John D. Neill

Inter- and Intragenus Structural Variations in Caliciviruses and Their Functional Implications

ABSTRACT The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.

Maternal Antibody Blocks Humoral but Not T Cell Responses to BVDV

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.

Prevalence and Antigenic Differences Observed between Bovine Viral Diarrhea Virus Subgenotypes Isolated from Cattle in Australia and Feedlots in the Southwestern United States

Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genus, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). Further phylogenetic analysis has revealed subgenotype groupings within the 2 types. Thus far, 12 BVDV-1 subgenotypes (a–l) and 2 BVDV-2 subgenotypes (a and b) have been identified. The purpose of the current study was to determine the prevalence of BVDV subgenotypes in the United States and Australia and to determine if there are detectable antigenic differences between the prevalent subgenotypes. To determine prevalence, phylogenetic analysis was performed on 2 blinded panels of isolates consisting of 351 viral isolates provided by the Elizabeth Macarthur Laboratory, New South Wales, and 514 viral isolates provided by Oklahoma State University. Differences were observed in the prevalence of BVDV subgenotypes between the United States (BVDV-1b most prevalent subgenotype) and Australia (BVDV-1c most prevalent subgenotype). To examine antigenic differences between the subgenotypes identified in samples from the United States and Australia, polyclonal antisera was produced in goats by exposing them at 3-week intervals to 2 noncytopathic and 1 cytopathic strain of either BVDV-1a, BVDV-1b, BVDV-1c, BVDV-2a, or Border disease virus (BDV). Virus neutralization (VN) assays were then performed against 3 viruses from each of the 5 subgenotypes. Comparison of VN results suggests that there are antigenic differences between BVDV strains belonging to different subgenotypes. The present study establishes a foundation for further studies examining whether vaccine protection can be improved by basing vaccines on the BVDV subgenotypes prevalent in the region in which the vaccine is to be used.

Distribution of Viral Antigen and Development of Lesions after Experimental Infection of Calves with a BVDV 2 Strain of Low Virulence

To examine the virus-host interaction in subclinical bovine viral diarrhea virus (BVDV) infections, the spread of a BVDV 2 strain of low virulence to different organs and the development of lesions were investigated. Eight colostrum-deprived, clinically healthy, 2–3-month-old calves were intranasally inoculated with 106 tissue culture infective dose of the naturally occurring BVDV 2 strain 28508–5 of low virulence, and 2 served as controls. Two calves each were euthanized at days 3, 6, 9, and 13 postinoculation (pi). Representative tissues were processed for histology and immunohistology. Signs of overt clinical disease were absent. However, a mild temperature elevation at days 7 or 8 pi and a moderate decrease of circulating lymphocytes occurred in all inoculated calves. The BVDV antigen was detected at day 3 pi in several lymphoid tissues. At day 6 pi, BVDV antigen was found widespread in lymphoid tissues and multifocally in intestinal epithelial cells but was associated with no or subtle lesions only. At day 9 pi, much less BVDV antigen was detectable, but there was severe depletion of lymphoid tissues. At day 13 pi, BVDV antigen had been cleared from most lymphoid tissues that were at variable phases of depletion and recovery. In conclusion, the BVDV strain of low virulence spread to lymphoid tissues and intestinal epithelial cells but was rapidly eliminated. Transient depletion of lymphoid tissues was followed by recovery.

Distribution of Viral Antigen and Tissue Lesions in Persistent and Acute Infection with the Homologous Strain of Noncytopathic Bovine Viral Diarrhea Virus

Viral distribution and lesions were compared between calves born with persistent infection (PI) and calves acutely infected with the same bovine viral diarrhea virus (BVDV) isolate. Two PI calves from 1 dairy herd were necropsied. The PI viruses from these calves were isolated, characterized by sequencing, and found to be identical. This virus strain, designated BVDV2-RS886, was characterized as a noncytopathic (ncp) type 2 BVDV. To establish acute infections, BVDV2-RS886 was used to inoculate clinically healthy, seronegative calves which were 3 weeks to 3 months old. Nine calves received 106–107 tissue culture infective dose of BVDV2-RS886 intranasally. Four additional age-matched animals served as noninfected controls. Infected calves were necropsied at 3, 6, 9, or 13 days postinoculation (dpi). Viral antigen was detected by immunohistochemistry in frozen sections, and lesions were evaluated in hematoxylin eosin–stained paraplast sections. In the PI calves, a wide distribution of viral antigen was found in all tissues and was not associated with lesions. In the acutely infected calves, viral antigen was widespread in lymphoid tissues at 6 dpi but had been mostly eliminated at 9 and 13 dpi. Depletion of lymphoid tissues was seen at 6, 9, and 13 dpi and repopulation at 9 and 13 dpi. In 1 of the calves at 13 dpi, severe arteritis was present in lymph nodes and myocardium. This comparison shows that an ncp BVDV strain that causes no lesions in PI animals is able to induce marked depletion of lymphoid tissues in calves with acute infection. Therefore, the failure to eliminate PI cattle from a herd causes problems not only in pregnant cattle but may also affect other age groups.

Evaluation of the reverse transcription-polymerase chain reaction/probe test of serum samples and immunohistochemistry of skin sections for detection of acute bovine viral diarrhea infections.

Bovine viral diarrhea viruses (BVDV) cause both acute and persistent infections. While diagnostic tests have been designed to detect animals persistently infected (PI) with BVDV, the reliability of these tests in detecting acute BVDV infections is not known. It is also possible that acute BVDV infections may be confused with persistent infections in surveys for PI animals. In this study, 2 tests presently in use in diagnostic laboratories to test for PI animals, polymerase chain reaction amplification followed by probe hybridization (RT-PCR/probe) of serum samples and immunohistochemical detection of viral antigen in skin biopsies (IHC), were evaluated for their ability to detect acute BVDV infections. Sixteen colostrum-deprived, BVDV-free, and BVDV-antibody-free calves were infected with 6 different BVDV strains. Clinical signs, seroconversion, and virus isolation indicated that inoculated animals did replicate virus. Virus could be detected in 19% (3/16) of acutely infected animals by the RT-PCR/probe technique. No acutely infected animals were positive by IHC.

Induction of T lymphocytes specific for bovine viral diarrhea virus in calves with maternal antibody.

Passive antibody to bovine viral diarrhea virus (BVDV) acquired through colostrum intake may interfere with the development of a protective immune response by calves to this virus. The objective of this study was to determine if calves, with a high level of maternal antibody to bovine viral diarrhea virus (BVDV), develop CD4(+), CD8(+), or gammadelta T lymphocyte responses to BVDV in the absence of a measurable humoral immune response. Colostrum or milk replacer fed calves were challenged with virulent BVDV at 2-5 weeks of age and/or after maternal antibody had waned. Calves exposed to BVDV while passive antibody levels were high did not mount a measurable humoral immune response to BVDV. However, compared to nonexposed animals, these animals had CD4(+), CD8(+), and gammadelta T lymphocytes that were activated by BVDV after exposure to in vitro BVDV. The production of IFNgamma by lymphocytes after in vitro BVDV exposure was also much greater in lymphocytes from calves exposed to BVDV in the presence of maternal antibody compared to the nonexposed calves. These data indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4(+), CD8(+), and gammadelta T lymphocytes in the absence of an active antibody response. A manuscript presented separately demonstrates that the calves with T lymphocytes specific for BVDV in this study were also protected from virulent BVDV genotype 2 challenge after maternal antibody became undetectable.

Simultaneous rapid sequencing of multiple RNA virus genomes.

Abstract Comparing sequences of archived viruses collected over many years to the present allows the study of viral evolution and contributes to the design of new vaccines. However, the difficulty, time and expense of generating full-length sequences individually from each archived sample have hampered these studies. Next generation sequencing technologies have been utilized for analysis of clinical and environmental samples to identify viral pathogens that may be present. This has led to the discovery of many new, uncharacterized viruses from a number of viral families. Use of these sequencing technologies would be advantageous in examining viral evolution. In this study, a sequencing procedure was used to sequence simultaneously and rapidly multiple archived samples using a single standard protocol. This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. This conferred sequence independence by random priming both first and second strand cDNA synthesis. Viral stocks were treated with a nuclease cocktail to reduce the presence of host nucleic acids. Viral RNA was extracted, followed by single tube random-primed double-stranded cDNA synthesis. The resultant cDNAs were amplified by primer-specific PCR, pooled, size fractionated and sequenced on the Ion Torrent PGM platform. The individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. This procedure consistently resulted in near full length, if not full-length, genomic sequences and was used to sequence multiple bovine pestivirus and coronavirus isolates simultaneously.

Comparison of acute infection of calves exposed to a high-virulence or low-virulence bovine viral diarrhea virus or a HoBi-like virus

OBJECTIVE To compare acute infection of cattle exposed to a high-virulence (HV) bovine viral diarrhea virus (BVDV), low-virulence (LV) BVDV, or HoBi-like virus. ANIMALS 24 Holstein bull calves. PROCEDURES Colostrum-deprived 2- to 4-week-old calves, free of BVDV antigen and antibodies, were allocated into 4 groups (6 calves/group). Calves in 3 groups were exposed to an LV BVDV strain (BVDV2-RS886), an HV BVDV strain (BVDV2-1373), or a HoBi-like virus (D32/00 HoBi), whereas calves in the fourth group were not exposed to a virus but were cohoused with calves exposed to the HoBi-like virus. Circulating WBCs, platelets, rectal temperature, and presence of virus in the blood were monitored. RESULTS Infection of calves with any of the 3 viruses resulted in reduced numbers of circulating WBCs. Pyrexia was detected in all calves exposed to HV BVDV or LV BVDV but in only 3 of 6 calves exposed to the HoBi-like virus. Diarrhea was observed in 0 of 6 calves exposed to the HoBi-like virus, 2 of 6 calves exposed to the LV BVDV, and 6 of 6 calves exposed to the HV BVDV. The HoBi-like virus was transmitted from acutely infected calves to naïve cohorts. CONCLUSIONS AND CLINICAL RELEVANCE The HoBi-like viruses are an emerging species of pestivirus isolated from water buffalo and cattle in South America, Southeast Asia, and Europe but not from cattle in the United States. Understanding the clinical course of disease caused by HoBi-like pestiviruses will be important for the design of surveillance programs for the United States.

Detection and Characterization of Genetic Recombination in Cytopathic Type 2 Bovine Viral Diarrhea Viruses

ABSTRACT In cytopathic bovine viral diarrhea virus genotype 1 (BVDV1) isolates, insertions are reported at position A (amino acid [aa] 1535) and position B (aa 1589). Insertions at position B predominate. In this survey it was found that in BVDV2, insertions at position A predominate. Possible reasons for this difference in relative frequency are discussed.