Evolution of protection after maternal immunization for respiratory syncytial virus in cotton rats

Maternal anti-respiratory syncytial virus (RSV) antibodies acquired by the fetus through the placenta protect neonates from RSV disease through the first weeks of life. In the cotton rat model of RSV infections, we previously reported that immunization of dams during pregnancy with virus-like particles assembled with mutation stabilized pre-fusion F protein as well as the wild type G protein resulted in robust protection of their offspring from RSV challenge. Here we describe the durability of those protective responses in dams, the durability of protection in offspring, and the transfer of that protection to offspring of two consecutive pregnancies without a second boost immunization. We report that four weeks after birth, offspring of the first pregnancy were significantly protected from RSV replication in both lungs and nasal tissues after RSV challenge, but protection was reduced in pups at 6 weeks after birth. However, the overall protection of offspring of the second pregnancy was considerably reduced, even at four weeks of age. This drop in protection occurred even though the levels of total anti-pre-F IgG and neutralizing antibody titers in dams remained at similar, high levels before and after the second pregnancy. The results are consistent with an evolution of antibody properties in dams to populations less efficiently transferred to offspring or the less efficient transfer of antibodies in elderly dams.

Evolution of protection after maternal immunization for respiratory syncytial virus in cotton rats

Maternal anti-respiratory syncytial virus (RSV) antibodies acquired by the fetus through the placenta protect neonates from RSV disease through the first weeks of life. In the cotton rat model of RSV infections, we previously reported that immunization of dams during pregnancy with virus-like particles assembled with mutation stabilized pre-fusion F protein as well as the wild type G protein resulted in robust protection of their offspring from RSV challenge (Blanco, et al Journal of Virology 93: e00914-19, https://doi.org/10.1128/JVI.00914-19). Here we describe the durability of those protective responses in dams, the durability of protection in offspring, and the transfer of that protection to offspring of two consecutive pregnancies without a second boost immunization. We report that four weeks after birth, offspring of the first pregnancy were significantly protected from RSV replication in both lungs and nasal tissues after RSV challenge, but protection was reduced in pups at 6 weeks after birth. However, the overall protection of offspring of the second pregnancy was considerably reduced, even at four weeks of age. This drop in protection occurred even though the levels of total anti-pre-F IgG and neutralizing antibody titers in dams remained at similar, high levels before and after the second pregnancy. The results are consistent with an evolution of antibody properties in dams to populations less efficiently transferred to offspring or the less efficient transfer of antibodies in elderly dams.

HUMAN ADENOVIRUS TYPE 4 COMPRISES TWO MAJOR PHYLOGROUPS WITH DISTINCT REPLICATIVE FITNESS AND VIRULENCE PHENOTYPES

Human adenovirus type 4 (HAdV-E4) is the only type (and serotype) classified within species Human mastadenovirus E that has been isolated from a human host to the present. Recent phylogenetic analysis of whole genome sequences of strains representing the spectrum of intratypic genetic diversity described to date identified two major evolutionary lineages designated phylogroups (PG) I, and II, and validated the early clustering of HAdV-E4 genomic variants into two major groups by low resolution restriction fragment length polymorphism analysis. In this study we expanded our original analysis of intra- and inter-PG genetic variability, and used a panel of viruses representative of the spectrum of genetic diversity described for HAdV-E4 to examine the magnitude of inter- and intra-PG phenotypic diversity using an array of cell-based assays and a cotton rat model of HAdV respiratory infection. Our proteotyping of HAdV-E strains using concatenated protein sequences in selected coding regions including E1A, E1B-19K and -55K, DNA polymerase, L4-100K, various E3 proteins, and E4-34K confirmed that the two clades encode distinct variants/proteotypes at most of these loci. Our in vitro and in vivo studies demonstrated that PG I and PG II differ in their growth, spread, and cell killing phenotypes in cell culture and in their pulmonary pathogenic phenotypes. Surprisingly, the differences in replicative fitness documented in vitro between PGs did not correlate with the differences in virulence observed in the cotton rat model. This body of work is the first reporting phenotypic correlates of naturally occurring intratypic genetic variability for HAdV-E4. IMPORTANCE Human adenovirus type 4 (HAdV-E4) is a prevalent causative agent of acute respiratory illness of variable severity and of conjunctivitis and comprises two major phylogroups that carry distinct coding variations in proteins involved in viral replication and modulation of host responses to infection. Our data show that PG I and PG II are intrinsically different regarding their ability to grow and spread in culture and to cause pulmonary disease in cotton rats. This is the first report of phenotypic divergence among naturally occurring known genetic variants of a HAdV type of medical importance. This research reveals readily detectable phenotypic differences between strains representing phylogroups I and II, and it introduces a unique experimental system for the elucidation of the genetic basis of adenovirus fitness and virulence and thus for increasing our understanding of the implications of intratypic genetic diversity in the presentation and course of HAdV-E4-associated disease.

Comparison of Ovarian Morphology and Follicular Disturbances between Two Inbred Strains of Cotton Rats (Sigmodon hispidus)

Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.

CX3CR1 is a Receptor for Human Respiratory Syncytial Virus in Cotton Rats

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo , we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wildtype virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs four days post-infection. In contrast, growth of RSV was not affected after pre-incubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day four post-infection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats, and in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. Importance The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals either directed against the receptor molecule on the cell or the receptor-binding protein of the virus. Amongst a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here we report that the cotton rat CX3CR1 which is similar to the human molecule acts as receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.

Microbial community structure and composition is associated with host species and sex in Sigmodon cotton rats

Background The cotton rat (genus Sigmodon) is an essential small animal model for the study of human infectious disease and viral therapeutic development. However, the impact of the host microbiome on infection outcomes has not been explored in this model, partly due to the lack of a comprehensive characterization of microbial communities across different cotton rat species. Understanding the dynamics of their microbiome could significantly help to better understand its role when modeling viral infections in this animal model. Results We examined the bacterial communities of the gut and three external sites (skin, ear, and nose) of two inbred species of cotton rats commonly used in research (S. hispidus and S. fulviventer) by using 16S rRNA gene sequencing, constituting the first comprehensive characterization of the cotton rat microbiome. We showed that S. fulviventer maintained higher alpha diversity and richness than S. hispidus at external sites (skin, ear, nose), but there were no differentially abundant genera. However, S. fulviventer and S. hispidus had distinct fecal microbiomes composed of several significantly differentially abundant genera. Whole metagenomic shotgun sequencing of fecal samples identified species-level differences between S. hispidus and S. fulviventer, as well as different metabolic pathway functions as a result of differential host microbiome contributions. Furthermore, the microbiome composition of the external sites showed significant sex-based differences while fecal communities were not largely different. Conclusions Our study shows that host genetic background potentially exerts homeostatic pressures, resulting in distinct microbiomes for two different inbred cotton rat species. Because of the numerous studies that have uncovered strong relationships between host microbiome, viral infection outcomes, and immune responses, our findings represent a strong contribution for understanding the impact of different microbial communities on viral pathogenesis. Furthermore, we provide novel cotton rat microbiome data as a springboard to uncover the full therapeutic potential of the microbiome against viral infections.

Small mammals killed in discarded bottles along roadsides in central Nebraska

Littered debris along roadways traps and kills small vertebrates. In Nebraska, at least 20 species of small mammals are small enough to enter openings of discarded bottles and cans. We surveyed roadsides for littered bottles and cans containing vertebrate remains in central parts of the state. We observed 459 bottles and 278 aluminum cans along 17.6 km of roadsides in Nebraska. Littered bottles contained 41 vertebrate remains representing nine taxa of small mammals. Glass bottles contained the majority of individuals, plastic bottles had a few individuals, and no vertebrate remains were detected in aluminum cans. Harvest mice (Reithrodontomys spp.) were the most frequently observed taxa trapped in bottles, followed by short-tailed shrews (Blarina spp.). Remains of a juvenile Hispid Cotton Rat (Sigmodon hispidus) in a glass bottle suggest that more species are at risk when individuals are young. Bottles with openings higher than their base contained more vertebrates (21.1%) than those lying flat (3.8%) or those with openings facing downward (1.2%). Overall, about 5.7% of bottles had vertebrate remains in Nebraska, which is slightly greater than studies in the eastern United States. Densities of littered bottles were much lower along roads in Nebraska than roadways in the eastern United States, resulting in fewer mortalities per km. Human population density appears associated with littered debris, thus numbers of vertebrates killed in these roadside hazards likely is greater in eastern Nebraska and near population centers where most of the state’s population reside. In Nebraska, six species of small mammals are species of conservation need, and thus, at risk from littered debris. Reduction and removal of litter along roadsides not only is aesthetically pleasing, but it can also reduce mortality of small mammals and other animals, such as small insects.

Immunogenicity and inflammatory properties of respiratory syncytial virus attachment G protein in cotton rats

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide. The attachment (G) protein of RSV is synthesized by infected cells in both a membrane bound (mG) and secreted form (sG) and uses a CX3C motif for binding to its cellular receptor. Cell culture and mouse studies suggest that the G protein mimics the cytokine CX3CL1 by binding to CX3CR1 on immune cells, which is thought to cause increased pulmonary inflammation in vivo. However, because these studies have used RSV lacking its G protein gene or blockade of the G protein with a G protein specific monoclonal antibody, the observed reduction in inflammation may be due to reduced virus replication and spread, and not to a direct role for G protein as a viral chemokine. In order to more directly determine the influence of the soluble and the membrane-bound forms of G protein on the immune system independent of its attachment function for the virion, we expressed the G protein in cotton rat lungs using adeno-associated virus (AAV), a vector system which does not itself induce inflammation. We found no increase in pulmonary inflammation as determined by histology and bronchoalveolar lavage after inoculation of AAVs expressing the membrane bound G protein, the secreted G protein or the complete G protein gene which expresses both forms. The long-term low-level expression of AAV-G did, however, result in the induction of non-neutralizing antibodies, CD8 T cells and partial protection from challenge with RSV. Complete protection was accomplished through co-immunization with AAV-G and an AAV expressing cotton rat interferon α.

Unique histological features of the tail skin of cotton rat (Sigmodon hispidus) related to caudal autotomy

ABSTRACTCaudal autotomy in rodents is an evolutionarily acquired phenomenon enabling escape from predators, by discarding the tail skin after traumatic injuries. The histological mechanisms underlying caudal autotomy seem to differ among species. Cotton rats (Sigmodon hispidus), which are important laboratory rodents for human infectious diseases, possess a fragile tail. In this study, we compared the tail histology of cotton rats with that of laboratory rats (Rattus norvegicus), which have no fragility on their tail, to elucidate the process of rodent caudal autotomy. First, the cotton rats developed a false autotomy characterized by loss of the tail sheath with the caudal vertebrae remaining without tail regeneration. Second, we found the fracture plane was continuous from the interscale of the tail epidermis to the dermis, which was lined with an alignment of E-cadherin+ cells. Third, we found an obvious cleavage plane between the dermis and subjacent tissues of the cotton-rat tail, where the subcutis was composed of looser, finer, and fragmented collagen fibers compared with those of the rat. Additionally, the cotton-rat tail was easily torn, with minimum bleeding. The median coccygeal artery of the cotton rat had a thick smooth muscle layer, and its lumen was filled with the peeled intima with fibrin coagulation, which might be associated with reduced bleeding following caudal autotomy. Taken together, we reveal the unique histological features of the tail relating to the caudal autotomy process in the cotton rat, and provide novel insights to help clarify the rodent caudal autotomy mechanism.