The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids.

The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.

Zoonotic tuberculosis in a high bovine tuberculosis burden area of Ethiopia.

Background: Tuberculosis (TB) is a major cause of ill health and one of the leading causes of death worldwide, caused by species of the Mycobacterium tuberculosis complex (MTBC), with Mycobacterium tuberculosis being the dominant pathogen in humans and Mycobacterium bovis in cattle. Zoonotic transmission of TB (zTB) to humans is frequent particularly where TB prevalence is high in cattle. In this study, we explored the prevalence of zTB in central Ethiopia, an area highly affected by bovine TB (bTB) in cattle. Method: A convenient sample of 385 patients with pulmonary tuberculosis (PTB, N = 287) and tuberculous lymphadenitis (TBLN, N = 98) were included in this cross-sectional study in central Ethiopia. Sputum and fine needle aspirate (FNA) samples were obtained from patients with PTB and TBLN, respectively, and cultures were performed using BACTEC™ MGIT™ 960. All culture positive samples were subjected to quantitative PCR (qPCR) assays, targeting IS1081, RD9 and RD4 genomic regions for detection of MTBC, M. tuberculosis and M. bovis, respectively. Results: Two hundred and fifty-five out of 385 sampled patients were culture positive and all were isolates identified as MTBC by being positive for the IS1081 assay. Among them, 249 (97.6%) samples had also a positive RD9 result (intact RD9 locus) and were consequently classified as M. tuberculosis. The remaining six (2.4%) isolates were RD4 deficient and thereby classified as M. bovis. Five out of these six M. bovis strains originated from PTB patients whereas one was isolated from a TBLN patient. Occupational risk and the widespread consumption of raw animal products were identified as potential sources of M. bovis infection in humans, and the isolation of M. bovis from PTB patients suggests the possibility of human-to-human transmission, particularly in patients with no known contact history with animals. Conclusion: The detected proportion of culture positive cases of 2.4% being M. bovis from this region was higher zTB rate than previously reported for the general population of Ethiopia. Patients with M. bovis infection are more likely to get less efficient TB treatment because M. bovis is inherently resistant to pyrazinamide. MTBC species identification should be performed where M. bovis is common in cattle, especially in patients who have a history of recurrence or treatment failure.

SARS-CoV-2 Prevalence and Variant Surveillance among Cats in Pittsburgh, Pennsylvania, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh (n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats (n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40-89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Little Brown Bats (Myotis lucifugus) Support the Binding of SARS-CoV-2 Spike and Are Likely Susceptible to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), believed to have originated from a bat species, can infect a wide range of non-human hosts. Bats are known to harbor hundreds of coronaviruses capable of spillover into human populations. Recent studies have shown a significant variation in the susceptibility among bat species to SARS-CoV-2 infection. We show that little brown bats (LBB) express angiotensin-converting enzyme 2 receptor and the transmembrane serine protease 2, which are accessible to and support SARS-CoV-2 binding. All-atom molecular dynamics (MD) simulations revealed that LBB ACE2 formed strong electrostatic interactions with the RBD similar to human and cat ACE2 proteins. In summary, LBBs, a widely distributed North American bat species, could be at risk of SARS-CoV-2 infection and potentially serve as a natural reservoir. Finally, our framework, combining in vitro and in silico methods, is a useful tool to assess the SARS-CoV-2 susceptibility of bats and other animal species.

Complete Genome Sequence of a Bovine Coronavirus Isolated from a Goat in Pennsylvania, USA.

We report a complete genome sequence of bovine coronavirus (BCoV) isolated from a goat in the state of Pennsylvania in 2022. BCoV often causes calf scours and winter dysentery in cattle.

SARS-CoV-2 Omicron (B.1.1.529) Infection of Wild White-Tailed Deer in New York City.

There is mounting evidence of SARS-CoV-2 spillover from humans into many domestic, companion, and wild animal species. Research indicates that humans have infected white-tailed deer, and that deer-to-deer transmission has occurred, indicating that deer could be a wildlife reservoir and a source of novel SARS-CoV-2 variants. We examined the hypothesis that the Omicron variant is actively and asymptomatically infecting the free-ranging deer of New York City. Between December 2021 and February 2022, 155 deer on Staten Island, New York, were anesthetized and examined for gross abnormalities and illnesses. Paired nasopharyngeal swabs and blood samples were collected and analyzed for the presence of SARS-CoV-2 RNA and antibodies. Of 135 serum samples, 19 (14.1%) indicated SARS-CoV-2 exposure, and 11 reacted most strongly to the wild-type B.1 lineage. Of the 71 swabs, 8 were positive for SARS-CoV-2 RNA (4 Omicron and 4 Delta). Two of the animals had active infections and robust neutralizing antibodies, revealing evidence of reinfection or early seroconversion in deer. Variants of concern continue to circulate among and may reinfect US deer populations, and establish enzootic transmission cycles in the wild: this warrants a coordinated One Health response, to proactively surveil, identify, and curtail variants of concern before they can spill back into humans.

Development and Validation of Indirect Enzyme-Linked Immunosorbent Assays for Detecting Antibodies to SARS-CoV-2 in Cattle, Swine, and Chicken.

Multiple domestic and wild animal species are susceptible to SARS-CoV-2 infection. Cattle and swine are susceptible to experimental SARS-CoV-2 infection. The unchecked transmission of SARS-CoV-2 in animal hosts could lead to virus adaptation and the emergence of novel variants. In addition, the spillover and subsequent adaptation of SARS-CoV-2 in livestock could significantly impact food security as well as animal and public health. Therefore, it is essential to monitor livestock species for SARS-CoV-2 spillover. We developed and optimized species-specific indirect ELISAs (iELISAs) to detect anti-SARS-CoV-2 antibodies in cattle, swine, and chickens using the spike protein receptor-binding domain (RBD) antigen. Serum samples collected prior to the COVID-19 pandemic were used to determine the cut-off threshold. RBD hyperimmunized sera from cattle (n = 3), swine (n = 6), and chicken (n = 3) were used as the positive controls. The iELISAs were evaluated compared to a live virus neutralization test using cattle (n = 150), swine (n = 150), and chicken (n = 150) serum samples collected during the COVID-19 pandemic. The iELISAs for cattle, swine, and chicken were found to have 100% sensitivity and specificity. These tools facilitate the surveillance that is necessary to quickly identify spillovers into the three most important agricultural species worldwide.

Mitigating the Impact of Emerging Animal Infectious Disease Threats: First Emerging Animal Infectious Diseases Conference (EAIDC) Report.

From 29 November to 1 December 2021, an "emerging animal infectious disease conference (EAIDC)" was held at the Pennsylvania State University. This conference brought together distinguished thought leaders in animal health, veterinary diagnostics, epidemiology and disease surveillance, and agricultural economics. The conference's primary objective was to review the lessons learned from past experiences in dealing with high-consequence animal infectious diseases to inform an action plan to prepare for future epizootics and panzootics. Invited speakers and panel members comprised world-leading experts in animal infectious diseases from federal state agencies, academia, professional societies, and the private sector. The conference concluded that the biosecurity of livestock operations is critical for minimizing the devastating impact of emerging animal infectious diseases. The panel also highlighted the need to develop and benchmark cutting-edge diagnostics for rapidly detecting pathogens in clinical samples and the environment. Developing next-generation pathogen agnostic diagnostics will help detect variants of known pathogens and unknown novel pathogens. The conference also highlighted the importance of the One Health approach in dealing with emerging animal and human infectious diseases. The recommendations of the conference may be used to inform policy discussions focused on developing strategies for monitoring and preventing emerging infectious disease threats to the livestock industry.

A CNN model for predicting binding affinity changes between SARS-CoV-2 spike RBD variants and ACE2 homologues.

The cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the association of its receptor binding domain (RBD) with human angiotensin converting enzyme 2 (hACE2) as the first crucial step. Efficient and reliable prediction of RBD-hACE2 binding affinity changes upon amino acid substitutions can be valuable for public health surveillance and monitoring potential spillover and adaptation into non-human species. Here, we introduce a convolutional neural network (CNN) model trained on protein sequence and structural features to predict experimental RBD-hACE2 binding affinities of 8,440 variants upon single and multiple amino acid substitutions in the RBD or ACE2. The model achieves a classification accuracy of 83.28% and a Pearson correlation coefficient of 0.85 between predicted and experimentally calculated binding affinities in five-fold cross-validation tests and predicts improved binding affinity for most circulating variants. We pro-actively used the CNN model to exhaustively screen for novel RBD variants with combinations of up to four single amino acid substitutions and suggested candidates with the highest improvements in RBD-ACE2 binding affinity for human and animal ACE2 receptors. We found that the binding affinity of RBD variants against animal ACE2s follows similar trends as those against human ACE2. White-tailed deer ACE2 binds to RBD almost as tightly as human ACE2 while cattle, pig, and chicken ACE2s bind weakly. The model allows testing whether adaptation of the virus for increased binding with other animals would cause concomitant increases in binding with hACE2 or decreased fitness due to adaptation to other hosts.

Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Its performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India, taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains), and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management, and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance. IMPORTANCE Vaccination against tuberculosis with bacillus Calmette-Guérin (BCG) can lead to adverse events, including a rare but life-threatening complication of disseminated BCG. This complication often occurs in young children with immunodeficiencies and is associated with an ∼60% mortality rate. A rapid method of reliably identifying BCG infection is important because BCG requires treatment unique to tuberculosis. BCG is resistant to the first-line antituberculosis drug pyrazinamide. Additionally, diagnosis of BCG disease would lead to further investigation of a possible underlying immune condition. We have developed a diagnostic assay to identify BCG which improves upon previously published methods and can reliably identify BCG from bacterial culture or directly from infected tissue. This assay can also differentiate between strains of BCG, which have been suggested to be associated with different rates of adverse events. This assay was validated on 19 clinical isolates collected at Christian Medical College in Vellore, India.

Prevalence and distribution of multilocus sequence types of Staphylococcus aureus isolated from bulk tank milk and cows with mastitis in Pennsylvania.

A total of 163 S. aureus isolates; 113 from mastitic milk (MM) and 50 from bulk tank milk (BTM) (2008, 2013-2015) submitted for bacteriologic analysis at the Penn State Animal Diagnostic Laboratory were examined for their phenotypic and genotypic characteristics. Multi-locus sequence typing (MLST) analysis identified 16 unique sequence types (STs) which belonged to eight clonal complexes (CCs). Majority of the isolates were variants of CC97 (68.7%) and CC151 (25.1%). CC97 comprised of seven STs, of which two were new STs (ST3273, ST3274), while CC151 comprised of three STs of which ST3272 was identified for the first time. Several farms had more than one ST type that were either members of the same clonal complex or unrelated STs. On one farm, six different STs of both categories were seen over the years within the farm. It was observed that ST352 and ST151 were the two main clonal populations in cattle not only in Pennsylvania but also globally. Most isolates were susceptible to all the antibiotics evaluated. 6.7% of isolates showed resistance to vancomycin and penicillin. Two isolates of ST398 showed multidrug resistance (>3 antibiotics) against clindamycin, erythromycin, tetracycline, and penicillin. It was noted that 59 of 163 (36.2%) isolates encoded for enterotoxigenic genes. Enterotoxin genes seg/sei accounted for ~85% of enterotoxin positive isolates. Toxic shock syndrome gene tsst-1 alone was positive in two isolates (ST352, ST 2187). 97.5% of CC151 isolates were enterotoxin seg/sei positive. Most isolates were positive for lukED (95%) and lukAB (96.3%) leukotoxin genes. Bovine specific bi-component leucocidin lukMF' was present in 54% of isolates. A prominent observation of this study was the explicit association of lukMF' with lineages ST151 and ST352. In conclusion, the findings of the study, suggest that small number of S. aureus STs types (ST352, ST2187, ST3028, and ST151) are associated with majority of cases of bovine mastitis in Pennsylvania dairy farms. It was observed that one ST of S. aureus predominated in the herd and this ST can coexist with several other ST types of S. aureus strains. When STs were interpreted along with virulence, leucocidin genes and antimicrobial resistance, ST-variants allowed better interpretation of the S. aureus molecular epidemiologic findings specifically for tracing recurrence or persistence of infections in cow over time, among cows in the herd, and between herds in Pennsylvania.

Complete Genome Sequence of Mycobacterium orygis Strain 51145.

We report the complete 4,352,172-bp genome sequence of Mycobacterium orygis strain 51145 assembled into a single circular chromosome. Comparative genomic analyses with other lineages of the Mycobacterium tuberculosis complex can provide insights into the biology, evolution, and epidemiology of this important group of pathogenic mycobacteria.

Influenza C and D viral load in cattle correlates with bovine respiratory disease (BRD): Emerging role of orthomyxoviruses in the pathogenesis of BRD.

Bovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Orthomyxoviruses, influenza C virus (ICV) and influenza D virus (IDV) have recently been implicated to play a role in BRD. However, there are contradicting reports about the association of IDV and ICV to BRD. Using the largest cohort study (cattle, n = 599) to date we investigated the association of influenza viruses in cattle with BRD. Cattle were scored for respiratory symptoms and pooled nasal and pharyngeal swabs were tested for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, ICV and IDV by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, 2 logs higher IDV viral RNA in BRD-symptomatic cattle that are co-infected animals than those infected with IDV alone. Our results strongly suggest that ICV and IDV may be significant contributors to BRD.

Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study.

BACKGROUND: Zoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India. METHODS: We did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). FINDINGS: The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. INTERPRETATION: M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis. FUNDING: Bill & Melinda Gates Foundation, Canadian Institutes for Health Research.

NLRC5 Serves as a Pro-viral Factor During Influenza Virus Infection in Chicken Macrophages.

Avian influenza viruses (AIVs) cause major economic losses to the global poultry industry. Many host factors have been identified that act as regulators of the inflammatory response and virus replication in influenza A virus (IAV) infected cells including nucleotide-binding oligomerization domain (NOD) like receptor (NLR) family proteins. Evidence is emerging that NLRC5, the largest NLR member, is a regulator of host immune responses against invading pathogens including viruses; however, its role in the avian immune system and AIV pathogenesis has not been fully explored. In this study, we found that NLRC5 is activated by a range of low and highly pathogenic AIVs in primary chicken lung cells and a chicken macrophage cell line. Further, siRNA mediated NLRC5 knockdown in chicken macrophages resulted in a significant reduction in AIV replication which was associated with the upregulation of genes associated with activated NFκB signaling pathway. The knockdown of NLRC5 enhanced the expression of genes known to be associated with viral defense and decreased innate cytokine gene expression following AIV infection. Overall, our investigation strongly suggests that NLRC5 is a pro-viral factor during IAV infection in chicken and may contribute to pathogenesis through innate cytokine regulation. Further studies are warranted to investigate the IAV protein(s) that may regulate activation of NLRC5.

Iminosugars With Endoplasmic Reticulum α-Glucosidase Inhibitor Activity Inhibit ZIKV Replication and Reverse Cytopathogenicity in vitro.

Zika virus (ZIKV), a vector-borne virus of the family Flaviviridae, continues to spread and remains a significant global public health threat. Currently, there are no approved vaccines or antivirals against ZIKV. We investigated the anti-ZIKV ability of three iminosugars with endoplasmic reticulum α-glucosidase inhibitor (ER-AGI) activity, namely deoxynojirimycin (DNJ), castanospermine, and celgosivir. None of the three iminosugars showed any significant cytotoxicity in Vero or human microglia CHME3 cells when applied for 72 h at concentrations up to 100 µM. Iminosugar treatment of Vero or CHME3 cells prior to ZIKV infection resulted in significant inhibition of ZIKV replication over 48 h. Reduction in ZIKV replication in iminosugar-treated cells was not associated with any significant change in the expression levels of key antiviral genes. Following infection with three different strains of ZIKV, iminosugar-treated Vero or CHME3 cells showed no cell death, whereas vehicle-treated control cells exhibited 50-60% cell death at 72 h post-infection (hpi). While there was no significant difference in apoptosis between iminosugar-treated and control cells, iminosugar-treated cells exhibited a substantial reduction of necrosis at 72 hpi following ZIKV infection. In summary, iminosugars with ER-AGI activity inhibit ZIKV replication and significantly reduce necrosis without altering the antiviral gene expression and apoptosis of infected human cells. The results of this study strongly suggest that iminosugars are promising anti-ZIKV antiviral agents and such warrant further in vivo studies.

Whole-genome sequence analysis reveals unique SNP profiles to distinguish vaccine and wild-type strains of bovine herpesvirus-1 (BoHV-1).

Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and the association of MLVs with clinical disease in cattle.

Complete Genome Sequences of Three Related Avian Avulavirus 1 Isolates from Poultry Farmers in Pakistan.

Avian avulavirus 1 infects multiple avian hosts, and rare reports of human infection have been noted throughout the last century. Here, we report the complete genome sequences of three isolates of avulavirus 1 collected from poultry farmers in Pakistan exhibiting mild respiratory signs.