Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia.

Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::blaCTX-M-2) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.

Efficacy of prepartum vaccination against neonatal calf diarrhea in Nelore dams as a prevention measure.

BACKGROUND: Neonatal calf diarrhea (NCD) is the leading cause of calf morbidity and mortality in beef cattle. Cow's vaccination in last stage of pregnancy is one of the most important measures to mitigate the risk of NCD outbreaks. The aim of this study was to evaluate the efficacy of prepartum single dose vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore dams and offspring. A total of 117 pregnant cows (n = 81) and heifers (n = 36) were distributed in two groups, vaccinated (VAC: cows = 40; heifers = 19) and non-vaccinated (NVAC: cows = 41; heifers = 17). Vaccination occurred between 60 to 50 days before the expected calving date with a single dose of a water-in-oil (W/O) vaccine, and NVAC group received a dose of saline solution 0.9%. Blood samples were collected before vaccination and 30 days after to evaluate the antibody (Ab) response. Specific IgG1 Abs against BoRVA and BCoV were measured by using an Enzyme Linked Immuno Sorbent Assay (ELISA). Calves' births were monitored, and the transference of passive immunity was evaluated. Diarrhea was monitored in the first 30 days of age, and fecal samples were collected for identification of the etiological agent. RESULTS: Higher titers of IgG1 Ab against BoRVA and BCoV was observed in the VAC group than NVAC group in the cow (P < 0.0001) and total dams categories (P < 0.0001). The titer of specific IgG1 Abs in the calves' serum reflected the dams response, observing higher IgG1 Ab titers for BoRVA (P < 0.0016) and BCoV (P < 0.0095) in the offspring born to VAC cows and higher IgG1 Ab titers for BoRVA(P < 0.0171) and BCoV (P < 0.0200) in the offspring born to VAC total dams. The general incidence of diarrhea observed was 18.6% (11/59) and 29.3% (17/58) in the calves born to the VAC and NVAC group, respectively. CONCLUSIONS: Prepartum vaccination with a single dose of the vaccine tested increased the titers of IgG1 Ab against BCoV and BoRVA, and it could be used as a preventive strategy to decrease the NCD occurrence in Nelore calves.

Molecular characterization of group A rotavirus strains detected in alpacas (Vicugna pacos) from Peru.

The alpaca is a very important social and economic resource for the production of fibre and meat for Andean communities. Peru is the main producer of alpacas. Group A rotavirus (RVA) has been sporadically detected in alpacas. In this study, a total of 1423 faecal samples from alpacas from different locations of the Puno department in Peru were collected and analysed by an antigen-capture ELISA in order to detect RVA. Four per cent of the samples were RVA-positive (57/1423). The genotype constellation of three selected alpaca RVA strains were G3/8 P[1/14]-I2-R2/5-C2/3-M2/3-A17-N2/3-T6-E3-H3. Two of the analysed strains presented a bovine-like genotype constellation, whereas the third strain presented six segments belonging to the AU-1-like genogroup (G3, M3, C3, N3, T3 and E3), suggesting reassorting events. Monitoring of the sanitary health of juvenile alpacas is essential to reduce the rates of neonatal mortality and for the development of preventive health strategies.

Development of a platform process for the production and purification of single-domain antibodies.

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus.

BACKGROUND: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. RESULTS: We describe a holistic approach for the molecular design of recombinant protein antigens-considering both their manufacturability and antigenicity-informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. CONCLUSIONS: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

A new passive immune strategy based on IgY antibodies as a key element to control neonatal calf diarrhea in dairy farms.

BACKGROUND: Neonatal diarrhea remains one of the main causes of morbi-mortality in dairy calves under artificial rearing. It is often caused by infectious agents of viral, bacterial, or parasitic origin. Cows vaccination and colostrum intake by calves during the first 6 h of life are critical strategies to prevent severe diarrhea but these are still insufficient. Here we report the field evaluation of a product based on IgY antibodies against group A rotavirus (RVA), coronavirus (CoV), enterotoxigenic Escherichia coli, and Salmonella sp. This product, named IgY DNT, has been designed as a complementary passive immunization strategy to prevent neonatal calf diarrhea. The quality of the product depends on the titers of specific IgY antibodies to each antigen evaluated by ELISA. In the case of the viral antigens, ELISA antibody (Ab) titers are correlated with protection against infection in calves experimentally challenged with RVA and CoV (Bok M, et al., Passive immunity to control bovine coronavirus diarrhea in a dairy herd in Argentina, 2017), (Vega C, et al., Vet Immunol Immunopathol, 142:156-69, 2011), (Vega C, et al., Res Vet Sci, 103:1-10, 2015). To evaluate the efficiency in dairy farms, thirty newborn Holstein calves were randomly assigned to IgY DNT or control groups and treatment initiated after colostrum intake and gut closure. Calves in the IgY DNT group received 20 g of the oral passive treatment in 2 L of milk twice a day during the first 2 weeks of life. Animals were followed until 3 weeks of age and diarrhea due to natural exposure to infectious agents was recorded during all the experimental time. RESULTS: Results demonstrate that the oral administration of IgY DNT during the first 2 weeks of life to newborn calves caused a delay in diarrhea onset and significantly reduced its severity and duration compared with untreated calves. Animals treated with IgY DNT showed a trend towards a delay in RVA infection with significantly shorter duration and virus shedding compared to control calves. CONCLUSIONS: This indicates that IgY DNT is an effective product to complement current preventive strategies against neonatal calf diarrhea in dairy farms. Furthermore, to our knowledge, this is the only biological product available for the prevention of virus-associated neonatal calf diarrhea.

Dynamics of neutralizing antibodies against Bovine respiratory syncytial virus in a dairy herd from Santa Fe Province, Argentina.

Bovine respiratory syncytial virus (BRSV) is one of the most relevant agents responsible for respiratory disease in cattle from both dairy and beef farms. BRSV is spread by horizontal contact causing a constant presence of seropositive animals that favors viral circulation throughout the year. Moreover, reinfections with BRSV are frequent between animals regardless of their age as BRSV does not confer long-lasting protective immunity. Several studies have demonstrated the circulation of BRSV in cattle from different regions of the world; however, little is known about the dynamics of BRSV infection in cows before and after they begin lactation. The aim of this work was to study the dynamics of BRSV neutralizing antibodies from birth up to 36 months of age in a closed dairy herd of Argentina specifically around the lactation period. Passive maternal antibodies against BRSV started to decrease monthly and became almost undetectable at 8 months of age. We detected two potential infection points at months 11 and 27 after birth, in which 30% and 45% of the animals showed seroconversion, respectively. Specifically, an increase in the proportion of seropositive cows after the start of lactation suggests that they became reinfected around the time they began lactating. We demonstrate the importance of understanding BRSV dynamics in a closed dairy herd to review the vaccination schedule of the animals to achieve protection against BRSV infection.

El virus respiratorio sincitial bovino (Bovine respiratory syncytial virus, [BRSV]) es uno de los principales agentes responsables de la enfermedad respiratoria en bovinos, tanto de tambos como de cría. El virus se transmite horizontalmente y causa la presencia constante de animales seropositivos, lo cual favorece la circulación viral a lo largo del año. A su vez, las reinfecciones por BRSV son frecuentes entre animales independientemente de su edad, dado que el virus no confiere inmunidad protectora a largo plazo. Numerosos estudios han demostrado la circulación de BRSV en bovinos de diferentes regiones del mundo, sin embargo, poco se conoce acerca de la dinámica de infección en vacas antes y después del inicio de la fase de lactancia. El objetivo de este trabajo fue estudiar la dinámica de anticuerpos neutralizantes anti- BRSV en vacas lecheras desde el nacimiento hasta los 36 meses de vida en un tambo cerrado de Argentina, específicamente, en el período de lactancia. Los anticuerpos pasivos específicos para BRSV comenzaron a declinar mensualmente hasta ser casi indetectables a los 6 meses. Detectamos dos potenciales puntos de infección a los meses 11 y 27 luego del nacimiento, momentos en los que el 30 y el 45% de los animales mostraron seroconversión, respectivamente. El incremento en la proporción de vacas seropositivas luego del comienzo de la lactancia sugiere que estas se reinfectaron en el inicio de dicha etapa. Demostramos la importancia de entender la dinámica de circulación del BRSV en un tambo cerrado, a fin de revisar el esquema de vacunación de los animales para que estén protegidos frente a la posible infección por este virus.

Bovine coronavirus in Uruguay: genetic diversity, risk factors and transboundary introductions from neighboring countries.

Bovine coronavirus (BCoV) is a recognized cause of severe neonatal calf diarrhea, with a negative impact on animal welfare, leading to economic losses to the livestock industry. Cattle production is one of the most important economic sectors in Uruguay. The aim of this study was to determine the frequency of BCoV infections and their genetic diversity in Uruguayan calves and to describe the evolutionary history of the virus in South America. The overall detection rate of BCoV in Uruguay was 7.8% (64/824): 7.7% (60/782) in dairy cattle and 9.5% (4/42) in beef cattle. The detection rate of BCoV in samples from deceased and live calves was 10.0% (6/60) and 7.6% (58/763), respectively. Interestingly, there was a lower frequency of BCoV detection in calves born to vaccinated dams (3.3%, 8/240) than in calves born to unvaccinated dams (12.2%, 32/263) (OR: 4.02, 95%CI: 1.81-8.90; p = 0.00026). The frequency of BCoV detection was higher in colder months (11.8%, 44/373) than in warmer months (1.5%, 3/206) (OR: 9.05, 95%CI: 2.77-29.53, p = 0.000013). Uruguayan strains grouped together in two different lineages: one with Argentinean strains and the other with Brazilian strains. Both BCoV lineages were estimated to have entered Uruguay in 2013: one of them from Brazil (95%HPD interval: 2011-2014) and the other from Argentina (95%HPD interval: 2010-2014). The lineages differed by four amino acid changes, and both were divergent from the Mebus reference strain. Surveillance should be maintained to detect possible emerging strains that can clearly diverge at the antigenic level from vaccine strains.

First detection of Ostreid herpesvirus 1 in wild Crassostrea gigas in Argentina.

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus of the genus Ostreavirus (Malacoherpesviridae family, Herpesvirales order). Worldwide, OsHV-1 and its microvariants have been associated with increased mortality of Pacific oysters, Crassostrea gigas. Adult asymptomatic oysters also have shown a high prevalence of viral infection. As a consequence, surveillance is needed to better describe OsHV-1 diversity, pathogenicity, clinical signs, and geographical distribution. We examined Crassostrea gigas sampled in October 2017 from the inner zone of the Bahía Blanca Estuary, Argentina, and found that 8 of 30 specimens (26.7%) presented macroscopic lesions in mantle tissues. Histological analysis revealed abnormal presentation of mantle epithelial cells and connective tissues. Conventional and real-time PCR conducted on the oyster samples revealed 70% to be positive for presence of OsHV-1 DNA. The nucleotide sequence of the amplicon obtained from one sample using the primer pair IA1/IA2 (targeting ORF 42/43) was 99% identical to OsHV-1 reference as well as µVar strains B and A (KY271630, KY242785.1), sequenced from France and Ireland. This finding represents the first detection of OsHV-1 DNA in a wild population of C. gigas in Argentina in association with gross mantle lesions.

Passive immunity to control Bovine coronavirus diarrhea in a dairy herd in Argentina.

Bovine coronavirus (BCoV) is a viral enteric pathogen associated with calf diarrhea worldwide being, in Argentina, mostly detected in dairy husbandry systems. The aim of the present work was to study if maternal IgG1 antibodies (Abs) to BCoV acquired by colostrum intake modulate the development of BCoV infection in calves reared in a dairy farm in Argentina. Thirty Holstein calves were monitored during their first 60 days of age. Animals were classified into two groups depending on their initial BCoV IgG1 Ab titers. The "failure of passive transfer" (FPT) group had significantly lower IgG1 Abs to BCoV than the "acceptable passive transfer" (APT) group of calves (log10 1.98 vs. 3.38 respectively) (p<0.0001). These differences were also observed when the total protein levels in both groups were compared (p=0.0081). Moreover, 71% (5/7) of calves from the FPT group showed IgG1 seroconversion to BCoV compared to 29.4% (5/17) of animals from the APT group. Regarding viral circulation, BCoV was detected in 10% (3/30) of all calves and BCoV IgG1 Ab seroconversion was detected in 42% of the total animals showing that almost half of the calves were infected with BCoV. In conclusion, calves with high titers of specific BCoV IgG1 (≥1024) were mostly protected against viral infection, while animals with low titers of IgG1 (<1024) were mostly infected with BCoV. IgG1 Abs from colostrum origin are critical for prevention of BCoV infection.

In vitro comparison of acyclovir, ganciclovir and cidofovir against equid alphaherpesvirus 3 and evaluation of their efficacy against six field isolates.

Equid alphaherpesvirus 3 (EHV3) is the etiological agent of equine coital exanthema (ECE), which is a venereal, highly contagious disease, characterized by the formation of papules, vesicles, pustules and ulcers on the external genitalia of mares and stallions. EHV3 remains in a latent state after a successful infection and there are latently infected animals in which the virus is reactivated and generally re-excreted subclinically. There are no available vaccines for this condition and prevention is based on the clinical examination of mares prior to mating, which allows to segregate those showing clinical signs. As this approach does not eliminate the risk of contagion in stallions from subclinically infected mares, there is a need for a specific EHV3 treatment. Nowadays, there exist various antiviral compounds of proven effectiveness for other alphaherpesviruses affecting humans and animals. The aim of the present study was to compare the efficacy of three antiviral compounds, acyclovir, ganciclovir and cidofovir against EHV3 in vitro, and to assess their efficacy against six EHV3 Argentinian field isolates. To determine the efficacy of these compounds in vitro, three parameters were analyzed: reduction of plaque number, reduction of plaque size and reduction of viral production. Additionally, the effectiveness of the three compounds at an optimum concentration previously determined in this study was investigated for the EHV3 field isolates. Based on our results, ganciclovir was the most potent antiviral compound to reduce EHV3 replication in vitro and may thus be a valuable candidate for treatment and prevention of ECE in mares and stallions.

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects.

[Serological survey of antibodies against viral diseases of public health interest in llamas (Lama glama) from Jujuy province, Argentina]. / Relevamiento serológico de anticuerpos contra enfermedades virales de interés sanitario en llamas (Lama glama) de la provincia de Jujuy, Argentina.

Llama population from Argentina is mainly concentrated in the Andean Puna, Jujuy. Llamas represent an important economic resource for the Andean communities. The aim of this study was to investigate the prevalence of antibodies against viral antigens associated to viral diseases of economic impact (neonatal diarrhea, reproductive and respiratory syndromes). A total of 349 serum samples from adult llamas were analyzed. The obtained antibody prevalence was 100 % for Rotavirus A and 70 % for Bovine parainfluenza virus 3. In contrast, no reactors were detected to Bovine herpesvirus 1, Bovine viral diarrhea virus 1, Human influenza A virus (H1N1) and Equine influenza virus (H3N8). These results confirm the wide circulation of rotavirus and parainfluenza virus in Argentinean llamas and suggest that susceptibility to infection with bovine herpesvirus, pestivirus and influenza A viruses is low. This serologic survey provides novel information regarding the epidemiology of viral diseases affecting llamas from the Argentinean Andean Puna.

Equine Rotavirus A under the One Health Lens: Potential Impacts on Public Health.

Group A rotaviruses are a well-known cause of viral gastroenteritis in infants and children, as well as in many mammalian species and birds, affecting them at a young age. This group of viruses has a double-stranded, segmented RNA genome with high genetic diversity linked to point mutations, recombination, and, importantly, reassortment. While initial molecular investigations undertaken in the 1900s suggested host range restriction among group A rotaviruses based on the fact that different gene segments were distributed among different animal species, recent molecular surveillance and genome constellation genotyping studies conducted by the Rotavirus Classification Working Group (RCWG) have shown that animal rotaviruses serve as a source of diversification of human rotavirus A, highlighting their zoonotic potential. Rotaviruses occurring in various animal species have been linked with contributing genetic material to human rotaviruses, including horses, with the most recent identification of equine-like G3 rotavirus A infecting children. The goal of this article is to review relevant information related to rotavirus structure/genomic organization, epidemiology (with a focus on human and equine rotavirus A), evolution, inter-species transmission, and the potential zoonotic role of equine and other animal rotaviruses. Diagnostics, surveillance and the current status of human and livestock vaccines against RVA are also reviewed.

Serological Responses of Guinea Pigs and Heifers to Eight Different BoAHV-1 Vaccine Formulations.

Bovine alphaherpesvirus 1 (BoAHV-1) infection affects the production and reproductive performance of dairy and beef livestock, resulting in considerable economic losses. In addition to biosecurity measures, vaccination programs are effective strategies for controlling and preventing BoAHV-1 infection and transmission. We evaluated the serological immune response against BoAHV-1 induced by eight different formulations of commercial vaccines: three modified live vaccines and five killed vaccines containing BoAHV type 1 or types 1 and 5. In the first experiment, 50 BoAHV-1-seronegative guinea pigs were assigned to eight groups; each individual in the treatment groups received two doses (one-fifth of the bovine dose). The second experiment was conducted using 29 crossbred Holstein × Gir heifers in four groups of six to nine animals each. The serological immune response against BoAHV-1 was measured using virus neutralization and enzyme-linked immunosorbent assays to measure the total IgG against BoAHV. We evaluated the effects of the vaccine, time, and interaction of the vaccine and time on neutralizing antibodies against BoAHV-1. Killed vaccines produced low levels of antibodies against BoAHV-1, whereas modified live vaccines produced high levels of antibodies capable of providing neutralizing titers in the vaccinated animals, with the thermosensitive modified live vaccine showing the highest levels of antibodies.

mRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea.

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves.

Bovine Coronavirus (BCoV) is a major pathogen associated with neonatal calf diarrhea. Standard practice dictates that to prevent BCoV diarrhea, dams should be immunized in the last stage of pregnancy to increase BCoV-specific antibody (Ab) titers in serum and colostrum. For the prevention to be effective, calves need to suck maternal colostrum within the first six to twelve hours of life before gut closure to ensure a good level of passive immunity. The high rate of maternal Ab transfer failure resulting from this process posed the need to develop alternative local passive immunity strategies to strengthen the prevention and treatment of BCoV diarrhea. Immunoglobulin Y technology represents a promising tool to address this gap. In this study, 200 laying hens were immunized with BCoV to obtain spray-dried egg powder enriched in specific IgY Abs to BCoV on a large production scale. To ensure batch-to-batch product consistency, a potency assay was statistically validated. With a sample size of 241, the BCoV-specific IgY ELISA showed a sensitivity and specificity of 97.7% and 98.2%, respectively. ELISA IgY Abs to BCoV correlated with virus-neutralizing Ab titers (Pearson correlation, R2 = 0.92, p < 0.001). Most importantly, a pilot efficacy study in newborn calves showed a significant delay and shorter duration of BCoV-associated diarrhea and shedding in IgY-treated colostrum-deprived calves. Calves were treated with milk supplemented with egg powder (final IgY Ab titer to BCoV ELISA = 512; VN = 32) for 14 days as a passive treatment before a challenge with BCoV and were compared to calves fed milk with no supplementation. This is the first study with proof of efficacy of a product based on egg powder manufactured at a scale that successfully prevents BCoV-associated neonatal calf diarrhea.