H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA1. Rapid dissemination to more than 190 farms in 13 states followed2. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 isolates. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Design of Optimal Rainfall Monitoring Network Using Radar and Road Networks.

Uncertainty in the rainfall network can lead to mistakes in dam operation. Sudden increases in dam water levels due to rainfall uncertainty are a high disaster risk. In order to prevent these losses, it is necessary to configure an appropriate rainfall network that can effectively reflect the characteristics of the watershed. In this study, conditional entropy was used to calculate the uncertainty of the watershed using rainfall and radar data observed from 2018 to 2019 in the Goesan Dam and Hwacheon Dam watersheds. The results identified radar data suitable for the characteristics of the watershed and proposed a site for an additional rainfall gauge. It is also necessary to select the location of the additional rainfall gauged by limiting the points where smooth movement and installation, for example crossing national borders, are difficult. The proposed site emphasized accessibility and usability by leveraging road information and selecting a radar grid near the road. As a practice result, the uncertainty of precipitation in the Goesan and Hwacheon Dam watersheds could be decreased by 70.0% and 67.9%, respectively, when four and three additional gauge sites were installed without any restriction. When these were installed near to the road, with five and four additional gauge sites, the uncertainty in the Goesan Dam and Hwacheon Dam watersheds were reduced by up to 71.1%. Therefore, due to the high degree of uncertainty, it is necessary to measure precipitation. The operation of the rainfall gauge can provide a smooth site and configure an appropriate monitoring network.

Independent evolution of porcine reproductive and respiratory syndrome virus 2 with genetic heterogeneity in antigenic regions of structural proteins in Korea.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that affects the global swine industry. The continuous evolution of this virus has made control and prevention difficult, which emphasizes the importance of monitoring currently circulating PRRSV strains. In this study, we investigated the genetic characteristics of whole structural genes of 35 PRRSV-2 isolates that circulated between 2012 and 2017 in Korea. Genetic and phylogenetic analysis demonstrated that a recently identified PRRSV-2 shared a relatively low level of nucleotide sequence identity that ranged from 86.2% to 92.8%; however, they were clustered into four distinct Korean field clades, except KU-N1702, in ORF2-7-based phylogeny. KU-N1702 was closely related to the NADC30-like strains that were identified in the USA and China. Amino acid sequence analysis showed that the GP5 neutralizing epitope was conserved among the KU viruses. In contrast, the viruses had genetic mutations in key residues for viral neutralization within GP5 and M. For minor structural proteins, neutralizing epitopes, aa 41-55 of GP2, 61-75 of GP3, and 51-65 of GP4, were variable among the KU viruses. Bioinformatics demonstrated diversifying evolution within the GP2 and GP4 neutralizing epitopes and the emergence of a novel glycosylation site within the GP3 and GP4 neutralizing epitopes. Taken together, these data provide evidence that Korean PRRSV-2 evolved independently in Korea, with genetic heterogeneity in antigenic regions of structural proteins.

Classical Swine Fever Outbreak after Modified Live LOM Strain Vaccination in Naive Pigs, South Korea.

We report classical swine fever outbreaks occurring in naive pig herds on Jeju Island, South Korea, after the introduction of the LOM vaccine strain. Two isolates from sick pigs had >99% identity with the vaccine stain. LOM strain does not appear safe; its use in the vaccine should be reconsidered.

Bovine highly pathogenic avian influenza virus stability and inactivation in the milk byproduct lactose.

A bovine isolate of highly pathogenic avian influenza H5N1 virus was stable for 14 days in a concentrated lactose solution at under refrigerated conditions. Heat or citric acid treatments successfully inactivated viruses in lactose. This study highlights the persistence of HPAIV in lactose and its efficient inactivation under industrial standards.

Bovine Highly Pathogenic Avian Influenza Virus Stability and Inactivation in the Milk Byproduct Lactose.

The recent incursion of highly pathogenic influenza viruses into dairy cattle opens new insights for influenza virus ecology and its interspecies transmission and may have a significant impact on public health and agriculture. The aim of this study was to determine the stability of a bovine highly pathogenic avian influenza H5N1 virus isolate in the milk byproduct lactose and to evaluate two inactivation methods using industrial procedures. The bovine isolate of the highly pathogenic avian influenza H5N1 virus was stable for 14 days in a concentrated lactose solution under refrigerated conditions. Heat or citric acid treatments successfully inactivated the virus in lactose. This study highlights the persistence of HPAIV in lactose and its efficient inactivation under industrial standards.

Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b.

ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.

Adjuvant Use of the Invariant-Natural-Killer-T-Cell Agonist α-Galactosylceramide Leads to Vaccine-Associated Enhanced Respiratory Disease in Influenza-Vaccinated Pigs.

Invariant natural killer T (iNKT) cells are glycolipid-reactive T cells with potent immunoregulatory properties. iNKT cells activated with the marine-sponge-derived glycolipid, α-galactosylceramide (αGC), provide a universal source of T-cell help that has shown considerable promise for a wide array of therapeutic applications. This includes harnessing iNKT-cell-mediated immune responses to adjuvant whole inactivated influenza virus (WIV) vaccines. An important concern with WIV vaccines is that under certain circumstances, they are capable of triggering vaccine-associated enhanced respiratory disease (VAERD). This immunopathological phenomenon can arise after immunization with an oil-in-water (OIW) adjuvanted WIV vaccine, followed by infection with a hemagglutinin and neuraminidase mismatched challenge virus. This elicits antibodies (Abs) that bind immunodominant epitopes in the HA2 region of the heterologous virus, which purportedly causes enhanced virus fusion activity to the host cell and increased infection. Here, we show that αGC can induce severe VAERD in pigs. However, instead of stimulating high concentrations of HA2 Abs, αGC elicits high concentrations of interferon (IFN)-γ-secreting cells both in the lungs and systemically. Additionally, we found that VAERD mediated by iNKT cells results in distinct cytokine profiles and altered adaptation of the challenge virus following infection compared to an OIW adjuvant. Overall, these results provide a cautionary note about considering the formulation of WIV vaccines with iNKT-cell agonists as a potential strategy to modulate antigen-specific immunity.

Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the haemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in a significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to humans.

Development and optimization of sampling techniques for environmental samples from African swine fever virus-contaminated surfaces with no organic contaminants.

African swine fever (ASF) is a highly contagious diseases in domestic pigs and wild boars with up to 100% mortality. ASF virus (ASFV) is a causative agent responsible for ASF and highly resistant in environments, which creates a significant challenge for the control and eradication of the virus. Despite the geographical expansion of ASFV and international movement of products to sustain the swine production system, there is limited knowledge on the use of environmental samples to perform surveillance to prevent the introduction of ASFV into ASFV-free areas and for control of transmission in affected areas. Therefore, this study aimed to develop and optimize sampling techniques for environmental samples for ASFV detection. The stainless steel surfaces were contaminated with ASFV-infected blood, swabbed using different devices, and then processed through different techniques. The environmental samples were processed and tested using qPCR analysis. The results showed that the use of pre-moistened gauze surgical sponges, sweeping pads, and sponge sticks resulted in increased sensitivity, when compared to either dry sampling devices or Dacron swab. In particular, the combination of the sponge stick and the commercial nucleic acid preservative supported the best detection of ASFV DNA on the clean stainless steel surfaces evaluated. Pre-incubation for the short period of time and centrifugation at low speed were sufficient to provide satisfactory diagnostic sensitivity of ASFV detection using qPCR for environmental samples. Our findings contribute to the development of techniques for environmental samples for ASFV surveillance to prevent the introduction and dissemination of ASFV.

Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to WT pigs. Our findings could support the commercial use of GE pigs to minimize (i) the economic losses caused by IAV infection in pigs, and (ii) the emergence of novel IAVs with pandemic potential through genetic reassortment in the "mixing vessel", the pig.

Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Generation and Genetic Stability of a PolX and 5' MGF-Deficient African Swine Fever Virus Mutant for Vaccine Development.

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo.

Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern.

Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Outcome of H5N1 clade 2.3.4.4b virus infection in calves and lactating cows.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Fomite Transmission of SARS-CoV-2 and Its Contributing Factors.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has drastically changed our lives, from our personal freedoms and habits to public health and socioeconomics [...].

Ancestral Lineage of SARS-CoV-2 Is More Stable in Human Biological Fluids than Alpha, Beta, and Omicron Variants of Concern.

SARS-CoV-2 is a zoonotic virus first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of the Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. IMPORTANCE Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel virus variants, posing a significant concern to global public health. Five of these variants have been classified to date into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.

Stability of SARS-CoV-2 in Biological Fluids of Animals.

Since its first emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve genetically, jump species barriers, and expand its host range. There is growing evidence of interspecies transmission including infection of domestic animals and widespread circulation in wildlife. However, knowledge of SARS-CoV-2 stability in animal biological fluids and their role in transmission is still limited as previous studies focused on human biological fluids. Therefore, this study aimed to determine the SARS-CoV-2 stability in biological fluids from three animal species, cats, sheep and white-tailed deer (WTD). Saliva, feces, 10% fecal suspensions, and urine of cats, sheep, and WTD were mixed with a known concentration of virus and incubated under indoor and three different climatic conditions. Our results show that the virus was stable for up to 1 day in the saliva of cats, sheep, and WTD regardless of the environmental conditions. The virus remained infectious for up to 6 days in feces and 15 days in fecal suspension of WTD, whereas the virus was rather unstable in cat and sheep feces and fecal suspensions. We found the longest survival of SARS-CoV-2 in the urine of cats, sheep, and WTD. Furthermore, side-by-side comparison with different SARS-CoV-2 strains showed that the Alpha, Delta, and Omicron variants of concern were less stable than the ancestral Wuhan-like strain in WTD fecal suspension. The results of our study provide valuable information for assessing the potential role of various animal biological fluids in SARS-CoV-2 transmission.

High-harmonic generation from a flat liquid-sheet plasma mirror.

High-harmonic radiation can be generated when an ultra-intense laser beam is reflected from an over-dense plasma, known as a plasma mirror. It is considered a promising technique for generating intense attosecond pulses in the extreme ultraviolet and X-ray wavelength ranges. However, a solid target used for the formation of the over-dense plasma is completely damaged by the interaction. Thus, it is challenging to use a solid target for applications such as time-resolved studies and attosecond streaking experiments that require a large amount of data. Here we demonstrate that high-harmonic radiation can be continuously generated from a liquid plasma mirror in both the coherent wake emission and relativistic oscillating mirror regimes. These results will pave the way for the development of bright, stable, and high-repetition-rate attosecond light sources, which can greatly benefit the study of ultrafast laser-matter interactions.