Biology subjects

Trujillo, J. D.

Publications and source records attributed to Trujillo, J. D..

14 recordsLinked to original sources

bioRxiv2026

Compartmentalized cytokine networks and systemic immune remodeling in bovine mammary H5N1 infection.

Highly pathogenic avian influenza A H5N1 has recently expanded its mammalian host range; in 2024, genotype B3.13 emerged in U.S. dairy cattle with pronounced mammary tropism. In the past, Influenza A virus immunology has been characterized primarily in respiratory infection models, whereas this study delineates immune responses after intramammary infection. An intramammary H5N1 challenge in Jersey cows in the early dry-off period enabled integration of dose- and compartment-resolved (alveoli versus teat cistern) cytokine and chemokine profiles with peripheral leukocyte dynamics and H5/N1-specific antibody responses. Infection-induced quarter-restricted, monophasic inflammatory networks peaking at 3-7 days post-infection, coordinated peripheral myeloid expansion and IFN-{gamma}-competent lymphocyte activation, and rising antibody titers across quarters.

microbiology↗

bioRxiv2026

Pathogenesis of H5N1 Clade 2.3.4.4b in dry Jersey cows following intramammary inoculation shows within-host compartmentalization

Dairy cattle have emerged as a prolific amplifying host for highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b and a new source for cross-species and zoonotic transmission. Independent introductions of H5N1 with unclear exposure routes have been reported in several dairy herds across the U.S. These events escalate the pandemic potential of HPAIV H5N1 as transmission within and between mammalian species present opportunities for mammalian adapted H5N1 viruses to emerge. Although more than 1000 herds have been infected, bovine H5N1 influenza virus pathogenesis, transmission, and evolution in dairy cattle remains not well characterized. Working with H5N1-infected lactating cattle in high containment has been a major challenge due to the required infrastructure and logistics associated with housing, husbandry, and waste management for this model. Thus, developing alternative bovine models that maintain biological relevance while reducing operational complexity is warranted. Here, we evaluate the susceptibility of lactating Jersey cattle in the dry-off period, and characterize the effect of inoculation dose on the mammary pathogenicity of HPAIV H5N1 genotype B3.13. The results of this study demonstrate that dairy cows 21 days into the dry-off period are highly susceptible to HPAIV H5N1, recapitulating the severe clinical and pathological outcomes observed in infected lactating cows under experimental conditions and in field cases. We also observed an association between virus dose and the onset and severity of mastitis in individual udder-quarters. Overall, this study demonstrates that dry cows can provide a feasible model to study H5N1 virology, pathology, and humoral immunology in dairy cows.

microbiology↗

bioRxiv2025

Temporal dynamics of cytokine, leukocyte, and whole blood transcriptome profiles of pigs infected with African swine fever virus

African swine fever virus (ASFV) is an important transboundary animal pathogen with significant impacts on the global swine industry. Overwhelming proinflammatory responses are a major virulence mechanism for ASFV, but the dynamics of these changes during clinical disease are not completely understood. We constructed a detailed portrait of the innate immune responses during acute African swine fever (ASF) at the cellular, transcriptomic, and cytokine levels. Samples serially obtained from infected piglets show progression of acute ASF is characterized by rapid increases in plasma type I interferons, TNF-, IL-12p40, and IL-10, which coincide with the manifestation of clinical disease and viral DNAemia. Lymphocytes and natural killer (NK) cells progressively declined, with fluctuations in B cell, CD8+ T cell, and CD4+/CD8+ T cell populations. Blood monocytes and macrophages were highly variable throughout infection, with an abrupt spike in CD203+ mature macrophages immediately prior to death. Transcriptomic analysis of blood showed downregulation of cellular translation as early as 1 day post challenge (DPC), and significant upregulation of antiviral immune processes at 5 DPC and 7 DPC which overlapped with the onset of clinical disease. Together, these results present a highly detailed delineation of fatal ASF as involving an initial infection and damage of susceptible myeloid cells prior to symptomatic disease characterized by pro-inflammatory immune responses, lymphoid depletion, and clinical deterioration.

microbiology↗

bioRxiv2025

Targeted Whole Genome Sequencing of African Swine Fever Virus and Classical Swine Fever Virus on the MinION Portable Sequencing Platform

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170-190 kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with genome size of approximately 12 kB belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS), allows for the establishment of vital NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10kB and 6kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the 3 known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with 2 different genotype 1 viruses. ASFV and CSFV amplicons from optimized PCR reactions were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1000X for ASFV with 99% of the genome covered, and 10,000X-20,000X for CSFV with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation as well as genetic characterization of these high consequence swine viruses, globally and within the United States, should an outbreak occur.

molecular biology↗

bioRxiv2025

Pathogenicity and transmissibility of bovine-derived HPAI H5N1 B3.13 virus in pigs

Since the first emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses in dairy cattle, the virus has continued to spread, reaching at least 17 states and at least 970 dairy herds in the United States. Subsequently, spillovers of the virus from dairy cattle to humans have been reported. Pigs are an important reservoir in influenza ecology because they serve as a mixing vessel in which novel reassortant viruses with pandemic potential can be generated. Here, we show that oro-respiratory infection of pigs resulted in productive replication of a bovine-derived HPAI H5N1 B3.13 virus. Infectious virus was mainly identified in the lower respiratory tract of principal infected pigs, and sero-conversion was observed in most of the principal pigs at later time points. In one animal, we detected the emergence of a mutation in hemagglutinin (HA) previously associated with increased affinity for "mammalian-type" 2,6-linked sialic acid receptors, but this mutation did not reach consensus levels. Sentinel contact pigs remained sero-negative throughout the study, indicating lack of transmission. The results support that pigs are susceptible to a bovine-derived HPAI H5N1 B3.13 virus, but this virus did not replicate as robustly in pigs as mink-derived HPAI H5N1 and swine-adapted influenza viruses.

microbiology↗

bioRxiv2024

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.

microbiology↗

bioRxiv2023

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

Rapid 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 mammal 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 moderate to severe interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract of principal-infected pigs and 100% seroconversion. Principal-infected pigs shed limited amount of virus through the nasal and oral cavities, and importantly, there was no transmission to contact sentinel pigs. Notably, critical mammalian-like mutations such as PB2-E627K and HA-Q222L emerged at low frequencies in clinical samples and tissues derived from 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 HPAI viruses to acquire mammalian-like adaptations. This work is critical for further risk assessment on the ability of the newly emerging H5N1 clade 2.3.4.4b HPAIVs to cross the species barriers into mammals.

microbiology↗

bioRxiv2021

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived cell cultures support SARS-CoV-2 replication. Furthermore, experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs primarily at 1-day post challenge (DPC), and also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was also observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naive sheep was not highly efficient; however, viral RNA was detected in some of the respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern (VOC), to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection, and that the alpha VOC outcompeted the ancestral lineage A strain.

microbiology↗

bioRxiv2021

Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization

SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization. Author summaryCells stably expressing ACE2 from various animals and a lentivirus-based SARS-CoV-2 pseudotyped virus assay were established to study SARS-CoV-2 cell entry. The results demonstrated that ACE2 from a wide range of animal species facilitate S-mediated virus entry into cells, which is supported by in silico data as well as natural and experimental infection studies. Pseudotyped viruses containing mutations in the RBD of S representative of the Alpha, Gamma, and especially Beta, variants of concern demonstrated that certain mutations are associated with increased viral entry compared to the parental S. The Beta variant was also observed to have a replicative advantage in vitro and in vivo compared to the prototype virus. Pseudotyped viruses containing combinatorial substitutions of 501Y-484K-417K, 614G-501Y-484K and 614G-501Y-484K-417N increased viral entry via ACE2 across multiple species. The 501Y or 478K single substitution did not significantly affect neutralizing capacity of immune sera compared to the prototype strain, but the addition of 484K or 452R substitutions significantly reduced the neutralizing titers.

microbiology↗

bioRxiv2021

Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer

SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

microbiology↗

bioRxiv2021

Experimental re-infected cats do not transmit SARS-CoV-2

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naive cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Naive sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naive sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.

microbiology↗

bioRxiv2020

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

microbiology↗

bioRxiv2020

Susceptibility of swine cells and domestic pigs to SARS-CoV-2

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

microbiology↗

bioRxiv2020

SARS-CoV-2 infection, disease and transmission in domestic cats

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility and transmission studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, associated disease and transmission dynamics in domestic cats. Six 4- to 5-month-old cats were challenged with SARS-CoV-2 via intranasal and oral routes simultaneously. One day post challenge (DPC), two sentinel contact cats were co-mingled with the principal infected animals. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding throughout the 21 DPC observation period. Postmortem examinations were performed at 4, 7 and 21 DPC to investigate disease progression. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats on 4 and 7 DPC. Serology showed that both, principal and sentinel cats, developed SARS-CoV-2-specific and neutralizing antibodies to SARS-CoV-2 detectable at 7 DPC or 10 DPC, respectively. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels within 2 days of comingling. The results of this study are critical for our understanding of the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment of the maintenance of SARS-CoV-2 in felines and transmission to other animals and humans.

microbiology↗