Comparative Pathogenesis of Bovine and Porcine Respiratory Coronaviruses in the Animal Host Species and SARS-CoV-2 in Humans.

Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).

Interaction of coronavirus nucleocapsid protein with the 5'- and 3'-ends of the coronavirus genome is involved in genome circularization and negative-strand RNA synthesis.

Synthesis of the negative-strand ((-)-strand) counterpart is the first step of coronavirus (CoV) replication; however, the detailed mechanism of the early event and the factors involved remain to be determined. Here, using bovine coronavirus (BCoV)-defective interfering (DI) RNA, we showed that (a) a poly(A) tail with a length of 15 nucleotides (nt) was sufficient to initiate efficient (-)-strand RNA synthesis and (b) substitution of the poly(A) tail with poly(U), (C) or (G) only slightly decreased the efficiency of (-)-strand synthesis. The findings indicate that in addition to the poly(A) tail, other factors acting in trans may also participate in (-)-strand synthesis. The BCoV nucleocapsid (N) protein, an RNA-binding protein, was therefore tested as a candidate. Based on dissociation constant (Kd ) values, it was found that the binding affinity between N protein, but not poly(A)-binding protein, and the 3'-terminal 55 nt plus a poly(A), poly(U), poly(C) or poly(G) tail correlates with the efficiency of (-)-strand synthesis. Such an association was also evidenced by the binding affinity between the N protein and 5'- and 3'-terminal cis-acting elements important for (-)-strand synthesis. Further analysis demonstrated that N protein can act as a bridge to facilitate interaction between the 5'- and 3'-ends of the CoV genome, leading to circularization of the genome. Together, the current study extends our understanding of the mechanism of CoV (-)-strand RNA synthesis through involvement of N protein and genome circularization and thus may explain why the addition of N protein in trans is required for efficient CoV replication.

Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 104 to 1.7 × 106 throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.

[The specific features of the reproduction of the bovine coronavirus strain ARRIAH in the continuous RBT cell culture].

Cultivation of the bovine coronavirus strain ARRHIAH in the continuous RBT versus MDBK and Taurus cell cultures made it possible to reduce the time taken to reproduce the virus and to increase its hemagglutination titer. The RBT cell line is suitable for determining the infectious activity of the coronavirus reproduced in the other cell cultures. Optimization of a manufacturing process for diagnostic agents and vaccines has been achieved while applying a roll tube method for both RBT cell cultivation and coronavirus reproduction.

Fatal winter dysentery with severe anemia in an adult cow.

An adult dairy cow fatally affected with winter dysentery was investigated pathologically and virologically. The cow had severe anemia and diarrhea with massive blood. Pathologically, the loss of surface epithelial cells and necrosis of crypt epithelial cells in the large intestine were observed. Bovine coronavirus (BCV) antigen was observed in necrotic crypt epithelial cells of the large intestine. Virus particles were found in the necrotic epithelial cells of the large intestine. Virologically, BCV was isolated from the feces of the dead cow. The dead cow had no serum antibody against BCV although the co-habitants did. These suggest that severe infection of BCV in the cow without the BCV antibody accompanied by severe hemorrhagic anemia resulted in the cow's death.

[Bovine coronavirus reproduction in the continuous cell cultures at a temperature of 34 degrees C].

Cultivation of bovine coronavirus at 34 degrees C showed the relationship of the degree of its reproduction to the cellular system. The possibility of obtaining the double yield of bovine coronavirus was demonstrated in MDBK and Vero cell cultures at 34 degrees C.

Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation.

The genetic diversity of 2 pairs (AH65 and AH187) of wild type bovine coronaviruses (BCoV) sequenced directly from nasal (respiratory) and rectal (enteric) swabs of two feedlot calves with respiratory and enteric symptoms [Hasoksuz, M., Sreevatsan, S., Cho, K.O., Hoet, A.E., Saif, L.J., 2002b. Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res. 84 (1-2), 101-109.]. was analyzed. Sequence analysis of the complete genomes revealed differences at 123 and 149 nucleotides (nt) throughout the entire genome between the respiratory and enteric strains for samples AH65 and AH187, respectively, indicating the presence of intra-host BCoV quasispecies. In addition, significant numbers of sequence ambiguities were found in the genomes of some BCoV-R and BCoV-E strains, suggesting intra-isolate quasispecies. The tissue culture (TC) passaged counterparts of AH65 respiratory BCoV (AH65-R-TC) and enteric BCoV (AH65-E-TC) were also sequenced after 14 and 15 passages and 1 plaque purification in human rectal tumor cells (HRT-18), respectively. Compared to the parental wild type strains, tissue culture passage generated 104 nt changes in the AH65-E-TC isolate but only 8 nt changes in the AH65-R-TC isolate. Particularly noteworthy, the majority of nucleotide changes in the AH65-E-TC isolate occurred at the identical positions as the mutations occurring in the AH65-R strain from the same animal. These data suggest that BCoV evolves through quasispecies development, and that enteric BCoV isolates are more prone to genetic changes and may mutate to resemble respiratory BCoV strains after tissue culture passage.

Evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle.

OBJECTIVE: To assess the relationship between shedding of bovine coronavirus (BCV) via the respiratory tract and enteric routes and the association with weight gain in feedlot cattle. ANIMALS: 56 crossbred steers. PROCEDURES: Paired fecal samples and nasal swab specimens were obtained and were tested for BCV, using antigen-capture ELISA. Paired serum samples obtained were tested for antibodies to BCV, using antibody-detection ELISA. Information was collected on weight gain, clinical signs, and treatments for enteric and respiratory tract disease during the study period. RESULTS: Number of samples positive for bovine respiratory coronavirus (BRCV) or bovine enteric coro navirus (BECV) was 37/224 (17%) and 48/223 (22%), respectively. Some cattle (25/46, 45%) shed BECV and BRCV. There were 25/29 (86%) cattle positive for BECV that shed BRCV, but only 1/27 (4%) cattle negative to BECV shed BRCV. Twenty-seven of 48 (56%) paired nasal swab specimens and fecal samples positive for BECV were positive for BRCV. In contrast, only 10/175 (6%) paired nasal swab specimens and fecal samples negative for BECV were positive for BRCV. Only shedding of BECV was associated with significantly reduced weight gain. Seroconversion to BCV during the 21 days after arrival was detected in 95% of the cattle tested. CONCLUSIONS AND CLINICAL IMPLICATIONS: Feedlot cattle infected with BCV after transport shed BCV from the respiratory tract and in the feces. Fecal shedding of BCV was associated with significantly reduced weight gain. Developing appropriate control measures for BCV infections could help reduce the decreased weight gain observed among infected feedlot cattle.

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves.

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks. In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.

Structural and functional analysis of the S proteins of two human coronavirus OC43 strains adapted to growth in different cells.

The receptor-binding activity of strain CU (grown in MDCK I cells) and of strain VA (adapted to Vero cells) of human coronavirus OC43 was analyzed and compared with the binding activity of bovine coronavirus (BCV) and of the OC43 strain provided by the American Type Culture Collection (AT). Results obtained with resialylated erythrocytes indicated that the ability of the viruses to recognize 9-O-acetylated sialic acid in an alpha 2,6-linkage decreased in the following order: AT > CU > BCV > VA. Only minor differences were observed with respect to the alpha 2,3-linkage. The amino acid sequence of the S protein of strain CU and VA was derived from the nucleotide sequence of the cloned gene. Strain VA differed from strain CU in 34 positions, 18 in the S1 and 16 in the S2 subunit.

Evaluation of shell vial cell culture technique for the detection of bovine coronavirus.

The effect of blind passage and centrifugation on the isolation of bovine coronavirus in human rectal tumor cells cultured in shell vials was investigated. A total of 68 fecal samples known to be positive for bovine coronavirus by transmission electron microscopic (TEM) examination were used. The samples were centrifuged onto human rectal tumor cell monolayers and incubated in the presence of trypsin. The growth of bovine coronavirus in infected cells was demonstrated by fluorescent antibody staining, and the extracellular virus was detected and confirmed by hemagglutination and hemagglutination-inhibition tests, respectively. Of the 68 TEM-positive samples, 51 (75%), 58 (85%), and 61 (90%) grew in shell vial cell cultures at first, second, and third passages, respectively. Of the 51 cultures positive on first passage, 19 were examined by TEM; 18 of these were positive for bovine coronavirus. The shell vial technique was also compared with direct detection of bovine coronavirus by staining cryostat sections of infected tissues in a direct fluorescent antibody assay. The results of direct fluorescent antibody assay were available for 54 of the 68 samples, of which 53 (98%) and 43 (80%) were positive by shell vial technique and direct fluorescent antibody assay, respectively. For identification of bovine coronavirus, shell vials using human rectal tumor cells in the presence of trypsin is more sensitive than direct fluorescent antibody assay but is relatively less sensitive than transmission electron microscopy.

Role of subgenomic minus-strand RNA in coronavirus replication.

Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgenomic mRNAs) probably function in mRNA amplification by serving as templates for transcription from internal (intergenic) promoters, rather than by faithful (full-length) mRNA replication.