccine strain circulated. The aim of this research was to study the duration of LSD immune response of subclinical and clinical animals after natural infection in dairy cattle. Sixty-six dairy cattle from ten farms in central and western regions of Thailand were investigated. Antibody was detected by virus neutralization test and ELISA. Cell mediated immunity (CMI)-related cytokine gene expressions were evaluated. Antibody was detected until at least 15 months after the noticeable symptom. Cattle with subclinical disease had lower antibody levels compared to animals which had clinical disease. IFN-γ and TNF-α levels were increased, while IL-10 level was decreased in the infected animals compared to the controls. This study elucidated immune responses in dairy cattle herd affected by recombinant LSDV.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100.

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Complete Genomic Characterization of Lumpy Skin Disease Virus Isolates from Beef Cattle in Lopburi Province, Central Thailand, during 2021-2022.

Lumpy skin disease (LSD) is a viral infection that impacts the cattle industry. The most efficient approach to prevent disease involves the utilization of live-attenuated LSD vaccines (LAVs), which stands out as the most successful method. However, LAVs might be subjected to changes to their genomes during replication that increase viral infectivity or virulence. The objective of this study was to monitor alterations in the genetic characteristics of the lumpy skin disease virus (LSDV) in beef cattle following the administration of LAVs in Lopburi Province of Central Thailand. A total of four skin samples from LSD cases were collected from non-vaccinated animals that exhibited LSD clinical symptoms from two distinct districts, spanning three subdistricts within the region. The samples of cattle were analyzed using real-time PCR targeting the LSDV074 p32 gene, the virus was isolated, and the entire genome sequences were evaluated through a single nucleotide polymorphisms (SNPs) analysis, and phylogenetic trees were assembled. The investigations revealed that LSDVs from two isolates from Chai Badan district exhibited significant mutations in the open reading frame (ORF) 023 putative protein, while another two isolates from Lam Sonthi district had a change in the untranslated region (UTR). For a result, the most proficient disease diagnosis and control should be evaluated on viral genetics on a regular basis.

Rapid Spread and Genetic Characterisation of a Recently Emerged Recombinant Lumpy Skin Disease Virus in Thailand.

The emergence of the lumpy skin disease virus (LSDV) was first detected in north-eastern Thailand in March 2021. Since then, the abrupt increase of LSD cases was observed throughout the country as outbreaks have spread rapidly to 64 out of a total of 77 provinces within four months. Blood, milk, and nodular skin samples collected from affected animals have been diagnosed by real-time PCR targeting the p32 gene. LSDV was isolated by primary lamb testis (PLT) cells, followed by Madin-Darby bovine kidney (MDBK) cells, and confirmed by immunoperoxidase monolayer assay (IPMA). Histopathology and immunohistochemistry (IHC) of a skin lesion showed inclusion bodies in keratinocytes and skin epithelial cells. Phylogenetic analyses of RPO30 and GPCR genes, and the whole genome revealed that Thai viruses were closely related to the vaccine-derived recombinant LSDV strains found previously in China and Vietnam. Recombination analysis confirmed that the Thai LSDV possesses a mosaic hybrid genome containing the vaccine virus DNA as the backbone and a field strain DNA as the minor donor. This is an inclusive report on the disease distributions, complete diagnoses, and genetic characterisation of LSDV during the first wave of LSD outbreaks in Thailand.

Thermal inactivation of African swine fever virus in feed ingredients.

African swine fever virus (ASFV) causes a fatal infectious disease affecting domestic pigs and wild boars. ASFV is highly stable and easily transmitted by consumption of contaminated swine feed and pork products. Heat treatment of feed ingredients is a means to minimize the risk of contamination through swine feed consumption. The objectives of this study were to determine the thermal inactivation of ASFV in non-animal and animal origin feed ingredients. The rate of thermal inactivation is represented by decimal reduction time (DT) or time required to reduce ASFV per 1 log at temperature T. The mean D60, D70, D80 and D85 of meat and bone meal (MBM), soybean meal (SBM), and maize grain (MZ) are in the ranges 5.11-6.78, 2.19-3.01, 0.99-2.02, and 0.16-0.99 min, respectively. DT is used to compare the heat resistance of ASFV in the feed ingredient matrices. The mean DT of ASFV in MBM, SBM and MZ was not statistically significant, and the heat resistance of ASFV in MBM, SBM, and MZ was not different at 60, 70, 80, or 85 °C. The multiple DT was used to develop a DT model to predict DT at various inactivation temperatures. The DT models for MBM, SBM, and MZ are log DT = - [Formula: see text] + 2.69, log DT = - [Formula: see text] + 2.55, and log DT = - [Formula: see text] + 4.01. To expand and ease the field applications, a spreadsheet predicting the DT and the inactivation time (with 95% confidence interval) from these DT models is available to download.

Persistence of African swine fever virus on porous and non-porous fomites at environmental temperatures.

BACKGROUND: African swine fever (ASF) is a lethal contagious disease affecting both domestic pigs and wild boars. Even though it is a non-zoonotic disease, ASF causes economic loss in swine industries across continents. ASF control and eradication are almost impossible since effective vaccines and direct antiviral treatment are not available. The persistence of ASFV on fomites plays an important role in the indirect transmission of ASFV to pigs encountering ASFV-contaminated fomites. ASFV persistence on porous and non-porous fomites (glass, metal, rubber, and cellulose paper) at different environmental temperatures was determined. The persistence of ASFV of fomites was determined by the rate of ASFV inactivation in terms of DT, or the time required to reduce ASFV per 1 log at each selected environmental temperature (T). DT is used to compare the persistence of ASFV on the fomites. RESULTS: The mean D25, D33, and D42, of dried infectious ASFV on glass, metal, rubber, and paper were in the ranges 1.42-2.42, 0.72-1.94, and 0.07-0.23 days, respectively. The multiple DT were used to develop a DT model to predict the DT for some other environmental temperatures. The DT models to predict the persistence of dried infectious ASFV on glass, metal, rubber, and paper are log DT = (- T/21.51) + 1.34, log DT = (- T/20.42) + 1.47, log DT = (- T/14.91) + 2.03, and log DT = (- T/10.91) + 2.84, respectively. A spreadsheet as a quick and handy tool predicting the persistence time of dried infectious ASFV on fomites at various environmental temperatures based on these DT models is available for public to download. CONCLUSION: Persistence of dried infectious ASFV on paper are significantly the longest at lower environmental temperatures whereas that of dried infectious ASFV on paper is significantly the shortest at higher environmental temperature.

Thermal Inactivation of African Swine Fever Virus in Swill.

The indirect transmission of the African swine fever virus (ASFV) is through contaminated fomite, feed ingredients, pork- and pig-derived products, including swill, as ASFV is highly stable within suitable organic material. Some previous studies have indicated that ASFV outbreaks were associated with swill feeding, particularly in smallholder pig farms. These outbreaks emphasize the significance of the appropriate heat treatment of swill to eliminate ASFV residual titer. The World Organization for Animal Health (OIE) recommended the heat treatment of swill at a temperature of at least 90°C for at least 60 min, with continuous stirring, while the Food and Agriculture Organization (FAO) recommended heat treatment at 70°C for 30 min. The lack of scientific evidence regarding ASFV inactivation by heat treatment of swill leads to such inconsistent recommendations. Therefore, the objectives of this study were to assess the thermal inactivation of ASFV in three swill formulae and to develop a D T model to predict D T at some other inactivation temperatures. The significant reduction of ASFV in swill occurred at temperatures as low as 60°C. D T or decimal reduction time (DRT) is defined as the time required to reduce the virus titer by 1 log, and this was also used as a comparative index of heat resistance. The mean D 60, D 70, D 75, and D 80 of ASFV in three swill formulae were in the ranges 23.21-33.47, 5.83-10.91, 2.15-2.22, and 1.36-1.47 min, respectively. These D T could be widely used for any nutritive composition of swill other than the three swill formulae in this study since there was no statistical difference of all D T of ASFV across three swill formulae. Based on D 70 and the predicted D 90 from the D T model in this study, including the highest ASFV titer in pork products, the calculated inactivation times at 70 and 90°C were 119 and 4 min, respectively.

Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell-specific manner.

Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell-specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell-specific Hem-1-deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) - alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

The actin-regulatory protein Hem-1 is essential for alveolar macrophage development.

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific actin-regulatory protein. Loss-of-function (LOF) variants in the NCKAP1L gene encoding Hem-1 have recently been found to result in primary immunodeficiency disease (PID) in humans, characterized by recurring respiratory infections, asthma, and high mortality. However, the mechanisms of how Hem-1 variants result in PID are not known. In this study, we generated constitutive and myeloid cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in lung immunity. We found that Hem-1-deficient mice accumulated excessive surfactant and cell debris in airways (pulmonary alveolar proteinosis) due to impaired development of alveolar macrophages (AMs) and reduced expression of the AM differentiation factor Pparg. Residual Hem-1-deficient AMs shifted to a proinflammatory phenotype, and Hem-1-deficient neutrophils and monocytes failed to migrate normally. Myeloid cell-specific Hem-1-deficient mice exhibited increased morbidity following influenza A virus or Streptococcus pneumoniae challenge. These results provide potential mechanisms for how LOF variants in Hem-1 result in recurring respiratory diseases.