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Nooruzzaman, M.

Publications and source records attributed to Nooruzzaman, M..

12 recordsLinked to original sources
bioRxiv2026

Hematogenous neuroinvasion and genotype-dependent transmission of influenza A H5N1 viruses in the cat host

The spillover of highly pathogenic avian influenza (HPAI) A H5N1 virus to mammalian hosts raises major concerns due to its pandemic potential. Cats are frequently affected mammals, often succumbing to systemic and neurological disease. Here, we characterized the pathogenesis and transmissibility of two H5N1 genotypes, B3.13 and D1.1, in cats. Infected cats exhibited high-level viremia and virus shedding in nasal, oral, and fecal secretions were consistently detected. The virus replicated initially in the upper respiratory tract and lungs, followed by systemic dissemination and neuroinvasion. Notably, the virus crossed the blood-brain-barrier by infecting endothelial cells, spreading to astrocytes and neurons, causing multifocal encephalitis. D1.1-virus infection caused protracted disease with lower shedding and no transmissibility, whereas B3.13 virus caused rapid onset with efficient shedding and transmission. These findings reveal critical H5N1 neuropathogenesis mechanisms and highlight mammalian transmission potential in a species with close human contact.

microbiology↗
bioRxiv2026

Recombinant influenza A H5N1 viruses expressing fluorescent and luminescent reporter proteins

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the United States. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication facilitating high-throughput screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural (NS) gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro, presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titers in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against Oseltamivir in vitro. Collectively, these results establish the TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis to be used in serological and antiviral drug screens against H5N1 viruses.

microbiology↗
bioRxiv2026

Infection and transmission dynamics of bovine and human influenza A H5N1 viruses in mouse and hamster models

Here we investigated the pathogenesis and contact transmission of bovine- and human-derived highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 viruses in mammalian models. Using reverse genetics, we rescued three naturally occurring viruses: rTX2/24 (bovine-derived), rTexas/37 and rMichigan/90 (both human-derived), and compared their infection dynamics, replication and pathogenicity with the wild-type bovine TX2/24 strain in vitro and in vivo. All four viruses demonstrated comparable replication kinetics in four mammalian cell lines. However, the rMichigan/90 strain exhibited significantly smaller plaques in bovine and human cells. In vivo studies showed that mice infected with any of the viruses succumbed to infection within 4-5 days; however, mice infected with the rMichigan/90 virus exhibited slightly lower viral replication and shedding compared to the other strains. Similarly, as in the mouse experiments, in hamsters, all viruses induced body weight loss and oral shedding, with robust virus replication observed in tissues, but the rMichigan/90 virus presented reduced replication and shedding. Contact transmission studies in hamsters revealed limited transmissibility for these viruses, with only one out of four animals inoculated with the rMichigan/90 virus transmitting it to a naive contact. These findings indicate that both bovine- and human-derived H5N1 genotype B3.13 viruses present high pathogenicity in mammals, though the overall transmissibility remains low. Significance StatementInfluenza A H5N1 virus spilled over from wild birds into dairy cattle in the US in 2024. Since then, an increased number of human infections - with at least 71 confirmed cases - were confirmed. In the present study we evaluated and compared the pathogenicity and transmissibility of a bovine and two human H5N1 viruses of the genotype B3.13. Our results show that while all viruses are pathogenic in mouse and hamster models, the bovine isolate TX2/24 presents a broader tissue tropism than the human derived viruses. Contact transmission studies revealed a limited ability of these viruses to transmit in the hamster contact transmission model. These findings highlight the high pathogenicity of H5N1 viruses in mammals and demonstrate that that their transmissibility potential remains low.

microbiology↗
bioRxiv2025

The ORF6 accessory protein contributes to SARS-CoV-2 virulence and pathogenicity in the naturally susceptible feline model of infection

Here the infection dynamics, replication, and pathogenicity of a recombinant virus containing a deletion of ORF6 (rWA1{Delta}ORF6) on the backbone of the highly virulent SARS-CoV-2 WA1 virus (rWA1) were investigated and compared to the parental rWA1 virus. While both rWA1 and rWA1{Delta}ORF6 viruses replicated efficiently in cultured cells, the rWA1{Delta}ORF6 virus produced smaller plaques, suggesting reduced cell-to-cell spread. Luciferase reporter assays revealed immune suppressing effects of ORF6 on interferon and nuclear factor kappa B (NF-{kappa}B) signaling pathways. Pathogenesis assessment in cats revealed that animals inoculated with rWA1 were lethargic and presented with fever on days 2 and 4 post-infection (pi), whereas rWA1{Delta}ORF6-inoculated animals developed subclinical infection. Additionally, animals inoculated with rWA1{Delta}ORF6 presented reduced infectious virus shedding in nasal and oral secretions and broncho-alveolar lavage fluid when compared with the rWA1-inoculated cats. Similarly, the rWA1{Delta}ORF6-inoculated cats presented reduced virus replication in the respiratory tract as evidenced by lower viral loads and reduced lung inflammation on day 3 and 5 pi when compared to rWA1-inoculated animals. Host gene transcriptomic analysis revealed marked differences in differentially expressed genes (DEG) in the nasal turbinate of animals infected with rWA1 when compared to rWA1{Delta}ORF6. Importantly, type I interferon signaling was significantly upregulated in rWA1{Delta}ORF6 infected cats when compared to rWA1-inoculated animals, which is correlated to the reduced replication of rWA1{Delta}ORF6 in the upper and lower respiratory tracts of infected animals. Collectively, these results demonstrate that the SARS-CoV-2 ORF6 is an important virulence determinant of the virus contributing to the modulation of host antiviral immune responses. IMPORTANCESARS-CoV-2 encodes several proteins that inhibit host interferon responses. The accessory protein ORF6 antagonizes interferon signaling by blocking the nucleocytoplasmic trafficking of key transcription factors. In this study, we showed that ORF6 plays an important role in SARS-CoV-2 pathogenesis. While both rWA1 and rWA1{Delta}ORF6 viruses replicated efficiently in cell culture, the rWA1{Delta}ORF6 presented impaired cell-to-spread and reduced innate immune inhibition as compared to the parental rWA1. Pathogenicity study in the feline model revealed an attenuated phenotype of the rWA1{Delta}ORF6 indicating that the ORF6 is a major virulence determinant of SARS-CoV-2. These results also demonstrate that ORF6 contributed to the feline host range of SARS-CoV-2.

microbiology↗
bioRxiv2025

Stability of influenza A H5N1 virus in raw milk cheese

Here we evaluated the stability of highly pathogenic avian influenza (HPAI) H5N1 virus in raw milk cheeses using a mini cheese model prepared with HPAI-spiked raw milk under varying pH levels (pH 6.6, 5.8 and 5.0) and in commercial raw milk cheese inadvertently produced with naturally contaminated raw milk. We observed a pH-dependent survival of the virus, with infectious virus persisting throughout the cheese making process and for up to 60 days of aging in the pH 6.6 and 5.8 cheese groups. Whereas at pH 5.0, the virus did not survive the cheese making process. These findings were validated using the commercial raw milk cheese samples in which infectious virus was detected for up to 60 days of aging. Our study highlights the potential public health risks of consuming raw milk cheese, underscoring the need for additional mitigation steps in cheese production to prevent human exposure to infectious virus.

microbiology↗
bioRxiv2024

Naturally occurring N-terminal mutations in SARS-CoV-2 nsp1 impact innate immune modulation but do not affect virus virulence

The non-structural protein 1 (nsp1) of SARS-CoV-2 plays a key role in host innate immune evasion. We identified two deletion variants ({Delta}82-85 and {Delta}83-86) in the N-terminal region of the nsp1 of a SARS-CoV-2 BA.5.2.1 variant recovered from a human patient. Analysis of the sequence databases revealed a frequency of 0.5% of these mutations amongst available SARS-CoV-2 sequences. Structural analysis of the deletion mutant nsp1{Delta}82-85 and nsp1{Delta}83-86 revealed a distortion in the protein pocket when compared to the wild-type nsp1 which may affect protein function. To evaluate the functional relevance of these mutations, we cloned the mutant BA.5.2.1 nsp1{Delta}82-85 and nsp1{Delta}83-86 and wild-type nsp1 proteins in expression plasmids and performed luciferase reporter-based assays to assess activation of the interferon and nuclear factor kappa B (NF-{kappa}B) signalling pathways. Both nsp1{Delta}82-85 and nsp1{Delta}83-86 mutants showed marked decreased ability to inhibit the interferon beta (IFN-{beta}) and NF-{kappa}B pathway activation. To assess the relevance of these deletions in the context of SARS-CoV-2 infection, we generated recombinant viruses carrying the wild type BA.5.2.1 nsp1 or the BA.5.2.1 nsp1{Delta}82-85 and nsp1{Delta}83-86 deletions in the backbone of WA1 strain. In vitro characterization of the recombinant SARS-CoV-2 viruses revealed that the recombinant viruses containing the nsp1{Delta}82-85 and nsp1{Delta}83-86 deletions presented similar plaque size and morphology to those produced by the wild-type rWA1-BA.5.2.1-nsp1 virus, indicating a similar ability of the mutant viruses to spread from cell to cell. Importantly, pathogenesis studies revealed that these mutations did not affect virus virulence and pathogenesis in a hamster model of SARS-CoV-2 infection.

microbiology↗
bioRxiv2024

Thermal inactivation spectrum of influenza A H5N1 virus in raw milk

The spillover of highly pathogenic avian influenza (HPAI) H5N1 virus to dairy cows and shedding of high amounts of infectious virus in milk raised public health concerns. Here, we evaluated the decay and thermal stability spectrum of HPAI H5N1 virus in raw milk. For the decay studies, HPAI H5N1 positive raw milk was incubated at different temperatures and viral titers and the thermal death time D-values were estimated. We then heat treated HPAI H5N1 virus positive milk following different thermal conditions including pasteurization and thermization conditions. Efficient inactivation of the virus was observed in all tested conditions, except for thermization at 50{degrees}C 10 min. Utilizing a submerged coil system with temperature ramp up times that resemble commercial pasteurizers, we showed that the virus was rapidly inactivated by pasteurization and most thermization conditions. These results provide important insights on the food safety measures utilized in the dairy industry.

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↗
bioRxiv2024

From birds to mammals: spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle led to efficient intra- and interspecies transmission

Infections with the highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b virus have resulted in the death of millions of domestic birds and thousands of wild birds in the U.S. since January, 20221-4 Throughout this outbreak, spillovers of the virus to mammals have been frequently documented5-12. Here, we report the detection of HPAI H5N1 virus in dairy cattle herds across several states in the U.S. The affected cows displayed clinical signs encompassing decreased feed intake, altered fecal consistency, respiratory distress, and decreased milk production with abnormal milk. Infectious virus and RNA were consistently detected in milk collected from affected cows. Viral staining in tissues revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Analysis of whole genome sequences obtained from dairy cows, birds, domestic cats, and a racoon from affected farms indicated multidirectional interspecies transmissions. Epidemiologic and genomic data revealed efficient cow-to-cow transmission after healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface and to a new and highly relevant livestock species, underscoring the ability of the virus to cross species barriers.

microbiology↗
bioRxiv2023

The SARS-CoV-2 Spike is a virulence determinant and plays a major role on the attenuated phenotype of Omicron virus in a feline model of infection

To assess the role of the Omicron BA.1 Spike (S) protein in the pathogenesis of the severe acute respiratory coronavirus 2 (SARS-CoV-2), we generated recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 S gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 strain genome. The recombinant viruses were characterized in vitro and in vivo. Viral entry, cell-cell fusion, viral plaque size, and viral replication kinetics of the rWA1-Omi-S virus were markedly impaired when compared to the rWA1-D614G virus, demonstrating a lower fusogenicity and ability to spread cell-to-cell of rWA1-Omi-S. To assess the contribution of the Omicron BA.1 S protein to SARS-CoV-2 pathogenesis the pathogenicity of rWA1-D614G and rWA1-Omi-S viruses were compared using a feline model of infection. While the rWA1-D614G-inoculated cats became lethargic and showed increased body temperatures on days 2 and 3 post-infection (pi), rWA1-Omi-S-inoculated cats remained subclinical and gained weight throughout the 14-day experimental period. Animals inoculated with rWA1-D614G presented higher levels of infectious virus shedding in nasal secretions, when compared to rWA1-Omi-S-inoculated animals. In addition, tissue replication of the rWA1-Omi-S was markedly reduced compared to the rWA1-D614G, as evidenced by lower in situ viral RNA and lower viral load in tissues on days 3 and 5 pi. Histologic examination of the nasal turbinate and lungs revealed intense inflammatory infiltration in rWA1-D614G-inoculated animals, whereas rWA1-Omi-S-inoculated cats presented only mild to modest inflammation. Together, these results demonstrate that the S protein is a major virulence determinant for SARS-CoV-2 playing a major role for the attenuated phenotype of the Omicron virus. Author summaryThe SARS-CoV-2 Omicron sublineage BA.1 spread rapidly across the globe in late 2021/early 2022. Experimental studies have shown an overall lower pathogenicity of Omicron BA.1 when compared to the ancestral SARS-CoV-2 lineage B.1 (D614G). Recently, we have demonstrated that the Omicron BA.1.1 variant presents lower pathogenicity when compared to D614G (B.1) lineage in a feline model of SARS-CoV-2 infection. There are over 50 mutations in the Omicron genome, of which more than two thirds are present in the S gene. To assess the role of the Omicron BA.1 S on virus pathogenesis, recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 Spike gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 were characterized in vitro and in vivo. While the Omicron BA.1 S gene results in early entry into cells, the rWA1-Omi-S presents impaired cell-cell spread and fusogenic activity. Inoculation of cats with the recombinant viruses revealed an attenuated phenotype of rWA1-Omi-S, demonstrating a critical role for S protein on the pathogenicity of SARS-CoV-2 and indicating that the Omi-S is a major determinant of the attenuated disease phenotype of Omicron strains.

microbiology↗
bioRxiv2022

The Omicron variant BA.1.1 presents a lower pathogenicity than B.1 D614G and Delta variants in a feline model of SARS-CoV-2 infection

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern (VOC), which emerged in late 2021 and rapidly achieved global predominance in early 2022. In this study, we compared the infection dynamics, tissue tropism and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2) and Omicron BA.1.1 sublineage (B.1.1.529) variants in a highly susceptible feline model of infection. While D614G- and Delta-inoculated cats became lethargic, and showed increased body temperatures between days 1 and 3 post-infection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID50.ml-1), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID50.ml-1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by in situ viral RNA detection, in situ immunofluorescence, and quantification of viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main - but not the only - sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats had severe pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. Author SummaryThe SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VOC) emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations, suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern (VOC) presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological and pathological evaluations revealed lower disease severity, viral replication and lung pathology in Omicron-infected cats when compared to D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.

microbiology↗